Antibody responses of Macaca fascicularis against a new inactivated polio vaccine derived from Sabin strains (sIPV) in DTaP-sIPV vaccine.

Antibody responses of Macaca fascicularis against a new tetravalent vaccine composed of diphtheria toxoid, tetanus toxoid, acellular pertussis antigens, and inactivated poliovirus derived from Sabin strains (sIPV) was investigated to predict an optimal dose of sIPV in a new tetravalent vaccine (DTaP-sIPV) prior to conducting a dose-defined clinical study. Monkeys were inoculated with DTaP-sIPVs containing three different antigen units of sIPVs: Vaccine A (types 1:2:3 = 3:100:100 DU), Vaccine B (types 1:2:3 = 1.5:50:50 DU), and Vaccine C (types 1:2:3 = 0.75:25:25 DU). There was no difference in the average titers of neutralizing antibody against the attenuated or virulent polioviruses between Vaccines A and B. The average neutralizing antibody titers of Vaccine C tended to be lower than those of Vaccines A and B. The sIPV antigens did not affect the anti-diphtheria or anti-tetanus antibody titers of DTaP-sIPV. Furthermore, the average neutralizing antibody titers of Vaccine A against the attenuated and virulent polioviruses were comparable between M. fascicularis and humans. These results suggest that M. fascicularis may be a useful animal model for predicting the antibody responses to sIPVs in humans, and that it may be likely to reduce the amount of sIPVs contained in DTaP-sIPVs, even for humans.

The essential region for assembly and particle formation in hepatitis B virus surface antigen produced in yeast cells.

The hepatitis B virus (HBV) genome carries a HBV surface antigen (HBsAg) gene that can encode a polypeptide of 226 amino acids (aa). This gene can be expressed in the yeast, Saccharomyces cerevisiae, and the products can be assembled into 22-nm particles indistinguishable from those recovered from a patient's serum. We constructed a set of deletion derivatives of the HBsAg gene, and examined the particle-forming ability of the resulting polypeptides by expressing the gene in yeast. Elimination of 9 aa from the N terminus had no effect, whereas the elimination of 21-80 aa decreased the ability to form particles, and the particles formed were correspondingly smaller. Elimination of 100 aa that delete the major hydrophobic domain of the molecule abolished the ability to form particles completely. Deletion of 53 aa from the C terminus showed little effect. However, deletions proceeding further toward the center of the molecule rendered the polypeptides unstable.

Production of hepatitis B virion-like particles in yeast.

We constructed a yeast strain that simultaneously expresses four genes encoding the major S, middle S, large S hepatitis B viral envelope proteins and the core protein under the control of the yeast glyceraldehyde-3-phosphate dehydrogenase promoter and terminator. The lysate from this cell line, examined by immunological, physicochemical methods and electron microscopy, was found to contain spherical particles with a diameter of about 40 nm and a density of 1.25 g/ml. These particles reacted with anti-envelope antibodies, but not with anti-core antibodies. However, core antigenicity appeared upon treatment with 3% Nonidet P-40 that eliminates an outer envelope. These observations suggest production of a virion-like complex structure, or at least its DNA-less analog, consisting of core particle enveloped by antibody-reactive envelope. Such a structure was made only when all the four gene products were synthesized in a yeast cell. This system may be useful for the study of virus structure and assembly, and for improved vaccine development.

Identification of a novel alpha-amylase by expression of a newly cloned human amy3 cDNA in yeast.

A novel amylase gene (amy3) that differs in nucleotide sequence from salivary amylase gene (amy1) and pancreatic amylase gene (amy2) has been described [Tomita et al., Gene 76 (1989) 11-18], but whether this gene can ever code for an active enzyme has not been shown. We prepared cDNA of this gene from an mRNA obtained from lung carcinoid tissue, and expressed it in Saccharomyces cerevisiae under the control of an acid phosphatase promoter. The product was secreted into culture media, and showed enzymatic activity, demonstrating that this novel alpha-amylase gene (amy3) can code for a functional isozyme. We purified this enzyme, and compared its biological properties with those of salivary and pancreatic human amylases similarly expressed in yeast. We observed that the novel amylase isozyme is more heat-sensitive than others, and that its substrate specificity is different from the other two isozymes.

A novel type of human alpha-amylase produced in lung carcinoid tumor.

