Société Belge de Biochimie; Belgische Vereniging Voor Biochemie

Archives Internationales de Physiologie, de Biochimie et de Biophysique, 1991, 99, B23-B46

REVUE TRIMESTRIELLE ( I ) , MARS 1991 DRIEMAANDELIJKS TIJDSCHRIFT ('), MAART 1991

SOCIETE BELGE DE BIOCHIMIE BELGISCHE VERENIGING VOOR BIOCHE-

1. CONFI?RENCES

Thtme : Reversed genetics

P. RAEYMAEKERS (UIA)

E. BAKKER (Leiden)

Reversed genetics : from chromosomes to genes.

The genetics of Duchenne muscular dystrophy.

MEMBRES PROTECTEURS BESCHERMENDE LEDEN AMERSHAM Belgium ANALIS APPLIED BIOSYSTEMS

BOEHRINGER-MANNHEIM (Belgium) CANBERRA PACKARD CERESTAR gruppo Ferruzzi DENLEY Europe

DU PONT DE NEMOURS HEWLETT PACKARD H.V.L. JANSSEN PHARMACEUTICA

LAMECO LAND& INTECHMIJ

BEUN-DE RONDE

DEVOS-FRANCOIS

LABAZ-SANOFI

MEDECINE/SCIENCE-JOHN LIBBEY EUROTEXT

MERCK PHARMACIA LKB PLEUGER PROMEGA ROCHE

SMITHKLINE BEECHAM BIOLOGICALS UCB VAN HOPPLYNUS WATERS Chromatography

SIGMA-ALDRICH

Division/MILLIPORE

Th. ARNOULD, Carine MICHIELS, Andrke HOUBION, M. DEU and J. b a U C L E (Loboratoire de Biochimie Cellulaire, Facull& Univem~taires ND de la Pa&, 61 rue de Bnuelles. ESOOO Namur).

Hypoxia-reoxygenation increases lipid mediator production by human endothelial cells.

Ischemia-reperfusion is a major phenomenon involved in several diseases such as myocardium infarc- tion and venous pathology. It has been recently demonstrated that ischemic tissue is rapidly transformed into an inflammatory area characterized by polymorphonuclear neutrophil accumulation.

In order to determine the role played by endothelial cells (EC) in this phenomenon, a hypoxia (H)- reoxygenation (R) model upon human cells in culture was developed which mimicks the situation in vivo. The activation state of EC was investigated by assaying prostaglandin release into the medium by gas chromatography-mass spectrometry (GC-MS). Pro- stacyclin (PGI,) is the main prostaglandin synthesized by EC at rest. We found that oxygen depletion leads to an increase in prostaglandin synthesis. PGD, and PGE, are more induced compared to prostacyclin. This increase in prostaglandin synthesis starts after 30 min of hypoxia, which seems to be a critical time and coin- cides with an increase in the cytosolic calcium concentration. Indomethacin and BW755 C, two cyclooxygenase inhibitors, prevent this increase in a dosedependent manner suggesting that prostaglandins measured in the medium result from a de novo synthesis. Reoxygenation is without effect on PG synthesis after a hypoxia period and does not increase the production of these mediators.

In conclusion, using this model, we observed an increase in PG release only during hypoxia. Furthermore, using a specific fluorescent probe, we were able to detect and quantify a rise in calcium concentration during hypoxia which could be explained by arrest of ionic pumps. These results suggest that calcium could be responsible for PG increase by activation of PLA, which releases arachidonic acid as precursor of biologically active compounds.

Carine MICHIELS is a Senior Research Assistant of FNRS.

2. - COMMUNICATIONS

(I) Publication subsidike par le Minkt8re de I'Education Natio- nale et de la Culture. Publikatie gesubsidieerd door het Minkterie vun Nationale Opvoeding en Cultuur.

Archives Internationales de Physiologie, de Biochimie et de Biophysique, 1991, 99 ( 3 )

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B24 SOCIJ~TE BELGE DE BIOCHIMIE, ANVERS UIA, 2 MARS 1991

J . B E ~ N , P. COUCKE, I. BUNTINX, B. VAN DER A m and P. J. WlLLEMS (MedicalGenetics, Univer- sity of Antwerp - U.I.A., Antwerp. Belgium).

Molecular investigation of the DNA mutations leading to the Angelman syndrome.

Angelman syndrome (AS) was first described by ANGELMAN (1%5) in 3 children with mental retardation and puppet-like movements. The syndrome is also called “happy puppet” syndrome because of the peculiar way of moving like a puppet-on-a-string and easily provoked paroxysms of laughter. AS is characterized by severe mental retardation, absence of speech, seizures, inappropriate bouts of laughter, micro-and brachycephaly and ataxic gait (ANGELMAN, 1965; WILLEMS et al., 1987).

In more than 50% of AS patients, a deletion of chromosome 15 in the 15ql lq13 region is found which causes AS. These chromosomal microdeletions can be detected cytogenetically by high-resolution banding techni- ques of prophase chromosomes. We have collected DNA of 25 AS patients and their parents. Six patients are of Belgian ancestry and 19 of Dutch ancestry.

We used classical Southern blotting analysis and pulsed-field gel electrophoresis with 15ql l q 1 4 DNA markers : MN55, CMW-1, pML34, pTD3-21, pIR4-3R, pIR10-1, pTD189-1, MN6, MN7, MN8, MN47, PW66, PW71, MN43, p3-16, pIR29-1, pIR39, ~ 1 3 5 , ~190-2, MN28, MN60.

The classical Southern blotting analysis transfers the fragmented genomic DNA that has been subjected to gel electrophoresis, to nylon membranes. Radio-labeled DNA markers hybridize with the complementary nucleotide sequences of the DNA on the membrane. By comparison of DNA of the AS patient with the DNA of the parents with specific polymorphic DNA markers localized in the l5ql l q 1 4 region, it was possible to detect deletions in the AS patients (1kuuuj.a et al., 1989).

Another technique used to detect molecular deletions, is pulsed-field analysis. Large DNA fragments obtained by digestion with rarecutting restriction endonucleases are subjected to a special electrophoresis with pulsed field. Pulsed-field gel electrophoresis is very appropriate to determine accurately the size of molecular deletions. This technique also allows the localization of the relative position of the different DNA-markers and allows the production of a physical map of a chromosomal region (SMITH et nl., 1987; LN el nl., 1989).

By comparison of the different molecular deletions in AS patients, we have tried to determine the exact localization of the AS gene in respect to the 15ql lq14 DNA markers. These data will be presented at the symposium.

References ANGELMAN, H. (1965) Dev. Med. Child. Neurol. 7, 681-683. I t&uznu. K., TAKADA, F., KUROKI. Y., N m o l a , K., W, J.

& NIIKAWA, N. (1989) Am. J. Med. Genet. 35, 314-318. LN, E., BURIER. B. W., CLARK, S. M., SIMON, M. I . & HOOD, L.

(1989) Biotechniques 7, 3442. S m , C. L. & CANTOR, C. R. (1987) Meth. Enqymol. 155,449467. WILLEM, P. J. , D m s m , I. , BROUWER, 0. F. & S m , G . P. A.

(1987) Am. J. Med. Genet. 27, 773-780.

E. CAPIEAUX, S. ULASZEWSKI, E. BALZI and A. GOF- EEAU (Unite de Biochimie Physiologique. Universite de Lou- vain, Place Croir du Sud I (Boite 8). 81348 Louvain-la-Neuve).

Physical, transcriptional and genetic mapping of a 24-kb DNA fragment located between the P u A 2 and ATE2 loci on cbromosorne VII from Sac- charomyces cerevisiae.

We have subcloned 60 kb of DNA, extending from the centromere to TRPS on the left arm of the chromosome VII of S. cerevkiae strain IL125-2B. The restriction maps of both cosmids were established, and subclones were used to locate the genes PMAI. PDRI, L E U , TRPS ( U m w s m et al.. 1987; BALZI et 01.. 1987). scll (BALZI et al., 1989) and ATE1 (BALZI et al., 1990) by complementation of mutant strains.

Within a 31-kb region, a total of 12 transcription products ranging from 0.6 to 3.6 kb were identified on rich medium. Nine of these transcripts were unambigously mapped over 24 kb of DNA. One transcript, of 2.3 kb average length, is present every 2.7 kb of DNA. Eighty seven per cent of the DNA investigated is transcribed. Previous reports of high density of transcription ranging from 67% to 85% have been made for regions located on chromosome I, XV, and X of S. cerevisiae (STRUIIL & DAVIS, 1981; COLEMAN et al., 1986; STEENSPM et al.. 1987; BARRY et al., 1987). These published studies, including ours, encompass a total of 83 kb of yeast DNA in which a total of 35 transcripts, covering 80% of the DNA, were identified. An average transcript of 1.9 kb was detected every 2.4 kb. If we assume that this gene density is representative for the entire yeast DNA, which aproximates 12 650 kb (rDNA excluded), a haploid S. cerevisiae should express a minimal of 5 300 genes.

Classical tetrad analysis was carried out for genetic mapping of the mutations p d r l , pmal, leul, trpS and ade6. Our data predict the following distances between the tested markers : centromere VII (4.0 cM)pmal (less than 0.12 tM) feu1 (2.3 cM) p d r l (9.1 cM) trp5. When the physical distances between he markers CEN7, PMAI, LEUI, PDRI and TRPS (spread over 60 kb of chromosome VII) are compared to the genetic distance, one reaches the conclusion that in this centromeric region a recombination frequency of 1 cM unit corresponds to an average distance of 3.3 kb between alleles. This is slightly smaller than the estimate of 3.5 kb/cM obtained previously by a much more global approach, considering all genetic markers on all regions from all chromosomes (MORTIMER & SCHILD, 1985). We observe an average rate of recombination of 0.30 cM/kb between all gene pairs examined on the centromeric region of chromosome VII. This rate is only slightly higher than the 0.28 cM/kb estimated for the entire genome by MORTIMER & SCEEILD (1985).

References BALZI. E., -N, W., ULASZEWSKI, s., CAPIEAUX, E. & GOFFEAU,

BALZI, E., CHEN, W., CAPIEAUX, E., MCCUSKER, J . H. , HABER, J .

BALZI, E., CHODER, M., CHEN, W. , VARSHAVSKY, A. & GOFFEAU,

BARRY, L., S m s , J. I., PEW, D. F., MELNICK, L. & S m w ,

COEW, K . G., STEENSMA, H. Y., KABACK, D. B. & PRINGLE, J .

MORTMER, R. K. & SCHILD, D. (1985) Microbiol. Rev. 49, 181-212. STEENSMA, H . Y., ~RO\KLEY, J . C. & KABACK, D. B. (1987) Mol.

STRUHL, K. & DAVIS, W. (1981) J. Mol. Biol. 152, 535-552. Uuszewsm, S., BALZI, E. & GOFFEAU, A. (1987) Mol. Gen. Genet.

A. (1987) J. Biol. Chem. 262, 16871-16879.

E. & G o m u , A. (1989) Gene 83. 271-279.

A. (1990) J. Biol. Chem. 265, 74647471.

F. (1987) Mol. Cell. Biol. 7, 632-638.

R. (1986) Mol. Cell. Biol. 6 , 4516-4525.

Cell. Biol. 7, 410-419.

207, 38-46.

Archives Internationales de Physiologie, de Biochimie et de Biophysique, 1991. 99 (3)

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SOCIl?l% BELGE DE BIOCEIIMIE, ANVERS UIA, 2 MARS 1991 B25

P. COUCKE, B. VAN DER AUWERA, P. PELCKMANS (l),

H. FIERENS (l), and P. J. WILLEMS [apartments of Medical Genetics and (I) Gastroenterology, Universitoire In- stelling Antwerpen. Belgium].

DNA linkage analysis in a family with familial adenomatous polyposis.

Familial adenomatous polyposis (Polyposis coli, FAP) is an autosomal dominant disorder characterized by the presence of hundreds of adenomatous polyps involving the entire large bowel at the age of fourty. The incidence of FAP is estimated at about 1 in 7 OOO. Unless colectomy is performed, death from colon carcinoma approaches 100% by age 55. Therefore it is important to detect affected persons at a presymptomatic stage.

Molecular evidence is consistent with the Knudson anti-oncogene model. (KNUDSON et al., 1985; VOGELS- TEIN et al., 1988). Knudson originally proposed a simple two-allele model in which two genetic events take place in the two alleles of the same genetic locus encoding a tumor suppressor gene or anti-oncogene. This leads to loss of function of the tumor suppressor protein, which induces carcinoma.

Once the FAP gene had been mapped to chromosome 5q21-q22 (BODMER et al., 1987), a series of polymorphic DNA markers flanking the FAP gene were developed allowing carrier detection by DNA analysis. Accurate presymptomatic diagnosis can be of- fered to FAP families combining results of coloscopy, eye fundus examination for congenital hypertrophy of the retinal pigmented epithelium, Rx of the mandible and the shull for osteomas with DNA data.

We performed DNA analysis of 93 members from a family with 24 patients with a clinical diagnosis of FAP. Seven probes flanking the FAP gene were used : YN5.120 (D5S83), C11P11 (D5S71), Pi227 (D5S37), ECB27 (D5S98), YN5.48 (D5S81), MC5.61 (D5S84), and EW5.5 (D5S86). No recombination over the inter- val Pi227-ECB27- FAP gene-YN5.48 was observed and it was possible to predict which family members carried the FAP gene with an accuracy of more than 99% (COUCKE et al., 1990). Two-point and multipoint analysis between the DNA markers and the FAP gene allowed us to generate a locus order. These data will be presented at the symposium.

References c

BODMER, W., BAILEY, C., BODMER, J . , BUSSEY, H., ELLIS, A., GOR- MAN, P., LUCBELLO, F., MURDAY, V. , RIDER, S., &AMBLER, P., SHEER, D., SOLOMON, E.&SPURR, N. (1987)Nature328,614.

COUCKE, P. , VAN DER AUWERA, B., V m , L. & WILLEMS, P. (1990) Tijakhr. Geneesk., in press.

KNUDSON, A. G . (1985) Cancer Res. 45, 1437. VOGELSTEM, B., FEARON, E., HAMILTON, S., KERN, S., PREISPIGER,

A., LEPPERT, M., NAKAMURA, Y., WHITE, R., S m , A. & BOS, J . (1988) New Engl. J. Med. 319, 525.

A. DE BRUYN (’), P. RAEYMAEKERS c), L. A. SAND= (z), G. RAES (l), D. HERSCH (9, V. DELVENNE (3),

J. MENDLEWICZ (7 and C. VAN BROECKHOVEN ( I )

[(I) Neurogenetics Lab, Dept. of Biochemistry, Born Bunge Foundation, University of Antwerp (UIA), Belgium, ( I ) Dept. of Clinical Genetics, Dijkzigt Uhiversity Hospital, Rotterdam, The Netherlands and (‘) Ermme Hospital, Dept. of Psychiatry. University of Brussek (ULB). Belgium].

Genetic linkage studies in families with bipolar illness.

Manic depressive illness (MDI), a severe cyclic mental illness with an estimated prevalence of 2%, is expressed in different forms. The two major forms of MDI are bipolar and unipolar illness referring to patients who have recurrent episodes of, respectively depression and mania, or episodes of depression only. The depressive and manic periods may be cyclic, in- termixed or separated by periods of normal mood. A minor form of MDI is cyclothymia, a chronic mood disturbance involving both depression and hypomania but of insufficient severity to meet the criteria for major depression or mania. Cyclothymia is sometimes viewed as a precursor of bipolar illness (RICE & RISCH, 1989). Family, twin and adoption studies suggested the involvement of genetic factors in the aetiology of MDI (MENDLEWICZ, 1988). Genetic linkage studies indicated the presence of a gene predisposing to MDI on the X chromosone (BARON et al., 1987; MENDLEWICZ et al., 1987) and on chromosome 11 (EGELAND ei al., 1987). However, several other linkage studies failed to confirm linkage to these loci in MDI families.

We examined linkage in eleven nuciear MDI families with chromosome 11. The DNA markers used are pEJ6.6, phins 310, pHGTH4 and HD2G1 belonging respectively to the Harvey-Ras I (HRASl) cellular oncogen, the insulin (INS) gene and the candidate genes tyrosine hydroxylase (TH) and D2 dopamine receptor (DRD2). Two-point and multipoint lod scores were calculated using the “LINKAGE” computer package assuming autosomal dominant inheritance of MDI, a MDI gene frequency of 0.015 and equal male and female recombination rates. The penetrance of the disease for the unaffected individuals was estimated according to the age penetrance classes of EGELAND et al. (1987). The overall linkage results were negative for each of the markers tested suggesting that a gene on chromosome 11 does not play an important role in the MDI families analysed.

References BARON, M., RISCH, N., HAMBURGER. R. , MANDEL, B., KUsENER, S.,

N E W , M., DRUMI~R, D. & BEL~UKER, R. H. (1987) Nature 326, 289-292.

EGELAND, J . A., GERRARD, D. S., PA=, D. L.. SuSsix, J. N., ~ D D , K . K . , ALLEN, C. R. , H O S T E ~ R , A. M. & HOUSMAN, D. E. (1987) Nature 325, 783-787.

MENDELWICZ, J . (1988) in Depression andMania (GEOROOTAS, A. & CANCRO, R. , eds) pp 197-212. Elsevia, Amsterdam.

MENDELWICZ. J . , SIMON, P.. SEW, s., RON, F., BROCAS, H., LEGROS, s. & VASSART, G. (1987) The Luncet i. 1230-1232.

RICE, J . & m a , N. (1989) Genetic Epidemiology 6 , 161-177.

