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At least 829 records · Page 46Linked to original sources
PubMed2004

Significance of the 3'-terminal region in minus-strand RNA synthesis of Hibiscus chlorotic ringspot virus.

RNA-dependent RNA polymerase (RdRp) was solubilized from crude extracts of Hibiscus cannabinus infected by Hibiscus chlorotic ringspot virus (HCRSV), a member of the Carmoviridae. After treatment of the extracts with micrococcal nuclease to remove the endogenous templates, the full-length genomic RNA and the two subgenomic RNAs were efficiently synthesized by the partially purified RdRp complex in vitro. When the full-length RNAs of Potato virus X, Tobacco mosaic virus, Odontoglossum ringspot virus and Cucumber mosaic virus were used as templates, no detectable RNA was synthesized. Synthesis of HCRSV minus-strand RNA was shown to initiate opposite the 3'-terminal two C residues at the 3' end in vitro and in vivo. The CCC-3' terminal nucleotide sequence was optimal and nucleotide variations from CCC-3' diminished minus-strand synthesis. In addition, two putative stem-loops (SLs) located within the 3'-terminal 87 nt of HCRSV plus-strand RNA were also essential for minus-strand RNA synthesis. Deletion or disruption of the structure of these two SLs severely reduced or abolished RNA synthesis. HCRSV RNA in which the two SLs were replaced with the SLs of Turnip crinkle virus could replicate in kenaf protoplasts, indicating that functionally conserved structure, rather than nucleotide sequence, plays an important role in the minus-strand synthesis of HCRSV. Taken together, the specific sequence CCC at the 3' terminus and the two SLs structures located in the 3'UTR are essential for efficient minus-strand synthesis of HCRSV.

3' Untranslated Regions↗
PubMed1989

In situ nucleoprotein structure at the SV40 major late promoter: melted and wrapped DNA flank the start site.

New in situ probing methods have been developed and used to probe the nucleoprotein structures at the SV40 major late promoter in infected monkey cells. The region that contains the three proximal transcription elements was probed with DNase I and micrococcal nuclease in transcriptionally active, permeabilized cells, and with the single-strand selective reagent KMnO4 in intact cells. The downstream element is included in a region of enhanced DNase I reactivity at 10- to 11-bp intervals for approximately 140 bp, presumably because of DNA wrapping around a specifically positioned nucleosome particle. The two other proximal DNA elements appear to be mostly melted, with a protecting factor bound primarily to the template DNA strand. The protecting factor directly borders the wrapped particle. These observations provide an initial description of parts of the biological transcription machinery and suggest that the SV40 major late promoter elements are part of a higher order nucleoprotein complex that involves wrapped and melted DNA.

Animals↗
PubMed1978

Evaluation of three test procedures for identification of Staphylococcus aureus from clinical sources.

A total of 520 clinical and environmental isolates of the family Micrococcaceae that fermented glucose anaerobically were tested for their ability to produce coagulase, thermostable nuclease, and deoxyribonuclease. Of these, 450 isolates coagulated rabbit plasma, produced thermostable nuclease, and were identified as Staphylococcus aureus, 447 of which produced a 3+ to 4+ clot. The remaining three isolates produced a 2+ clot, deoxyribonuclease, and thermostable nuclease. It was found that three of the S. aureus isolates failed to produce deoxyribonuclease. A total of 70 isolates which did not coagulate rabbit plasma and which were thermostable nuclease negative were identified as S. epidermidis. Three of them produced deoxyribonuclease. It is suggested that the thermostable nuclease test be performed on all isolates producing a 2+ (or 1+) clot in the coagulase test before identifying them as S. aureus.

Anaerobiosis↗
PubMed2006

Evaluation of a single-locus real-time polymerase chain reaction as a screening test for specific detection of methicillin-resistant Staphylococcus aureus in ICU patients.

The aim of the present study was to determine the diagnostic value of a single-locus real-time polymerase chain reaction (PCR) recently proposed for rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) from clinical samples (IDI-MRSA; Infectio Diagnostic, Sainte-Foy, Québec, Canada). This test, which was developed on the basis of studies of the sequence analysis of the mecA gene carried by staphylococcal cassette chromosome mec (SCCmec), was used to screen nasal swabs of 320 intensive care unit (ICU) patients at admission. The results were compared with those of conventional culture of swabs from several body sites. When compared with culture of swabs from the nose, throat, and wounds, the diagnostic values of the real-time PCR test from nasal swabs were as follows: 92.3% sensitivity, 98.6% specificity, 75.0% positive predictive value, and 99.6% negative predictive value. Fifteen (4.7%) samples could not be evaluated because the PCR reaction was inhibited, even after the samples were frozen and thawed for retesting. Culture of nasal swabs showed that 78 of the patients were colonized with methicillin-susceptible S. aureus. Unexpectedly, 4 (5.1%) of these samples gave false-positive results in the IDI-MRSA. These isolates were all single clones, as shown by pulsed-field gel electrophoresis and spa typing. Reliable results were obtained with the IDI-MRSA assay, even in a patient population with a low prevalence (approximately 4%) of MRSA and even when compared with swabs of different body sites. Nevertheless, further work is needed to reduce the inhibition rate of the PCR and to explain why false-positive results were obtained with methicillin-susceptible S. aureus.

