A new class II restriction endonuclease,
AsnI, with a novel sequence specificity was isolated from the Gram-positive eubacterium
Arthrobacter species, strain N-CM.
AsnI recognizes the unambiguously ...defined palindromic hexanucleotide
▪ consisting of A- and T-residues. The novel enzyme in the presence of Mg
2+ cleaves specifically both strands as indicated by the arrows. The staggered cuts generate 5′-protruding ends with single-stranded 5′-TA-3′ dinucleotide extensions. The novel enzyme may be a useful tool for cloning experiments by complementation of the few enzymes such as
PstI and
PvuI cutting only once in the Amp
r-gene of plasmids pBR322 and pBR328.
The pathogenesis of coxsackievirus B3 (CVB3)-induced myocarditis was investigated in adult Han:NMRI mice. The outbred model, in comparison with inbred models, represents better the natural variable ...susceptibility of the human population.
We analyzed the replicating virus titer, the antibody response in the acute and chronic phase of disease, the histology of myocardial injury, and the persistence of viral RNA.
NMRI mice infected with 5000 plaque-forming units (PFU) of the CVB3 variant "P"D, a lytic variant to human fibroblast lines, showed a peak of virus replication at day 14 and developed a severe acute myocarditis. The chronic myocarditis was characterized by progressive fibrosis, small foci of infiltrates, persistent viral RNA in the heart, and detectable anti-CVB3 IgG production and neutralizing antibody response up to day 98 postinfection.
CVB3"P"D is able to induce chronic myocarditis in NMRI mice. This model provides a method for examining and proving the mechanisms of myocardial pathogenesis and of developing therapeutic strategies.
A search for a dark matter (DM) annihilation signal into gamma -rays toward the direction of the Canis Major (CMa) overdensity is presented. The nature of CMa is still controversial and one scenario ...represents it as a dwarf galaxy, making it an interesting candidate for DM annihilation searches. A total of 9.6 hr of high-quality data were collected with the H.E.S.S. array of Imaging Atmospheric Cherenkov Telescopes, and no evidence for a very high energy gamma -ray signal is found. Upper limits on the CMa dwarf galaxy mass of the order of 109 M are derived at the 95% confidence level (CL) assuming neutralino masses in the range 500 GeV-10 TeV and relatively large annihilation cross sections. Constraints on the velocity-weighted annihilation cross section sigma v are calculated for specific weakly interacting massive-particle scenarios, using a Navarro-Frenk-White model for the DM halo profile and taking advantage of numerical simulations of hierarchical structure formation. Assuming a total halo mass of 3 X 108 M , 95% CL exclusion limits of the order of 5 X 10 -24 cm3 s -1 are reached in the 500 GeV-10 TeV DM particle mass interval.
The phenomenon that rHuIFN-alpha1(D) displays an apparently higher antiviral activity when assayed on bovine cells as compared to human cell lines was applied to the elucidation of the nature of ...recombinant HuIFN prepared in our institute. These investigations were carried out by using a microtitre test, which defines biological activity as the IFN concentration leading to 50% inhibition of the cytopathic effect of vesicular stomatitis virus (VSV). In addition, the ability of IFN to diminish the reproduction of infectious viruses was monitored. The two methods yielded similar results. With bovine cells, antiviral activities of the same order of magnitude were observed, regardless of the interferon types applied, i.e. rHuIFN-alpha 1, rHuIFN-alpha 2 and human leukocyte interferon. On human fibroblasts, however, rHuIFN-alpha 1 had an apparently 45 to 165 times lower activity than the other two interferons. On human WISH cells, the differences in apparent activity between the respective IFNs were even greater, with factors of up to 212 fold being observed. Still more distinctive were the effects on murine L 929 cells where an antiviral effect could be confirmed only for rHuIFN-alpha 1 whereas the other two interferons proved completely inactive.
We report on the first completely simultaneous observation of a gamma-ray burst (GRB) using an array of Imaging Atmospheric Cherenkov Telescopes, which is sensitive to photons in the very high energy ...(VHE) gamma -ray range ( 100 GeV). On 2006 June 2, the Swift Burst Alert Telescope (BAT) registered an unusually soft gamma -ray burst (GRB 060602B). The burst position was under observation using the High Energy Stereoscopic System (HESS) at the time the burst occurred. Data were taken before, during, and after the burst. A total of 5 hr of observations were obtained during the night of 2006 June 2-3, and five additional hours were obtained over the next three nights. No VHE gamma -ray signal was found during the period covered by the HESS observations. The 99% confidence level flux upper limit (> 1 TeV) for the prompt phase (9 s) of GRB 060602B is 2.9 X 10-9 erg cm-2 s-1. Due to the very soft BAT spectrum of the burst compared with other Swift GRBs and its proximity to the Galactic center, the burst is likely associated with a Galactic X-ray burster, although the possibility of it being a cosmological GRB cannot be ruled out. We discuss the implications of our flux limits in the context of these two bursting scenarios.
The antiviral effect of nitric oxide (NO)-releasing compounds was investigated. Using bacterially expressed and purified proteinases 2A and 3C of coxsackievirus B3, in vitro assays demonstrated the ...inhibition of the 2A proteinase activity in the presence of S-nitroso- N-acetyl-penicillamine (SNAP), 3-morpholinosydnonimine (SIN-1), 4-phenyl-3-furoxancarbonitrile (PFC), glyceryl trinitrate (GTN), and isosorbide dinitrate (ISDN). Sodium nitroprusside (SNP), which releases NO after metabolization, had no effect. The 3C proteinase was inactivated by SNAP, GTN, and ISDN. The vasodilators GTN and ISDN, widely used in the treatment of angina pectoris, exhibited antiviral activity in CVB3-infected GMK cells. CVB3-infected NMRI outbred mice showed significantly reduced signs of myocarditis after treatment with GTN or ISDN. Inhibitors of the cellular inducible NO synthase (iNOS) such as N(G)-nitro-L-arginine methyl ester (L-NAME), N(G)-nitro-L-arginine (L-NNA), and S-methyl-isothiourea (SMT), had no deleterious effect on CVB3-infected NMRI mice, indicating that endogenous NO synthesis is unlikely to be a major defense mechanism after enterovirus infection of outbred mice.