A novel type of alpha-amylase was detected in a lung carcinoid tissue after surveying the cDNA library constructed from this tumor mRNA. Nucleotide sequence analysis showed that the amylase expressed in this carcinoid tumor has 13 and 6 amino acid substitutions when compared with salivary amylase (Amy1) and pancreatic amylase (Amy2), respectively. The nucleotide sequence homologies of cDNAs between this carcinoid amylase and amy1, amy2 are 97.5% and 98.2%, respectively. The nucleotide sequence comparison strongly suggests that this new amylase is the product of the amy3 gene that has been detected in human genome [Emi et al., Gene 62 (1988) 229-235]

Potential therapeutic use of the selective dopamine D1 receptor agonist, A-86929: an acute study in parkinsonian levodopa-primed monkeys.

The clinical utility of dopamine (DA) D1 receptor agonists in the treatment of Parkinson's disease (PD) is still unclear. The therapeutic use of selective DA D1 receptor agonists such as SKF-82958 (6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzaze pine hydrobromide) and A-77636 ([1R, 3S] 3-[1'-admantyl]-1-aminomethyl-3,4-dihydro-5,6-dihydroxy-1H-2-benzo pyran hydrochloride) seems limited because of their duration of action, which is too short for SKF-82958 (< 1 hr) and too long for A-77636 (> 20 hr, leading to behavioral tolerance). We therefore conducted the present acute dose-response study in four 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-exposed cynomolgus monkeys primed to exhibit levodopa-induced dyskinesias to evaluate the locomotor and dyskinetic effects on challenge with four doses (from 0.03 to 1.0 mg/kg) of A-86929 ([-]-[5aR,11bS]-4,5,5a,6,7,11b-hexahydro-2-propyl-3-thia-5-+ ++azacyclopent-1- ena[c]phenathrene-9-10-diol), a selective and full DA D1-like receptor agonist with an intermediate duration of action. Levodopa and the DA D2-like receptor agonist, LY-171555 ([4aR-trans]-4,4a,5,6,7,8,8a,9-o-dihydro-5n-propyl-2H-pyrazo lo-3-4-quinoline hydrochloride) were also used for comparison. Acute administration of A-86929 was as efficacious in alleviating MPTP-induced parkinsonism as levodopa and LY-171555, but was less likely to reproduce the levodopa-induced dyskinesias in these animals than with either LY-171555 or subsequent challenge of levodopa. Selective stimulation of the DA D1 receptor may provide better integration of neural inputs transmitted to the internal segment of the globus pallidus (referred to as the basal ganglia output) compared with levodopa and selective DA D2 receptor agonist. Potent DA D1 receptor agents with an intermediate duration of efficacy such as A-86929 (approximately 4 hr at higher doses tested) are potential therapeutic tools in PD and merit further attention.

Minor structural differences in Boc-CCK-4 derivatives dictate affinity and selectivity for CCK-A and CCK-B receptors.

We previously reported novel Boc-CCK-4 (Boc-Trp-Met-Asp-Phe-NH2) derivatives possessing the general structure Boc-Trp-Lys[N epsilon-CO-NH-(R-Ph)]-Asp-Phe-NH2 (Shiosaki et al. J. Med. Chem. 1991, 34, 2837-2842). In contrast to Boc-CCK-4, which is 70-fold selective for the CCK-B receptor, the modified lysine-bearing tetrapeptides were highly potent and selective full agonists at the CCK-A receptor. Further investigation of the structure-activity profile following modification of the substituted phenylurea moiety appended off the lysine revealed that moving certain substituents, e.g. nitro or acetyl, from the 2- or 3-position on the phenyl ring to the 4-position, a relatively minor and subtle structural modification within the tetrapeptide, resulted in loss of CCK-A receptor selectivity and development of a trend toward CCK-B selectivity. These tetrapeptides, e.g. Boc-Trp-Lys[N epsilon-CO-NH-(4-NO2-Ph)]-Asp-Phe-NH2 and Boc-Trp-Lys[N epsilon-CO-NH-(4-Ac-Ph)]-Asp-Phe-NH2, were full agonists relative to CCK-8 in stimulating intracellular calcium mobilization in a cell line that expresses the CCK-B receptor.

Boc-CCK-4 derivatives containing side-chain ureas as potent and selective CCK-a receptor agonists.