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SOC&T& BELGE DE BIOCHIMIE, ANVERS UIA, 2 MARS 1991 B26

N. D~IZBNNB, p. BUC-CALDERON, H. TAPER and M. ROBB-OW (Unite de Biochimie Toxicologique el cad tdog ique , UCL 7369. B-1200 BnureNes).

Comparative studies in vitro and in vivo of the hepatotoxicity of secondary bile acids ia rats.

The aim of this study was to compare the hepatoxi- city of lithocholic acid (3a-hydroxycholanoic acid) and deoxycholic acid (3a, 12a-dihydroxycholanoic acid), which are present in small amount in the liver, but whose concentration may increase in cholestatic syndrome or hepatobiliary disease (PALMER et af., 1972).

Studies in vitro were performed to compare the cell lysis induced by both compounds, as measured by the release of cytosolic enzymes from isolated hepatocytes surviving in suspension.

Experiments in vivo were aimed at studying : 1) the acute toxicity resulting from feeding rats for two weeks with deoxycholic acid or lithocholic acid (0.5% or 1 Yo in diet), by analyzing the following parameters : serum markers of liver disturbance or cholestasis (tran- saminases activity, bilirubine, total bile acids); histological and histochemical alterations as well as lipid peroxidation in liver tissue; 2) the effect of the administration of the secondary bile acids on rat liver cancer development.

Results indicate that deoxycholic acid is by far more hepatotoxic than lithocholic acid. Indeed, deoxycholic acid causes an important lysis of hepatocytes (leading in vivo to a subsequent cell proliferation) and an increase in lipid peroxidation in the liver, two phenomena related to carcinogenesis (Prro~ et ul., 1980). Moreover, when given chronically after the fully carcinogenic treatment described by SOLT and FARBER (1976), it increases the incidence and the yield of benign and malignant liver tumors in rats, while lithocholic acid administration has no such effects. Such differences between these two bile acids could partidy be explained by the differences in their metabolic pathways : lithocholic acid is largely transformed in rat liver into the trihydroxylated- and thus considered as less toxic- muricholic acids, although deoxycholic, when given orally to the rat, becomes the major bile acid in the liver.

References PALMER, R. H. (1972) Arch. Intern. Med. 130, 606-617. P m , H. & Snuc~, A. (1980) Biochim. Biophys. Acta605, 191-215. SOLT, D. & FARBER, E. (1976) Nature 263, 701-703.

E. DE ROS and P. BUC-CALDERON (Unite B ~ c , UCL 7369. Universitt! Catholique de Louvain. 8-1200 Brussels).

Preventive effects of thiols against the oxidative stress induced by organic hydroperoxides.

The metabolization of an organic hydroperoxide such as tert-butyl hydroperoxide (tBOOH), involves the participation of the glutathione (GSH) redox cycle and its enzymes GSH-peroxidase and GSH-reductase (RUSH et al., 1985). The strategy to increase the intracellular antioxidant defenses, e.g. the GSH content by the use of N-acetylcystein (NAC) has given some promising results. Indeed, when associated with doxorubicin, it decreased in a substantial way the cardiac toxicity of the anticancer drug (DOROSHOW et al., 1981).

We have tested the ability of some thiols (cystein, NAC, cysteamine, GSH) to reduce the cytotoxicity of tBOOH to isolated hepatocytes as measured by the lost in membrane integrity (LDH leakage) and the decrease in both protein synthesis (incorporation of 14C-leucine) and GSH content.

The results show that only the addition of GSH was able to inhibit the oxidative stress induced by tBOOH in all the parameters measured, whereas the other thiols showed partial protection. At 0.5 f l l ~ tBOOH, the effect of GSH was concentration-dependent. Since exo- genous GSH is not incorporated by the hepatocyte (GRIFFITH & MEISTER, 1979), the mechanism(s) of this protection require more data to be elucidated.

References DOROSHOW. J. H., LOCKER, G . Y., IFRIM, 1. &MYERS, C. E. (1981)

J. Clin. Invest. 68, 1053-1064. G~UFFITH, 0. W. & MELSTER. A. (1979) Proc. Nat. Acad. Sci. USA

76, 5606-5610. Rum. G. F., GORSKI, J . R., RIPPLE, M. G., SOWINSKI, J., BUGELSKI,

P. & HEW, W. R. (1985) Toxicol. Appl. Pharmacol. 78, 473483.


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SOCIkTE BELGE DE BIOCHIMIE, ANVERS UIA, 2 MARS 1991 B27

G. DE SMLANX, H. HOOGENBOOM, J. ROBBEN and G. VOLCKAERT (Loboratory of Gene Technology, KULeuien, de Croylaan 42, 8-3001 h v e n ) .

Construction and expression of singlechain antibody fusion proteins with chloramphenicol acetyltransferase in E. COU.

The binding capacity of an antibody is basically determined by the complementarydetermining regions (CDR) in the variable domains of light and heavy chains. These binding activities can be engineered in a variety of ways by expression of the corresponding cloned genes either as separate light and heavy chains or as a single chain or fragments thereoff. Expression of the variable regions is sufficient to reconstitute the binding activity. In E. coli efficient expression of antibody variable regions usually is obtained only by secreting the releant chain@) (PL~CKTHUN & S n m , 1989; WARD et al., 1989).

We have explored the feasibility of expressing an antibody variable domain as a fusion to a chloramphenicol acetyltransferase (Cat). Therefore, we in- troduced the coding sequence of a singlechain antibody against the human antitransferrin receptor (H. HOOGENBWM et al., unpublished) into the 3'-terminal part of Tn9-cat. The latter gene is expressed at high level in a vector system derived from pGV451 (VOLCWRT, 1987), and based on tuc as promoter (ROBBEN et al., 1991). This fusion resulted in cytoplasmic expression in E. coli of a Cat: : sc-antibody chimaeric protein with Cat activity. In a similar construct the antibody part of the fusion was restricted to the light chain of the variable domain. Although in both cases the expression level was lower than obtained with intact Cat, substantial amounts were produced so as to allow visualisation of the protein sizes by SDS- PAGE. The solubility and binding properties of the antibody derivatives are being analysed.

J . ROBBEN is a postdoctoral fellow of the Instituut tot Aun- moediging van her WetemhappeIijk Ondenoek in Nijverheid en Landbouw. This project was funded in part by contract ETC-007 of the Ylaams Actieprogramma Biotechnologie.

References PLOCKTEUN, A. & SKERRA, A. (1989) Methods Enzymol. 178,

ROBBEN, J . , B o s w s , E., WELLENS, B. & VOLCKAERT, G . (1991)

VOLCKAERT, G . (1987) Methods Entvmol. 155, 231-250. WARD, E. S., GUssow, D., GNFFITHS, A. D., JONES, P. T. & WINTER,

497-5 15.

Arch. in!. PhysioI. BigEhim. 99, I342.

G . (1989) Nuture 341, 544-546.

Bhtrice DEWEZ, C. C m m o (') and M. ROBERFROID [Unit6 BCTC, UCL 7369, Ecole de Phurmacie. Universitt! Catholique de Louvain et (I) Unit6 MEXP, ICP 7439, B-1200 Bruxelles).

Interaction between actin and DNase I : a molecular basis to expldn chromatographic and enzymatic properties of human serum slkaline DNase.

Alkaline DNase is present in human serum but, as yet no general agreement has been reached concerning its functional role. We have recently reported that measuring this enzyme activity has predictive value in monitoring cancer therapy (ECONOMIDOU-KARAOGLOU et al., 1988).

Several authors have identified inhibitor(s) of alkaline DNase among which monomeric actin has received much attention (LAZARIDES & LJNDBERG, 1974). However, the presence of actin in circulating serum is still controversial and the nature of the inhibitor(s) of serum alkaline DNase still remains unknown.

We have previously reported that human serum alkaline DNase is detected, by chromatography on Ultrogel AcA 34, at a mol.wt of about 130 kd even- though purified DNaseI has a mol.wt of 35 kd. In addition, using the same experimental conditions, we have shown that human serum contains, in the 1Wkd fractions, protein(s) which bind(s) DNaseI to form a 13@kd complex and/or inhibit(s) the enzyme (DEWEZ et al., 1989).

The objective of the present study was to test the hypothesis that actin is part of/or is this 1Wkd protein.

In the same experimental conditions set up for gel filtration of serum we report that : - bovine monomeric actin is recovered at a mol.wt of 100 kd; - by mixing bovine monomeric actin and bovine DNaseI at a molar ratio 2:1, a complex forms which elutes in the same fractions as endogenous human serum alkaline DNase (130 kd); - in this chromatographed high-molecular-weight complex, enzymatic DNase activity is not inhibited. In addition we report that by mixing bovine monomeric actin and bovine DNaseI at molar ratios of 16:l or higher the enzymatic activity is inhibited and that this inhibition becomes stronger as the ratio increases.

Monomeric actin has some of the properties that make it a good candidate to be part of/or to be the 1Wkd protein we identified in human serum and which interacts with alkaline DNase. Further work is in progress to identify actin in human serum and to demonstrate that it interacts with alkaline DNase in the ways we have described.

References DEW, B., C m w , C., LANS. M., TAPER, H., ECONOMIDOU-

KrJUoomu, A. & ROBERFROID, M. (1989) Arch. int. Physiol. Biochim. 97, B25.

E c o ~ o ~ u - I u ~ A., Lms, M.. TAPER, H., M ~ c m x , J . & ROBERFROID, M. (1988) Cuncer 61, 1838-1843.

LAZARIDBS, E. & LINDBERG, U. (1974) Proc. Nat. Acad. Sci. 71, 47424746.

Archives Internationalm de Physiologie, de Biochimie et de Biophysique, 1991. 99 (3)

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B28 S O C I ~ T ~ BELGE DE BIOCHIMIE, ANVERS UIA, 2 MARS 1991

P. J. DIERICKX and M. M. ALMAR (l) [ht i tuut voor Hygiene en Epidemiologie, A fdeling Toxikologie. Wytsmamtraat 14, 3-1050 i?~-ussel and ( I ) Bepartamento de Fkiologia, Farmacologia y Toxicologia, Universidad de Leon, Spain].

In vitro interaction of heavy metals with human glutathione transferase 'K.

Glutathione transferase (GST) catalyzes the con- jugation of glutathione (GSH) with xenobiotics and their metabolites. Other important detoxication functions of GST include its peroxidase activity and its direct binding capacity of toxic compounds. The interaction of both inorganic (DIERICKX, 1982) and organic (DIERICKX, 1985) heavy-metal compounds with rat liver GST isoenzymes has been previously described. More recently the human GST 'K was recognized as a tumor marker in different tissues (SEA et ul.. 1988). Here we report on the interaction of heavy metals with human GST u.

GST r was purified from PLC/PRF/5 cells by affinity chromatography of the 100 OOO x g supernatant (DIERICKX, 1989). The GST activity was measured with GSH and l-chloro-2,4-dinitrobenzene (CDNB) as the substrates. The in vitro interaction of mercury-, copper (11)- and cadmium chloride with GST was investigated as described (DJERICKX, 1985).

The GST u inhibition by heavy metals was dose- dependent but not linear. With mercury and copper (11) the GST 'K inhibition was smaller using higher GSH concentrations. The sulfhydryl amino-acid cysteine showed also a dose-dependent protective function against the heavy-metal inhibition. In enzyme kinetics experiments a parabolic inhibition was found for mercury and copper (11) with GSH as the variable substrate. An intermediate kinetic pattern between uncompetitive and noncompetitive inhibition towards CDNB was observed. A kinetic pattern of competitive inhibition was never found. This indicates that the inhibition of the GST u activity can not be explained by the heavy metals competing with CDNB as a substrate for GST r.

The results demonstrate that heavy metals interact with human GST T by direct binding to this protein. Abortive complex formation with products as an ex- planation for the parabolic inhibition is in agreement with the observation that heavy metals were very well spontaneously conjugated with GSH. Quantitative but no qualitative differences were observed with rat liver GST. We have demonstrated that human GST 'K can directly bind heavy metds. The in vitro interaction of GST 'K with these compounds suggests that it could have a protective function against them.

We thank the Spanish Minktry of Education and Science for financial support.

References DERICKX, P. J . (1982) Enzyme 27, 25-32. DIERICKX, P. J. (1985) Pharmacol. Res. Commun. 17, 489-500. DIE~CKX, P. J. (1989) Biomed. Res. 10, 301-306. SEA, T. C., KELLEY. S . L. & HENNJX, W. D. (1988) Cancer Res. 48, 527-533.

~ _____ ~~~ ____ ~~


F. DUPONT, L. TENENBAUM and J. ROMMELAERE (Department of Molecular Biology, Universitk Libre de Bru- xelltx, 8-1640, Rhode St Genke).

Negative control of replication of a MVM/SV40 hybrid genome in COS-1 cells by the MVM non- structural protein NS1.

Parvoviruses are a group of small nuclear-replicating DNA viruses which infect animal cells and whose genome is a linear single-stranded DNA molecule of about 5 kb with palindromic terminal sequences ( T S ) (COTMORE et al., 1987).

In this study, use was made of pMM984, a molecular clone of the entire genome of the autonomous parvovirus minute of mice (MVM) inserted into the bacterial plasmid pBR322 (MERCHLINSKY et al., 1983). When pMM984 is transfected into permissive cells, it expresses non-structural (NS) and capsid (VP) proteins, replicates and produces infectious MVM particles after excision of the MVM in- sert from the vector. Both excision and replication steps require an intact NS1 gene in trum and functional genomic ends in cis.

pMM984 was modified by substituting part of the MVM capsid genes with the simian virus 40 (SV40) regulatory region that includes the origin of replication (NSoriTS'plasmid). When the chimeric plasmid was in- troduced by transfection into COS-1 cells (expressing the SV40 large T antigen required to initiate replication from SV40 or& it replicated with a similar efficiency as pMM984, giving rise to the typical parvoviral DNA in- termediates. A 97-base-pair deletion within the right hand palindrome (NS'ori'TS-) abolished the parvoviral mode of DNA replication, resulting in a residual level of SV40-like replication that depended on a functional ori. Interestingly, the NS'oriTS- plasmid replicated poorly compared with a SV40 replicon that did not contain parvoviral sequences (pSV2neo). A frameshift mutation within the NS1 gene (NS1-ori'TS- plasmid) significantly increased the efficiency of replication of the chimera, suggesting that the MVM non-structural protein NSl is responsible for the down-regulation of SV40 ori-driven DNA replication. This inhibitory effect was mediated in trans by NSI since replication of the NS1-ori'TS- plasmid was inhibited in a dose-dependent fashion upon cell cotransfection with a NSl -competent vector, pMM984. It should also be stated that NSl did not inhibit the replication of pSV2neo and therefore appeared to exert its inhibitory effect through a direct or indirect interaction with parvoviral sequences. Consistently, terminal sequences of a defective parvovirus (AAV) were also reported to be the target for the inhibition of the replication of AAV/SV40 hybrids expressing parvoviral non-structural proteins (LABOW et al., 1988).

Studies are presently in progress in our laboratory to unravel the interference of NSl with SV40-driven DNA replication. The analysis of this process may be relevent to our understanding of the mode of action of NSl, in particular its involvement in the replication of parvoviral DNA and in the disturbance of host cells.

References COTMORE, S . F.&TATTERSALL, P. (1987)Adv. VimRes. 33,91-174. LABOW, M. A. & BERNS, K. I . (1988) J. Virol. 62, 1705-1712. MERCELINSKY, M. J. , T A ~ R S A L L , P. J . , LEARY, J . J . , COTMORE,

S. F., GARDINEX, E. M. & WARD, D. C. (1983) J. Virol. 47, 221-232.

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C . FAGNY (I), A. MICHEL (*), I . LEONARD c), G . BERKENBOOM (3), J. FONTAINE (7 and M. DESCHODT-LANCKMAN (') I(') Laboratoire Pluridisci- plinaire de Recherche Erperimentale Biornddicale, Facultt! de Mddecine, Universitc! Libre de Bruxelles, B-1070 Bruxelles, (') Laboratoire de Chirnie Biologique. Facultt! des Sciences, Universitt! & I'Etat, B-7Ixx) Mom and (') Wprtement de Phar- macologie. Institut de Pharrnacie, Universitt! Libre de Brux- elles, B- 1050 Bruxelles] . Degradation in vitro of endothelin-1 by endopeptidase 24.11 (enkephalinase).

Recently a vasoactive peptide was isolated from vascular endothelial cells and named endothelin-1 (ET-I). This 21-amino-acid peptide containing two disulfide bridges was initially reported to possess potent contractile activity on isolated vascular smooth muscle (YANAGISAWA et al., 1988).

Autoradiographic studies of rat, porcine and human tissues indicated that endothelin receptors are present not only in the arteries and heart but also in other organs such as kidney, lung, adrenal gland and brain. In brain, receptors for endothelin are present in different areas but are particularly dense in the choroid plexus.

As the kidney and choroid plexus are rich in endopeptidase 24.11 (enkephalinase, neutral endopeptidase, EC 3.4.24.1 l), a peptidase known to inactivate small peptide hormones, we investigated whether this enzyme was able to hydrolyse ET-1 in virro, using crude membranes derived from human kidney and choroid plexus and a purified preparation of human renal endopeptidase 24.1 1 (DESCHODT-LANCKMAN er ul., 1988). We also tested the vasoconstrictor activity of some of the metabolites produced by the enzyme on isolated rat aortic rings.

Incubation of 'zSI-labelled endothelin-1 with both human kidney and choroid plexus membranes led to the generation of several labelled fragments that were separated by HPLC. This degradation could be completely abolished in the presence of 1 p~ phosphoramidon. These results suggest an involvement of endopeptidase 24.11 in the metabolism of ET-1 by these membranes in vitro.

