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An exposure to GB virus C/hepatitis G virus (GBV-C/HGV) was studied among populations at risk for blood and sexual exposure to analyze risk factor of the transmission of the virus. Blood samples were drawn from 98 intravenous drug users (IVDU), 100 female high-class commercial sex workers (CSW) and 50 male outpatients (MOP) at a sexually transmitted diseases (STD) clinic in Chiang Mai, Thailand. These blood samples were analyzed for GBV-C/HGV RNA; antibodies against second envelope protein of GBV-C/HGV (anti-E2); anti-hepatitis C virus antibody (HCV-Ab); hepatitis B core antibody (HBcAb); and antibodies against human immunodeficiency virus (HIV-Ab). Prevalences of GBV-C/HGV RNA, anti-E2, HCV-Ab, HBcAb and HIV-Ab were 27.6%, 16.3%, 84.7%, 76.5% and 45.0% in IVDU; 0%, 21.5%, 2.0%, 72.0% and 11.0% in CSW; 6.0%, 13.6%, 0%, 64.0% and 14.0% in MOP. While the prevalence of GBV-C/HGV RNA was higher in IVDU than in CSW and MOP, comparable prevalences of anti-E2 among the three populations were found. Intravenous drug injection showed association with GBV-C/HGV RNA, while history of STD associated with anti-E2. In conclusion, intravenous drug injection and STD were found to be risk factors for the previous exposure to GBV-C/HGV, but STD did not increase the risk of the GBV-C/HGV viraemia.

To evaluate viral interference between hepatitis B and C, we studied coinfected patients serologically and molecular biologically. Twenty-seven patients positive for hepatitis B surface antigen (HBsAg) and anti-hepatitis C virus (HCV) antibody, were classified into Groups BC-L and BC-H according to DNA-polymerase activity (less or greater than 100 cpm, respectively). Patients with hepatitis B or C alone were also enrolled as controls. HCV-RNA was detected more often in Group BC-L than in Group BC-H. Genotype 1b of HCV was determined in 75% of Group BC-H, 87.5% of Group BC-L, and 70.7% of hepatitis C-only patients. Activity of DNA-polymerase in coinfected patients was lower in patients positive for HCV-RNA as compared with those negative. HBsAg titers tended to be lower in coinfected patients than in patients with hepatitis B virus (HBV) alone. In conclusion, in coinfection, HBV may suppress the replication of HCV and HCV appears to reduce the expression of HBsAg and probably suppresses HBV replication.</P>

Rat liver and simian virus 40 (SV40) chromatin were reconstituted in vitro under physiological conditions of ionic strength and temperature. The nucleosome assembly under the conditions was mediated in the presence of chromatin extracts, rich in nicking-closing activity, from rat liver or cultured CV-1 cells. The frequency of nucleosome assembly on DNA was dependent on both the incubation time and the weight ratio of histone to DNA. The regulatory effects of host cellular histones on the transcription of SV40 DNA were investigated by using reconstituted SV40 chromatin containing or lacking histone H1. The cellular histones composing the chromatin were prepared from permissive CV-1 cells. Transcription of chromatin was analyzed in vitro using Escherichia coli DNA-dependent RNA polymerase. The rate of incorporation of ribonucleoside triphosphates into RNA synthesized on SV40 chromatin containing Hl as the template was 5 to 10% of the rate for RNA synthesized on supercoiled SV40 DNA. The rate of incorporation for SV40 chromatin lacking Hl was approximately 40 to 50% of that for SV40 DNA. RNA products transcribed from both these chromatin and SV40 DNA were fairly homogeneous 16 to 28S species with several identical peaks.

Using the erythroid cells of Rana nigromaculata the hemoglobin synthesis has been studied in the relation of DNA and RNA contents. Results showed that the hemoglobin synthesis starts in the early stage of erythroblast but becomes marked just before the complete maturation. RNA contents drops markedly in the later stage of maturation. Measurement of DNA contents by Feulgen reaction suggested the termination of the mitosis just before the prematuration. From these results the author concludes that the RNA which will act as the template for the globin synthesis, develops from the early stage of erythroblast but the templation is accelerated in the terminal stage of maturation and the marked acceleration in hemoglobin synthesis in this stage.