Bacterial Proteins↗
PubMed2006

Molecular identification and characterization of mannitol-negative methicillin-resistant Staphylococcus aureus.

We report on the isolation, molecular identification, and characterization of 5 mannitol-negative methicillin-resistant Staphylococcus aureus (MRSA) from clinical samples in KwaZulu-Natal (KZN) province, South Africa. Identification based on phenotypic testing and polymerase chain reaction detection of the S. aureus species-specific nuc gene and the coagulase gene indicated that the mannitol-negative isolates were S. aureus. Furthermore, they were mecA positive, and SCCmec typing demonstrated that all the isolates harbored type IV SCCmec. API STAPH (Biomerieux, Marcy-l'Etoile, France) misidentified 2 mannitol-negative MRSA that belonged to the major clone in KZN province, as Staphylococcus lugdunensis. Although the prevalence and mechanism of mannitol-negative MRSA is unknown, laboratories are encouraged to investigate S. aureus with atypical characteristics.

Bacterial Proteins↗
PubMed1970

Replicative form II DNA of phi X174; resistance to exonucleolytic cleavage by E. coli DNA polymerase.

The progeny replicative form II DNA isolated from E. coli cells infected with varphiX174 is resistant to the exonucleolytic activity associated with E. coli DNA polymerase. A limited endonucleolytic cleavage with micrococcal endonuclease renders the replicative form II molecule susceptible to the exonucleolytic activity associated with the E. coli DNA polymerase. These experiments suggest that the resistance of the replicative form II DNA to the exonucleolytic activity is a result of the structure of these molecules at their 5'-terminus.

Chemical Phenomena↗
PubMed1976

Behaviors of a vigorous protease-producing strain of Staphylocollus aureus in the skin tissue of mice.

The behaviors of a protease-producing strain of Staphylococcus aureus, including the production of alpha- and beta-hemolysin, protease, and nuclease in the skin tissue of mice, were examined by the fluorescent antibody technique and hematoxylin and eosin staining. When about 10(6) viable cells were subcutaneously inoculated into a mouse, they were phagocytized by leukocytes and a suppurative focus developed in situ. No active extracellular substances other than alpha-hemolysin were demonstrated in this mouse. When 10(7) viable cells were inoculated, most of them were also phagocytized by leukocytes, and a rather large suppurative focus was formed. In this focus a low degree of multiplication of the organisms was observed. Protease and alpha-hemolysin could be detected in the neighborhood of the leukocytes. When 10(8) to 10(9) cells were inoculated, they multiplied transitorily in the subcutaneous tissue and produced the four substances mentioned above. The multiplication of bacterial cells as well as the production of extracellular substances ceased within a comparatively short period and the transitory state was similar to that in a batch fermenter. Necrotic and histolytic lesions were observed only in the mice inoculated with 10(8) to 10(9) viable cells. Similar dermatolytic lesions were also found in mice injected with 0.5 to 1.0 mg of pure protease.

Animals↗
PubMed1997

Rapid detection of oxacillin-resistant Staphylococcus aureus in blood cultures by an impedance method.

The feasibility of using an impedance method for direct detection of oxacillin-resistant Staphylococcus aureus (ORSA) in blood cultures was evaluated. An aliquot (0.1 ml) of the positive blood culture, which showed growth of gram-positive cocci and demonstrated thermonuclease activity, was inoculated into the module well of a Bactometer incubator (bioMerieux Vitek, Hazelwood, Mo.) containing 0.6 ml of Mueller-Hinton agar supplemented with oxacillin (2 microg/ml). The modules were incubated at 37 degrees C, and the change in impedance in each well was continuously monitored by the instrument at 6-min intervals for 24 h. ORSA strains from blood cultures could multiply in the oxacillin-containing medium, and a time point (detection time [DT]) at which an accelerating change of impedance occurred in the medium was obtained, with an average of 5.5 h. The growth of oxacillin-sensitive S. aureus (OSSA) strains was largely inhibited, and no DT was obtained for these strains within an incubation period of 24 h. For 96 positive blood cultures (38 ORSA and 58 OSSA) tested, 36 and 57 were found to be oxacillin resistant and oxacillin sensitive, respectively, by the impedance method. The impedance method had a sensitivity and specificity of 94.7 and 98.3%, respectively, for the detection of ORSA and had an agreement of 96.9% with the disc diffusion method. Comparable results were obtained by the testing of 235 clinical stock cultures of S. aureus (149 ORSA and 86 OSSA). The impedance test is simple for detecting ORSA in blood cultures and may allow proper antimicrobial treatment almost 36 h before the results of the conventional culture methods are available.

Bacteremia↗