The influence of propofol (CAS 2078-54-8 (intravenous propofol emulsion) on cell membrane properties was investigated in vitro with techniques of cell electrofusion and cell electroporation. Human ...lymphoma cells and plant protoplasts were chosen as a model system. Propofol (intravenous propofol emulsion) decreased the electrofusion yield of the cells and their membrane permeability. A 50% decrease in relative electrofusion was observed in human lymphoma cells in the presence of about 0.05 mmol/l propofol (intravenous propofol emulsion) and in plant protoplasts in the presence of about 0.1 mmol/l. The fusion of human lymphoma cells was inhibited to 100% at concentrations higher than 0.2 mmol/l propofol and 0.4 mmol/l intravenous propofol emulsion. The membrane permeability of human lymphoma cells decreased by the factor of two with increasing propofol concentrations up to about 0.1 mmol/l. The effects of electroporation were highly reversible. Propofol (intravenous propofol emulsion) was more effective than tetracaine. These sensitive techniques are suitable for the investigation of interactions between anesthetic drugs and the cell membrane.
Aside from enteroviruses and other viruses, e.g., adenoviruses, which are known to be associated with idiopathic dilated cardiomyopathy (IDC), a cardiac tropism is also attributed to parvovirus B19 ...(PVB19). The purpose of the present study was to determine the prevalence of enterovirus, adenovirus and PVB19 genomes in the myocardium of adult patients with IDC and to analyze the significance of PVB19 with regard to the course of the disease, as compared to the other cardiotropic viruses. In 52 adult patients with IDC and 10 control patients with normal left ventricular ejection fraction (> or =55%) undergoing coronary artery bypass surgery, myocardial tissue samples were investigated for enteroviral RNA using polymerase chain reaction (PCR) and Southern blot hybridization of the PCR product. Specific nested PCR was used to assess the prevalence of adenovirus and PVB19 DNA, in addition to sequencing of the latter. The clinical and echocardiographic course of the disease was followed for a mean (+/- SD) period of 21.1+/-9.5 months. Fourteen of the 52 patients (27%) were enterovirus-positive, 2/52 (4%) patients were adenovirus-positive, 14/52 (27%) patients were PVB19-positive, 8/52 (15%) patients were enterovirus plus PVB19-positive, and in 14/52 (27%) patients no viral genomes were found. Six patients died during the follow-up period, without any significant difference between the patient groups: 1/14 (7%) in the enterovirus-positive, 0/2 (0%) in the adenovirus-positive, 2/14 (14%) in the PVB19-positive, 1/8 (12.5%) in the enterovirus plus PVB19-positive, and 2/14 (14%) in the virus-negative group. PVB19 genome was found in 4 of the 10 (40%) control patients, but no enterovirus or adenovirus genomes were detected in these patients. In conclusion, in the myocardium of patients with IDC, PVB19 is detectable as frequently as enteroviral genome. PVB19-positive patients with IDC have a rather favorable prognosis and do not differ significantly from the other virus-positive or virus-negative patient groups with respect to survival. Finally, the pathogenetic and prognostic significance of PVB19 in IDC still remains unclear.
The procedures generally used for the in vitro induction of interferon exploit fibroblast or leucocyte culture suspensions. Several factors and conditions influencing the capacity for IFN induction ...were studied, with the following results. In contrast to previous findings, optimum IFN induction was obtained at 37 degrees C, rather than 30 degrees C, when human fibroblasts were induced with 100 micrograms/ml of Poly (IC). There was a direct correlation between the dose of the inducing agent applied and the amount of IFN produced. Also, the cells were excellently super-inducible with metabolic inhibitors like actinomycin D and cycloheximine. In addition, priming procedures enhanced IFN induction in human fibroblasts. Comparing different cell types with respect to their ability to be induced for IFN production, we found that different cell types show great differences in their inducer sensitivity. Finally, the response of different cell culture systems was highly variable when induced with different compounds.
Coxsackievirus B3 (CVB3) infections are the most frequent causes of human myocarditis, often resulting in chronic stages characterized by fibrosis and loss of function. This disease is called dilated ...cardiomyopathy (DCM). Persistent virus in the myocardium may lead to chronic activation of fibroblasts, and subsequently, to fibrosis of the myocardium. Studies with immunodeficient mice have shown that certain defects of the immune system retard the rate at which virus is eliminated from the heart, thus leading to viral persistence. Therefore, we followed the immune response of two immunocompetent mouse strains (C57BL/6 and Balb/c) to CVB3 infection. These two strains have been reported to develop different immune responses to infections and we expected a similar reaction to viral infections as well. The two mouse strains recovered completely from CVB3 infection and expressed identical levels of cytokine mRNA in the heart. However, the virus in heart tissue decreased more slowly in Balb/c than in C57BL/6 mice. This was accompanied by a strong virus-specific IgG and weak IgM response in the C57BL/6 mice, in comparison to the Balb/c mice. We conclude, therefore, that viral-specific IgG is of importance for CVB3 elimination from infected hearts.
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