2-Azido[alpha-32P]adenosine diphosphate (2-azido[alpha-32P]ADP) has been used to photolabel the ADP/ATP carrier in beef heart mitochondria. In reversible binding assays carried out in the dark, this photoprobe was found to inhibit ADP/ATP transport in beef heart mitochondria and to bind to two types of specific sites of the ADP/ATP carrier characterized by high-affinity binding (Kd = 20 microM) and low-affinity binding (Kd = 400 microM). In contrast, it was unable to bind to specific carrier sites in inverted submitochondrial particles. Upon photoirradiation of beef heart mitochondria in the presence of 2-azido[alpha-32P]ADP, the ADP/ATP carrier was covalently labeled. After purification, the photolabeled carrier protein was cleaved chemically by acidolysis or cyanogen bromide and enzymatically with the Staphylococcus aureus V8 protease. In the ADP/ATP carrier protein, which is 297 amino acid residues in length, two discrete regions extending from Phe-153 to Met-200 and from Tyr-250 to Met-281 were labeled by 2-azido[alpha-32P]ADP. The peptide fragments corresponding to these regions were sequenced, and the labeled amino acids were identified. As 2-azido-ADP is not transported into mitochondria and competes against transport of externally added ADP, it is concluded that the two regions of the carrier which are photolabeled are facing the cytosol. Whether the two photolabeled regions are located in a single peptide chain of the carrier or in different peptide chains of an oligomeric structure is discussed.

Prostate specific antigen (PSA) is a protease which is characteristic of the prostate. It is widely used as a serum marker for the early diagnosis of prostate cancer (PCa). Nevertheless, for concentrations between 4 and 10 ng/mL, PSA does not enable PCa to be distinguished from benign diseases, such as benign prostate hyperplasia (BPH). In sera, the use of a ratio between free PSA (PSA uncomplexed with protease inhibitor) and total PSA (free PSA and PSA bound to alpha-1 anti-chymotrypsin) enables the "gray zone" to be reduced, but an important proportion of patients are still wrongly classed. Using two-dimensional electrophoresis, we demonstrated using 52 PCa and 40 BPH well-documented clinical cases that BPH sera show a significantly greater percentage of low-molecular-weight free PSA elements (IwPSA) than PCa sera. In our study, the use of a ratio between IwPSA and standard free PSA enables the correct diagnosis of 100% of PCa and 82.5% of BPH cases as against when 73.1% and 42.5% respectively were correctly diagnozed using the total PSA and the free/total PSA ratio. This important finding may be related to differences in the mechanism secreting PSA from the prostate into the bloodstream. We have shown how a tissue marker may be turned into a powerful tumor marker by events probably unrelated to its expression.

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Monoclonal antibodies (MAbs) were generated by immunizing mice with a truncated recombinant protein corresponding to the immunodominant region (residues 1-120) of hepatitis C virus (HCV) nucleocapsid protein. The specific recognition by either human sera or mouse monoclonal antibodies of overlapping peptides spanning the core region 1-120 as well as the comparison with epitopes described earlier allowed the fine mapping of HCV core. Within the region 1-120, the major antigenic domain could be restricted to the first 45 amino acids. Indeed, the peptide S42G (residues 2-45) allowed the detection of an anti-HCV core response by all anticore-positive human sera examined. According to their epitope localization, three groups of mouse MABs could be evidenced that were directed against different regions of core. Group II MAbs recognized a strictly linear epitope (QDVKF, residues 20-24), whereas group I MABs were directed against a conformational epitope mainly located at the amino acid residues (QIVGG, 29-33). The epitope of group III MABs was also conformational (PRGRRQPI, residues 58-65). These three epitopes appeared close but different from the three major human epitopes RKTKRNTN, VYLLPR, and GRTWAQPGYPWPLY (residues 7-17, 34-39, and 73-86, respectively). Group II MAB 7G12A8 and group I MAB 19D9D6 were used in a sandwich ELISA for the capture and the detection, respectively, of viral core antigen in sera of patients with chronic HCV infection. After treatment of sera with triton x 100 in acidic conditions, amounts of viral antigen as low as 20 pg/ml of sera could be detected.

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4Corresponding author (Utrecht) Communicated by T.HuntWe have cloned and sequenced the cDNAs encoding Patella vulgata cyclins A and B. The cDNA clones contain an open reading frame of 426 and 408 amino acids respectively, which present similarity with cyclins from other species. Cyclin A and B RNAs are present as polyadenylated and non-polyadenylated RNA in prophase oocytes and are completely polyadenylated in metaphase I. During the first cleavages after fertilization the level of cyclin A and B mRNAs is high and drops when the free swimming stage is reached. Using pl3sucl-Sepharose bead precipitation we demonstrate that cyclin synthesis is triggered during maturation and that inhibition of protein synthesis makes the cyclins disappear rapidly from the metaphase I oocytes, which shift to interphase condition. By microinjecting antisense oligonucleotides into metaphase I oocytes, we demonstrate that in vivo ablation of cyclin A and B messengers together gives the same result, whereas microinjection of only one oligonucleotide does not show any effect.

A cyclic peptide that spans the major antigenic determinant of the human immunodeficiency virus (HIV) glycoprotein 41 (gp41) has been synthesized according to various strategies. For immunodiagnostic applications, biotin was added at the N‐terminus of the peptide and aminohexanoic acid was used as a spacer. Polymer‐supported oxidations were carried out in a variety of ways with thallium (III) trifluoroacetate. The biotinylcyclic peptide was released from the support using trimethylsilyl trifluoromethane sulfonate and various scavengers. The efficacy of these different cyclization and cleavage procedures was compared. Side reactions were studied, and a simple and efficient procedure was set up to monitor peptide cyclization by mass spectrometry. In a second series of syntheses the disulfide bridge was replaced by an amide bond. For this purpose, an aspartic acid derivative and a diaminopropionic acid were introduced during the synthesis in place of the two cysteine residues in the parent sequence. On‐resin cyclization was performed and led to a major side‐product identified as a piperidide. This undesired base‐mediated side reaction was prevented when, instead of piperidine, 1,8‐diazabicyclo‐[5.4.0]undec‐7‐ene was used for fluorenylmethyl ester deprotection. Reactivity of these peptides with different patients'sera and with a monoclonal antibody directed against the whole gp41 was tested using an enzyme‐linked immunosorbent assay.

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