Both the cornea and muscle cornins have no effect at all on oxygen uptake of tissue, and likewise they catalase affect Qctivity not in any way. The corneacornin has an effect to reduce P/O ratio to about one half, but the muscle cornin does not show such an effect. Both comins decrease thc incorporation of P³² into nucleic acid fraction and DNA synthesis. In the ultracentrifugal analysis of nucleic acids during development of sea urchin eggs, cornins inhibit the polymerization of nucleic acids. In addition, both of these comins depress the incorporation of P³² into DNA and ribosome RNA of regenerating rat liver. Both comins inhibit the increase of -SH quantities before the cleavage of sea urchin eggs.

For the purpose to clarify the control mechanism of erythroid cell differentiation, the author observed morphologic changes in bone-marrow cells and circulating red cells in phenylhydrazine anemia of rabbits by introducing a mass of red cells into vein at one time and reached the following conclusions. 1. After red cell transfusion in a mass to animal showing a marked hematopoietic activity, anisocytosis or macrocytosis becomes distinct with the appearance of big reticulocytes and red cells as large as four times the normal in volume. This suggests, judging from their volume, the accelerated denucleation of erythroblast as early as at the late basophilic stage. 2. Observations on bone marrow at this stage revealed the reduction in the number of erythroblasts of undifferentiated type with the increase of rather differentiated ones. In erythroid islet, undifferentiated cells are found surrounding a reticulum cell located in the center, while well differentiated ones in the outskirt area are situated near the sinusoid. Such a cell arrangement suggests that the erythroid cell requires a high oxygen tension for its differentiation. 3. From these observations and other results obtained from the studies on reticulocyte maturation and RNA synthesis of erythroblast, the author stresses that erythroid cells can differentiate as long as it is provided with a certain level of oxygen, even though it may develop m-RNA for differentiation. In other words, there should be two steps in the differentiation of erythroblast, the first is m-RNA synthesis induced by the information and the second is the somatic protein synthesis with oxygen supply. This seems to be directly connected to the control mechanism of hematopoiesis by oxygen.

The mechanism of induction of anisocytosis was studied with experimental anemia of rabbits induced by blood depletion and phenylhydrazine chloride injection. The Price-Jones' curves of erythrocytes from anemic animals showed a large variety of red cell size, indicating the appearance of abnormally large sized cells. RNA contents of some reticulocytes in anemia were comparable to those of polychromatic and late basophilic erythroblasts. The result proved that in severe anemia a large number of erythroblasts are denucleated at earlier maturation stages, in most cases at polychromatic, and some at late basophilic and some at orthochromatic stages, resulting in anisocytosis.

Partially separated double-stranded RNA from purified Rous sarcoma virus, Schmidt.Ruppin strain, was observed by electron microscopy utilizing 8.M urea and protein monolayer technique. Furthermore, viruses in pair were frequently and viruses with two nuc1eoids were occasionally observed in ultrathin. sectioned specimens of chick cells transformed by RSV. From these results taking other reports in consideration, a possible mechanism of RNA replication in Rous sarcoma virus is proposed.

Recently, factors predicting the response to interferon (IFN) therapy against hepatitis C virus (HCV) have received much attention. To evaluate the usefulness of the quantitation of intrahepatic HCV RNA as a predictive marker of the response to IFN therapy, we compared the amount of intrahepatic HCV RNA with serum levels in 16 patients. Eleven patients who had 10(10) copies/g or more of intrahepatic HCV RNA had increased level of serum alanine aminotransferase (ALT) after IFN therapy, while 4 of 5 patients who had less than 10(10) copies/g of intrahepatic HCV RNA achieved sustained normalization of serum ALT level and were designated as complete responders. Four complete responders possessed significantly less HCV RNA in the liver parenchyma than partial and nonresponders (P = 0.010, Mann-Whitney U-test), but the amount of HCV RNA in the serum was not significantly different between those groups. In conclusion, the results suggest that the quantitation of intrahepatic HCV RNA is a better indicator of the response to IFN therapy than serum HCV RNA.

