Seroepidemiology of hepatitis C virus infection in Japan and HCV infection in haemodialysis patients.

Since January 1990, Japanese Red Cross Blood Centres have introduced hepatitis C virus screening with a first-generation ELISA. From April to December 1992, approximately 0.98% among 10,905,489 blood donations screened by a second-generation assay were anti-HCV-positive in all Japan. Seropositivity of anti-HCV increased with the age and serum transaminase value in both sexes. In blood donors having a history of transfusion, the anti-HCV reactive rate was 7.4%. The results of the study made by the Japanese Red Cross Non-A, Non-B Hepatitis Research Group show the effectiveness of implementation of HCV screening to prevent posttransfusion hepatitis. Consecutive haemodialysis patients with chronic renal failure are at risk for infection by a variety of blood-borne agents transmitted within dialysis units. Because of their immunocompromised state, they frequently also have an unusual susceptibility to a variety of nosocomial infections, such as HBV, HCV, and HTLV-I. We tested the prevalence of anti-HCV in 1423 (848 males and 575 females) haemodialysis patients from 18 hospitals in Kumamoto Prefecture, Japan, using the Ortho first generation anti-HCV screening assay. There were 316 patients (22.2%) positive for HCV antibodies. The second-generation test was positive in most haemodialysis patients who were reactive to the first-generation assay. The prevalence of HCV infection increased with the duration of haemodialysis, yet there was a high frequency of HCV seropositivity even without blood transfusion. Acquisition of HCV in dialysis patients could be explained by HCV infection within the unit other than by blood (all haemodialysis are done with disposable kits, syringes, and needles), by secondary HCV infection after the immunodeficiency of haemodialysis, or by HCV infection of the kidney or glomerular deposition of immune HCV/anti-HCV complexes leading to chronic renal failure (as with HBV infection of the liver and kidney.

In vivo effects of deoxyspergualin (NKT-01) on lymphocyte activation in response to alloantigens.

We studied the effects of deoxyspergualin (NKT-01) on the events of lymphocyte activation in vivo by inoculating mice in the footpad with allogeneic spleen cells, and compared the effects with those of cyclosporin A (CyA). The administration of NKT-01 increased the numbers of cells recovered from the popliteal lymph node (PLN) 7 days after inoculation, but inhibited the proliferation of these cells in the presence of exogenous interleukin 2 (IL-2). NKT-01 enhanced IL-2 production, but suppressed the production of macrophage activating factor (MAF) in the mixed lymphocyte reaction between the PLN cells and allogeneic spleen cells treated with mitomycin C. CyA decreased the numbers of PLN cells little, and suppressed the response to exogenous IL-2 and the production of both IL-2 and MAF. Results with tumor cells used as allogeneic cells suggested that there was a close relationship between the suppression of MAF production by NKT-01 and its inhibition of allograft rejection. The findings showed that NKT-01 inhibited both the MAF production by and the response to IL-2 of PLN cells, and that these effects were involved in the suppression of allograft rejection by NKT-01.

Feedback analysis of the behavior of the renin-angiotensin system under inhibition of angiotensin-converting enzyme.

It was previously shown that autoregressive modeling can be used for feedback analysis in the body. We used this method to investigate the dynamic changes in and around the renin-angiotensin system in vivo induced by angiotensin-converting enzyme (ACE) inhibition. Long-term studies were performed on rabbits, which were given a daily injection of one of the following ACE inhibitors: captopril, foroxymithine, or histargin. For comparison, other rabbits received injections of saline or the renin inhibitor pepstatin. Autoregressive coefficients were computed from the raw data thus observed and were used to simulate the impulse-response function proper to each animal. The response of each animal estimated in this way exposed the effects of ACE inhibitors in vivo which were obscured by the feedback regulation of the renin-angiotensin system. Also, it was suggested that histargin has a peculiar action, blocking the negative feedback that would be elicited by the usual ACE inhibition. Feedback analysis seems to be essential to elucidate the in vivo effects of enzyme inhibitors.

OF4949, new inhibitors of aminopeptidase B. IV. Effects of OF4949 and its derivatives on enzyme systems.

OF4949-I and II inhibited aminopeptidase B from Ehrlich ascites carcinoma in a competitive way and the Ki value for both against L-arginine-beta-naphthylamide was 8 X 10(-9) M. Inhibition by I and II of various exopeptidases and endopeptidases was examined. OF4949-I and II both strongly inhibited leucine aminopeptidase and enkephalin-degrading aminopeptidase; I also inhibited enkephalinase B. The inhibitory effects of various derivatives of I and II on aminopeptidase B activity, showed that the terminal amino and carboxamide groups are essential for activity.

OF4949, new inhibitors of aminopeptidase B. V. Effect on the murine immune system.