Identification of the cleavage sites by purified human renal endopeptidase 24.11 in the sequence of endothelin-1 was carried out by determination of the amino-acid composition of the fragments produced and/or coelution with standard peptides. It appears that bonds involving the amino side of the hydrophobic amino acids (Ser4, Leu6, Val12, Phel4, Hisl6, Leul7, Ile19) were susceptible to cleavage. The (1-161 N-terminal fragments were tested for their constrictor activity-on rat aortic rings. They were devoid of bioactivity at a 0.1 p~ concentration.

Endothelin-1 appears thus to behave as a substrate for endopeptidase 24.11 in vitro. It is degraded at multiple sites producing inactive fragments. The physiological role of this inactivation process remains to be established.

This work was supported by grant 3.4503.89 from the Fonds de la Recherche Scientifique Mddicale (Belgium) and by the Banque Nationale de Belgique.

References DESCHODT-LANCKMAN, M. , PAWLS, S., NNDOVSKI, T., hd"E,

R. & DOCKRAY, G. J . (1988) Gastroenterology 94, 712-721. YANAGISAWA, M.. KURIHARA, H. , K m , S., TOMOBE, Y.,

KOBAYASEII, M., Mrrsm. Y., YAZAKI, Y., GOTO, K. 8i Wm, T. (1988) Nature 332, 411-415.

B. FOCANT (I), F. SLUSE ( 2 ) , F. H w u x (I), C. DWCKAERTS ( 2 ) and M. RADERMECKER c) [(I)

Laboratoire de Biologie Cellulaire et Tissulaire (86). (l)

Laboratoire de Bidnergdtique (B6) and (7 Laboratoire de Chirurgie Cardiovasculaire (835). Universitt! de Liege, Salt- Tilman, B4OOO Liege].

Kinetics of biochemical and bioenergetic adaptations during skeletal muscle transformation induced by chronic electrical stimulation via intramuscular leads.

The transformation of a fast-twitch fatigue-prone muscle into a slow-twitch fatigue - resistant one (rich in type I fibers) is a well-known phenomenon. The biochemical modifications of the contractile proteins have been extensively documented but some discrepancies remain especially at the level of the respective rates of transformation of myosin heavy (HC) and light (LC) chains and on their distribution after prolonged stimulation. Metabolic adaptations have been well studied but exhibit heterogeneous behaviours in important enzymic reactions ("key" enzymes of several pathways) : a synchronism in the reaching to thei maximal activities is far to be observed and if some of these enzymes conserve their highest activities others show subsequent and important decline sometimes called de-transformation. We presently are achieving the follow-up of the relations between the events occurring at the level of the contractile machine and the adap- tation of the oxidative energy production.

We analysed the events during 200 days in goat latksimus dorsi (LD) submitted to a low frequency electrical stimulation by intramuscular leads (Medtronic Itrel) at a voltage of 2-4 V in order to elicit a significant see-saw motion of the animal limb (GEORGES et al., 1990). Samples of LD were immediately frozen for enzyme activity measurements and used fresh for analysis of LDH isozymes and myosin HC and LC subunits by native and dissociating PAGE.

A complete transformation of the contractile machine occurs within 60 days (from 16 to 100% for HCI and LC2 slow) and remains stable until 200 days. It should be noted that myosin HC and LC evolve in a totally parallel way. The H- LDH isoforms are still increasing after that time (from 12 to 80%).

On the other hand, the maximal activities of the respiratory-chain oxidases increase very fast until peak values after 30 days (when the machine is 75% transformed) : from 0.4 to 4 nmol min-' mg-I wet weight for NADH-cyt c oxido- reductase, from 0.8 to 3.4 for succinateqt c oxido-reductase and from 4 to 21 for cyt c oxidase. After the synchronous peak activities of the oxidases, a steep decrease was observed during the completion of the contractile machine transformation (from 75 to 100%) leading to stable activities between 100 and 200 days. Total LDH activity steeply decreases till 60 days (from 720 to 130 nmol min-' mg-' ww) and then slowly from 60 to 200 days (from 130 to 70 nmol min-' mg-' ww).

It can be concluded that a new stable steady-state is reached after 100 days between the energy consuming contractile machine and the oxidative energy producing process.

B.F. is research Associate of the FNRS. This research was partly supported by the contract no 3.4516.89 of the FRSM and by grants from the FNRS, the Fonds Sp&ial de la Recherche dam les Univer- sit& and the Fonds de Recherche de la Facultt! de Mddecine. Our thanks are also due to Prof. R. L m for his help.

Reference GEORGES, P., RADERMECKER, M., FOCANT, B., DWCKAERTS, C.,

CAMUS. G., REZNIK, M. & SLUSE, F. E. (1990) Arch. int. Physiol. Biochim. 98, B29.

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G. GEIB~SEN and M. VAN MONTAGU (Laboratorium voor &mtiw, Rijhniversiteit, Gent).

Analysis of plant DNA rearrangements associated with T-DNA integration.

The interaction of the soil bacterium Agrobacterium tumefaciem with plants is characterized by a unique kind of genetic transformation : the bacterium transfers part (the T-DNA) of its tumor-inducing plasmid to plant cells where it is integrated into the nuclear genome. To get some insight into the integration mechanism, we have analysed nine plant target DNA sites before and after T-DNA integration.

DNA sequence determination of the plant targets did not reveal remarkable features or a consensus sequence. A comparison between the target and the T- DNA sequences shows that the recombination joints occur between unrelated DNA ends that contain only few identical nucleotides. This is a typical feature for illegitimate recombination processes such as those described for integration of viral or transfected DNA into mammalian cells. Another similarity with illegitimate recombination in mammalian cells is the presence of additional nucleotides at the recombinant junctions. These fillers are found at six of the 15 sequenced T-DNA junctions (40070) and are three- to 44-nucleotides long. The 40% frequency is much higher than the 10% found at the junctions after SV40 cir- cularization and rearrangements in non-immune cells (compiled by ROTH et al., 1989). This might simply reflect the small sample size or it might be typical for plant cells since BAKICEREN et al. (1989) and WESSLER et al. (1990) reported similarly high frequencies in other plant recombinational events.

Most of the nine T-DNA integration events were accompanied by a small deletion, usually in the range of 13 to 28 nucleotides (GHEYSEN et a[., 1991) but a 1Wnucleotide deletion was also found. In addition to the 158-base-pair target duplication described before (GHEYSEN et al., 1987), two T-DNA insertions were associated with larger target rearrangements, most probably a large insertion ( L 1 kb) in the one and a large (> 3 kb) direct duplication of plant DNA in the other case.

References BAKKEREN. G., KOUKOL~KOVA-NICOLA, Z., GRIMSLEY, N. & Horn,

GHEYSEN. G., VAN MONTAGU,~. & ZAMBRYSKI, P. (1987) Prm. Natl.

GHEYSEXN, G., VILLARROEL, R. & VAN MONTAGU, M. (1991) Genes

ROTH, D. B., CHANG, W. B. & WWN, J . H. (1989) Mol. CeK Biol.

WF.SSLER, S . , TARPLEY, A., FWRUOGANAN, M., SPELL, M. & OFIAGAKI,

B. (1989) Cell 57, 847-857.

Acad. Sci. USA 84, 6169-6173.

& Development, in press.

9, 3049-3057.

R. (1990) Proc. Natl. Acad. Sci. USA 87, 8731-8735.

V. GILLIQUET, G. BERBEN and F. HILGER (Unite de Microbiologie, Facult6 des Sciences agronomiques de Gembloux, 6, avenue MarPchal Juin, B-5030 Gembloux). PHO80 and PH085 are pleiotropic genes in Sac- charomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, transcriptional regulation of PH0.5 encoding the repressible acid phosphatase is under the control of inorganic phosphate (Pi) via a complex regulatory system : PH080 and PH085 encode negative regulatory factors of this system (OSHIMA. 1982). These genes have close functional relationship in the “PHO system” and genetic data indicate a protein-protein interaction between them (GILLIQLET et a/.. 1990). Combinatorial regulation of PH080 and PHO85 in diverse cellular functions has been investigated.

Beside its function in regulation of PH05, the “PHO system” is also involved in regulation of the low K , Pi transport and pho80 and pho85 mutants show derepressed Pi transport under repression conditions (TAMN et a/., 1985). Although pho80 mutants confer permeability to dTMP, wild-type strains andpho8.5 mutants are not permeable to this nucleotide (BXS.SON & THORNER, 1982).

Disruption of PH080 or PH08.5 in yeast is not lethal, indicating that PHOBO and PH08.5 are not essential for cell viability. However, analysis of the descendants of a pho85 heterozygote indicates that ph08.5 spores germinate more slowly than wild-type spores. T h i s result prompts with a possible involvement of PH085 in the life cycle. We have thus studied, inpho80 and ph08.5 mutants, a particular aspect of this life cycle, the process of mating : production of the a pheromone has been tested by the halo assay (HERSKOWZ, 1988) in p h d 0 and pho8.5 haploid a cells and their wild-type isogenic counterpart. A patch of hapoloid a cells, producing a factor, gives rise to a halo, corresponding to an area of inhibited growth, on a lawn of a strain supersensitive to the a factor. This test indicates that production of a factor is reduced strongly in a p h d 5 null mutant. Expression of MFaI, the major gene coding for a factor (KuR- JAN & HERSKOWITZ, 1982) has been examined, in pho80 and pho85 mutants, by means of lac2 fusion. Our results show that expression of MFaI-lac2 is enhanced in pho80 and pho85 mutants and that PH085 affects MFal-lac2 expression more strongly than does PHO8O. Enhanced expression of MFaI and diminished production of n factor could be explained by overexpression of the BAR1 protein which degrades the a factor (CIE~EK & THORNER, 1979; MACKAY el a/., 1988).

In order to investigate a possible function of PHO8O and PH08S in glucose repression, we have tested the ability of the mutants and their wild-type isogenic counterparts of diverse genetic background to grow on diverse carbon sources. The mutants show reduced ability to grow on glycerol, maltose, ethanol, galactose and acetate. Weak in pho80 mutants, this phenotype is strongly pronounced in pho85 mutants. Due to the reduced ability to grow on non fermentable carbon sources, we could observe in phOS0 and ph085 mutants isogenic to strain W303-1B ( M T a , a, hid, leu.?, t p I , ura3> cad) a lowering in the accumulation of the red pigment derived from phosphoribosylaminoimidazole, an intermediate of the adenine pathway, that accumulate in ode2 mutants.

PH080 and PH08.5 genes affect expression of phosphate- and glucose-regulated genes and of some cell-type-specific genes. TUPI and SNF5 genes are also involved in the regulation of the expression of this wide variety of genes (WILLUMS & TRUMBLY, 1990; LAURENT el al., 1990). Moreover, a tupl mutant, ljke a pho80 mutant, is permeable to dTMP (BISON & TAORNER, 1982). It would be worth studying a possible relationship between the negative PHO regulatory factors, TUPI and SNFS.

This work was suppoKed by the Belgian Notional Incentive Program on Fundomenrol R a m h in Life Sciences initiated by the Belgian State - Prime Minister’s Office - Science Policy Progmmming.

References BISON. L. F. & TEORNER. J. (1982) Genetics 102. 341-359. CIKIP.K. E. & TEORNER. J . (1979) Cell 18, 623435. GUQUET. V.. LEORUN. M.. B-N. G . & Hnom. F. (1%) Gene, in press. H ~ ~ s x o w m , 1. (1988) Micobiol. Rev. 52. 536553. KUPJAN. J. & H ~ ~ u o w m , 1. (1982) Cell 30. 933-947. LAWNT. 8. C.. T m L , M. A. 8 C~auorr, M. (1990) Mol. Cell. Biol. 10. 56165625. MACLAY, V. L.. WmcE. S. E.. INSlsu . M . Y., MN”Nr. T. R., Hour. J. . SMRI. G. C.

Ckmm~.Y.(1982)in ?lteMd~larBiologyofrhe Ymrrkharomyces.vol.2. 159-180.

TW. Y.. TOE-E. A. & Osmm. Y. (1985) 1. Bacfm’ol. 164.964968. WILLIAMS, F. E. 81 TRUMBLY. R. J. (1990) Mol. Cell. Biol. 10. 65004Sll.

& P m n , M. (1988) Roc, Natl. A d . Qi . USA 85, 55-59.

Cold Spnng Harbor Laboratory, Cold Spring Harbor N.Y.

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E. GOORMAGHTIGH (I), JoElle DE Wunm (I), F. SZOKA ('), Vkronique CABIAUX (I), Roberta A. PARENTE (') and J.-M. RWSSCHAERT ( I ) [(I) Universitk Libre de Bnuelles CP206/2, Laboratoire de Chimie Physique des Macrornolkcules aux Interfaces, Bld du Triomphe, 8-1050 Brussels and (*) University of California, SCHOOL of Phar- macy, Son Francisco, CA 941434446. U.S.A.].

Secondary structure and orientation of tbe amphipathic peptide GALA in lipid structures : an infrared spectroscopy approach.

A synthetic, amphipathic 30-amino-acid peptide based upon a Glu-Ala-Leu-Ala motive was designed to mimic the behaviour of viral fusion proteins (SUBBARAO et al., 1987). GALA is a water-soluble peptide with an aperiodic conformation at neutral pH and becomes an amphipathic a helix as the pH is lowered to pH 5 where it interacts with phospholipid bilayers. Attenuated total reflection infrared spectroscopy using polarized light provides information on the structure and orientation of the peptide and the lipids which is not subject to ar- tifacts due to light scattering on large particles (GOOR- MAGHTIGH et al., 1990). H/D exchange rate of the amide N-H group and analysis of the shape of the amide I' by Fourier self deconvolution and curve fitting indicate that the a helix content increases from 19% to 69% upon pH lowering. A further increase to 100% a-helix is observed after interaction with palmitoyloleoyl- phosphatidylcholine (POPC) vesicles. Dichroism data obtained on oriented bilayers of the POPC-GALA complex demonstrate that POPC hydrocarbon chains and the peptide helical axis are essentially perpendicular (f 15') to the membrane plane. At neutral pH, in the presence of dimyristoylphosphatidylcholine (DMPC), GALA is known to form discoidal structures similar to those formed under the same conditions by apolipopro- teins A1 and A2. In these discoidal complexes, the a helical content was estimated to be 65% with the rest of the structure being essentially unordered. No significant modification of the all-trans conformation of the hydrocarbon chain of dimyristoylphosphatidylcholine (DMPC) is detected upon disc formation. Dichroism measurements show that the helical axis is essentially parallel to the hydrocarbon chains. These data support a model in which a discoidal patch of the bilayer is sur- rounded by the amphipathic helices which shield the hydrophobic region of the bilayer from the aqueous environment. The infrared spectrum of GALA in this complex was found to be very similar to those of apolipoprotein A1 and A2 which form discoidal com- pIexes with DMPC but the spectrum is quite different from that of apolipoprotein Bl00 in low density lipoproteins which does not form discoidal complexes.

References GOORMAOHTIGH, E., Cm~ux, V. & RWSSCHAERT, J. M. (1990) Eur.

S m w o , N . K., P m m , R. A., S ~ M , Jr. F. C.. NADSDI, L. & J. Biochem. (in press).

PONGIUCZ, K. (1987) Biochemistry 26, 2964-2972.

M. HAMMARSTR~M-SCHMITZ, I. KAYSER, M. PAULY, M. DICATO and J.-M. MICHELS (Laboratoire de Recherche sur le Cancer et les Maladies du Sang, Z.I. Grasbusch. L-3370 Leudelange).

Oxygen scavengers and glutathione can modulate Saccharomyces cerevisiae sensitivity to cis-platin.

Cis-diamminedichloroplatinum(I1) has found a wide use in the chemotherapeutic treatment of a variety of cancers. Despite the therapeutic success of the drug, the existence of cis-platin-resistant cell lines have been described (KELLEY et al., 1988). Although DNA seems to be its major biological target, evidence has been presented that careful modifications of glutathione metabolism could potentiate the toxicity of the drug in resistant mammalian cell lies (CANON et al., 1990). Various wild-type and mutant haploid strains of S. cerevisiae were used as biological tools to gain further insight into the possible cytotoxic effects of cis- platin. Thus, the use of strains exhibiting reduced glutathione synthesis capabilities enabled us to show that mortality following cis-platin treatment is dramatically enhanced in these strains, compared to the wild-type. Treatment of the latter with concentrations of buthionine-sulfoximine (a GSH-synthesis inhibitor) greater than 1 nm is sufficient to reproduce the effects of the drug exhibited in the gsh- yeast strain. At the enzymatic level, our results show that the activity of GSSG reductase, a key enzyme in GSH metabolism is down-regulated when gsh- strains are exposed to exo- genous GSH. This inactivation is stopped when cis- platin (25 p) is added to the incubation medium, con- trary to the control. The modulation of enzyme activity is independent of the presence of cycloheximide.

We also show that the S. cerevisiae Q2MI mutant, deficient for the synthesis of 6-aminolevulenic acid (a porphyrin precursor) exhibits decreased sensitivity to cis-platin when 6-aminolevulinate is supplied to the growth medium, i.e. under conditions where Oz-scavenging enzymes are induced.

Our results show that i) S. cerevisiae cells are a useful tool for the study of intracellular cis-platin metabolism and ii) that GSH and 0, scavengers are likely to participate both in the detoxification of the chemotherapeutic drug.

We thank Profs. A. SEU and M. PKNNINCKX (ULB) for their kind gift of yeast strains and for stimulating discussions. This work is supported by a grant from the CRP-Santk of the Grand-Duchy of Luxembourg.

References CANON, J . L., HUMBLBT, Y. & S m , M. (1990) Eur. J. Cancer 26,

KELLEY, S . L., BASU. A., TEICHER, B. A., HACKER, M. P.. HAMER, 1-3.

