Action of Miracil D and related compounds on histone synthesis and phosphorylation in regenerating liver

The effect of Miracil D and hycanthone on ³H-amino acid incorporation into histones was studied under conditions known to cause a greater than 90% inhibition of thymidine incorporation into DNA of regenerating rat liver. A dose level of 50 mg of either drug per kg body weight administered 8 h after partial hepatectomy caused an approximate 50% inhibition of amino acid incorporation into fl, f2b and combined f2a plus f3 histone in 24-h regenerating liver. There was little or no effect on amino acid nitrogen concentration or incorporation of ³H-amino acid into the acid-soluble fraction, cytoplasmic proteins or acid-insoluble nuclear proteins. Under the same conditions, Miracil D caused a 65% inhibition of ³²P incorporation into lysirierich f1 histone whereas a structurally related compound, GE-99, did not have a significant inhibitory effect on this parameter nor on [³H]thymidine incorporation into DNA. Temporal studies with hycanthone revealed a suppression of the increased phosphorylation of fl histone in regenerating rat liver without influencing the phosphorylation of other histones. The data support the concept of coordinated control of DNA synthesis and phosphorylation of fl histone.     

Beneficial effects of population bottlenecks in an RNA virus evolving at increased error rate

RNA viruses replicate their genomes with a very high error rate and constitute highly heterogeneous mutant distributions similar to the molecular quasispecies introduced to explain the evolution of prebiotic replicators. The genetic information included in a quasispecies can only be faithfully transmitted below a critical error rate. When the error threshold is crossed, the population structure disorganizes, and it is substituted by a randomly distributed mutant spectrum. For viral quasispecies, the increase in error rate is associated with a decrease in specific infectivity that can lead to the extinction of the population. In contrast, a strong resistance to extinction has been observed in populations subjected to bottleneck events despite the increased accumulation of mutations. In the present study, we show that the mutagenic nucleoside analogue 5-azacytidine (AZC) is a potent mutagen for bacteriophage Qβ. We have evaluated the effect of the increase in the replication error rate in populations of the bacteriophage Qβ evolving either in liquid medium or during development of clonal populations in semisolid agar. Populations evolving in liquid medium in the presence of AZC were extinguished, while during plaque development in the presence of AZC, the virus experienced a significant increase in the replicative ability. Individual viruses isolated from preextinction populations could withstand high error rates during a number of plaque-to-plaque transfers. The response to mutagenesis is interpreted in the light of features of plaque development versus infections by free-moving virus particles and the distance to a mutation-selection equilibrium. The results suggest that clonal bacteriophage populations away from equilibrium derive replicative benefits from increased mutation rates. This is relevant to the application of lethal mutagenesis in vivo, in the case of viruses that encounter changing environments and are transmitted from cell to cell under conditions of limited diffusion that mimic the events taking place during plaque development.     

Structure of a bacterial photosynthetic membrane: integrity of reaction centers following proteolysis and detergent solubilization

cDNAs were molecularly cloned for proteins specifically expressed in embryo as well as in a chemically induced rat pancreatic B cell tumor in which virally related oncogenes such as v-myc, v-src, v-yes, v-mos and v-kis were previously demonstrated not to be expressed. A plasmid cDNA library consisting of 48,000 independent colonies was constructed from poly(A) containing cytoplasmic RNA isolated from 12 day rat embryo. The library was screened by hybridization with 32p-labelled cDNA synthesized from poly(A) containing RNA of rat pancreatic B cell tumor or normal islet B cells. Two clones were obtained which showed a clearly positive reaction only with tumor probe. Nucleotide sequence of one of them harboring insert of 615 nucleotides was determined and its amino acid sequence of 119 residues was deduced, which showed that the protein encoded by this mRNA is highly basic, basic residues/acidic residues being 1.63.     