Novel Boc-CCK-4 derivatives were communicated recently as having high potency and selectivity for the CCK-A receptor (Shiosaki et al. J. Med. Chem. 1990, 33, 2950-2952). While Boc-CCK-4 binds selectively to the CCK-B receptor, replacement of the methionine with an N epsilon-substituted lysine dramatically reversed receptor selectivity, leading to the development of this novel series of tetrapeptides. A detailed structure-activity analysis of a series of urea-substituted tetrapeptides, represented by the general structure Boc-Trp-Lys(N epsilon-CO-NHR)-Asp-Phe-NH2, revealed that a number of substituted phenyl, naphthyl, and aliphatic urea residues in the lysine side chain yielded potent and selective CCK-A ligands. These tetrapeptides elicit full agonist responses in stimulating pancreatic amylase release that are effectively blocked by a selective CCK-A receptor antagonist. Conversion of the urea to a thiourea significantly reduced CCK-A binding potency as did replacement of the lysine with the homologous ornithine or homolysine. Tetrapeptides that were partial agonists (less than 80% efficacy) in phosphoinositide (PI) hydrolysis relative to CCK-8 did not exhibit high-dose inhibition of amylase secretion in guinea pig acini.

Development of potent and selective CCK-A receptor agonists from Boc-CCK-4: tetrapeptides containing Lys(N epsilon)-amide residues.

A series of Boc-CCK-4 derivatives represented by the general structure Boc-Trp-Lys(N epsilon-COR)-Asp-Phe-NH2, where R is an aromatic, heterocyclic, or aliphatic group, are potent and selective CCK-A receptor agonists. These amide-bearing compounds complement the previously described urea-based tetrapeptides (Shiosaki et al. J. Med. Chem. 1991, 34, 2837-2842); structure-activity studies revealed parallel as well as divergent trends between these two series. A significant correlation was observed between pancreatic binding affinity and the resonance constant R of the phenyl substituent in one particular series of derivatives. Sulfation of phenolic amides appended onto the epsilon-amino group of the lysine did not affect affinity for the CCK-A receptor in contrast to the 500-fold increase in binding potency observed upon sulfation of CCK-8, suggesting that the lysine appendage and the sulfated tyrosine in CCK-8, both key structural elements that impart high affinity for the CCK-A receptor, are interacting differently with the receptor. The amide-bearing tetrapeptides are full agonists relative to CCK-8 in stimulating pancreatic amylase release while being partial agonists in eliciting phosphoinositide (PI) hydrolysis. Both effects were blocked by selective CCK-A receptor antagonists.

Potent and selective inhibitors of an aspartyl protease-like endothelin converting enzyme identified in rat lung.

Two structurally distinct series of potent and selective inhibitors of an aspartyl protease-like endothelin converting enzyme (ECE) activity identified in the rat lung have been developed. Pepstatin A, which potently inhibits the rat lung ECE, served as the basis for the first series. Alternatively, selected renin inhibitors containing the dihydroxyethylene moiety were shown to be inhibitors of rat lung activity. Subsequent modifications improved inhibition of the rat lung ECE while eliminating renin activity. Both series of ECE inhibitors demonstrated a range of selectivity over Cathepsin D. Water-solubilizing moieties were appended onto selected compounds to facilitate in vivo testing. Partial reduction of the pressor response to exogenously administered Big ET-1 was observed with selected rat lung ECE inhibitors.

Substituted hexahydrobenzo[f]thieno[c]quinolines as dopamine D1-selective agonists: synthesis and biological evaluation in vitro and in vivo.

A series of substituted 9,10-dihydroxyhexahydrobenzo[f]thieno[c]quinolines (TB[f]Q), varying with respect to the position of the thiophene relative to the benzo[f]quinoline core and the nature and position of the substituent on the thiophene, were prepared and evaluated for their affinity and selectivity for the dopamine D1-like receptor. The thieno[3,2-c]B[f]Q regioisomers bearing a small alky1 (C1-C3) substituent at the 2 position were potent (Ki < 20 nM) and selective (D2/D1 > 50) D1 agonists with close to full agonist activity (IA > 85%). The compounds were resolved and found to exhibit a high level of enantiospecificity in their interaction with the D1 receptor. Selected compounds were tested in vivo in the 6-OHDA rodent model of Parkinson's disease and for their liability to produce seizure-like activities in mice. (5aR)-trans-2-Propyl-4,5,5a,6,7, 11b-hexahydro-3-thia-5-azacyclopent-1-ena[c]phenanthrene-9,10-diol (5) emerged as the compound with the best overall in vivo profile in terms of potency (ED50 = 0.04 mumol/kg) and safety.

