Microbiology and Immunology
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Chromosome‐Determined Zinc‐Responsible Operon <i>czr</i> in <i>Staphylococcus aureus</i> Strain 912 Abstract A novel operon, czrAB (zinc‐responsible genes), was identified in the chromosome of Staphylococcus aureus . The operon consists of two genes, czrA and czrB . The czrA gene, coding for an 11.5 kDa protein, was homologous to cadC, arsR of S. aureus plasmid pI258 and smtB of Synechococcus PCC7942. The czrB , coding for a 36 kDa membrane spanning protein, was homologous to the czcD gene, cobalt, zinc and the cadmium‐resistant factor of Bacillus subtilis and Alcaligenes eutrophus . In the presence of zinc (0.1‐10 mM), the transcription of czrAB was enhanced in a concentration‐dependent manner. Other heavy metals, such as cobalt, copper, manganese and nickel showed no effect on czrAB expression. The disruptant of the czrB gene became sensitive to zinc ion (MIC, 2 mM; MBC, 10 mM), and the complementation with the plasmid recovered the resistance to zinc at the same concentration as a parental strain (MIC, 5 mM; MBC, 20 mM). The disruptant accumulated intracellular zinc up to 0.4 mg per g dry weight of the organism, while that of the parental strain was 0.25 mg per g dry weight. The findings indicated that the novel operon czrAB should play a role in the transportation of zinc across the cell membrane to maintain the proper intracellular concentration.
Microbiology and Immunology - Tập 43 Số 2 - Trang 115-125 - 1999
Transfer of Two <i>Burkholderia</i> and An <i>Alcaligenes</i> Species to <i>Ralstonia</i> Gen. Nov. Abstract Based on the results of phenotypic characterization, cellular lipid and fatty acid analysis, phylogenetic analysis of 16S rDNA nucleotide sequences and rRNA‐DNA hybridization, Burkholderia pickettii, Burkholderia solanacearum and Alcaligenes eutrophus are transferred to the new genus Ralstonia , and Ralstonia pickettii (Ralston, Palleroni and Doudoroff 1973) comb. nov., Ralstonia solanacearum (Smith 1896) comb, nov., and R. eutropha (Davis 1969) comb. nov. are proposed. The type species of the new genus is R. pickettii . Type strain of R. pickettii is ATCC 27511T , of R. solanacearum is ATCC 10696T , and of R. eutropha is ATCC 17697T .
Microbiology and Immunology - Tập 39 Số 11 - Trang 897-904 - 1995
Maternal Transmission and Dental Caries Induction in Sprague‐Dawley Rats Infected with <i>Streptococcus mutans</i> Abstract Thirty‐four female rats (18 days old) were infected with Streptococcus mutans MT8148R (serotype c ) or 6715 (g ). Diets containing different proportions of sucrose were used to prepare the dams which harbored various levels of S. mutans in their oral cavity. Around 66 days of age, the female rats were bred and 34 dams subsequently bore 322 offspring. The dams were killed upon weaning (20 days of age) of their respective litters. There were positive correlations between the recovery of inoculated S. mutans and the caries incidence in the dams. Transmission of S. mutans from a dam to her offspring was studied in 10‐, 15‐, 20‐, 27‐, 34‐, 41‐, 48‐, and 55‐day‐old rats by evaluating the recover of S. mutans from the offspring. Positive correlation between the magnitudes of recovered S. mutans MT8148R from dams and their offspring was found in all ages of young rats examined. Furthermore, caries incidence in young rats was found to be positively correlated with the recovery of both strains of S. mutans as well as with incidence of caries in their respective dams.