Chemical interactions with herpes simplex type 2 virus: Enhancement of transformation by hydrazine and 1,2-dimethylhydrazine

Quantitative assays for the morphological transformation of 3T3 Swiss mouse cells by herpes simplex type 2 virus (HSV-2) were employed to examine the effect on cell transformation of chemical carcinogens and suspected carcinogens. Exposure of the cells to the chemical compound, followed by virus infection, resulted in enhancement of transformation when compared to that observed with chemical or virus alone. Enhancement occurred in tests utilizing either UV light-inactivated HSV-2 (strain 333) or a temperature-sensitive (ts) mutant of HSV-2 [A₈(293)]. A series of seven ts-mutants were tested and exhibited varying degrees of transformation. Enhancement of transformation occurred in cells treated with hydrazine (HZ) and 1,2-dimethylhydrazine (SDMH). No enhancement occurred when cells were treated with monomethylhydrazine, 1,1-dimethylhydrazine and the jet fuels JP-5, JP-10, RJ-4 and RJ-5. A strong time dependence after treatment was demonstrated with some enhancement seen at 6 h after chemical treatment but the greatest enhancement appeared when virus infection began after 24 h of chemical exposure.     

Intimate coupling of creatine phosphokinase and myofibrillar adenosinetriphosphatase

ATPase and creatine phosphokinase (CPK) activities of isolated cardiac myofibrils were determined with ³²P γ-labeled ATP alone and with the addition of phosphorylcreatine (PC). With ATP and PC as substrates the label in the inorganic phosphate formed is greatly diluted indicating that the ATP formed by PC through CPK can reach the ATPase active site more readily than labeled ATP from the medium. The tight coupling of the ATPase and CPK activities further strengthens our view that PC serves an important role as high energy carrier between the energy producing sites (mitochondria) and the energy utilizing sites (myofibrils).     

H1N1流感病毒在微载体培养MDCK细胞上增殖的研究

目的探索MDCK细胞在微载体上的培养条件,并研究H1N1型流感病毒在MDCK细胞上的增殖条件。方法在微载体上培养好MDCK细胞上用H1N1型流感病毒在不同的病毒感染复数(MOI)、胰酶浓度两个关键的病毒增殖条件进行流感病毒在细胞上的增殖研究。结果微载体质量浓度为6 g/L时,MDCK细胞培养密度可以达到4.5×106cells/mL。在MOI为0.05接种流感病毒,胰酶质量浓度4μg/mL,流感病毒在MDCK细胞上可获得较高的滴度。结论 MDCK细胞用微载体培养可以达到较高的细胞密度,可以作为规模化生产新型流感病毒疫苗的主要细胞基质进行进一步的研究。     

Effect of streptovaricin on the incorporation of uridine into cellular and viral RNA.

Poxvirus replication is inhibited by streptovaricin. The most readily observed effect is the inhibition of incorporation of [3H]uridine into viral mRNA, suggesting an inhibition of RNA synthesis. Streptovaricin also inhibits the incorporation of [3H]uridine into cellular RNA but not as severely as viral RNA. On the other hand, [3H]uridine incorporation into the RNA of Semliki Forest virus (SFV), which contains a positive strand RNA genome, does not seem to be inhibited by streptovaricin. The inhibitory effect of streptovaricin is completely reversible after removal of the inhibitor. In addition to inhibiting RNA synthesis, streptovaricin also may inhibit the methylation of cellular RNA. Viral RNA is stable in the presence of streptovaricin.     

Effects of pH on plaque forming unit counts and aggregation of MS2 bacteriophage

AIM: The aim of this study was to determine whether aggregation processes in aqueous phase may explain the decrease in plaque forming unit (PFU) counts for pH close to the isoelectric point (pI) of viral particles (MS2 phages). METHODS AND RESULTS: Loss in PFU was observed for pH < or = pI (pI(MS2) = 3.9): for example, at pH 2.5, loss was approx. 3 log(10) PFU. Particle size analysis combining results of dynamic light scattering and flow particle image analysis was then applied to determine the aggregate state of viral suspensions by recording size distributions. The size of major population significantly changed to 30 nm at neutral pH to more than several micrometres when passing below the isoelectric point. CONCLUSIONS: Our study shows that MS2 phages exhibit significant aggregation processes for pH < or = pI leading to aggregate with sizes of few micrometres. This aggregation process can largely explain the decline in PFU counts. SIGNIFICANCE AND IMPACT OF THE STUDY: It is clear that viral aggregation can be a source of significant bias for PFU assays because in the presence of an aggregate the PFU count can be less than the sum of its constituent particles. Therefore, cautions should be taken in terms of conditions of storage (pH far from pI) to avoid such aggregation artefact.     