Tetrapeptide CCK-A agonists: effect of backbone N-methylations on in vitro and in vivo CCK activity.

N-Methylation of backbone amide bonds was conducted on a tetrapeptide that had been identified previously (Shiosaki, K.; et al. J. Med. Chem. 1991, 34, 2387-2842) as a potent and selective CCK-A agonist. N alpha-Methylation at the position corresponding to Asp32 (CCK-33 numbering) was consistent with high affinity, efficacy, and selectivity for the CCK-A receptor. Combination of this (N-Me)Asp with the (N-Me)Phe modification also provided a highly active analogue. The observation of parallel structure-binding affinity profiles with respect to sites of N-methylation in the C-terminal regions of tetrapeptide vs heptapeptide CCK analogues suggests that the two series interact similarly with the CCK-A receptor.

Actions of the D1 agonists A-77636 and A-86929 on locomotion and dyskinesia in MPTP-treated L-dopa-primed common marmosets.

Common marmosets show parkinsonian motor deficits following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration and develop dyskinesias during chronic L-dopa exposure. The D1 agonists A-77636 [(1R, 3S) 3-(1'-adamantyl)-1-aminomethyl-3, 4-dihydro-5, 6-dihydroxy-1H-2-benzopyran HCl] and A-86929 [(-)-trans 9, 10-hydroxy-2-propyl-4, 5, 5a, 6, 7, 11b-hexahydro-3-thia-5-azacyclopent-1-ena[c]phenanthrene hydrochloride] possess potent antiparkinsonian activity in the MPTP-treated marmoset and we now assess their influence on L-dopa-induced dyskinesias. MPTP-treated marmosets with stable motor deficits were treated with L-dopa plus carbidopa for 28 days to induce dyskinesias. Subsequently, they received A-86929 for 10 days, initially at 0.5 micromol/kg and then at 1.0 micromol/kg for a further 5 days. Several months later, L-dopa 12.5 mg/kg plus carbidopa 12.5 mg/kg was given orally twice daily for 7 days, followed by A-77636 1 micromol/kg for 10 days, and then both A-77636 and L-dopa plus carbidopa were given concurrently for 3 further days. In these L-dopa-primed animals, A-86929 effectively reversed akinesia and produced dose-dependent dyskinesias which were significantly less intense than those produced by L-dopa administration. A degree of behavioral tolerance was encountered, but antiparkinsonian activity was preserved and elicited behaviour was free of hyperkinesis and stereotypy and more naturalistic than that seen with L-dopa. After a week of twice-daily L-dopa dosing, administration of the long-acting D1 agonist A-77636 initially dramatically enhanced locomotion and reproduced dyskinesia with prominent dystonia, but after repeated administration of A-77636, dyskinesia and in particular chorea, gradually disappeared. Tolerance to locomotor stimulation greater than with A-86929 occurred, although activity remained significantly above baseline levels. There was a marked reduction in L-dopa-induced climbing, stereotypy and hyperkinesis and behaviour more closely resembled that of normal unlesioned marmosets. Upon reintroduction of L-dopa concurrently with continued A-77636 administration, dystonic, but virtually no choreic dyskinesias appeared and behaviour was once again free of stereotypy and hyperkinesis, contrasting dramatically with the presence of these behaviours along with abundant chorea when L-dopa is given alone. These results show a lesser liability of A-86929 and A-77636 to reproduce dyskinesia in L-dopa-primed MPTP-lesioned subjects while maintaining effective antiparkinsonian activity and producing a more naturalistic motor response. The differential effects of A-77636 on chorea and dystonia, with suppression of chorea and stereotypy on co-administration with L-dopa, may reflect an altered balance of activity in the direct and indirect striatofugal pathways. These results suggest a possible role for D1 agonists in the treatment of Parkinson's disease.

Actions of three human alpha-amylases expressed in yeast on modified substrates and the amino acid residues causing their different actions.