D. H. & L a o , J . S. (1988) Science 241, 1813-1815.

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J. HENDRICKX and P. J. W u m (MeiiicalGenetics, Univer- sity of Antwerp - U.I.A.. Belgium).

Diagnosis of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency by direct detection of the mutation with PCR.

Medium-chain acyl-CoA dehydrogenase (MCAD) is an enzyme which catalyzes the first reaction of the &oxidation of medium-chain fatty acids (ROE et al., 1989). Deficiency of MCAD is characterized by episodes of fasting intolerance, vomiting and sometimes coma accompanied by hypoketotic hypoglycemia, acidosis and mediumchain dicarboxylic aciduria (ROE et al., 1989). It is an autosomal recessive disorder with a relatively high frequency of 1 in 1O.OOO births. MCAD deficiency also accounts for a few percent of the cases of sudden infant death syndrome and Reye syndrome (EDITORIAL, 1986). The human cDNA has been cloned and characterized (MATSUBARA et al., 1987). Recently a A to G transition at nucleotide 985 of the cDNA was found in patients with MCAD deficiency resulting in the substitution of lysine by glutamic acid at amino acid position 329 of the MCAD protein (MATSUBARA et al., 1990; YOKOTA et al., 1990). This mutation accounts for approximately 91% of all MCAD mutations.

We have used the polymerase chain reaction (PCR) to detect this mutation in families with MCAD deficiency. The basic principle of this technique is the ex- ponential amplification of a target DNA sequence by Taq DNA polymerase in a cyclic process. The synthesis starts from two oligonucleotide primers flanking the target sequence. The primers used in this PCR span a 63-bp DNA fragment containing the MCAD mutation site (MATSUBARA et al., l w ) . They are designed to in- troduce an NcoI restriction site if the mutation is present, cleaving the PCR product to a 43-bp fragment. After digestion with NcoI the PCR products can be separated by electrophoresis on a polyacrylamide gel.

Detection of the most common mutation is a very fast and easy method for the prenatal diagnosis of MCAD deficiency since it can easily be applicated on chorion villi or amniocytes, and yields results in less then 2 days. Further it is a very reliable method in contrast to prenatal assay of MCAD enzyme activity or prenatal determination of organic acids.

References EDITORIAL (1 986) Lancet ii, 1073- 1075. MATSUBARA, Y., KRAUS, P. , &A, H., GLASSBERG, R., FMNoc-

c m o , G., IKEDA, Y., MOLE. J. . ROSENBKRC, L. E. & TANAKA, K . (1987) J. Biol. Chem. 262, 10104-10108.

MA-, Y.. NWWA, K.. M N A B O Y ~ , S., TADA, K., GATES, P. M., BACEMA", C., ELSAS, L. J. . P o r n , R. J. , WAD, W. J . L. & Roc, C. R. (1990) Biochem. Biophys. Res. Com- mun. 171, 498-505.

ROE, C. R. & COATES, P. M. (1989) in SCRIYER, C. R., BEAUDET, A. L., SLY, W. S., V m , D. eds. The metabolic basis of inherited disease, 6th ed., New York : McGraw-Hill, 889-914.

YOKOTA, I . , INDO, Y. , COATES, P. M., TANAKA, K. (1990) J. Clin. Invest. 86, 1000-1003.

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J. HENDRICKX, P. RAEYMAEKERS ( I ) and P. J. WILLEMS [Medical Genetics and ( I ) Neurogenetics, University of Ant- werp - U.I.A., Antwerp, Belgium].

Mapping of the gene for X-linked liver glycogenesis due to phosphorylase kinase deficiency to human chromosome region Xp22.

X-linked liver glycogenosis (XLG) (Mc Kusick 30600) is a benign glycogen storage disorder due to deficient activity of phosphorylase kinase (PHK). The clinical symp- toms include hepatomegaly, growth retardation, elevation of glutamate pyruvate transaminase and glutamate oxa- loacetate transaminase, hypercholesterolemia, hyper- triglyceridemia and fasting hyperketosis. With age these clinical and biochemical abnormalities gradually disappear and most adult patients are asymptomatic (WILLEMS et al., 1990).

PHK (E.C.2.7.1.38) is a regulatory protein kinase activating inactive phosporylase b to active phosphorylase a. The enzyme consists of 4 different subunits a,p, y and 6. The genes encoding the muscle-specific subunits of PHK have been cloned but it is unknown whether human liver subunits are encoded by the same genes (ZANDER et al., 1988). As the gene encoding the muscle a subunit of PHK (PHKA) was localized on the X chromosome in Xq12q13, it was considered a likely candidate gene for the mutation responsible for XLG (FRANCKE et al., 1989).

In the present study we investigated whether human liver glycogenosis due to phosphorylase kinase deficiency is indeed due to mutations in the muscle PHKA gene.

Two Dutch families affected with X-linked liver glycogenosis (22 patients and 13 obligate heterozygotes) were studied. Seventeen polymorphic DNA marker loci mapping between Xqter and Xpter were used in the present linkage study. A 5-point linkage analysis between XLG and ~58.1.) cX37.1, PGKl and pDP34 yielded lod scores beneath the exclusion limit -2 in each marker in- terval. This excludes the presence of the XLG gene in the Xq 12-q 13 region.

Combining several multipoint linkage analyses the presence of the XLG gene was excluded over a genetic distance of more than 150 cM of the X chromosome.

Positive two-point lod scores in both families were observed with the markers pTS247, pD2, pXUT23 and RC8. Combined peak lod scores (Zmm) for the two families were 6.64,3.75, 1.30 and 0.88 for pTS247, pD2, PXUT23 and RC8, respectively, all at recombination fraction (0) zero. A 6-point multipoint linkage analysis was performed between XLG and dic56, p99.6, pERT87-15 and pD2-pTS247 combined as a haplotype. This raised the peak lod score to 9.56 at 0% recombination relative

In conclusion, we have shown that XLG is not due to mutations in the PHKA gene as the Xq12-q13 region was excluded as the site of the XLG mutation. Furthermore the XLG gene was assigned to the Xp22 region, but candidate genes for XLG have not yet been reported in this region (WILLEMS et al., 1991).

to pD2-pTS247.

References FRANCKE, U., DARRAS, B. T., ZANDER, N. F. & KLLIMA", M. W.

(1989) Am. J. Hum. Gener. 45, 276-282. WILLJXS, P. J. , GERVER, W. J. M., BERCER, R. & FERNANDES, J .

(1990) Eur. J. Pediatr. 199, 268-27 1 . WILLEMS,~. J.,HENDRICKX, J.,VANDERAUWERA,B. J.eta1. (1991)

Genomics. In press. ZANDER, N. F., MEYER, H. E., HOFFMANN-POSORSKE, E., CRABB,

J. W., HEILMEYER, L. M. G . J r & K L L w , M. W. (1988)Proc. Natl. Acad. Sci. USA 85, 2929-2933.

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A. HERMAN, N. VAN CRAESBECK, L. KUTNOWSKI and G. PETIAu-DE V m (Chimie Gedraie I. CP 160, Univer- sit4 Libre de Bmelles, Avenue Franklin D. Roosevelt, 50, B-I050 Bmelles).

Chromatofocussing pattern of 0-galactosidase from murine myeloid leukemic cells and from a related tumor.

Malignant transformation, tumor growth and metastasis are accompanied by cell surface and intracellular modifications where glycosylation plays an important role. The purpose of this investigation was to test whether glycosylated constituents play a significant part in the development of a tumor. We have compared the activity of a lysomal enzyme, b-galactosidase - in murine leukemic cells (N122 obtained from E. Wright) - to that present in a tumor induced by sub- cutaneous passage of ten millions of these cells in a nude mouse. Cultured cells (conserved at - 80°C) were mixed to one volume (v/v) morpholinopropane- sulfonate buffer pH 7.4 containing 0.1 M glycine, 0.2 M NaCl and 2% Triton X-100 and disrupted in a Potter- Evelhjem system. The extract was put at 4°C for 2 hours and centrifuged at 2 900 x g. The supernatant was used as crude extract. The same procedure has been used for the preparation of a crude extract from the tumor. Both crude extracts were analysed by chromatofocussing on a Polybuffer exchanger 94 (Phurmuciu). The first eluting buffer was 25 m~ im- idazole, pH 7.4; the second buffer was the diluted Polybuffer 74, pH 4 (1:8 v/v). The presence of the enzyme was detected in an enzymatic assay containing 0.3 ml of a 0.2 M phosphate - 0.1 M citrate buffer pH 5, 0.3 ml substrate (1 m~ 4-methylumbeUiferyl-b-D- galactopyranoside) and 0.3 ml of enzyme. After incubation at 30°C for 30 min, the reaction was stopped by addition of 1.8 ml of a 0.5 M glycine buffer at pH 10.3. The fluorescence emission was measured after excita- tion at 370 nm. The presence of at least four isoforms was detected in both extracts (the pZ being respectively 7.2, 6.2, 5.4, 4.7 and a fraction with pZ below 4.5).

The chromatofocussing profiles for the 0- galactosidase activity were similar for the cells and the tumor but quantitative differences were observed. These results suggest that the injected tumor cells re- tain several properties. The quantitative differences can reflect different degrees of sialylation of the same proteins. Some modifications of the regulation of synthesis of these enzymes can occur during the passage in vivo. We have reported that a Golgi enzyme, /3-(1-4)- galactosyltransferase can also be resolved into several isoforms in the cell and tumor extracts and that the profiles are comparable (KUTNOWSKI et d., 1990).

This work was supported by a SMB-GALEPHAR grant.

Reference KUTNOWSKI, L., SCHNEK, A. G . & PETUU-DE VRIES, G . M. (1990)

Eur. J. Cell. Biol. 53, suppl. 31, 45.

I. INGELBRECHT, M. VAN MONTAGU and A. DEPICKER (Laboratorium voor Genetica, Rijksuniversiteit Gent).

Specific sequences downstream of the 3’- processing and polyadenylation signals are necessary for optimal expression in transgenic plants.

The nature of the 3’-untranslated region (3’UTR) clearly influences the level of expression of a reporter gene in plant cells. For example, in transgenic plants the steady-state mRA level of a reporter gene containing the 3’UTR of a chalcone synthase gene (3’ch) was approximately 20-fold lower than that of a gene containing the 3’UTR of a ribulose-l,5-bisphosphate car- boxylase gene (INGELBRECHT et d., 1989). In contrast, in electroporated protoplasts these two genes direct similar mRNA levels.

Now we have constructed a neomycin phosphotransferase I1 (nptZZ) reporter gene with a longer 3’ch fragment and compared the expression of this gene with that of the gene containing the shorter 3’ch. In transient expression the “ T I 1 enzymatic activity and the mRNA level was similar for both genes. Moreover, S1 -mapping experiments indicated that the two mRNAs were polyadenylated at the same site(s). This suggests that all information necessary for 3’ end processing and polyadenylation is present in the two genes. Both genes were subcloned in plant expression vectors and several independently transformed tobacco plants were analysed. Nearly all plants containing the gene with the longer 3’chs region had high NPTII enzymatic activity. In contrast, most plants containing the gene with the shorter 3’ch had a very low or undetectable enzymatic activity. Two plants of each series were analysed in more detail. The level of steady- state RNA present in leaf was approximately 20-fold lower in case of the gene containing the shorter 3’chs. In each case the RNA transcripts were polyadenylated and had the same length as estimated by Northern analysis. This implies that cytoplasmic stability for both mRNAs must be the same. Nuclear run-off analyses indicated a 2- to 4-fold lower transcriptional activity for the plants containing the gene with the longer 3’ch. These experiments also indicated that for this gene transcription terminates in the 3’-flanking sequences, which are absent in the other construct. We propose that this region contains specific information for transcription termination and that these sequences are important for maturation of a primary transcript.

Reference INGELBRECHT, I. L. W., HERMAN, L. M. F.. DEKEYSBR, R. A., VAN

MONTAGU, M. & DEPICKER, A. (1989) Plant Cell 1, 671-680.

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1. -CB;AERT, H. COEN, R. COLLUMBIEN, M. PAW= and F. ROELS (kfenseloke Anatomie, Vrije Universiteit Brussel, Laarbeeklaan 103, 8-1090 Brussel).

Computerized measurement of peroxisomal parameters in light microscopic sections (demonstration).

Human and rat peroxisomes are visualized by staining for catalase activity with diaminobenzidine. After embedding in Epon, 0.4 pm sections of liver are observed in transmitted tight and video images acquired by the VICOM image processor through a Bosch measuring camera at a resolution of 17 pixels/pm. Grey value resolution is improved by averaging up to 256 frames. For each pixel the optical density is computed log Io/I where 10 is the incident light intensity (no sample present); this corrects for heterogenous illumination. Subsequently stained organelles are separated from the background (segmentation) by an algorithm based on the local OD gradient (CLAEYS et d., 1989; KERCKAERT et ol., 1989). Manual exclusion of erythrocytes or sinusoids is optional. The following parameters are then measured by the image processor : number, area, form- factor, integrated OD, mean OD, mean OD in the central pixels only. The performance of the automated segmentation has been evaluated by comparing mor- phometric parameters at different levels of OD (catalase staining) (KERCKAERT et a/., 1989; 1990). Rare ag- gregated granules can be excluded by their form factor. By this method catalase activity and morphology have been measured in human adult (ROELS & COR- NELIS, 1989), foetal and postnatal liver (KERCKAERT, 1990), and in rat liver under the influence of thyroid hormones (KERCKAF~RT et d., 1989). Results were compared to data obtained by electron microscopy.

Supported by FGWO 3.0034.86 and 3.0018.89, and Nationale Loterc 9.0002.88. Presented at the 144th Meeting, May 1990.

References CLAKYS. A., CORNELIS. A., KERCKAERT, I . , COEN. H., ZUKOWSKI.

F., SHETS, G., & ROEIS, F . (1989) Gegenbaurs morphol. Jahrb. Leipzig (1989) 1, 83-90:

KERCKAERT, I . (1990) Them, Brussels. KERCKAERT, I . , CLAEYS, A., JUST, W., CORNELIS, A. & ROELS, F.

(1989) Micron and Microscopica Acto 20, 9-18. ROEIS, F. & CORNELIS, A. (1989) J. Histochem. Cytochem. 37,

331-337.

Isabelle LATOUR and P. BUC-CALDERON (Unit6 BCTC 7369, Universite Catholique de Louvain, 8-1200 Bruxelles).

Tert-butyl hydroperoxide inhibits RNA and protein synthesis in isolated rat hepatocytes.

The reaction of free -radicals with biological macromolecules like the polyunsaturated lipids (thus initiating a typical chain reaction such as lipid peroxidation) has been extensively reported (for a review see KAPPUS, 1985), but a free-radical-mediated effect on cellular functions has received less attention.

We reported that cells exposed to tert-butyl hydroperoxide (tBOOH) are damaged by peroxidation of their membrane phospholipids (Buc-CALDERON et d., 1989). At present time however, it remains still controversial whether this lipid peroxidation represents the principal mechanism of such cytotoxicity (MASAKI et QI., 1989; MCCONKEY et d., 1989).

In order to best understand the molecular mechanisms linked to the cytotoxicity of tBOOH, we have designed experiments to study its relationship with two major cell functions such as the protein synthesis ability and the maintain of the glycogen content.

This paper shows that tBOOH decreased both the RNA and the protein synthesis without loss of membrane integrity. In addition, the glycogen content was strongly decreased by tBOOH in a concentration- dependent way. Such oxidative effects were irreversi- ble after 5 min of incubation, suggesting a close relationship with the depletion of reduced glutathione and the increase in cytosolic free calcium.

The inhibition on protein synthesis by tBOOH could be mediated either by a covalent binding of a metabolite to DNA (e.g. an alkoxyl radical) with impairment of DNA template functions thus inhibiting the corresponding transfer and ribosomal RNA synthesis; or by the inhibition of RNA polymerase I1 (at -SH levels ?) resulting in some specific inhibition of messenger RNA synthesis.

References BUC-CALDERON, P., B u m , I . & ROBERFROID, M. (1989) Arch. inl.

KAPPUS, H. (1985) in Oxidative S t r m (Sm, H., ed.), pp 273-310.

MASAKI, N., KYLE, M. E. & FARBER, J. L. (1989) Arch. Biochem.

MCCONICEY, D. J . , H A R ~ L L , P., NICOTERA, P. & ORRENNS, S .

Physiol. Biochim. 97, B12.

Academic Press, London.

Biophys. 269, 390-399.

(1989) FASEB J. 3, 1843-1849.

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P. LIANG (l), A. L~FGREN (I) , W. VAN HUL (I) , J-J. MARTIN v), J. DUMON (3), J. LEROY (‘), I. LIEBAERS (”) and C. VAN BROECKHOVEN ( I ) [(I)

Neurogenetics Lab, ( I ) Neuropathology Lob and (’) Medical Genetics, University of Antwerp (UIA), (‘) Medical Genetics, University of Gent (RUG) and (”) Medical Genetics, Universi- ty of B m e l (WB) . Belgium].

Mutation and carrier analysis in Duchenne muscular dystrophy by pulsed-field gel electrophoresis.

Initially mutation analysis and carrier detection in Duchenne muscular dystrophy (DMD), a common X- linked myopathy, was performed by Southern-blot hybridization using polymorphic genomic probes either flanking or located inside the DMD gene (BAKKER et al., 1985). In 10% of the patients deletions were detected with the intragenic genomic probes (KUNKEL et al., 1986). The detection of deletions was greatly enhanced by the cloning of the entire 14-kb cDNA for the DMD gene (KOENIG et al., 1987). However, the cDNA clones could not detect deletions in 40Vo of the DMD patients. In some of these patients duplications are present in the DMD gene, which may easily be overlooked by Southern-blot hybridization. Further- more, the use of cDNA probes for the diagnosis of carrier females requires precise quantitative Southern-blot analysis. Some of these problems may be overcome by direct analysis of the DMD gene using pulsed-field gel electrophoresis (PFGE) (DEN DUNNEN et al., 1989).

