Expression of hepatitis B virus middle and large surface antigen genes in Saccharomyces cerevisiae.

The hepatitis B virus genome carries the surface antigen (SAg) gene and an open reading frame that encodes two SAg-related polypeptides: SAg with a 55-amino-acid N-terminal extension polypeptide and SAg with a 174-amino-acid N-terminal extension polypeptide. These are termed middle S and large S, respectively. These polypeptides or their glycosylated derivatives have been detected in Dane particles, but their chemical and biological properties have remained largely unknown because of their limited availability. We attempted to produce these proteins in Saccharomyces cerevisiae by placing the coding regions under the control of the promoter of the yeast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Yeast cells carrying middle S and large S coding sequences produced 33,000- and 42,000-dalton products, respectively, each of which reacted with anti-S antibody and bound to polymerized human serum albumin, in accordance with the known properties of pre-S proteins from particles in human sera (K. H. Heermann, U. Goldmann, W. Schwartz, T. Seyffarth, H. Baumgarten, and W. H. Gerlich, J. Virol. 52:396-402, 1984; A. Machida, S. Kishimoto, H. Ohnuma, K. Baba, Y. Ito, H. Miyamoto, G. Funatsu, K. Oda, S. Usuda, S. Togami, T. Nakamura, Y. Miyakawa, and M. Mayumi, Gastroenterology 86:910-918, 1984). The middle S polypeptide is glycosylated and can be assembled into particles whose size and density are similar to those of SAg. However, this polypeptide was highly susceptible to proteolytic degradation into 29,000- and 26,000-dalton polypeptides, of which only the former retained the binding activity to polymerized albumin. The large S polypeptides are nonglycosylated, relatively stable, and do not seem to assemble into particles by themselves.     

Expression of tumor necrosis factor receptors on human monocytes and internalization of receptor bound ligand

Tumor necrosis factor (TNF) is a polypeptide produced by monocytes and macrophages. Although TNF receptors have been identified on a variety of cell types, previous studies have not determined whether these receptors also exist on monocytes. In the present work, highly purified recombinant TNF was labeled with 125I. The 125I-labeled TNF bound specifically to receptors on human monocytes and monocyte membrane preparations. A curvilinear Scatchard plot indicated the presence of TNF-binding sites with two different affinities. The results also indicate that receptor-bound TNF is rapidly internalized by monocytes and then degraded intracellularly. These findings are in concert with recent studies demonstrating that TNF immunomodulates monocyte function by an autocrine mechanism.     

Purification of basic FGF receptors from rat brain

Receptor molecules for basic fibroblast growth factor (bFGF) were isolated from rat brain by a novel and rapid procedure and characterized. Purification was performed by wheatgerm agglutinin (WGA) gel affinity chromatography in combination with bFGF gel affinity chromatography, utilizing a novel elution method involving heparin. The eluted proteins were active in binding bFGF and were separated as two bands with respective molecular masses of 140 kDa and 110 kDa on SDS-PAGE. More than half of this bFGF-binding activity was lost after 16 h at 4 degrees C. Thus, bFGF receptors were purified as labile glycoconjugates.     

Chimeric heterosis in mandibular size in mice

Morphometrical observations were carried out on the mandibles of chimeras made from the embryos of C57BL/6 and BALB/c mice to compare with the two strains and their reciprocal F1 crosses. The results of the principal component analysis indicate that the first principal component (PC1) and the second principal component (PC2) extracted might be acceptable as size and shape factors, respectively. Variations of both PC1 and PC2 were generally larger in the chimeras than in the two component strains and their F1 crosses. The mean PC1 value of the chimeras was larger than that of the two component inbred strains, and it was similar to that of F1 crosses, or slightly larger. The overall size of the mandible represented by PC1 tended to be larger in the chimeras consisting of two component cells that were approximately equivalent than in those that shifted to either cell population. The above trend was observed in both sexes. These results indicate that chimeric heterosis due to the interaction between genetically different cells (C57BL/6 and BALB/c) has some relation to mandible size. The mean PC2 value, which was accepted as shape factor, was intermediate between the two inbred strains. The mandible size (PC1) and shape (PC2) were bilaterally symmetrical, except for the shape in the female chimeras and in (C57BL/6 x BALB/c)F1.     