Monensin blocks endocytosis of vesicular stomatitis virus

Monensin inhibits the infection of mouse cells by Vesicular Stomatitis Virus (VSV). At low drug concentrations (0.5 μM), endocytosis of VSV is inhibited whereas viral binding is unaffected. Monensin may be useful for analyzing the internalization of other viruses as well as soluble ligands.     

Overexpression of IRS2 in isolated pancreatic islets causes proliferation and protects human beta-cells from hyperglycemia-induced apoptosis

Studies in vivo indicate that IRS2 plays an important role in maintaining functional beta-cell mass. To investigate if IRS2 autonomously affects beta-cells, we have studied proliferation, apoptosis, and beta-cell function in isolated rat and human islets after overexpression of IRS2 or IRS1. We found that beta-cell proliferation was significantly increased in rat islets overexpressing IRS2 while IRS1 was less effective. Moreover, proliferation of a beta-cell line, INS-1, was decreased after repression of Irs2 expression using RNA oligonucleotides. Overexpression of IRS2 in human islets significantly decreased apoptosis of beta-cells, induced by 33.3 mM D-glucose. However, IRS2 did not protect cultured rat islets against apoptosis in the presence of 0.5 mM palmitic acid. Overexpression of IRS2 in isolated rat islets significantly increased basal and D-glucose-stimulated insulin secretion as determined in perifusion experiments. Therefore, IRS2 is sufficient to induce proliferation in rat islets and to protect human beta-cells from D-glucose-induced apoptosis. In addition, IRS2 can improve beta-cell function. Our results indicate that IRS2 acts autonomously in beta-cells in maintenance and expansion of functional beta-cell mass in vivo.     

Modeling, analysis, and validation of a novel HIV integrase structure provide insights into the binding modes of potent integrase inhibitors

It has been shown that L-731988, a potent integrase inhibitor, targets a conformation of the integrase enzyme formed when complexed to viral DNA, with the 3′-end dinucleotide already cleaved. It has also been shown that diketo acid inhibitors bind to the strand transfer complex of integrase and are competitive with the host target DNA. However, published X-ray structures of HIV integrase do not include the DNA; thus, there is a need to develop a model representing the strand transfer complex. In this study, we have constructed an active-site model of the HIV-1 integrase complexed with viral DNA using the crystal structure of DNA-bound transposase and have identified a binding mode for inhibitors. This proposed binding mechanism for integrase inhibitors involves interaction with a specific Mg^2 + in the active site, accentuated by a hydrophobic interaction in a cavity formed by a flexible loop upon DNA binding. We further validated the integrase active-site model by selectively mutating key residues predicted to play an important role in the binding of inhibitors. Thus, we have a binding model that is applicable to a wide range of potent integrase inhibitors and is consistent with the available resistant mutation data.     

Effects of pyrrol-carboxylic acid derivatives on the growth of coliphages.

Effects of 14 pyrrol-carboxylic acid derivatives and analogues (PY-compounds) on the growth of coliphage MS2 using E. coli E102 (Hfr) as the host were measured by the agar double-layer method. Enlargements of plaque size were observed with 7 PY-compounds but increase in plaque numbers was not induced. These enlargements of plaque size were specific to RNA coliphages MS2, GA and qbeta and not found with DNA coliphages delta AC and T4. Furthermore, the interaction between PY-compound PY-10 and the coliphage MS2 was dependent on the host bacterium (indicator strain). When E102 (Hfr) was used, the enlargement was marked, in the case of substrain W1895 (Hfr) it was less, while in the case of substrain W6 (F+) it was undetectable. The one-step growth of the phage MS2 and the production of intracellular phage MS2 were little affected by the PY-compound PY-10. However, the rate of one-step growth was increased in the early stage after infection. Accordingly, the enlargements of plaque size by the PY-compounds might be correlated with an increase in rate of release of phage particles.     