We analyzed SfiI-digested DNA of 34 patients by PFGE and hybridization with the cDNA probes : 1-2, 3b-5a, 5b-7, 8b and 13-14 plus the genomic probes : P20, GMGX11 and 566. In total 22 (65%) mutations were detected, 18 deletions and 4 duplications. The ma- jority of the mutations was detected with the probes 1-2 and 566, none with probe 13-14. All deletions had previously been visualized by cDNA analysis using con- ventional Southern blots. However, 3 out of the 4 duplications were first detected by PFGE. We also analysed by PFGE the DNA of 27 females at risk. In 19 females altered SfiI fragments were detected next to the normal fragments c o n f e n g their carrier status.

References BAKKER, E. et al. (1985) The Lancet i, 655-658. KOKNIG, M., HOFFMAN, E. P., BERTELSON, C. J., MONACO, A. P.,

FEENER, C. & KUNKEL, L. M. (1987) Cell 50, 509-517. KUNKEL, L. M., MONACO, 4 P., MIDD~SSWORTH, W., Ocm, H.

D. & Lam, S. A. (1985) Roc. Natl. Acad. Sci. 82,41784182. DEN DUN”, J . T., GROVIXHOLTEN, P. M., BAKKER. E., BLONDEN,

L. A. J . , GINIMR, H. B., WAPENMR, M. C., VAN PAASSEN, H. M. B., VAN BROECKHOVEN, C., PEARSON, P. L. & VAN OM- MEN, G. J . B. (1989) Am. J. Hum. Genet. 45, 835-847.

L. LINS ( I ) , B. VANLOO (’), J. M. RWSSCHAERT (I) , R. BRASSEUR (’) and M. ROSSENEU (’) [(I) Loboratoire de Chimie Physique des Macromol&ules aux Interfaces, Universitt! Libre de Bnurelles and ( I ) Department of Clinical Chemistry, A . Z . St Jan, B-8OOO Brugge].

Characterization and th&retical modelling of apo AIV/DMPC complexes.

Apolipoprotein AIV is a protein associated with chylomicrons, high density lipoprotein (HDL) and also occurring in the lipoprotein-free plasma fraction (LAGROST et al., 1989). It is activating the lecithin cholesterol acyltransferase enzyme (LCAT) (STEJNMETZ et ol., 1985; CHEN et al., 1985) and is presumed to play a role in the reverse cholesterol transport. However, its physiological function remains unknown.

We have obtained discoidal particules of apo AIV/DMPC (in a ratio 1/3 W/W) in vitro by using the method described by JONAS (1986).

After purification of these complexes on a Superose 6HR, the phospholipid and protein content and the complex size were determined. A 260/1 lip/prot molar ratio and a 71 f 4 A Stokes radius were found (each complex contained 2 apo AIV). The transition temperature of DMPC is higher in the discoidal complexes than in DMPC liposomes.

Concerning the orientation, the amphipathic helices associated to apo AIV are parallel to the acyl chains of the lipids.

On the other hand, we elaborated a theoretical model for the apo AIV/DMPC discoidal complexes using the approach applied to the apo AI/DMPC complexes modelling (BRASSEUR et al., 1990). The alpha helical segments present in apo AIV were identified by different predictive methods. The helical structure energy was then minimized at the lipid/water interface and the minimized segments were assembled together with phospholipids. The helices are amphipathic with a hydrophilic face larger than the hydrophobic face. In the disk, the helices hydrophobic domain is associated with the fatty acyl chains and the hydrophilic domain allows the solubilization of the complex by fa- cing the aqueous environment.

The calculated molar ratio and diameter obtained were respectively 275/1 lip/prot and 130 A, in good agreement with the experimental data.

References BUSSEUR, R., DE MEUTTER, J. , VANLOO, B., G~~IU~AGETIGH, E.,

RIJYSSCHAERT, J . M. & ROSSENEU, M. (1990) Biochim. Biophys. Acta 1043. 245-252.

CHEN, C. H. & ALBERS. J . J . (1985) Biochim. Biophys. Acta 836,

JONAS, A. (1986) in Methodr in Enumology, 128, A, IV, 553-582,

LAGROST, L., GAMBERT, P., B o s Q ~ N , M. & LALLEMANT, C. (1989)

STEINMETZ. A. & UTERMANN, G. (1985) J. Biol. Chem. 260,

279-285.

Academic Press, New York.

J. Lip. Research 30, 1525-1534.

2258-2264.

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A. LOFGREN ( I ) , P. LIANG ( I ) , P. STINISSEN ( I ) , J-J. MARTIN (*), J. DUMON (9, J. LEROY ('), I. LIEBAERS (5) and C. VAN BROECKHOVEN ( I ) [(I)

Neurogenetics Lab, (2) Neuropathology Lab and (7 Medical Genetics, University of Antwerp (UIA), (') Medical Genetics, University of Gent (RUG) and C) Medical Genetics, Universi- ty of B m e l (VUB), Belgium]. Mutation analysis in patients with Duchenne muscular dystrophy by multiplex PCR amplification and cDNA hybridization.

X-linked Duchenne muscular dystrophy (DMD) is a rapidly progressing degenerative disorder of skeletal muscle afflicting about 1 in 3500 live-born males. The less frequent Becker muscular dystrophy (BMD) is allelic to DMD and shows similar but less severe clinical features. The DMD gene is 2,5 million base-pairs in size and comprises at least sixty exons (KOENIG et al., 1987). In 60% of the DMD (BMD) patients the disease is caused by a deletion of one or more exons (KOENIO et al., 1987; FORREST et al., 1988). Although the DMD deletions are heterogeneous in size and location they are concentrated around two specific regions in the DMD gene (LIECHTI-GALLATI et al., 1989). The observation of deletion hotspots has been used in the development of kits for the efficient and rapid diagnosis of DMD (BMD) patients by multiplex polymerase chain reaction (PCR) analysis of nine exons (GIBBS et al., 1989; BEGGS et al., 1990).

In our lab we are performing deletion diagnosis using nineplex PCR amplification of exons 4, 8, 12, 17, 19, 44, 45, 48 and 51 (Gmrs et ul., 1989) com- plemented with threeplex PCR analysis of exons 1, 50 and 60 (BEGGS et al., 1990). Furthermore each patient DNA was analyzed by Southern-blot hybridization using the cDNA probes described by KOENIG el al. (1987). By the multiplex PCR analysis we missed only one patient with a very small deletion involving exons 10 and 11. In a total of 57 patients, 31 (54%) deletions and 3 (5,3%) duplications were detected. Of the deletions 68% were in DMD patients, 22% in BMD patients and 10% in patients with an unclear clinical picture. All duplications were in DMD patients. It should be stated however, that duplications can easily be overlooked by the detection methods used. It has been predicted that the phenotypic differences between BMD and DMD patients depend on whether the translational open reading frame is preserved or shifted (FORREST et al., 1988; MONACO et ul., 1988). In our deletion patients we found 88% correlation between the phenotype and the reading frame hypothesis.

References BE-, A. H., KOENIG, M., BOYCE. F. M. & KUNKEL. L. M. (1990)

Hum. Genet. 86, 4 5 4 . FORREST, S. M., CROSS. G. S., FLINT, T., SPEER, A., ROBSON, K.

J. H. & D a m . K. E. (1988) Genomics 2, 109-114. Gmm, R. A., CHAMBERLUN, J . S., CASKEY. C. T. (1989) in PCR

Technology : Principles and Applications for DNA Amplitica- tion. (ERLICH, H. A., ed.) pp. 171-191. Stockton Press, New York.

KOENIG, M., HOFFMAN, E. P., BERTELSON, C. J . , MONACO, A. P., FEENER, C. & KUNKEL, L. M. (1987) Cell 50, 509-517.

LIECHTI-GALLATI, S., KOENIG, M., KINKEL, L. M., FREY, D.. BOLTSHAUSER, E., SCHNEDER, V., BRAG& S. & MOSER, H. (1989) Hum. Genet. 81, 343-348.

MONACO, A. P., BERTELSON, C . 5 . . L m c m - G u n , S.. MOSER, H. & KUNKEL, L. M. (1988) Genomics 2, 90-95.

R. LORIS, Julie BOUCKAERT, Ingrid ZEGERS, E. VAN DDSSCHE and L. WmS (Vrge universiteit Brussel, In- stituut voor Moleculaire Biologie, Paardenstraat 65, B-I640 Sint-Genesius- Rode).

Crystallographic studies on legume lectins.

The legume lectins are a large family of homologous proteins capable of recognizing specific polysaccharide cell determinants. Although some of them have been studied extensively, their true role is still obscure and the residues involved in carbohydrate recognition have been identified with certainly in only a limited number of cases. We obtained crystals from the lentil (Lens culinaris) lectin in the orthorhombic space C222, with unit cell dimensions a = 55.3 A, b = 73.8 A and c = 86.9 A. These crystals diffract to a much higher resolution (2 A or better) than the form previously reported (LEBRUN et al., 1983). Data col- lection on a FAST area detector is in progress. The crystals will also be tes:ed on the SRS synchrotron source (Daresbury, U.K.) in order to improve the resolution still further.

Based on extensive sequence homology and a similar sugar specificity (VAN DRJESSCHE, 1988), the three dimensional structure of the lentil lectin has always been sup- posed to be very similar to that of pea lectin. However their different crystal forms together with the results from more recent lI3Cd NMR studies (MARCHETTI et al., 1988) suggest that there should be some interesting differences in structure.

In order to study the structure-function relationship in the legume lectins, we are currently trying to co- crystallize the lentil lectin and concanavalin A with polysaccharides. Although of high importance to improve our knowledge of protein carbohydrate interactions, only very few crystallographic studies dealing with this subject can be found in the literature (RINI et al., 1986; B o r n et ul., 1990). We also prepared metal-substituted forms of concanavalin A and of the lentil lectin. The metal-binding properties of these lectins are well studied in solution (BREWER et al., 1983) and should now be in- terpreted in terms of the three dimensional structure of their metal-binding rcgions and of conformational differences induced h . ~ substituting the native manganese and calcium by : h e r transition metals. A preliminary 2.3-A resolutiw data set of double manganese-substituted con- canasalin A has already been recorded on station 7.2 of the Daresbury synchrotron source. Crystals had the same space group (1222) as the native protein but diffract to a much higher resolution.

This work was supported by the Biotechnology project of the V.R. W.B. Rerny LORIS, Julie BOUCK~~SRT and Ingrid Z E m received a grant from the IWONL.

References BOURNE, Y., R o u ~ , P. & CAMLULUU. C. (1990) J. Biol. Chem. 265,

BREWER, C. F., BROWN, R. D. & KOENIG, S. H. (1983) J. Biomol.

LEBRUN, E., VAN RAPENBUSH, R., Forue~s. A. & HOEBEKE, J. (1983)

MARC HE^, P. S., BEAITACEARYYA, L., ELLIS, P. D. & BREWER,

RINI, J . M., CARVER, J . P. & HARDMAN, K. D. (1986) J. Mol. Biol.

VAN DRIESSCHE, R. (1988) in Advances in Lectin Research 1,83-134, VEB Verslag Volk und Gesundheit, Berlin; Springer Verlag, Heidelberg.

18 161-18 165.

Struct. Dynamics 1, 961-997.

J. Mol. Biol. 166, 99-100.

C. F. (1988) J. Magn. Res. 80, 417-426.

189, 259-260.

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P. MALC~RPS and J.-P. DUFOUR (Unit6 de Brasserie et des Industries Alimentaires, Univemitd Catholique de Louvain. Place Croix du Sud 3. 8-1348 Louvain-la-Neuve).

Purification of an alcohol : acetyl-CoA acetyltransferase (AAT) from Sacchromyces cerevisiae.

Aliphatic esters are important flavour active compounds which are mainly synthesized by the yeast under anaerobic conditions of fermentation. An alcohol : acetyl- CoA acetyltransferase (AAT) catalyses this synthesis from aliphatic alcohols and acetyl-CoA (M,ucoms et al., 1991). Until now, only crude purification procedures of this enzyme have been reported by others (Yosmow & HASHIMOTO, 1981; AKITA et al., 1990).

In the present work, an AAT was purified from the 100 OOO x g (1 h) supernatant of a cell-free extract obtained by disruption with glass beads. Previous report (MALCORPS et al., 1989) have shown that under such conditions the AAT was associated to high-molecular-weight structures, presumably non-sedimentable membrane fragments.

The AAT was purified 12 OOO fold from this fraction to an apparent homogeneity (as juged by SDS-PAGE electrophoresis) using a series of chromatography columns.

The enzyme was first precipitated by ammonium sulfate (60% saturation) and polyethylene glycol (PEG) 6OOO (10% p/v). After solubilization of the pellet with Thesit (a polyoxyethylene-type detergent), a second precipitation by PEG 6OOO was carried out. The resulting supernatant containing the AAT activity was concentrated by adsorption on a Bio-Gel HFT hydroxylapatite column. After elution with 200 ~ l l ~ NaH,PO, the AAT fractions were pooled and layered on a Sephacryl S300-SF. The gel permeation chromatography was followed by ion- exchange chromatography on DEAE Sephacel (elution with a 0 to 200 m NaCl gradient). AAT fractions were subsequently chromatographed on a second hydroxylapatite column (elution with a 0 to 200 m NaH2PO4 gradient) and loaded on a Blue Sepharose CL-6B column (elution with a 0 to 1 M NaCl gradient). AAT was finally purified with a final yield of 2% after a last purification step including a third hydroxylapatite column.

The AAT consists of a single peptide with a molecular weight of 55 kd and an isoelectric point of 5.5 as determined by chromatofocussing on the PBE 94 exchanger.

The enzyme required the presence of ethylene glycol (20% v/v) and a non-tonic detergent throughout the purification procedure for maintaining the activity.

Moreover, when solubilized with octylglucoside the AAT reassociated to the initial structure if the detergent concentration was decreased below its critical micellar concentration, confirming the highly hydrophobic character of the enzyme.

References MA, O., SUZUKI, S., OBATA, T. & I-Luw. S . (1990) Agric. Biol.

IlrIALcows, P., MALDAGUE, A., SCHADECK, N. & DUPOUR, J.-P. Chem. 54, 1485-1490.

(1989) Arch. int. Physiol. Biochim. 97, B164.

J. Am. Soc. Brew. Chem. (in press). MALCORPS, P., CHEVAL, J.-M., Jw, S. & DLWOUR, J.-P. (1991)

Yosmow, K . & HASHIMOTO, N. (1981) Agric. Biol. Chem. 45. 21 83-2190.

B. MICHELET, C. PEREZ, P. BOGAERTS and M. BOmy (Unit6 de Biochimie Physiologique, Univemitd Catholique de Louvain, Place Croix du Sud 2-20.8-1348 Louvain-la-Neuve).

Regulation of the genes encoding the plasma membrane H + -ATPase of NicotiaM plumbaginifolia. All living organisms rely, for the transport of solutes

at the cellular level, on a plasma membrane ATPase that creates an electrochemical gradient across that membrane. Different ATPases exist, they pump specific cations against their gradient and find the energy for doing this in the cleavage of ATP.

Nicotianaplumbaginofolia, as all the other plants, has its plasma membrane electrochemical gradient built up by a H+-ATPase. This enzyme was discovered to be encoded by a multigenic family of at least 5 genes (BOUTRY et al., 1989). Three of these pma genes (for plasma membrane ATPase) were sequenced and found to be 95% homologous at the amino-acid level. S 1 -nuclease-mapping of their transcripts revealed the existence of a long transcribed and untranslated sequence upstream of the PMA-coding sequence. This long leader contains in each case a small open reading frame of five to nine amino acids, which is reminis- cent of translational control systems found in other organisms. These characteristics were also found in a gene coding the same enzyme in Arabidopsis thaliana (HARPER et al., 1990). We are studying the effect that these peculiar leaders could have on the expression of the pma genes.

We also study the transcriptional regulation of the pma genes. Hybridization with specific probes indicated a differential expression of threepma genes at the organ level. A more sensitive approach to this problem is to transform tobacco with fusions of prna promoter regions and a reporter gene that permits the histochemical analysis of transformed plants (JEFFER- SON et al., 1987). This should help us to detect the cells and tissues where the pma genes are transcribed. Our initial approach on the pmal gene will be reported.

B. M. and P. B. hold a fellowship from IRSIA. M. B. is research associate of the Belgian Fund for Scientific Research. This work was supported by grant BIO-02 from the (( Service de la Programmation de la Politique Scientifique N.

References & m y , M., &RELET, B. & G o m u , A. (1989) Biochem. Biophys.

HARPER, J., Mmm, L., DE Wm, N.. Yoo, M. & Swssw, M.

JEFFERSON. R., KAVANAGH, T. & BEVAN. M. (1987) EMBO J. 6,

Res. Comm. 162, 561-574.

(1990) J. Biol. Chem.. in the press.

3901-3907.

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B. ~VLISEREZ, L. VAN LAER, J. DE BLOCK and W. P. DE POlTER (Luboratoty of Neurophannacology, Dept. of Medicine, University of Antwerp (UIA). Universiteitsplein I , B-2610 Wilrijk).

Presence and subcellular distribution of proteins of large dense cored vesicles in bovine adrenergic cell bodies.

Large dense cored vesicles (LDV) from adrenergic neurons are assembled in the neuronal perikaryon and consequently transported towards the varicosities where their secretory products are released by regulated exo- cytosis. Several soluble and membrane-associated components of LDV have been characterized in detail in sympathetic axons and nerve terminals.