OF4949-I inhibited the growth of the solid form of IMC carcinoma and protected against pulmonary metastases of Lewis lung carcinoma. It also augmented the cytostatic activity of mouse peritoneal macrophages, the natural killer activity, and the antibody-dependent cell-mediated cytotoxicity of mouse spleen cells. This substance was not cytotoxic to cultured tumor or normal cells even at high concentrations. These results suggest that a cell-mediated immune response stimulated by this compound might account for its antitumor activity.

Nucleotide sequence and expression of the cloned gene of bacteriophage SP6 RNA polymerase.

The coding region of the gene for bacteriophage SP6 RNA polymerase was cloned into pBR322, and its entire nucleotide sequence was deduced. The predicted amino acid sequence for the polymerase consists of 874 amino acid residues with a total molecular weight of 98,561 daltons. Comparison of the amino acid sequence with that of T7 RNA polymerase reveals that regions with partial homology are present along the sequence. The coding region of SP6 RNA polymerase was inserted into an E. coli expression vector. The polymerase gene was efficiently expressed in E. coli cells, and the enzymatic properties of the expressed polymerase were very similar to those of the enzyme synthesized in SP6 phage-infected Salmonella typhimurium cells.

OF4949, new inhibitors of aminopeptidase B. I. Taxonomy, fermentation, isolation and characterization.

New aminopeptidase B inhibitors that we named OF4949-I, II, III and IV were isolated from the culture broth of a fungus, Penicillium rugulosum OF4949. The molecular formula of I was C23H26N4O8 and that of II, C22H24O8, judging from elemental analysis and secondary ion mass spectrometry. The concentrations of I, II, III and IV required for 50% inhibition of aminopeptidase, using Ehrlich ascites carcinoma cells as the source of the enzyme, were 0.0054, 0.0048, 3.4 and 1.7 micrograms/ml, respectively. Components I and II augmented delayed-type hypersensitivity in mice to sheep red blood cells.

OF4949, new inhibitors of aminopeptidase B. II. Elucidation of structure.

The structures of OF4949-I, II, III and IV were identified by analysis of the products of their chemical degradation and by 1H NMR, 13C NMR, and mass spectrometry. These compounds were new cyclic peptides containing diphenyl ether as a chromophore. OF4949-I had two amino acids, beta-hydroxy-L-asparagine and 4-methylisodityrosine. The structural differences between I and II and between III and IV lay solely in the diphenyl ether moiety; the phenolic hydroxyl group in II and IV was methylated in I and III. OF4949-III and IV contained L-asparagine instead of the beta-hydroxy-L-asparagine moiety of I and II.

OF4949, new inhibitors of aminopeptidase B. III. Biosynthesis.

To elevate production of OF4949 by Penicillium rugulosum OF4949 and to elucidate the pathway of its biosynthesis, mutants were selected on the basis of their resistance to growth inhibition by phenylalanine analogs. A mutant resistant to m-fluorophenylalanine, strain No. M414, had 3-fold the production of the parent. In a study of the biosynthesis of OF4949-I and II, several 14C-labeled compounds were examined as possible precursors of OF4949. L-[14C]Tyrosine and L-[14C]asparagine were incorporated efficiently. Most of the radioactivity of L-[14C]tyrosine was found in the 4-methylisodityrosine (B2) or isodityrosine (B1) moieties, and that of L-[14C]asparagine was in the beta-hydroxyasparagine moiety.

Regulation of inter- and intramolecular ligation with T4 DNA ligase in the presence of polyethylene glycol.

Polyethylene glycol (PEG) stimulates ligation with T4 DNA ligase. In 10% (w/v) PEG 6,000 solutions, only intermolecular ligation is enhanced by monovalent cations, while both inter- and intramolecular ligation occur without their presence. Similar stimulation was also caused by divalent cations or polyamines in the PEG 6,000 solutions. Such properties of the ligase could be applied to control the extent of inter- and intramolecular ligation. Ligation with cations or polyamines in 10% PEG 6,000 solutions was effective for intermolecular ligation. Ligation without cations or polyamines in 6.0% to 10% PEG 6,000 solutions was effective for intramolecular ligation.

NADP+ production using thermostable NAD+ kinase of corynebacterium flaccumfaciens AHU-1622.

A sonicate of Corynebacterium flaccumfaciens AHU-1622 had the highest NAD+ kinase activity (1.22 mU/mL culture broth) of the strains of bacteria we investigated. This enzyme was thermostable, with activity maintained at 50 degrees C for 1 h. This treatment inactivated phosphatase activity. Resting cells of the bacterium also had NAD+ kinase activity when treated at 60 degrees C for 30 min with 0.2% Triton X-100. NADP+ production was achieved using 8 mumol NAD+, 8 mumol ATP, 16 mumol MgCl2, 1.6 mumol NaN3, and 12 mU NAD+ kinase (0.1 g of permeabilized wet cells) in 2 mL of 0.1 M phosphate buffer, pH 7.5. The conversion ratio of NADP+ from NAD+ was 75% after 10 h of incubation at 50 degrees C, and the amount of accumulated NADP+ was 3 mumol/mL of reaction mixture. The NAD+ kinase activity of the permeabilized cells was stable and did not decrease after repeated use.