Synthesis and in vitro anti-HSV-1 activity of a novel Hsp90 inhibitor BJ-B11

In this study, a novel Hsp90 inhibitor BJ-B11, was synthesized and evaluated for in vitro antiviral activity against several viruses. Possible anti-HSV-1 mechanisms were also investigated. BJ-B11 displayed no antiviral activity against coxsackievirus B₃ (CVB₃), human respiratory syncytial virus (RSV) and influenza virus (H1N1), but exhibited potent anti-HSV-1 and HSV-2 activity with EC₅₀ values of 0.42 ± 0.18 μM and 0.60 ± 0.21 μM, respectively. Additionally, the inhibitory effects of BJ-B11 against HSV-1 were likely to be introduced at early stage of infection. Our results indicate that BJ-B11 with alternative mechanisms of action is potent as an anti-HSV clinical trial candidate.     

Inhibition of postreplication repair and the enhancement of induction of SV40 virus from transformed hamster kidney cells.

The induction by ultraviolet light of simian virus 40 (SV40) from two SV40--transformed hamster kidney cell lines is enhanced by caffeine. In order to investigate the mechanism responsible for this enhancement, the effect of caffeine on postreplication repair of DNA damaged by UV light was studied utilizing alkaline sucrose-gradient sedimentation. Caffeine at concentrations of 0.5, 1.0 or 2.0 mM inhibited the filling of gaps during postreplication repair. In addition, caffeine was found to potentiate cell killing by mitomycin C, an alkylating agent, and to enhance SV40 induction by mitomycin C. We postulate that the persistence of gaps in DNA, caused by the presence of caffeine, results in the enhancement of SV40 virus induction.     

Cofilin-1 is involved in regulation of actin reorganization during influenza A virus assembly and budding

Influenza A virus (IAV) assembly and budding on host cell surface plasma membrane requires actin cytoskeleton reorganization. The underlying molecular mechanism involving actin reorganization remains unclarified. In this study, we found that the natural antiviral compound petagalloyl glucose (PGG) inhibits F-actin reorganization in the host cell membrane during the late stage of IAV infection, which are associated with the suppression of total cofilin-1 level and its phosphorylation. Knock-down of cofilin-1 reduces viral yields. These findings provide the first evidence that cofilin-1 plays an important role in regulating actin reorganization during IAV assembly and budding.     

Inhibitory role of neutrophils on influenza virus multiplication in the lungs of mice.

The protective role of neutrophils on intranasal infection of influenza virus was investigated in 3 strains of tumor-bearing mice with neutrophilic leukocytosis. In vitro multiplication of influenza virus was inhibited by neutrophils from both normal and tumor-bearing mice, and the inhibitory effect of neutrophils was augmented by an addition of fMLP to the culture. Pulmonary virus infectivities in the early phase after infection decreased in such ICR and BALB/c mice, and virus elimination in the late phase was accelerated in the ICR mice. However, no decrease in pulmonary virus infectivity was observed in tumor-bearing C57BL/6 mice. Intranasal administration of fMLP into normal and tumor-bearing C57BL/6 mice after infection significantly inhibited the virus propagation in the lungs. The decrease in neutrophil infiltration into the lung in tumor-bearing C57BL/6 mice was confirmed from histological observations of the lung and lung lavage after infection and from analysis of the neutrophil chemotactic activity induced by fMLP. This might be responsible for the high level of pulmonary virus titer in tumor-bearing C57BL/6 mice. Phagocytic activities of alveolar macrophages and productions of neutralizing antibody were suppressed in the 3 strains of tumor-bearing mice. These observations indicated that neutrophils could be significant effector cells as a host defense mechanism against influenza virus infection in vivo, and infiltration and functional activation of neutrophils could play a significant role in virus elimination from the infected site. Furthermore, the inhibition of virus propagation by neutrophils in vitro was almost completely abrogated by an addition of ZnSO4, suggesting that calprotectin could inhibit influenza virus multiplication.     