Therefore we were interested in the subcellular localization of LDV-specific proteins in the adrenergic cell body. Are chromogranin A (CGA), dopamine 8- hydroxylase (DBH) and cytochrome b,,, (CYT) localized in LDV, SDV, immature vesicles or Golgi-fractions? The subcellular distribution of these components is compared to the distribution of noradrenaline (NA) and neuropeptide Y (NPY).

Homogenates of bovine stellate ganglia were subjected to differential centrifugation to investigate their NA-storing organelles. A low speeci supernatant was layered on sucrose-deuterium-oxide density gradients to isolate a vesicle-containing fraction, while, in other experiments, a microsomal fraction was subjected to sucrose gradient centrifugation. The subcellular fractions were analysed by immunoblotting in order to study the presence of vesicle-specific antigens in large dense cored vesicles and other putative NA-storing organelles. Markers of Golgi-complex and dense cored vesicles were also assayed biochemically.

In microsomal fractions of sympathetic ganglia, only one type of NA-storing vesicles was observed, according to the distribution of NPY and NA. However, the distribution of DBH is quite different from that of NA and IWY with a large shoulder of enzymatic activity in the lower density range. This broad peak can be attributed to Golgi-derived immature vesicles representing an incomplete stage of the NA-storage par- ticle development. Immunoblotting with anti-DBH sup- ports these findings. The distribution of CYT-immunoreactivity perfectly parallels the distribution of NA and NPY. Therefore it is unlikely that the DBH-activity in the lower density fractions is derived from small NA-vesicles. The distribution of CGA reveals that this component is not primarily localized in the large dense cored vesicles at the level of the neuronal cell body.

Immunoblotting-experiments with isolated large dense cored vesicles from sympathetic cell bodies have also demonstrated that DPH is not exclusively localized in the LDV-containing fraction of the gradient but also in a lower density fraction significantly enriched by Golgi-membranes.

We can conclude from these results that, in contrast to various morphological studies, only one type of NA-storing vesicle, i.e. the large dense cored vesicle, can be demonstrated in adrenergic cell bodies. Vesicle-specific antigens to small dense cored vesicles could provide additional evidence.

D. MWSHONT, N. HALBOT, P. SOLEIL and A. MICHEL (hboratory of Biological Chemistry, Faculty of Sciences. University of Mom, 21, Avenue Maistrim, 57000 Mom).

Study of interaction be!ween the synthetic peptides analogs of signal-peptides and liposomes of DMPC and DPPC.

Almost all secretory proteins in both eukaryotic and prokaryotic cells are initially synthesized in a precursor form with an amino- terminal extension peptide named leader (or signal) sequence. Typical- ly, these peptides are 15-25 amino acid residues long and are com- prised of three distinct regions : a basic amino N-terminus, a central apolar domain and a polar C-terminus region with Pro residue (AUSTEN, 1979; AN- et al.. 1985). In order to investigate the interaction mechanism between leader sequences and membranes, two model peptides were synthetized according to the classical solid- phase method and purified on DEAEcellulose column and by reverse- phase HPLC (C4 silica-gel column). The sequences of synthetic peptides were chosen to mimick those of natural leader-sequences. The two following peptides were studied : R401 NH&UCLLEAFLELFAEFLEAWGPGGC-COOH R399 NH,-KKFAAFLEAFAELLELELFAEFL~WGPGGC-COH

Interaction of peptides with small unilamellar vesicles (SUV) of DMPC (250 pg/ml) and of DPPC (250 pg/ml) were investigated at 37°C. In order to discriminate between aggregation and fusion of vesicles induced by peptides R399m (m = monomer, 25pg/ml), R399 (d =dimer, oxydation of C-terminal Cys), R401rn and R401d, two fluorescent probes, NBD (energy donor) and Rh (energy acceptor) were inserted into the bilayers of lipid vesicles and their fluorescence spectra were analyzed in terms of fusion and aggregation mechanisms. Fusion and aggregation were distinguished using two different procedures. The fust one involves donor and acceptor-labelled vesicles mixed separately and then fused in the presence of peptides. This method measures both aggregation and fusion. The second method involves donor and acceptor chromophores being premixed in the same population of vesicles (in this case fusion is induced by peptides in the presence of unlabelled vesicles); this method measures only fusion phenomenon. The results for SUV of DMPC indicate that peptide R3% would induce 72% of aggregation + fusion (25% of fusion), that R399 would induce 63% of aggregation + fusion (17% of fusion), that peptide R401m would induce 80% of aggregation + fusion (17% of fusion) and that R401d would induce 77% of aggregation + fusion (31% of fusion) at pH 7.4. At pH 5.0 however, the following data were observed: - R399m : 43% of aggregation + fusion (22% of fusion); - R399d : 37% of aggregation + fusion (16% of fusion); - R401m : 40% of aggregation + fusion (12% of fusion); - R401d : 36% of aggregation + fusion (18% of fusion).

Concerning the liposomes SUV of DPPC, only the aggregation + fusion phenomenons were investigated at pH 7.4. The results indicated that peptides R399m. R399d. R401m and R401d would induce respectively 43%, 31%, 53% and 46% of aggregation + fusion.

On the other hand the release of calcein from DPPC vesicles (UAUSNER et al., 1981) was also measured at the two pH values in presence of peptides R399 and R401 (dimeric forms). The calcein- containing liposomes were incubated at 30°C for 30 min and the release of calcein was monitored by fluorescence at 520 nm. The results indicate that peptide R399 induces 19% of release at pH 7.4 and 17% at pH 5.0 while as much as 98% and 34% of release were observed for peptide R401 respectively at pH 7.4 and pH 5.0.

These results suggest that the model peptides (R399 and R401) can interact with bilayers of SUV of DMPC (Tc = 23°C) and of DPPC (Tc = 41°C) at pH 7.4 and pH 5.0. However the aggregation phenomenon is very important at pH 7.4. This effect probably arises from electrostatic interactions between the negative charges of Glu residues and the positive charges of quaternary ammonium of choline moiety of DMPC and DPPC.

This work was supported in part by grants from~RS(1.5 .117 .87F) and IRSIA.

References ANANTBARAUAUII. G . M., Jom. J . L.. BROUJUETIX. C. G.. SCHMIDT. C.

F., CHLMG, B. H., Hwcvas, T. A. & S m m . J. P. (1985) J. Biol. Chem. 260, 4311-4317.

AUSTEN, B. M. (1979) FEBS k t t . 103, 308-313. K~USNER, R. D.. K-, N.. WEINSTEIN, J . N., BLWM~NTHAL, R. & F u n ,

M. (1981) J . Biol. Chem. 256, 5879-5885.

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E. NELIS (I) , H. BACKHOVENS (Is2), C. VAN DUYN v), L. HENDRIKS (I) , M. CRUTS (I) , A. WEHNERT (Is’),

A. HOFMAN (”) and C. VAN BROECKHOVEN ( I ) [(I)

Neurogenetics Lab, Dept. of Biochemktty, Born Bunge Foun- dation, University of Antwerp (VIA), v) Innogenetics Inc., Belgium and (’) a p t . of Epidemiology. Erasmus University Rotterdam, The Netherlands].

Analysis of chromosome 21 DNA markers in families with Alzheimer’s disease.

Alzheimer’s disease (AD) is the most common form of senile dementia. AD is a degenerative disorder of the central nervous system characterized by progressive impairment of memory and intellectual functioning starting in the middle or late adult life. The major pathological hallmarks found in the brain of AD patients are loss of neurons, intraneuronal neurofibrillary tangles and extracellular amyloid plaques. Although the etiology of AD is unknown, inheritance plays an important role in the pathogenesis of the disease. Several families have been described in which AD seems to be caused by a genetic defect in a single autosomal dominant gene. In two separate studies, linkage was detected between AD and chromosome 21 markers (ST. GEORGE-HYSLOP et al., 1987; GOATE ef al., 1989) whereas in two other studies, linkage could not be confirmed suggesting genetic heterogeneity in familial AD (SCHELLENBERG et al., 1988; PERICAK-VANCE et al., 1988). The results of a recent collaborative study con- f m e d the chromosome 21 linkage in early onset AD families (onset <65 years) (ST. GEORGE-HYSLOP et ol., 1990). In the late onset AD families (onset >65 years) the disease may be caused by other gene(s) or by non-genetic factors.

We examined five AD families for linkage with four DNA loci on proximal 21q : D21S16, D21S13, D21S1 and D21Sll. The loci D21S16 and D21S13 and the loci D21S1 and D21S11 are tightly linked and physically close and may be considered as one locus, D21S13/D21S16 and D21SVD21Sll respectively, in the linkage analysis (ST. GEORGE-HYSLOP et a/., 1987; STINSSEN et al., 1990). The five AD families belong to an epidemiological study carried out in Rotterdam, The Netherlands ( H o w et 01.. 1990). In four of the families the AD patients had a mean age-of-onset of 64 years, in one family the age-of-onset was 45 years. The lod scores (logarithm of the likelihood ratios for linkage) in the four late onset AD families were negative at both the loci D21Sl/D21Sll and D21S13/D21S16. The early onset family showed with D21S13/D21S16 a slightly positive lod score of 0.44 at recombination fraction 0.0. The lod scores sum- mated over the five AD families were negative, but not significantly enough to exclude linkage to chromosome 21. Our non-informative results can be explained by the poor pedigree structure typically for late onset diseases such as AD and the low informativeness of the DNA markers used. However, the minorly positive results obtained in the early onset AD family and the‘overall negative results in the late onset AD families is compatible with the observation of genetic heterogeneity between the two types of families.

E.N. is holder of an IWONL sholarship.

References H O W , A., Scmm, W.. TANJA, T. A., VAN D~DN, C. M.. HAAXMA, R.,

LAYERIS, A. J.. &TEN, V. M. & SAM, R. J. (1989) Neumlogv 39. 1589-1592.

GOATE, A. M. et 01. (1989) Luncef 1, 352-355. PERICM-VANCE, M. A. er 01. (1988) Exper. Neurol. 102, 271-279. S~HELLENBERG, G. D. et at. (1988) Science 241, 1507-1510. ST. GEORGE-HYSUIP, P. H . el at. (1987) Science 235, 885-890. ST. GEORCE-HYSUIP, P. H. ef a/. and other members of the FAD (1990) Nclrurp

STINISSEN, P.. VAN Ha. W., VAN CAMP, G., B A C ~ ~ O W N S , H.. WE-T, A.. VAN DEN BERCHE. A. & VAN BROECKKOWN. C. (1990) Genomics

347, 194-197.

J. NORTIER (I) , P. VANDENABEELE e), E. NOEL (3), Y. BOSSELOIR (I), M. GOLDMAN (I) and M. DESCHODT- LANCKMAN ( I ) [(I) Laboratoire Plundkiplindre de Recher- che Exp&rimentale Biombdicale, Facultb de Mbdecine. Univer- sit6 Libre de Bruxelles. B-I070 Bruxelles, (l) Laborafory of Molecular Biology. State Uriivemily. B-9ooo Ghenf and (7 Departement de Chimie Biologique, HGpital Erasme. B-1070 Bnuelles] . Enzymatic degradation of tumor necrosis factor by activated human neutrophils : role of elastase.

Beside its role in the pathophysiology of septic shock and tumor-associated cachexia, tumor necrosis factor-a! (TNF) is also involved in local inflammatory responses. This latter effect is at least in part related to the action of TNF on polymorphonuclear neutrophils (PMN). Indeed, several functions of mature granulocytes have been shown to be activated by TNF, such as respiratory burst, chemotaxis, phagocytic activity and degranulation leading to the release of proteolytic enzymes in the pericellular area.

In the present study, we investigated whether TNF could be a substrate for leukocyte hydrolytic agents, in particular elastase and cathepsin G, two neutral proteases contained in azurophilic granules and known to degrade protein components of the extracellular matrix. The observation of a proteolytic digestion of [IZ5I)TNF in vifro led us to examine the biological activity of the degradation fragments of recombinant human TNF (rhTNF).

Human PMN were prepared and stimulated with phor- bol 12-myristate 13-acetate (PMA). This treatment resulted in a significant increase in elastase secreted in the supernatant of PMN, as compared to unstimulated PMN.

The incubation of [IZ5I]TNF with PMA-activated PMN produced several radioactive metabolites, whereas resting PMN did not degrade the peptide. The nearly complete recovery obtained after incubation in the presence of al-antitrypsin (al-AT), a well-known endogenous inhibitor of neutral serine proteases, suggested the involvement of neutrophil proteolytic agents in this degradative pathway. Degranulation of previously activated PMN was thus a necessary process to promote hydrolysis of TNF by intracellular proteases released into the medium. The comparison of HPLC profiles obtained from incubation of [12s1]TNF either with supernatant of PMA-activated PMN or with purified leukocyte elastase or cathepsin G led us to attribute a predominant role to elastase in the degradation process of TNF. Indeed, from the five peaks generated upon incubation of [‘2s1]TNF with the supernatant of PMA- activated PMN, four could be generated by incubation of the peptide with purified leukocyte elastase.

Finally, rhTNF incubated with either purified elastase or cathepsin G produced several fragments that were tested for their biological activity. None of the fractions corresponding to the degradation products was found biologically active in our cytotoxicity assay.

Our results suggest that an enzymatic degradation of TNF maYOCCUratinflamma tory sites, mostly by neutrophil elastase released by activated PMN and escaping regulation by al-AT, since the major part of this latter agent is rendered inactive by oxidation. This catabolic process affecting TNF might be regarded as ~ L I effective extracellular pathway for biological inactivation of TNF, limiting its pleiotropic and potentially toxic effects by negative feed-back.

This work was supported by grant 3.4503.89 from the FRSM, by the Banque Nationale de Belgique and the FG WO.

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L. OVERBERGH, S. TORREKENS and F. VAN LEUVEN (Center for Human Genetics, Campus Gasthuirberg. Univer- sity of Leuven, 8-3000 Leuven).

Molecular characterisation of the proteinase inhibitors of the alpha-%macroglobulin family in the mouse.

Proteinase inhibitors of the alpha-2-macroglobulin (A2M) type are characterized by the typical feature that inhibition is initiated by proteolysis at the bait region, which activates the inhibitor. The proteinase is sterically (in the tetrameric and dimeric members of the family) trap- ped and/or is linked covalently to the thiolester of the inhibitor by a Glu-Lys isopeptide bond or by an ester bond. These inhibitors are closely related to the complement C3, C4 and C5. The complex is cleared by receptor-mediated endocytosis in cells like macrophages and fibroblasts.

Indication for an important physiological role for this type of proteinase scavengers is their ubiquitous occur- rence (mammals, birds, amphibians, arthropods), their presence as multiple, different molecular forms in all the species examined and their evolutionary conservation. We believe that their physiological importance can only be studied in a transgenic animal model since no clinical clues are available to the functioning in vivo of these proteinase inhibitors. It is necessary then to characterise at the molecular level all the members of the A2M family in the animal model selected, the mouse.

Screening of mouse liver cDNA libraries with a cDNA clone corresponding to human A2M and subsequntly with murine-specific clones, yielded nearly hundred cDNA clones, analysed by restriction and partial sequencing. We identified 48 clones coding for mouse A2M (MAM), including a full-length of 4.6 kilobases, yielding a consensus MAM cDNA sequence. The predicted sequence confirmed protein micro-sequencing analysis of the N- terminal of both the 165 kd and the 35 kd subunits of MAM isolated from mouse plasma (VAN LEUVEN et al., 1987, and unpublished results). This demonstrates that both subunits are encoded by one gene and that the smaller subunit represents the carboxy-terminus, including the receptor-binding domain.

Other cDNA clones were isolated coding for other members of the A2M family. Two highly homologeous full-length cDNA sequences were derived. The encoded proteins showed sequence homology with human, rat and mouse A2M, and with rat Al-inhibitor3. In the mouse one single-chain inhibitor of the A2M type, named murinoglobulin, was described (SAITO & SINOHARA, 1985). We have isolated this inhibitor from mouse plasma and its N-terminal sequence matched one of the cDNA clones. A third related cDNA contained two introns, which ap- parently escaped proper splicing. Moreover a frame-shift mutation, caused by a one-base insertion indicates that this cDNA clone could be derived from a pseudogene.

The observed, unsuspected diversity is currently being further investigated at the protein level and at the genomic level, to obtain all the necessary information needed for genetic manipulation.

References SAITO, S. & SINOHMA, H. (1985) J. Biol. Chem. 260, 775-781. VAN LmrvW, F., W Y N M , P., CASSIMAN, J. J. & VAN DEN BERGHE,

H. (1987) J. Eiochem. 101, 1181-1189.

K. PETIT (I) , G. VAN DESSEL (’), W. DE POTTER ( I ) and w. DIERICK (’) [UZA-Laboratory for ( I ) Neurophar- macologv and c) Pathological Biochemktty, University of Ant- werp, Universiteirsplein I , 261 0 Wilrijk] . Characterization of phosphatidylinositol-4,5bis phosphate phospholipase C in chromaffin cells.

The coupling of the initial stimulus to secretion in many secretory cells involves the activation of a phosphatidylinositol-4,56isphosphate(PIP~)-specific phospholipase C (PLC) (EBERHARD et al., 1990).

Recently a tight correlation between polyphosphoinositide levels and secretion was found in chromaffin cells by manipulating the levels of these lipids in digitonin-treated cells with a bacterial PLC and ATP depletion. FISCHER et al. (1981) and EBERHARD & HOLZ (1987) reported on respectively phosphoinositides and inositolphosphates synthesis in bovine adrenal chromaffin cells. Previously a PLC ac- ting on PI was reported to be present in these cells (ZAEILER et al., 1986).