A new restriction endonuclease from Spirulina platensis.

Three restriction endonucleases, Sp1I, Sp1II and Sp1III have been purified partially from Spirulina platensis subspecies siamese and named. Sp1I cleaves bacteriophage lambda DNA at one site, phi X 174 RF DNA at two sites, but does not cleave pBR322 DNA. This enzyme recognizes the sequence 5'CGTACG3' 3'GCATCG5' and cuts the site indicated by the arrows. Sp1II is an isoschizomer of Tth111I and Sp1III is an isoschizomer of HaeIII.

Influence of angiotensin-converting enzyme inhibitor, foroxymithine, on dynamic equilibrium around the renin-angiotensin system in vivo.

To understand the in vivo actions of angiotensin-converting enzyme (ACE) inhibitors, a prolonged study was performed in rabbits over a half year, using one of such inhibitors, foroxymithine. During the initial 2 months of the inhibitor administration, the serum level of ACE was suppressed. Thereafter, probably triggered by the consequent sharp rise in the plasma renin activity (PRA) level, the ACE level regained its initial value. Thus the close correlation between the levels of PRA and ACE seen in the control animal was entirely broken by this inhibitor. A multivariate study indicated that the inhibitor drastically changed the normal networks of peptide metabolism in vivo. These results are compatible with the notion that the ACE inhibitor blocks the regulatory mechanisms of the renin-angiotensin system in vivo.

AccIII, a new restriction endonuclease from Acinetobacter calcoaceticus.

A new site-specific restriction endonuclease, AccIII, was isolated from Acinetobacter calcoaceticus. AccIII recognizes T/CCGGA and cleaves at the position shown by the arrow. AccIII activity was inhibited by adenine methylation at the overlapping dam methylase recognition sequence.

Stimulation of intermolecular ligation with E. coli DNA ligase by high concentrations of monovalent cations in polyethylene glycol solutions.

In the presence of high concentrations of the nonspecific polymer polyethylene glycol (PEG), intermolecular cohesive-end ligation with the DNA ligase from Escherichia coli was stimulated by high salt concentrations: 200 mM NaCl or 300 mM KCl in 10% (w/v) PEG 6000 solutions, and 100-200 mM NaCl or 150-300 mM KCl in 15% PEG 6000 solutions. Intermolecular blunt-end ligation with this ligase was also stimulated at 100-150 mM NaCl or 150-250 mM KCl in 15% PEG 6000 solutions. The extent of such intermolecular ligation increased and the salt concentrations at which ligation was stimulated extended to lower concentrations when we raised the temperature from 10 to 37 degrees C.

Demalonyl derivatives of guanidylfungin A and copiamycin: their synthesis and antifungal activity.

Guanidylfungin A was chemically modified by alkylation, reduction and/or demalonylation. Demalonylmethylguanidylfungin A became soluble in water and showed approximately eight-fold higher activity against fungi and Gram-positive bacteria than guanidylfungin A along with strongly fungicidal effect. Similarly, copiamycin was converted to demalonylmethylcopiamycin, which also showed higher antifungal activity than copiamycin itself.

Interaction of the restriction endonuclease ScaI with its substrates.

The kinetic constants of the site-specific endonuclease, ScaI, for various substrates were determined. We estimated Vmax and Km for octa-, deca-, dodeca-, and hexadecanucleotides and for plasmid pBR322 DNA. Vmax for these substrates were close, but Km were quite different (in decreasing order, octa- greater than deca-, dodeca-, hexadeca- greater than pBR322). The results were discussed with respect to the tertiary structure of substrate.

Influence of monovalent cations on the activity of T4 DNA ligase in the presence of polyethylene glycol.

Monovalent cations such as Na+ and K+ inhibit the activity of T4 DNA ligase. However, the extent of inhibition varies with the terminal sequence of the duplex DNA used as substrate; in many cases, ligation of DNA is completely inhibited at 200 mM. The activity of the ligase is stimulated by raising the concentration of polyethylene glycol 6000 from 0 to 15% (w/v) when NaC1 and KC1 were both absent. Ligation was reduced as the concentration of NaC1 or KC1 was raised in a mixture containing 5 or 15% PEG 6000. With 10% PEG 6000, both cohesive- and blunt-end ligation of this ligase increased at high concentrations of salt (150-200 mM NaC1, or 200-250 mM KC1). Further, with 10% PEG 6000, inter- and intramolecular ligation occurred at low salt concentrations (0-100 mM NaC1, or 0-150 mM KC1); only linear oligomers were formed by intermolecular ligation at the high concentrations.

Solid-phase synthesis of 5'-phosphorylated oligodeoxynucleotides.

We used morpholino groups to protect phosphate during the phosphorylation of the 5'-terminal ends of oligodeoxynucleotides, via phosphotriester and phosphoramidite intermediates. These groups could be removed selectively.