A rapid detection method using flow cytometry to monitor the risk of Legionella in bath water

Legionella species are the causative agents of human legionellosis, and bathing facilities have been identified as the sources of infection in several outbreaks in Japan. Researchers in Japan have recently reported evidence of significant associations between bacterial counts and the occurrence of Legionella in bathing facilities and in a hot tub model. A convenient and quantitative bacterial enumeration method is therefore required as an indicator of Legionella contamination or disinfection to replace existing methods such as time-consuming Legionella culture and expensive Legionella-DNA amplification. In this study, we developed a rapid detection method (RDM) to monitor the risk of Legionella using an automated microbial analyzing device based on flow cytometry techniques to measure the total number of bacteria in water samples within two minutes, by detecting typical patterns of scattered light and fluorescence. We first compared the results of our RDM with plate counting results for five filtered hot spring water samples spiked with three species of bacteria, including Legionella. Inactivation of these samples by chlorine was also assessed by the RDM, a live/dead bacterial fluorescence assay and plate counting. Using the RDM, the lower limit of quantitative bacterial counts in the spiked samples was determined as 3.0 × 10³ (3.48 log) counts mL^− 1. We then used a laboratory model of a hot tub and found that the RDM could monitor the growth curve of naturally occurring heterotrophic bacteria with 1 and 2 days' delayed growth of amoeba and Legionella, respectively, and could also determine the killing curve of these bacteria by chlorination. Finally, samples with ≥ 3.48 or < 3.48 log total bacterial counts mL^− 1 were tested using the RDM from 149 different hot tubs, and were found to be significantly associated with the positive or negative detection of Legionella with 95% sensitivity and 84% specificity. These findings indicated that the RDM can be used for Legionella control at bathing facilities, especially those where the effectiveness of chlorine is reduced by the presence of Fe²⁺, Mn²⁺, NH₄⁺, skin debris, and/or biofilms in the water.     

Influence of increased CD4 cell counts on the genetic variability of hepatitis C virus in patients co-infected with human immunodeficiency virus I.

In order to study the effect of increased CD4 cell counts on the biology of hepatitis C virus (HCV), we analyzed the genetic variability of HCV generated over 8 y in eight human immunodeficiency virus-1 (HIV-1) and HCV co-infected patients. This was a retrospective study in which HIV patients were selected who had profound immune impairment evident over four years and were co-infected with HCV genotype 1 and who then went on highly active antiretroviral therapy (HAART). These patients achieved different degrees of immune reconstitution, measured as increased CD4 cell counts during a 4- to 8-y period, following initiation of HAART. HCV genetic variability was determined by measuring the genetic diversity (Hamming distance, HD), and complexity (number of viral variants) in plasma samples collected at yearly intervals just before and after the initiation of HAART. The parameters were assessed by molecular cloning and sequencing of a 575-bp fragment including the HCV envelope 1 and envelope 2 genes (E1/E2), containing the hypervariable region 1 (HVR1). significantly increased HVR1 genetic diversity was observed in analyzed samples where the patients' CD4 cell counts were > or =100 compared with CD4 cell counts <100. A significant increase in genetic diversity in HVR1 was detected in co-infected patients whose CD4 cell counts increased from <100 to >400 over a period of more than 4 y of HAART therapy. This was in contrast to a minimal increase in HCV genetic diversity of HVR1 occurring in patients whose CD4 cell counts failed to rise much over 200 over 7 y of follow up. Insertion and deletion of HCV genomic fragments in the E1/E2 region was documented in one patient who developed fulminant hepatitis C.