In this paper we like to report on the presence and characterization of a PIP,-PLC in chromaffin cells using an inositol-labelled substrate. Plasma membranes were isolated from bovine adrenal chromaffin cells as described by KIDRONI et a/. (1980). PLC assays were performed as described previously (VAN DESSEL et al., 1981). Enzymatic activity was found to be linear over 5 min and directly proportional to the concentration of protein up to 60 pg. Optimal activity was found at pH 7.5. The PLC displayed an absolute requirement for Ca2* ions (optimal at 1 m). Treatment with EGTA abolished the activity. Other divalent cations (e.g. Cu, Zn) were inhibitory. Lowering the ionic strength has no effect on the enzymatic activity; replacement of K’ with Na’ or Li’ at constant ionic strength resulted in no significant activity changes. The PLC was found to be dependent on cholate (0.5%) for optimal activity. Non-ionic detergents were inactive. The “apparent” K, for the substrate was 4 x M and the specific activity was 100 nmol min-I mg-I protein. The phosphatase was inhibited by neomycin and by agents which react with sulphydryl groups, but the enzymatic activity was unaffected by dithiothreitol. Studying the nucleotide specificity of stimulation, a slight increase (150%) in activity was found for GTP (7)s.

References EBERHARD, D., COOPER, C., Low, M. & HOLZ, R . (1990) Biochem.

J . 268, 15-25. EBERHARD, D. & Hou, R . (1987) J. Neurochem. 49, 1634-1643. FISCHER, S., HOLZ, R. & AGRANOFF, B. (1981) J. Neurochem. 37,

491-497. KIDRONI, G., SPIRO, M. & SPIRO, R. (1980) Arch. Biochem. Biophys.

203, 151-160. VAN DESEL, G., DE WOLF, M., LAGROU, A., HILDERSQN, H., VOETS,

R. & DIERICKX, W. (1981) Arch. int. Physiol. Biochim. 89, B205-206.

ZAHLER, P., REVIT, M., PILARSKA, M. & ROSENHECK, K . (1986) Biochim. Biophys. Acta 077, 312-379.

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J. PINXTEREN, D. VAN REETH, W. ANNAERT, L. VAN LAER, E. COEN and W. P. DE POTTER (Laboratory of Neuropharmacology, Department of Medicine, University of Antwerp (UIA), B-2610 Wilrijk).

Mitotic stimulation of bovine adrenal medullary chromaffin cells in culture by oxytocin.

Adrenomedullary chromaffin cells of adult animals are generally regarded as essentially postmitotic and ter- minally differentiated. Recently, it was shown that oxytocin enhanced the total number of chromaffin cells in adult rats (PLECAS et al., 1990). The presence of oxytocin receptors was also shown in this tissue (NUSSEY et al., 1987).

Cells were prepared by digestion of bovine adrenal glands with 0,2070 collagenase by a modification of the procedure of L m m et al. (1987). Purity of the preparations ranged from 95 to 98070, viability was determined by trypan-blue exclusion and was generally >%To. Cells were plated at a density of 105 cells/cm,/ml on Costar 24-well plates in Dulbecco’s modified Eagle medium/F12 (1:l) containing 10% fetal calf serum and supplemented with 100 p g / d penicilline, 100 p g / d gentamycine, 100pg/ml streptomycine, 25 pg am- photericine B, 50 p g / d ascorbic acid, 5 p g / d insuline and 50 pg/ml dexamethasone. Medium was changed on the third day after isolation, growth measurements started 24 hours later. Growth was detected by determination of [3H]-thymidine incorporation and counting of cells. Incubations were at 37°C under 5 % CO, atmosphere.

Growth response to oxytocine was shown to be dose-dependent. Duplicate culture media without oxytocin were used as control. No incorporation of radioactivity was detected when media were supplemented with 2.5 p g / d cytosine arabinoside and 2.5 pg/ml fluorodeoxyuridine. Dexamethasone was supplemented in the experiments because it was shown to inhibit cell differentiation without affecting proliferation (STEMPLE et al., 1988). It is also shown to stimulate survival of cells after digestion. Cells generated by mitosis during incubation are smaller and contain less granules (NUSSEY et a/., 1987). Within four days, cell number rises by at least 5% in medium containing 1 p g / d oxytocin. Mean [3H]-thymidine incorporation on the fourth day was found to be 22 f 0.6 pmol h-’ per 105 cells.

Oxytocin is shown to stimulate mitosis in cultured chromaffin cells. It becomes possible now to transfect these cells successfully because the absence of growth is shown to be a limiting step in previous trials.

J.-L. PREGALDIEN and P. BUC-CALDERON (Unite BCTC, UCL 7369. Universitk Catholique de Louvain. 8-1200 Bruxelles).

Enhanced cytotoxicity of hydrogen peroxide to hepatocytes by the addjtion of iron salts.

The catalytic decomposition of hydrogen peroxide by transition metals like iron or copper, yielding the strong oxidizing agent hydroxyl radical, is know as the Fenton reaction. However, due to the absence of catalytically active iron in physiological conditions, the real existence of such reaction remains still controversial (TIEN et al., 1982). The formation of reactive oxygen species such as superoxide anion or hydrogen peroxide during an oxidative stress has been extensively reported (SIES, 1985). Furthermore, a decrease in the affinity of iron to ferritin, thus yielding a free catalytically active iron, might lead to a Fenton chemistry as a real entity with toxicological implica- tions. Such situation may result by decreasing the cytoplasmic pH, or by iron overload (MASINI et al., 1989; S H A R ~ ~ A et al., 1990).

The aim of this work was to study the influence of iron salts in the cytotoxicity of hydrogen peroxide to isolated hepatocytes. Parameters such as LDH leakage, glycogen content and MDA formation were measured to analyse the contribution of mixtures with different ratios between femc and ferrous salts, and the influence of chelating agents (EDTA, ADP, Desferal) as well as free-radical scavengers such as mannitol, thiourea.

As expected, the results show that the addition of iron salts into suspensions of hepatocytes enhanced the toxic effects of hydrogen peroxide. Neither the redox status of iron salts, nor the ratio in a mixture of ferric and ferrous salts have a significant influence in such increased toxicity. The preincubation of hepatocytes with Desferal abolished totally the contribution of iron salts. No inhibitory action was observed by the addition of free-radical scavengers, thus suggesting that hydroxyl radicals may not be involved as the yoxic ultimate agent.

References -”I, A., ~BCCARELLI, D., TRENTI, T., CORONOW, F. P. &

MUSCATELLO, U. (1989) Biochim. Biophys. Acta 1014, 133-140. SEARMA. B. K., BACON, B. R. , BRITTON, R. S., PARK, C. H.,

MAQIERA, C. J. , O’Ntiru, R. , DALTON, N., SMANXK, P. & SPEROFF. T. (1990) Hepatology 12, 31-39.

SIBS, H. (1985) in Oxidative Stress (SIES. H. , ed.), Academic Press, London.

TIEN, M., SVINQEN, B. A. & AUST, S. D. (1982) Arch. Biochem. Biophys. 216, 142-151.

J . PINXTEREN holds a grant of IWONL.

References LIVETT, B. G. , MITCHELFULL, K. I . & DEAN, D. M . (1987) in In vitro

methods for studying secretion, chap. 1 1 , 171-175. NUSSEY, S. S., PRYSON-JONES, R. A., T A ~ O R , A., h a , V. T. Y.

& JENKINS, J . S. (1987) J. Endocrinology 115, 141-149. PLECAS, B., HIUSTIC, M., POPOVIC, A. & JAVOWC, D. (1990) Horn.

meta. Res. 22, 402-403. STEMPLE, D. L., MAHANTHRAPPA, N. K., ANDERSON, D. J . (1988)

Neuron 1, 517-525.

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J. ROBBEN (I), E. BOSMANS (2), B. WELLENS (2) and G. VOLCKAERT (') [(I) Laboratory ol Gene Technology, KULeuven. de Croyhan 42.8-3001 and v) Eurogenetics N. V.. Industriezone 24, Industriaone West, 8-3980 Teswnderlo] . Construction and high level expression of rubella- specific antigenic determinants as fusion proteins with active chloramphenicol acetyltransferase in E. coli.

Stable expression of peptides usually requires linkage to a carrier protein. We developed a multicopy vector for high level expression of fusion proteins with chloramphenicol acetyl transferase (Cat) in E. coli. This vector is of extremely small size (1386 bp), and carries a functional fragment of the pBR322 origin of replication, the cat gene derivative with multiple cloning sites near the 3'-terminus, and the tac promoter controlling both cat expression and vector replication. In batch cultures, Cat levels produced from this vector can reach up to 50% of the E. coli soluble proteins. The vector system takes advantage of the fact that carboxyl- terminal fusions can be carried out without loss of Cat enzyme activity, and, hence, transformants can be selected under chloramphenicol pressure. A powerful strategy in the construction of peptide fusions consists of cloning and assembling synthetic oligonucleotides coding for the peptide of choice. Computer-assisted reverse translation of the peptide amino-acid sequence allows the introduction of a series of unique restriction recognition sites in the coding sequence. Due to the small size of the vector only a limited number of restriction enzymes are excluded from this analysis. The presence of multiple recognition sites in the peptide gene sequence is a useful tool for cassette mutagenesis and protein engineering. Three antigenic determinants of the Rubella virus El-membrane protein which occur in a linear cluster in the native protein (TERRY et al., 1988) were fused to Cat, in separate constructs as well as in different combinations. The resulting fusion proteins react specifically with human anti-Rubella an- tisera. We also demonstrate that such chimaeric proteins can be affinity-purified via the Cat-moiety on chloramphenicol caproate agarose.

J . ROBBEN is a postdoctoral fellow of the Inrtituut tot Aan- moediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw.

Reference TERRY, G. M., Ho-TERRY, L., LONDESLIOROUOH, P. & REES, K . R.

(1988) Arch. Virol. 98, 189-197.

H. K. STALS and P. E. DECLERCQ (Laborutorium voor Klinkche Chemie, Instituut voor Farmaceutische Wetenschap- pen, KULeuven, Van Evenstraat 4. 8-3000 Leuven).

A convenient assay for radiolabeled long-chain acyl-CoA's in isolated hepatocytes.

Long-chain acyl-coenzyme A thioesters (acyl-CoA) are the substrates of &oxidation and glycerolipid synthesis in the liver. The determination of the steady-state level of acyl-CoA is therefore an important aspect of the study of the regulatory mechanisms in lipid metabolism.

HPLC analysis (CORKEY et al., 1990) is regarded as the method of choice for the assay of these acyl- CoA's. However, this technique is not suitable for handling large numbers of routine samples.

In the course of our investigation of triacylglycerol synthesis in permeabilized rat hepatocytes, a simple procedure has been developed to compare 14C- palmitoyl-CoA levels in permeabilized and intact cells, incubated with "C-palmitic acid.

Hepatocyte suspensions (1 ml) were acidified with 0.2 ml of glacial acetic acid and washed twice with 4.5 ml of light petroleum ether (b.p.40-65"C) : isopropanol (8:1, v/v) to remove free fatty acids (MANCHA et al., 1975). Based on the method described by CAUSEY et al. (1986), each sample was extracted twice with 4 ml of methanol : chloroform (2:1, v/v). Addition of saturated ammonium sulfate (0.2 ml) improved the acyl-CoA recovery by more than 30'70, probably due to its chaotropic effect on the acyl-CoA-protein binding. A fraction of the water/methanol upper phase was directly chromatographed on silicagel-60 plates. The use of a double solvent system (first : chloroform : methanol : acetic acid, 65:25:2 v/v, 1/2 h at 4"C, and second : chloroform : methanol : sodium acetate buffer 1 M pH = 3.6,50:50:4 v/v, 4 112 h at 4°C) resulted in migration of neutral lipids, common phospholipids and acyl-camitines, leaving only acyl-CoA at the origin. The purity of this acyl-CoA was confirmed by another TLC system (n-butanol : water : acetic acid, 50:30:20) and spectral analysis (CONSTANTINIDES et al., 1985). The radioactvity in the acyl-CoA spot was determined by liquid scintillation counting in Ready Safe (Beckman). Overall recovery was approx. 70%.

References CAUSEY, A. G. & BARTLETT, K. (1986) Biochem. SOC. Truns. 14,

1175-1176. CONSTANTINIDES, P. P . & STEIM, J . M. (1985) J. Biol. Chem. 260,

7573-7580. CORKEY, B. E. & DEENEY, J . T. (1990) in Fatty Acid Oxidation :

Clinical, Biochemical, and Molecular Aspects, pp. 21 7-232, Alan R . Liss.

htmcm, M., STOKES, G . B. & S ~ F , P. K . (1975) Anal. Biochem. 68,600-608.

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H. K. STALS and P. E. DECLERCQ (Laboralorium voor Klinkhe Chemie, Instituut voor Farmaceutische Wetenschap pen, KULeuven, Van Eventstraat 4. 8-3000 Leuven).

Computation and statistical comparison of the estimates of the Micbaelis-Menten model using nonlinear optimization.

Fitting the Michaelis-Menten hyperbolic function to enzyme kinetic data has been attempted in many different ways. Linear transformation (e.g. Lineweaver- Burk, Eadie-Hofstee) is a class of commonly used methods, in spite of known statistical shortcomings (LEATHERBARROW, 1990).

An attractive alternative is nonlinear optimization, but this requires a serious computational effort (MOTULSKY et al., 1987).

To overcome this barrier, a user-friendly software package -ENZ 2.0- has been developed, that allows the computation and comparison of parameter estimates of the Michaelis-Menten equation.

Computation of the estimates follows a nonlinear least-squares regression routine based on the Davidon- Fletcher-Powell optimization method. The numerical report includes current and final estimates and residual sums of squares, calculation of the bias of the estimates, the asymptotic variance-covariance matrix, the degrees of freedom and the residual mean square. In addition, residual analysis is provided to test the ade- quateness of the model. The graphics routine provides export files for three- and two-dimensional plots, including confidence bands. The capability to handle two data files simultaneously allows the comparison of Michaelis-Menten constants originating from different experimental data sets. An F-statistic on the residual mean squares of the separate and pooled data can be applied to test the equivdence-hypothesis of the parameters (hvom et al., 1989).

The statistical inferences are strictly valid only when based on linear equations. However, these inferences can still be made in the nonlinear case to the extent that the model-data combination has a near-to-linear behaviour. As a measure for the degree of this behaviour, the bias of the estimates is used, as suggested by RATKOWSKY (1983).

References ALvom, W. G., DRIVER, J . H., -TON, L. &CREASON, P. (1989)

LEATHERBARROW, R. J. (1990) Anal. Biochem. 184, 274-278. MOTULSKY, H. J. & Rmmu, L. A. (1987) Faseb J. 1 , 365-374. RATKOWSKY, D. A. (1983) i? Nonlinear Regression Modelling, a

uni/ed andpractical approach, pp. 20-23, Marcel Dekker lnc.

Mutation Re. 240, 177-194.

V. TIMMERMAN ('), P. RAEYMAEKERS (I), P. DE JONGHE ('), G . DE WINTER (I) , J. GHEUENS (z), L. SWERTS ('), K. JACOBS e), A. VANDENBERGHE (I), J . 4 . MARTIN (') and C . VAN BROECKHOVEN ( I ) [(I)

Neurogenetics Lab, Depl. of Biochemistry and e) Neurology and Neuropathologv Lab, Dept. of Medecine, Born Bunge Foundation, University of Antwerp (UIA), Belgium].

Genetic fine-mapping of t6e gene for Charcot-Mane- Tooth neuropathy type 1 (CMT la) to chromosome 17~11.2.

Charcot-Marie-Tooth type 1 disease (CMT 1) is an autosomal dominant hereditary motor and sensory neuropathy (HMSN I), characterized by progressive atrophy and weakness of distal limb muscles and nerves resulting in loss of deep tendon reflexes (DYCK, 1984). Pathologically CMT I is associated with extensive de- and remyelination of the peripheral nerves leading to a major reduction in the nerve conduction velocities. In one family, designated CMT Ib, the genetic defect showed conclusive linkage to the Duffy blood group (FY) located on chromosome Iq (STEBBINS & CONNEALLY, 1982). Recently, in the latter family the CMT Ib gene was tightly linked to the FC gamma RII gene located in lq21.2q23 (LEBO et ol., 1990). However, in a large number of families, designated CMT la, linkage was detected with two proximal 17p marker loci D17S58 (pEW301) and D17S7l (pATIWl), proving that CMT 1 disease is genetically heterogeneous with a major gene locus on chromosome 17 (VANCE et a/.. 1989; WYMAEKERS el ul., 1989). Further, we localized in one extended family, CMT A, the CMT l a gene in bands 17p11.2-pl2 between the DNA marker D17S71 and the gene for myosine heavy chain (MyH2) (Tnmauwm et ul., 1990).

We analyzed the multiple-affected, five-generation CMT l a family, CMT A, with markers located between D17S71 and MYH2 in 17pll.2-pl2 in order to localize the CMT la gene more precisely. The markers used are in accordance with their genetic map order: centromere - D17S71 (pATlO-41) - D17S122 (pVAW409) - D17S125 (pVAW412) - D17S61 @EW401) - Dl75123 CpVAW410) - D17S67 (pEW503) - MYHZ Cp10.5) - telomere (WRIGHT el ol., 1990). LOD-scores with CMT la were calculated using the programs MLINK and LINKMAP of the computer package LINKAGE (LATHROP et ol., 1985) assuming a CMT 1 gene frequency of l/lO.OOO and equal male and female recombination rates (e). Recombinants were observed with D17S71 and D17S67/D17S123 with peak LOD-scores of 5.19 at 0 = 0.038 and 1.46 at 8 = 0.101 respectively, suggesting that these markers are flanking the CMT la gene. No recombinants were detected with D17S122, D17S125 and D17S61. The highest LOD score (16.18 at 8 = 0.00) was obtained with D17SI22, indicating that the CMT la gene is close to this marker. Two- and multipoint data will be presented.