Hepatitis C virus (HCV)-RNA in the blood was measured by polymerase chain reaction (PCR) in 37 subjects from eight families in which 2 or more persons tested seropositive for antibodies against C100-3 or CP9. HCV-RNA was positive in 17 of 37 subjects. Two or more HCV-RNA-positive subjects were observed in six of the families. Intrafamilial HCV infection was studied by determining the HCV-RNA type (I, II, III or IV) by PCR using type-specific primers. In two families, all of the subjects showed type III infection, and in three other families, all of the subjects showed type II infection, with different types of HCV infections being observed in only one family. The HCV type was uniform in all but one. These findings suggest a possibility of intrafamilial infection between husbands and wives and between members of the same household.

The covalently closed form of circular duplex SV40 DNA was separated from the open and linear form of SV40 DNA by agarose gel electrophoresis in a large-scale gel system. The closed circular DNA was recovered from agarose gels by re-electrophoresing the gel slices. The recovery of DNA was about 70%. Electron microscopic analysis showed that the recovered DNA did not have doube- or single-stranded breaks. The recovered DNA can be used without further purification for electron microscopy, as a substrate for experiments using restriction endonuclease and as a template for in vitro RNA synthesis.

The molecular defects in the catalase gene, levels of m-RNA and properties of the residual catalase studied by scientists are reviewed in human (Japanese, Swiss and Hungarian) and non-human (mouse and beagle dog) acatalasemia with reference to the bioinformatics. Japanese acatalasemia-I, the G to A transition at the fifth position of intron 4 of the catalase gene, limited the correct splicing of the mRNA and synthesized trace catalase with normal properties. Hungarian acatalasemia type C showed a splicing mutation. In the Japanese acatalasemia II and the type A and B of Hungarian acatalasemia, the deletion or insertion of nucleotides was observed in the coding regions, and the frame shift altered downstream amino acid sequences and formed truncated proteins. In the Hungarian acatalasemia D, the substitution of a nucleotide in the exon was found. In mouse and beagle dog acatalasemia, the substitution of nucleotides in the coding regions was also observed. Studies of residual catalase in Swiss, mouse and beagle dog acatalasemia showed that aberrant catalase protein degrades more quickly than normal catalase in cells. The experimental research in genetic toxicology concerning the effect of oxidative stressors (nitrogen monoxide, nitrogen dioxide and so on) on Japanese acatalasemic blood and acatalasemic mice is described. The clinical features of Japanese and Hungarian acatalasemic subjects are also described.

Native and heat-treated RNAs from the purified Schmidt-Ruppin strain of Rous sarcoma virus (RSV) were fractionated by sucrose density gradients in the presence of ribonuclease inhibitor diethyl-pyrocarbonate and observed by electron microscopy. The structure of native 60-70S RNA was classified into two forms: tanglefolded type and linear type. In the tangle-folded type double stranded portions were observed in several sites. A high frequency of 60-70S RNA were 1.0 mum and 3-3.5 mum in length. Molecules with length about 9mum were of the tangle-folded type while molecules shorter than 6 mum were of the linear form. The structure of heat-treated RNA(30-40S) was linear with the most frequent length being 1-1.5 mum. These results indicate that native 60-70S RNA is folded with the total molecular length being in the order of 6 to 9 mum. Molecules about 3mum long are likely to be the main subunits of 60-70S RNA, and they are fragmented further into smaller subunits of about 1 mum length.

DNA-dependent RNA polymerases (EC 2.7.7.6) were extracted and partially purified form the nuclei of rat ascites hepatoma cells (AH-130) induced by 4-dimethylaminoazobenzene. The patterns of RNA synthesis and the properties of these enzymes were compared with enzymes from the nuclei of rat liver. The specific activity of RNA polymerase in the homogenate from the nuclei of AH-130 cells was the same as normal rat liver nuclei. RNA polymerase was solubilized from the homogenate at high ionic strength and separated into two forms by DEAE-Sephadex column chromatography. Enzymatic characterization showed that these enzymes corresponded to RNA polymerase I and II. RNA polymerase I more effectively transcribed native DNA than denatured DNA at low salt concentration, but at high salt concentration RNA polymerase I effectively transcribed denatured DNA. RNA polymerase II more effectively transcribed denatured DNA. In AH-130 cells the activity of RNA polymerase I was 4 to 5 times higher than RNA polymerase II, and in rat liver the activity of RNA polymerase I was 1.5 to 2 times higher than RNA polymerase II. The activity of RNA polymerase I in AH-130 cells may have increased by induction.

