Functional properties of recombinant factor V mutated in a potential calcium-binding site

Activated coagulation factor V (FVa) is a cofactor of activated factor X (FXa) in prothrombin activation. FVa is composed of a light chain (LC) and a heavy chain (HC) that are noncovalently associated in a calcium-dependent manner. We constructed a recombinant FV Asp111Asn/Asp112Asn mutant (rFV-NN) to abolish calcium binding to a potential calcium-binding site in FVa in order to study the specific role of these residues in the expression of FVa activity. Whereas thrombin-activated recombinant FV wild type (rFV-wt) presented with stable FVa activity, incubation of rFV-NN with thrombin resulted in a temporary increase in FVa activity, which was rapidly lost upon prolonged incubation. Loss of FVa activity was most likely due to dissociation of HC and LC since, upon chromatography of rFVa-NN on a SP-Sepharose column, the HC did not bind significantly to the resin whereas the LC bound and could be eluted at high ionic strength. In contrast, rFVa-wt adhered to the column, and both the HC and LC coeluted at high ionic strength. In the presence of phospholipid vesicles, the loss of rFVa-NN activity was partially prevented by FXa, active site inhibited FXa, and prothombin in a dose-dependent manner. We conclude that the introduced amino acid substitutions result in a loss of the high-affinity (calcium-dependent) interaction of the HC and LC of FVa. We propose that the introduced substitutions disrupt the calcium-binding site in FV, thereby yielding a FV molecule that rapidly loses activity following thrombin-catalyzed activation most likely via dissociation of the HC and LC.     

Treatment with leucine stimulates the production of hepatocyte growth factor in vivo

Hepatocyte growth factor (HGF) has pleiotropic effects. Up-regulation of HGF activity in vivo may be beneficial. Branched-chain amino acids (BCAAs) are known to modulate various cellular functions. When starved rats received intraperitoneal injections of valine, leucine or isoleucine, only leucine treatment increased both hepatic and circulating levels of HGF in a dose-dependent manner, up to 1.5 and 2.3 times higher, respectively, than in controls. When young growing rats with free access to food were injected with leucine once a day for a week, HGF levels and liver weights were significantly higher than those of control rats. Furthermore, 1 week of leucine treatment of adult rats resulted in elevated serum albumin levels with an increase in HGF levels. Taken together with our previous report showing that leucine stimulates HGF production by hepatic stellate cells in culture, leucine, among BCAAs, may induce an increase in HGF production by the liver in vivo.     

Enumeration of Mycobacterium leprae using real-time PCR

Mycobacterium leprae is not cultivable in axenic media, and direct microscopic enumeration of the bacilli is complex, labor intensive, and suffers from limited sensitivity and specificity. We have developed a real-time PCR assay for quantifying M. leprae DNA in biological samples. Primers were identified to amplify a shared region of the multicopy repeat sequence (RLEP) specific to M. leprae and tested for sensitivity and specificity in the TaqMan format. The assay was specific for M. leprae and able to detect 10 fg of purified M. leprae DNA, or approximately 300 bacteria in infected tissues. We used the RLEP TaqMan PCR to assess the short and long-term growth results of M. leprae in foot pad tissues obtained from conventional mice, a gene knock-out mouse strain, athymic nude mice, as well as from reticuloendothelial tissues of M. leprae-infected nine-banded armadillos. We found excellent correlative results between estimates from RLEP TaqMan PCR and direct microscopic counting (combined r = 0.98). The RLEP TaqMan PCR permitted rapid analysis of batch samples with high reproducibility and is especially valuable for detection of low numbers of bacilli. Molecular enumeration is a rapid, objective and highly reproducible means to estimate the numbers of M. leprae in tissues, and application of the technique can facilitate work with this agent in many laboratories.     

Seroepidemiology of hepatitis A virus in Japan

A seroepidemiologic study to detect class-specific antibody against hepatitis A virus (HAV) was made with 831 randomly collected sera (415 in 1973 and 416 in 1984) from healthy Japanese. Competitive-inhibition, IgG, IgA, and IgM anti-HAV enzyme-linked immunosorbent assays (ELISA) were used. Both collections showed a low prevalence of IgG anti-HAV in young age groups and it increased rapidly at middle age and plateued at greater than or equal to 94% prevalence in the older age groups. However, two age groups spanning ages 25-34 demonstrated statistically lower IgG anti-HAV age prevalences in 1984 vs 1973 (P less than 0.001), with an average 10-year prevalence shift. These data suggest that there has been no significant level of HAV infection to alter antibody prevalences in Japan from 1973 to 1984. The markedly decreased incidence of HAV infection in Japan has created a presently large and growing population of HAV susceptibles.     