Microbiology and Immunology - Tập 32 Số 8 - Trang 785-794 - 1988
Association of Rabies Virus Nominal Phosphoprotein (P) with Viral Nucleocapsid (NC) Is Enhanced by Phosphorylation of the Viral Nucleoprotein (N) Abstract We investigated possible role(s) of N protein phosphorylation in the rabies virus replication process. A large amount of P proteins are associated with the viral nucleocapsid (NC) in the infected cell, the amount which was greatly decreased by phosphatase‐treatment of the isolated NC, indicating that the phosphate group of N and/or P proteins is essential for their stable association with the NC. Immunoprecipitation studies were performed on the coexpressed normal N or phosphorylation deficient N(S389A) and P proteins, demonstrating that the P protein associated with phosphorylation‐deficient NC‐like structures was much less in amount than that associated with the wild type NC. Similar results were also obtained with a mutant P protein, PΔN19, which lacked the N‐terminal 19 amino acids and was capable of binding to the NC‐like structures but incapable of forming the RNA‐free N‐P complexes. Immunoprecipitation studies with mAb #402‐13 further suggested that the NC‐specific linear 402–13 epitope was exposed even on the P proteins which were associated with the phosphorylation‐deficient NC‐like structures, but such association was very weak as demonstrated by greatly decreased amounts of coprecipitated NC‐like structures. From these results, we assume that the phosphorylation of N protein enhances the association between the 402–13 epitope‐positive P protein and the NC probably by stabilizing such P‐NC binding.
Microbiology and Immunology - Tập 48 Số 5 - Trang 399-409 - 2004
Suppression of Hepatitis C Virus Replicon by RNA Interference Directed against the NS3 and NS5B Regions of the Viral Genome Abstract RNA interference (RNAi) is a phenomenon in which small interfering RNA (siRNA), an RNA duplex 21 to 23 nucleotides (nt) long, or short hairpin RNA (shRNA) resembling siRNA, mediates degradation of the target RNA molecule in a sequence‐specific manner. RNAi is now expected to be a useful therapeutic strategy for hepatitis C virus (HCV) infection. In the present study we compared the efficacy of a number of shRNAs directed against different target regions of the HCV genome, such as 5′‐untranslated region (5′UTR) (nt 286 to 304), Core (nt 371 to 389), NS3–1 (nt 2052 to 2060), NS3–2 (nt 2104 to 2122), and NS5B (nt 7326 to 7344), all of which except for NS5B are conserved among most, if not all, HCV subtype 1b (HCV‐1b) isolates in Japan. We utilized two methods to express shRNAs, one utilizing an expression plasmid (pAVU6+27) and the other utilizing a recombinant lentivirus harboring the pAVU6+27‐derived expression cassette. Although 5′UTR has been considered to be the most suitable region for therapeutic siRNA and/or shRNA because of its extremely high degree of sequence conservation, we observed only a faint suppression of an HCV subgenomic replicon by shRNA against 5′UTR. In both plasmid‐ and lentivirus‐mediated expression systems, shRNAs against NS3–1 and NS5B suppressed most efficiently the replication of the HCV replicon without suppressing host cellular gene expression. Synthetic siRNA against NS3–1 also inhibited replication of the HCV replicon in a dose‐dependent manner. Taken together, the present results imply the possibility that the recombinant lentivirus expressing shRNA against NS3–1 would be a useful tool to inhibit HCV‐1b infection.
Microbiology and Immunology - Tập 48 Số 8 - Trang 591-598 - 2004
Lambda‐Toxin of <i>Clostridium perfringens</i> Activates the Precursor of Epsilon‐Toxin by Releasing Its N‐ and C‐Terminal Peptides Abstract The effect of γ‐toxin, a thermolysin‐like metalloprotease of Clostridium perfringens , on the inactive ε‐prototoxin produced by the same organism was examined. When the purified ε‐prototoxin was incubated with the purified γ‐toxin at 37 C for 2 hr, the 32.5‐kDa ε‐prototoxin was processed into a 30.5‐kDa polypeptide, as determined by SDS‐polyacrylamide gel electrophoresis. A mouse lethality test showed that the treatment activated the prototoxin: the 50% lethal doses (LD50 ) of the prototoxin with and without γ‐toxin treatment were 110 and 70,000 ng/kg of body weight, respectively. The lethal activity of the prototoxin activated by γ‐toxin was comparable to that with trypsin plus chymotrypsin and higher than that with trypsin alone: LD50 of the prototoxin treated with trypsin and trypsin plus chymotrypsin were 320 and 65 ng/kg of body weight, respectively. The ε‐toxin gene was cloned and sequenced. Determination of the N‐terminal amino acid sequence of each activated ε‐prototoxin revealed that γ‐toxin cleaved between the 10th and 11th amino acid residues from the N‐terminus of the prototoxin, while trypsin and trypsin plus chymotrypsin cleaved between the 13th and 14th amino acid residues. The molecular weight of each activated ε‐prototoxin was also determined by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. The C‐terminus deduced from the molecular weight is located at the 23rd or 30th amino acid residue from the C‐terminus of the prototoxin, suggesting that removal of not only N‐terminal but also C‐terminal peptide is responsible for activation of the prototoxin.