Highly potent and specific inhibitors of human renin

We designed aldehyde derivatives of small peptides representing the C-terminal portion of angiotensin I sequence as an inhibitor of human renin. Among compounds that we synthesized, benzyloxycarbonyl (Z)-Phe-His-Leucinal (compound V), Z-Pro-Phe-His-Leucinal (Compound IV) and Z-[3-(1'-naphthyl)Ala]-His-Leucinal (compound VII) markedly inhibited human renin (IC50, 7.5 X 10(-7), 3.2 X 10(-7) and 8.0 X 10(-8) mol/l, respectively). Compound VII was shown to be noncompetitive (Ki = 2.4 X 10(-7) mol/l). It did not inhibit either cathepsin D or pepsin. Compound V had slight or no inhibitory effect at the concentration of 10(-5) mol/l on six animal renins except for monkey and rabbit renins. Results obtained show that these aldehyde compounds are highly selective and species specific inhibitors for human and monkey renins.     

Inheritance of hydronephrosis in the inbred mouse strain DDD

The mode of inheritance of hydronephrosis was investigated by crossing inbred DDD mice having a high incidence of hydronephrosis and C57BL/6 mice having normal kidneys. In the males, incidences of hydronephrosis in F1 animals were intermediate between the two parental strains at a rate of 32.6% in (DDD x C57BL/6)F1 and 23.4% in reciprocal F1. The same tendency was observed in F2 male animals. In BCF1 males, the number of affected mice was higher in (C57BL/6 x DDD) F1 x DDD (72.4%) than in (DDD x C57BL/6)F1 x C57BL/6 (11.1%). A few affected mice were found among the females of hybrids F1, F2 and BCF1. These results suggested that hydronephrosis in the DDD strain of mice was controlled by polygenes, and that male hormones may have some effect on the occurrence of hydronephrosis.     

Modification of batroxobin with activated polyethylene glycol: reduction of binding ability towards anti-batroxobin antibody and retention of defibrinogenation activity in circulation of preimmunized dogs

Amino groups of batroxobin (Bothrops atrox thrombic protease) were modified with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine (activated PEG2). The modified batroxobin had the reduced binding ability towards anti-batroxobin antibody but retained its enzymic activity in vitro and in vivo. Administration of modified batroxobin in which 29% of the total amino groups in the molecule had been modified, to beagle dogs preimmunized with native batroxobin gave rise to a marked reduction of the fibrinogen level in plasma, accompanied with an increased level of fibrinogen (fibrin) degradation products, FDP. On the other hand, no reduction of fibrinogen level was observed when native batroxobin instead of modified batroxobin was injected to immunized dogs.     

Whether Zn2+ for the polymerase system was selected inevitably or by historical accident

Zinc ion was found in various kinds of polymerases and involved in the activation of the 3'-hydroxyl group of primer chain's growing end. In order to investigate whether zinc ion was selected for nucleotide synthesizing system inevitably or by historical accident, ab initio molecular orbital calculations were carried out and the results suggest that zinc ion may have been selected inevitably.     

Quantification of gene expression over a wide range by the polymerase chain reaction.

We investigated the usefulness of the polymerase chain reaction (PCR) method for the relative quantification of gene expression using a simultaneously amplified sequence of beta-actin mRNA as an internal control for the target sequence of tax/rex mRNA of human T-cell leukemia virus type I. The PCR product of the internal control was reduced by delaying the addition of the primers for its sequence. The photostimulated luminescence of the bands was measured with a laser image analyzer, and the values were plotted against the cycle number. The cycle differences between the logarithmic phase of the curves for the target sequence and for beta-actin (delta cycle) showed a linear correlation with the initial concentration of the sample. This method is highly sensitive for evaluating gene expression over a wide range.