Cytological studies on the mosaic silkworms induced by low temperature treatment

Mosaic silkworms were induced when the hybrid eggs of two strains with different egg color and larval markings were exposed to low temperature. Cytological studies were conducted to find out the relation between mosaic larval pattern and ploidy in reproductive cells along with demonstration of chromosomes in the mosaic embryos. — Mosaic eggs eith the characters of both the father and the hybrid were two types, one with large serosa nuclei (LN-mosaic) and the other with small serosa nuclei (SN-mosaic). The cytological studies demonstrated that LN-mosaic individuals were 2n/4n, while SN-mosaic ones were n/2n. In both types of silkworms, cell ploidy level in nuclei of synkaryon origin was two times that of androgenic ones. — From the results obtained in the present studies as well as in the previous studies, a possible mechanism of induction of mosaicism in silkworms by cold shock is contemplated.     

Biosynthesis of poly(3-hydroxyalkanoate) from amino acids

It was found that an optically active copolyester, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), denoted as P(3HB-co-3HV), is synthesized by Alcaligenes eutrophus H16 from several amino acids under various fermentation conditions. The optimum condition for the biosynthesis from one amino acid, threonine, was investigated and its biosynthetic pathway was discussed on the basis of the relation between the fermentation condition and the co-monomer composition of the produced polyesters.     

Interactions between the subunits of transducin and cyclic GMP phosphodiesterase in Rana catesbiana rod photoreceptors

In bullfrog (Rana catesbiana) rods the activity of cyclic GMP (cGMP) phosphodiesterase was stimulated 10 times by washing disc membranes with an isotonic, GTP-containing buffer. This stimulation was maintained following hydrolysis of GTP and after removal of guanine nucleotides. At least 60-70% of the inhibitory gamma subunit of cGMP phosphodiesterase (P gamma) was physically released from membranes by these washing procedures. When cGMP phosphodiesterase was activated by a hydrolysis-resistant GTP analogue, P gamma was found in the supernatant complexed with the transducin alpha subunit (T alpha) using three chromatography systems. When GTP was used to activate cGMP phosphodiesterase, P gamma was also found in the supernatant complexed with GDP.T alpha. This complex was also isolated using the same three chromatography systems, indicating that P gamma remained tightly bound to T alpha even after bound GTP was hydrolyzed. Interaction with the beta,gamma subunits of transducin, which remained associated with disc membranes, was required for the release of P gamma from the GDP.T alpha complex, which resulted in the deactivation of active cGMP phosphodiesterase. We conclude that during activation of cGMP phosphodiesterase, P gamma is complexed with T alpha (both GTP and GDP forms) in the supernatant and that, following GTP hydrolysis, beta,gamma subunits of transducin are necessary for the release of P gamma from the complex and the resulting inactivation of cGMP phosphodiesterase in frog photoreceptors.     

A novel DNA polymerase inhibitor and a potent apoptosis inducer: 2-mono-O-acyl-3-O-(alpha-D-sulfoquinovosyl)-glyceride with stearic acid

Sulfo-glycolipids in the class of sulfoquinovosyl diacylglycerol (SQDG) including the stereoisomers are potent inhibitors of DNA polymerase alpha and beta. However, since the alpha-configuration of SQDG with two stearic acids (alpha-SQDG-C(18)) can hardly penetrate cells, it has no cytotoxic effect. We tried and succeeded in making a permeable form, sulfoquinovosyl monoacylglycerol with a stearic acid (alpha-SQMG-C(18)) from alpha-SQDG-C(18) by hydrolysis with a pancreatic lipase. alpha-SQMG-C(18) inhibited DNA polymerase activity and was found to be a potent inhibitor of the growth of NUGC-3 cancer cells. alpha-SQMG-C(18) arrested the cell cycle at the G1 phase, and subsequently induced severe apoptosis. The arrest was correlated with an increased expression of p53 and cyclin E, indicating that alpha-SQMG-C(18) induced cell death through a p53-dependent apoptotic pathway.