Microbiology and Immunology - Tập 41 Số 7 - Trang 527-535 - 1997
Two Distinct Cytotoxic T Lymphocyte Subpopulations in Patients with Vogt‐Koyanagi‐Harada Disease that Recognize Human Melanoma Cells Abstract The functional properties of cytotoxic lymphocytes from patients with Vogt‐Koyanagi‐Harada disease (VKH) specific for human melanoma cells (P‐36 melanoma cell line established from a patient with malignant melanoma) were investigated by using monoclonal antibodies specific for human T cell subsets. Peripheral blood lymphocytes (PBL) from patients with VKH showed significant cytotoxic activity against the P‐36 (SK‐MEL‐28) human melanoma cell line, but not against a human cervical carcinoma of the uterus cell line (HeLa‐S3 cell line) or against a mouse melanoma cell line (B‐16 cell line) originating from a C57BL/6 strain mouse or against the EL‐4 mouse lymphoma cell line from a C57BL/6 mouse. The cytotoxic activity of the patients' PBL against the P‐36 melanoma cell line was markedly reduced by pretreatment of the PBL with monoclonal anti‐human Leu‐1 antibody plus rabbit complement, but it was reduced to much less extent by pretreatment with either monoclonal anti‐human Leu‐2a or Leu‐3a antibody plus rabbit complement. The specific cytotoxic activity of the patients' PBL against the P‐36 human melanoma cell line is, therefore, mediated by T cells bearing Leu‐1+ Leu‐2a+ or Leu‐1+ Leu‐3a+ antigens. Furthermore, the cytotoxic activity was shown to be blocked not only by anti‐Leu‐2a antibody specific to human cytotoxic/suppressor T cells but also unexpectedly by anti‐Leu‐3a antibody which has previously been considered to be specific to human inducer/helper T cells. The results of this study suggest that at least two distinct subpopulations of cytotoxic T cells specific for P‐36 human melanoma cells are present in the peripheral blood of VKH patients. These cytotoxic T cells have different surface antigens, Leu‐2a and Leu‐3a.
Microbiology and Immunology - Tập 28 Số 2 - Trang 219-231 - 1984
An Evaluation of Serodiagnostic Tests in Patients with Candidemia: Beta‐Glucan, Mannan, Candida Antigen by Cand‐Tec and D‐Arabinitol Abstract The serodiagnostic tests, beta‐glucan, mannan, candida antigen by Cand‐Tec, and D ‐arabinitol were evaluated in 10 patients with candidemia, 14 patients with suspected fungemia, and 10 healthy persons. By blood culture or lysis centrifugation, C. albicans was isolated from 5 patients, C. parapsilosis from 4, and C. tropicalis from 1 patient; no organisms were isolated from the 14 patients with suspected fungemia or the 10 healthy subjects. Beta‐glucan was measured by the difference between two chromogenic limulus tests (Endotoxin test‐D® and Endospecy®), which was more than 60 pg/ml in 7 of 9 (78%) candidemic patients and 1 of 12 (8%) patients with suspected fungemia. Mannan was positive in 6 of 10 (60%) candidemic patients and 1 of 13 (8%) patients with suspected fungemia. Both antigens were very sensitive and highly specific for candidemia. However, the Cand‐Tec assay was less specific, because titers of more than 4 were observed in 5 of 14 (34%) patients with suspected fungemia. D ‐Arabinitol was the least sensitive, because a D ‐arabinitol/creatinine ratio greater than 2.0 μmol/mg was observed in only 2 of 7 (29%) candidemic patients. The titers of serodiagnostic tests decreased after successful treatment with an anti‐fungal agent. Our results show that the combined use of the assays in necessary for accurate serological diagnosis of candidemia.