In vivo inactivation of cystathionine gamma-lyase by D,L-propargylglycine, a suicide inhibitor, was found to be less profound in rat kidney than in the liver. We investigated the cause of this difference using rat tissues. We fractionated kidney extract to characterize the substance which protected enzyme, and found that cysteine exhibits protecting action. Addition of 0.3 mM L-cysteine to the incubation mixture containing dialyzed kidney supernatant and 0.5 mM D,L-propargylglycine resulted in the protection of cystathionine gamma-lyase from the inactivation by the inhibitor. The content of cysteine in the kidney was six-fold higher than that in the liver. Thus, we have concluded that one of the reasons why the in vivo inactivation of cystathionine gamma-lyase in rat kidney was less than that in the liver is the presence of a higher concentration of cysteine in the kidney. S-Carboxymethylcysteine, a cysteine derivative, exhibited a similar, but weaker, protective effect.

Further purification and characterization are reported on rat cytosol cornin (RLCC), an antimitotic substance. Fraction I (purified RLCC) was purified more than 10-fold from crude RLCC with Sephadex G-50 column chromatography and showed a remarkable inhibitory effect on division of inseminated sea urchin eggs and mouse fibroblast cells. Fraction I was observed as one spot, and the molecular weight was estimated to be about 25,000 by thin layer gel filtration. Fraction I contained protein (92%) and RNA (8%), but the antimitotic activity was scarcely affected by treatment by pancreatic RNase. The protein of Fraction I was separated into two bands by SDS-polyacrylamide gel electrophoresis, and the molecular weight was estimated as 10,000 and 15,000, respectively. The 50% inhibition dose of Fraction I on the first division of inseminated sea urchin eggs and on proliferation of mouse L cells was about 2.5 X 10(-5) g/ml and 5 X 10(-4) g/ml, respectively. The yield of fraction I was about 35 mg from 100 g rat liver.

The intracellular localization of the avian myeloblastosis virus (AMV) genome was studied. Nuclear and mitochondrial DNAs from myeloblasts were examined by hybridization with 32P labeled AMV-RNA of high molecular weight for the presence of virus specific DNA sequences. Nuclear DNA (nDNA) from myeloblasts specifically hybridized with viral RNA, whereas purified closed circular mitochondrial DNA (mtDNA) did not hybridize with viral RNA. It was therefore concluded that viral genome was present in nuclear DNA and not in mitochondrial DNA. Likewise, in normal chick cells, nDNA but not mtDNA hybridized with viral RNA.

Mitochondrial RNA (mtRNA) was synthesized from purine and pyrimidine nucleosides in coupling with oxidative phosphorylation using isolated mitochondria. The in vivo synthesized mtRNA was adenine-uracil rich and sedimented at about 20 S by sucrose density gradient centrifugation. A major part of the newly synthesized mtRNA was shown to be poly (A)-containing RNA by the resistance to the digestion with pancreatic RNase and RNase T1 and the affinity to poly (U)-Sepharose columns or Millipore filters.

Malignant lymphoma was induced in Japanese (JWY), New Zealand (NZY) and Dutch (DUY) white rabbits by oral spray of cell-free pellets of culture fluid (crude virus fraction) of Ts-B6 cells (cynomolgus monkey B-lymphoblastoid cells harboring Epstein Barr virus-related simian herpesvirus or Cyno-EBV). Nine of 11 inoculated rabbits developed malignant lymphomas within 42-160 days after oral inoculation (JWY, 2/3; NZY, 5/6; DUY, 2/2). In contrast, none of the control rabbits inoculated in the same fashion with B95-8 (EBV-producing marmoset cell line) cell-free pellets developed malignant lymphoma. Most rabbits showed increased anti-VCA IgG and anti-EA-DR IgG antibody titers after inoculation by oral spray of Ts-B6 cell-free pellets. EBV-encoded RNA-1 was revealed in the tumor cells by in situ hybridization. EBV DNA was detected in the rabbit peripheral blood leukocytes (PBL) by polymerase chain reaction; the earliest positive result was obtained only two days after oral inoculation. These data suggest that orally administered Cyno-EBV in Ts-B6 cells infects PBL and then induces malignant lymphoma in rabbits. The availability of this animal model promises to clarify the role of EBV in human lymphoma and provides a means for studying prophylactic and therapeutic regimens.