The mode of action of an alpha-amylase (yHXA) which was the gene product of a newly found human alpha-amylase gene expressed in yeast on synthetic substrates was compared with those of the gene products (yHSA and yHPA) of human salivary and pancreatic alpha-amylase gene in yeast. The substrates used were phenyl alpha-maltopentaoside (G5 phi) and its derivatives in which the CH2OH groups of the non-reducing-end glucose residues were converted to CH2NH2 (AG5 phi), COOH (CG5 phi), or CH2I (IG5 phi). The digests were subjected to HPLC to determine the amounts of products. The HPLC analysis revealed that yHXA and yHSA bound G5 phi to their active sites in similar manners to give the same products, while yHPA hydrolyzed it in a different way. Modifications of the non-reducing-end glucose of G5 phi caused change of the binding mode to the active sites of the enzymes. AG5 phi and CG5 phi were hydrolyzed by the enzymes to give more phenyl alpha-glucoside (G phi) and less phenyl alpha-maltoside (G2 phi), while IG5 phi gave more G2 phi and less G phi, compared with G5 phi. The substrate binding mode of yHXA changed more extensively than that of yHSA. The results suggested that there exists an amino acid replacement between yHXA and yHSA. The amino acid residues replaced are neither acidic nor basic, are located in subsite S3, and interact with the CH2OH residue of the non-reducing-end glucose residue of G5 phi.(ABSTRACT TRUNCATED AT 250 WORDS)

Differentiation of human non-salivary, non-pancreatic alpha-amylase from salivary and pancreatic alpha-amylases by use of FG5P.

Human non-salivary, non-pancreatic alpha-amylase (yHXA) is the gene product of a newly found human alpha-amylase gene expressed in yeast. Its mode of action on a fluorogenic derivative of p-nitrophenyl alpha-maltopentaoside, FG5P (FG-G-G-G-G-P), was examined at various pH values to elucidate the difference between yHXA and pancreatic or salivary alpha-amylase. The product analysis of the digests by HPLC showed that the enzyme hydrolyzed FG5P to FG3 (FG-G-G) and p-nitrophenyl alpha-maltoside (G-G-P) and to FG4(FG-G-G-G) and p-nitrophenyl alpha-glucoside (G-P), and the ratio of the two reactions changed with pH. The three enzymes differed from each other in the mode of action at pH 5.5. The molar ratio of FG4 to FG3 in the digest with yHXA was the largest. This suggested that the expression of the new gene in human can be detected by the use of FG5P as the substrate in the alpha-amylase assay.

Fine structure of hepatitis B virus surface antigen produced by recombinant yeast: comparison with HBsAg of human origin.

The ultrastructure of hepatitis B virus surface antigen (HBsAg) particles produced by recombinant yeast cells was examined using high-resolution negative staining, and ice embedding, electron microscopy. With negative staining, the HBsAg particles were spherical to slightly ovoid with a mean diameter of 27.5 nm and consisted of many subunits each 4 nm in diameter. Subunits were marked with a minute central pore. With ice embedding, particles were mostly spherical to ovoid, with a mean diameter of 23.7 nm and a 7-8 nm thick cortex surrounding an electron translucent core. Human HBsAg particles, examined using the same methods, were smaller, apparently because of molecular differences in polypeptide structure.

Induced expression of neuronal membrane attack complex and cell death by Alzheimer's beta-amyloid peptide.

beta-amyloid peptide (A beta) and complement-derived membrane attack complex (MAC) are co-localized in senile plaques of brains from Alzheimer's disease (AD) patients. But the relationship between A beta and complement activation is unclear. We have used human neurotypic cells, differentiated SH-SY5Y, as a model system to examine regulation of neuronal MAC expression and cell death by A beta. We demonstrated that mRNAs (C1q, C2, C3, C4, C5, C6, C7, C8 and C9) and proteins (C1q, C3 and C9) for the major components of the classical complement cascade are present in the SH-SY5Y neurotypic cells, indicating that neuronal cells can synthesize the necessary proteins required for MAC formation. Furthermore, immunocytochemical studies showed the A beta-induced neuronal MAC expression on the SH-SY5Y cells after CD59 was removed by PIPLC or blocked by anti-CD59 antibody. Meanwhile, increased A beta-induced neuronal cell death was observed following treatment with anti-CD59. Taken together, these results suggest that A beta activates neuronal complement cascade to induce MAC, and a deficiency of endogenous complement regulatory proteins, e.g., CD59, may increase the vulnerability of neurons to complement-mediated cytotoxicity.