Microbiology and Immunology - Tập 37 Số 3 - Trang 207-212 - 1993
Kinetics of Anti‐Mannan Antibodies Useful in Confirming Invasive Candidiasis in Immunocompromised Patients Abstract In studying the anti‐mannan antibodies longitudinally in serial serum samples of three immunocompromised patients, it was observed that anti‐mannan antibodies started to increase shortly after the moment that cultures of deep‐tissue sites became positive with Candida albicans . The mean anti‐mannan antibody titers determined in a group of 36 immunocompromised patients with invasive candidiasis increased within two weeks after the probable onset of invasive candidiasis. In contrast, anti‐mannan antibody levels in serial serum samples of 14 immunocompromised patients who were only colonized with C. albicans remained stable or decreased over time. The HA test measuring the anti‐mannan antibodies was 64% sensitive and 89% specific in determining invasive candidiasis. In contrast, antibodies specific for candidal cytoplasmic antigens or enolase alone were of little value in confirming invasive candidiasis in these immunocompromised patients.
Microbiology and Immunology - Tập 40 Số 2 - Trang 125-131 - 1996
Anti‐quorum sensing activity of <i>Psidium guajava</i> L. flavonoids against <i>Chromobacterium violaceum</i> and <i>Pseudomonas aeruginosa</i> PAO1 ABSTRACT Psidium guajava L., which has been used traditionally as a medicinal plant, was explored for anti‐quorum sensing (QS) activity. The anti‐QS activity of the flavonoid (FL) fraction of P. guajava leaves was determined using a biosensor bioassay with Chromobacterium violaceum CV026. Detailed investigation of the effects of the FL‐fraction on QS‐regulated violacein production in C. violaceum ATCC12472 and pyocyanin production, proteolytic, elastolytic activities, swarming motility and biofilm formation in Pseudomonas aeruginosa PAO1 was performed using standard methods. Possible mechanisms of QS‐inhibition were studied by assessing violacein production in response to N ‐acyl homoserine lactone (AHL) synthesis in the presence of the FL‐fraction in C. violaceum ATCC31532 and by evaluating the induction of violacein in the mutant C. violaceum CV026 by AHL extracted from the culture supernatants of C. violaceum 31532. Active compounds in the FL‐fraction were identified by liquid chromatography–mass spectrometry (LC–MS). Inhibition of violacein production by the FL‐fraction in a C. violaceum CV026 biosensor bioassay indicated possible anti‐QS activity. The FL‐fraction showed concentration‐dependent decreases in violacein production in C. violaceum 12472 and inhibited pyocyanin production, proteolytic and elastolytic activities, swarming motility and biofilm formation in P. aeruginosa PAO1. Interestingly, the FL‐fraction did not inhibit AHL synthesis; AHL extracted from cultures of C. violaceum 31532 grown in the presence of the FL‐fraction induced violacein in the mutant C. violaceum CV026. LC–MS analysis revealed the presence of quercetin and quercetin‐3‐O ‐arabinoside in the FL‐fraction. Both quercetin and quercetin‐3‐O ‐arabinoside inhibited violacein production in C. violaceum 12472, at 50 and 100 μg/mL, respectively. Results of this study provide scope for further research to exploit these active molecules as anti‐QS agents.
Microbiology and Immunology - Tập 58 Số 5 - Trang 286-293 - 2014
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