The availability of flanking and highly linked DNA markers is important not only for the eventual localization, isolation and identification of the CMT l a gene but also for prenatal and presymptomatic testing of individuals at risk for CMT l a disease.

V.T. is holder of an IWONL scholarship.

References DYCK. P. J. (1984) in DLFWWS of the peripheral nervous wstem. Peripheral

neuroputhy (DYCK, P. J . , THOMAS, P. K., LAMBERT, E. H. & BUNCE. R. eds.) 2nd edition. vol. 2. Saunders, Philadelphia, pp 16001655.

LATHROP, C. M., LAU)UEL, J . M., J u ~ m t , C. & OTT, J . (1985) Am. J . Hum. Genet. 37, 482498.

LEW, R., LYNCH, E., WEGMT, J . , MOORE. K., TROUNsTnrr, M. & V M DER PUIEC, M. (1990) J . Neurol. Sci. 98 suppl., A106.

RMYMAEKERS. P., T - W , v., DI7 JONCHE, P . , SWERTS, L., GHEUENS, J . , MARTIN, J.-J. , MWYLLE, L., DE WINTER, G.. VANDENBERGHE. A. & V m BROECKHOVEN, C. (1989) Am. 3. Hum. Genet. 45, 953-958.

S ~ ~ ~ B I N S . N. B. BCONNEALLY, P. M. (1982) Am. J . Hum. Genet. 34. 195A. T ~ M ~ F ~ M N . V.. m m . P.. DE JON-. P., D6 W m , G., S m n ,

BROECKHOVEN, C. (1990) Am. 3. Hum. Genet. 47, 680-685. VANCE, J . M., NICHOLSON, G . A., Y m o u , L. H., STAJICH, J . , STEWART.

C. S., SPEm. M. C., HUNG, W. Y. . R o s ~ s , A. D., B-R, D. & Prruca- VANCE, M. A. (1989) Exp. Neurology. 104, 1-4.

WRIGHT, E. C., GOLDOM, D. E., F m . P. R. , BARKER, D. F. & SKOLNICK. M. H . (1990) Genomics 7 , 103-109.

L . , ~ A C O ~ ~ , K . , G - N S , L . , ~ ~ , J.-J.,VANDENBERGHE,A.&VAN

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Sandrine TINTON and M. ROBERFROID (Unit6 BCTC, UCL 7369, Ecole de Pharmacie. Universit6 Calholique de Louvain, Av. E. Mounier- 73, 8-1200 Bwelles).

Quantitative analysis of DNA release by human peripheral blood lympbocytes by a nick translation assay.

Several authors have reported that human peripheral blood lymphocytes release in vitro, without any stimulation, a molecular complex containing DNA (ANKER el al., 1975; JACHERTZ el al., 1979; ANKER et af., 1984). In order to detect and to quantify DNA, they used either UV spec- trophotometry or colorimetry whith diphenylamine, two methods which are of low specificity and low sensitivity ( > 5 pg/ml) and thus require a large sample, usually a pool of lymphocytes from few individuals.

In order to confirm the previous observations concerning the release of DNA by lymphocytes and to get more quantitative data about this phenomenon, we have decided to use a more performant technique, the method of nick translation adapted by FOURND? et af. (1986) to measure plasma DNA. This assay is highly specific and it detects as little as 4 ng DNA nP. It can thus be applied to small volume @l) of individual cultures containing about 2 x 106 lymphocytes ml-I.

This assay was done as follows : lymphocytes were isolated from whole heparinized blood on Ficoll-Paque gradient and cultured in medium 199 in the absence of any stimulant, according to ANKER et af. (1984). At zero time as well as after different incubation times, a sample of cell suspension was collected, cells were counted and the cell viability was evaluated by measuring the percen- tage of cells excluding Erythrosin B. Lymphocytes were then separated from the culture medium and washed several times. The amount of DNA present in the cell-free supernatant was assayed by nick translation.

Using this technique, our results confirm that human DNA appears in the culture medium during incubation in vitro although it is not present at zero time. Moreover, for the same incubation time (48 h), the amount of DNA varies from one person to the other (20-100 ng DNA ml-') and the kinetics of release also varies amongst individuals.

The appearance of DNA in the culture medium is not due to cell lysis because no correlation was seen between cell death and the amount of DNA in the supernatant. The concentration of DNA measured after 38 h of incubation is dependent on the number of living lymphocytes in culture (from 0.5 to 5 x 106 cells ml-I). The nick translation assay is thus of great value to study the process of DNA release by non stimulated lymphocytes incubated in vitro. The technique we have adapted will be used to establish whether this release of DNA is specific for lymphocytes, if it is specific for some subpopulations of lymphocytes and if it is energy-dependent.

References ANKER, P.. STROUN, M. & MAURICE, P . (1975) Cancer Res. 35,

ANKER, P., JACHERTZ, D., MAURICE, P., HEW, J. , LEDERREY, C.

Fou&, G., GAYRAL-TAMINH, M., BOUCH& J . P . &Cow, J. (1986)

JACJERTZ. D., ANKER, P., MAURICE, P. & STROUN, M. (1979) Im-

2375-2382.

& STROUN, M. (1984) Exp. Cell. Biol. 52, 133-136.

Anal. Biochem. 158, 250-256.

munol. 31, 753-763.

B. CORNET, E. DECROLY, G. VANDENBUSSCHE, L. GIURGEA, J.-M. RWSSCHAERT and M. VANDEN- BRANDEN (Free University of Brussels CP206-2, Laboratoire de Chimie Physique des Macromol6cules a w interfaces, Bid du Triomphe, 1050 Brussels).

Properties of the envelopeglycoprotein (gp160) of the HIV-1 virus inserted into liposomes.

The envelope glycoprotein precursor (gp160) of the HIV-1 virus was inserted into the lipid bilayer of liposomes using a detergent dialysis procedure (COR- NET et al., 1990). The glycoprotein was expressed in mammalian cells using a vaccinia vector and was purified by affinity chromatography on lectin and by immunoaffinity. Liposomes bearing gpl60 were characterized by centrifugation on linear sucrose gradients and the protein-to-lipid ratio was optimized. Bin- ding assays of liposome-bound gp160 with recombinant soluble CD4 demonstrated that gpl60 has conserved its ability to bind to its cellular receptor (CD4). Com- petition assays with the CDCbinding antibodies OKT4 and OKT4a confirmed the specificity of this binding. Demonstration that a gp160 domain was really an- chored in the lipid bilayer and not simply adsorbed was evidenced by photolabelling with the hydrophobic probe 3-([125I]-trifluoromethhyl)-3-(indophenyl)di~irine. The secondary structure of gp160 prior to, and after, membrane insertion was determined using FT-IR spectroscopy (CABIAUX et al., 1989). The usefulness of gpl 60-liposomes as a model to study the fusogenic properties of gp160 involved in virus entry and syncytia formation is discussed.

This work was supported by grants from IRSIA (E.D.), NIH (NIAID grant A I-27136-01Al) (M.V.) and CEE (contract Sc1OOO195). We thank C. BRUCK, C. THIRUUU), A. DELERS and J . P. PRIEELS from Smith-Kline for gp160 supply and continuous support. Dr J . BRUNNER is acknowledged for collaboration in TID labell- ing experiments.

References Cheuux, V., BRASSEUR, R. , WATTKR, R. , FALMAGNE, P.,

RUYSSCHAERT, J . M. & G O O R M A G ~ G H , E. (1989) J. Biol. Chem. 264, 4928-4938.

CORNET, B., VANDENBRANDEN, M., COONUIJX, J . , GIURGEA, L., DEKEGEL, D. & RWSSCHAERT, J . M. (1990) Biochim. Biophys. Res. Commun. 161, 1.

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B. VAN DER AUWERA (l), P. WILLEMS (I), K. DE BOULLE (l), E. VAN DE KELFT (z) and V. TIMMERMAN (”) I(’) Department of Medical Genetics (l) Neurosurgery and (’) Biochemistry, University of Antwerp - UIA and University Hospital Ant werp] . Loss of constitutional heterozygosity in human glial tumors.

Inactivation of tumor suppressor genes or ‘anti- oncogenes’ as well as activation or amplification of dominant oncogenes seem te be important mechanisms in the pathogenesis of many solid tumors (MCDONALD & DOHRMANN, 1988). Human gliomas display astrocytic, oligodendrocytic or ependymal differentiation, and these different histophological findings indicate their biological aggressiveness (DAUMAS-DUPORT et ul., 1988). Tumor recurrence is frequent and the recurrent tumor is often less well differentiated. Cytogenetic analysis of direct preparations and short-term cultures of gliomas have provided information regarding the gross chromosomal changes taking place in these tumors. Malignant gliomas often show non-random numerical and structural abnormalities involving chromosomes 10, 17 and 22 (BIGNER et ul., 1984).

We compared constitutional and tumor genotypes at different restriction fragment length polymorphism (RFLP) loci on chromosomes 10 and 17 in IS unrelated individuals with astrocytomas grade I-IV (10 grade IV and 5 grade 1-111 samples). All tumor samples were removed surgically from the brain prior to chemotherapy and/or irradiation, and were immediately frozen in liquid nitrogen. Genomic DNA from human samples and from peripheral blood leukocytes was extracted using methods previously described (FIJLTS et al., 1989). DNA samples of approximately 10 pg were digested to completion with different restriction enzymes, fractionated by agarose gel electrophoresis, transferred to nylon and hybridized to probes radiolabeled by nick translation or oligopriming. Loss of heterozygosity (LOW in tumor DNA W ~ S detected for at least one chromosome 17 marker in 11 tumors (grade I - IV) whereas LOH for chromosome 10 loci was only detected in 3 gliomas grade IV Cglioblastoma multiforme). The control (nonchromosome 10 or 17 probes) used for the dosage determination, were chosen by revealing maintenance of constitutional heterozygosity in tumor DNA.

Chromosome 17 deletions are d t d with a number of known malignan~es esPedlY if the p53 tumor suppressor gene on the-short arm hv0lvtd (NI~RO et ul., 1989). S ine LOH for chromosome 10 loci k restricted

ble that recessive mutations at thm are engaged in tumor progression. These results also demonstrate that glioblastoma arises as the clonal expansion of an earlier staged precursor.

References

only to tumors of the highest malinnnncv gradc,itispossi-

BIGNER, s. H., W. J. , w, M. S. & h m , D. D. (1984)

DAW-DWRT, C., S c m A u w . B., O’FALLON. J. & KBLLY. p. Hereditas 101, 103-113.

(1988) cancer 62, 2152-2165.

R. (1989) Cancer Res. 49, 65726577. MCDONALD. J. D. & DOHRYA”. G. J. (1988) Neurasurgev 23,

537-544. N ~ ~ R O . J. M. el al. (1989) Nature 342. 705-708.

m m , D., ~ P E T S , R. H.. no-, G. A.. N-. Y. & WEITE,

Sylvie VANDERSTRAETEN and Francoise FOURY (unite de Biochimie Physiologique. Universitt! Catholique de Louvain, Place Croix du Sud 2-20. 8-1348 Louvain-la-Nave).

Mutations in the gene encoding the mitochondrial DNA polymerase of Sacc@romyces cerevisiae pro- duce a mutator phenotype.

The nuclear MIPI gene encodes the replicative mitochondrial DNA (mtDNA) polymerase of S. cerevkiue (FOLJRY, 1989). No other mtDNA polymerase gene has been identified so far and sequence analysis has shown that the primary structure of the 143.5-kd MIPl product highly diverges from that of prokaryotic and eukaryotic polymerases. However, the N-terminal region of MIPl possesses the three typical motifs associated with the 3’-5’ exonuclease proofreading activity present in many DNA polymerases (BERNAD et al., 1989). We have verified that mitochondrial extracts of a MIPl-overproducing strain exhibit an intense 3’-5’ exonuclease proofreading activity. Replacement by site- directed mutagenesis of a conserved aspartate residue in the exonucleolytic consensus I11 by an alanine residue dramatically decreases the 3’-5’ exonuclease activity in vitro and is associated, in vivo, with a 10-fold increase in the production of spontaneous point mutations in the mtDNA. These results show that the 3’-5’ exonuclease activity plays an important function in the cor- rections of errors made during DNA replication.

Interestingly, a mutant obtained by in vitro random mutagenesis which has been assigned to the N-terminal region of MIPl exhibits a 100-fold increase in the mutation rate of mtDNA and is accompanied by a ther- mosensitive growth phenotype on glycerol. Sequence and biochemical analyses will allow us to determine whether this dramatic increase of the mtDNA mutation rate is explained by the loss of the proofreading activity.

References BERNAD. A., BLANCO, L., LAZARO, J . M., MARTIN, G . & SALAS, M.

FOURY, F. (1989) J. Biol. Chem. 264, 20552-20560. (1 989) Cell 59, 2 19-228.


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B46 S O C ~ T E BELGE DE BIOCHIMIE, ANVERS UIA, 2 MARS 1991

G. VAN DESSEL (l), L. HEYTENS (2), J. MARTIN (") and W. DIERICK (') [UIA-Laboratory for (I) Pathological Biochemistry, ( I ) Malignant Hyperthermia and (I )

Neuropathology, Department of Medicine, University of Ant- werp, Universiteitsplein 1, 2610 Wilra)k].

Lipid analysis of muscles from humans susceptible to malignant hyperthermia.

Malignant hyperthermia is a genetically inherited disorder of the skeletal muscular system that occurs during anesthesia. An abnormality in the structure and function of the muscle membranes is suggested (MICKELSON et al., 1987).

In this study the lipid analysis of human MHS individuals were compared to controls. The classification (MHS vs normal) was performed as described by the European MH group (1984) after the halothane and caffein contracture test. Lipids of whole muscle (vastus lateralis) were extracted and analysed as described previously (VAN DJSSEL e? af., 1979; STEEN e? af., 1984).

On a wet weight basis there was no significant dif- ference in the levels for cholesterol and dolichol between normal and pathological tissue. However a 40% decrease in phospholipid concentration was noted in MHS. Apart from a reduction in the concentration of lysophospholipids and an increase in sphingomyelin content, two-dimensional TLC reveals no changes in the concentration of the major phospholipids. HPLC- analysis of the dolichol homologues shows a normal distribution with the 85C homologue as the major one followed by the lOOC dolichol. Recalculation of the lipid concentration with respect to protein concentration demonstrated a drastic decrease of all lipid classes in MHS due to the lower protein level (50Vo) in these pathological muscles.

Changes in phospholipid composition could result in changes in membrane characteristics and are in agreement with the changes in rigidity, fluidity and permeability described by CHEA & CHEAH (1985) for MHS muscles.

This work was partly supported by a Lotto grant # 9.0013.88 and by a FGWO grant # 3.0020.90.

References CHEAH, K. & CHEAH, A. (1985) Experientia 41, 656-661. EUROPEAN MH GROUP (1984) Br. J. Anaesth. 56, 1267-1269. MICKEISON, J., Ram, J. , HYSLOP, R. , GALLANT, E. & Lorn, F.

(1987) Biochim. Biophys. Acta 897, 364-376. STEEN, L., VAN DESSEL, G.,.DE WOLF, M., LAGROU, A., HILDER-

SON, H., DE KEUKGLEIRE. D.. PINKSE, F., FOKKENS, R. & DIERICK, W. (1984) Biochim. Biophys. Actu 141, 116-120.

VAN DFSEL, G., LAGROU, A., MARTIN, J., "IERICK, C. & DIERICK, W. (1979) J. Neurol. Sci. 40, 77-86.

H. VAN MEIRVENNE, G. VAN DESSEL, M. DE WOLF, A. LAGROU, H. J. HILDERSON and W. DIERICK (RUCA-Laboratory for Human Biochemistry and UIA- Laboratory for Pathological Biochemistry, University of Ant- werp, Groenenborgerlaan 171, 8-2020 Antwerp).

The uptake of dolichoi in Vero cells.

Previously uptake process for dolichol were studied both in vitro and in vivo, but no characteristics of the uptake mechanism or efflux data were provided (for a review : VAN DESSEL e? al., 1990).

Vero cells incorporate dolichol in a time-dependent and dose-dependent manner. Optimal uptake was found at 37°C and a pH of 7.4. The physico-chemical condition in which dolichol is presented to the cells influences the degree of uptake (DMSO > ethanol > liposomes). After a 20-h incubation, no metabolic products of dolichol could be identified. After differential pelleting nearly equivalent RSA values for incorporated dolichol were found for the M, L and P fractions. When expressed on the basis of organelle protein content the highest amounts of dolichol were found in the cytosol > mitochondria > light mitochondria > nuclei > microsomes. Dolichol was not taken up by bulk phase endocytosis, as sorbitol, a non- metabolizable label, was taken up poorly and in a time- independent way. Addition of excess amounts of cholesterol gave no inhibitory effect on the incorporation of dolichol indicating independent uptake mechanisms. Farnesol, geraniol, retinol, chloroquine, NaCN, NaF and NaAsO, inhibited the incorporation of dolichol in Vero cells.

When cells were enriched with dolichol and the efflux of the isoprenol was monitored, there was a very rapid phase of dolichol leakage (40% in +2-3 h) followed by a slower phase. The half-life time of dolichol in this second phase was found to be approximately 50 h. Addition of a lipid acceptor (serum, PC- liposomes) in the efflux medium increase the efflux by nearly 200-300%.

This work is partly supported by FGWO grant 3.0083.87.

Reference VAN DESSEL, G., DE WOLF, M., HILDERSON, H. J . , LAGROU, A. &

DIERICK, W. (1990) in Subcellular Biochemistry, Vol. 8 , pp. 227-278.

Archives Infernationales de Physiologie. de Biochimie ef de Biophysique, 1991, 99 (3)

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