Dopamine D1 receptor desensitization profile in MPTP-lesioned primates.

The motor effects of dopamine D1 receptor activation and the optimal way to stimulate these receptors were studied in a primate model of parkinsonism induced by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), using 2 selective full dopamine D1 receptor agonists: A-77636 ([1 R,3S] 3-(1'-adamantyl)-1-aminomethyl-3,4-dihydro-5,6-dihydroxy-1 H-2-benzopyran hydrochloride), and SKF 82958 (6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1 H-3-benzazepine hydrobromide). A-77636 was administered to one group of primed monkeys (N = 4) previously treated with levodopa and other dopamine receptor agonists, while SKF 82958 was given to another group of drug-naive monkeys (N = 3). These drugs have different durations of efficacy, lasting > 20 h and approximately 1 h, respectively, and were administered once daily (A-77636) or thrice daily (SKF 82958) for 7 days. Both drugs demonstrated excellent antiparkinsonian efficacy and locomotor stimulation. However, a rapid, functionally important, homologous (selective for D1 receptor agonists) desensitization process took place as early as on the second day with the longer-acting drug and a dose escalation of A-77636 failed to restore the initial benefit. Thrice daily dosing at a 4-h interval with the short-acting agent SKF 82958 maintained the maximal antiparkinsonian response but some shortening in the duration of response was observed after several days. These behavioral results show that dopamine D1 receptors are susceptible to desensitization after prolonged occupancy and can be desensitized profoundly and independently of dopamine D2 receptors in vivo in this model. Potent dopamine D1 receptor agonists with an intermediate half-life may prove to be better adjuncts in the treatment of Parkinson's disease. Clinical entities with pathologically enhanced dopamine D1 receptor-linked neural transmission might eventually also benefit from such desensitization.

Hyperactivity and behavioral seizures in rodents following treatment with the dopamine D1 receptor agonists A-86929 and ABT-431.

A-86929 ((-)-trans-9,10-dihydroxy-2-propyl-4,5,5a,6,7,11b-hexahydro-3- thia-5-azacyclopent-1-ena[c]phenanthrene) is a potent and selective full agonist at the dopamine D1 receptor. Both A-86929 and ABT-431 ((-)-trans-9,10-diacetyloxy-2-propyl-4,5,5a,6,7,11b- hexahydro-3-thia-5-azacyclopent-1-ena[c]phenanthrene hydrochloride), the diacetyl prodrug derivative of A-86929, were evaluated for their effects on behavioral excitability in rodents. In rats, A-86929 produced a dose-dependent increase in locomotor activity that was attenuated by the selective dopamine D1 receptor antagonist, SCH 23390, as well as by higher doses of the dopamine D2 receptor antagonist, haloperidol. Repeated administration of A-86929 over 6 days produced hyperactivity which did not change in magnitude across days. Acute administration of A-86929 and ABT-431 to mice produced behavioral seizure activity, with ED50 values of 7.1 and 2.7 mumol/kg, s.c., respectively, that was blocked by SCH 23390. Young rats (35-37 days) exhibited behavioral seizures following A-86929 and ABT-431 treatment (ED50 = 34.2 and 35.6 mumol/kg, s.c., respectively), but at doses higher than those required in mice. Moreover, adult rats (3 months) were less sensitive (ED50 = 345 mumol/kg, s.c.) to A-86929-induced seizures than young rats. Comparison of the ED50 values that produced behavioral seizure activity in rats with those previously established to produce contralateral rotation (ED50 = 0.24 mumol/kg, s.c.) in 6-hydroxydopamine-lesioned rat indicates that a significant dose separation exists between these two properties of A-86929.

A yeast system for stable expression of hepatitis B surface antigen.

We have constructed a yeast strain that has integrated into its chromosomal ribosomal RNA gene site two copies of Hepatitis B virus surface (HBS) antigen gene under the control of the yeast (Saccharomyces cerevisiae) glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter and terminator. The level of expression of HBS gene was low in the strain, but upon chemical and physical mutageneses, in combination with an immunological screening procedure, a mutant clone which expressed HBS protein at a high level was obtained. This mutant strain produces HBS antigen stably under non-selective conditions.