Journal of Microbiology

Volume 40(3); September 2002

Protection of Specific-pathogen-free (Spf) Foals from Severe Equine Herpesvirus Type-1 (Ehv-1) Infection Following Immunization with Non-infectious L-particles: Mohd Lila Mohd-Azmi , John Gibson , Frazer Rixon , John McLauchlan , Hugh John Field; J. Microbiol. 2002;40(3):183-192.

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Abstract: Cells infected with equine herpesvirus type-1 (EHV-1) produced both infectious and non-infectious virus-related particles. Compared to the whole virion, non-infectious particles termed L-particles were determined to lack 150 kDa protein, commonly known as nucleocapsid protein. The potential of L-particles to induce immune responses was studied in mice and foals. Intranasal immunization with L-particles or whole virions induced poor IgG antibody responses in mice. Interestingly, despite the poor antibody response, the conferred immunity protected the host from challenge infections. This was indicated by a significant reduction in virus titers in line with recovery towards normal body weight. Subsequently, the test on the usefulness of L-particles as immunizing agents was extended to foals. Immunization of specific-pathogen-free (SPF) foals resulted in similar results. As determined by a complement-fixing-antibody test (CFT), foals seroconverted when they were immunized either with inactivated L-particles or whole virions via intramuscular (i.m.) injections. The presence of the antibody correlated with the degree of protection. Beyond day 1 post challenge infection (p.i.), there was no virus shedding in the nasal mucus of foals immunized with whole EHV-1 virions. Virus shedding was observed in foals immunized with L-particles but limited to days 6 to 8 p.i. only. In contrast, extended virus shedding was observed in non-immunized foals and it was well beyond day 14 p.i. Viremia was not detected for more than four days except in non-immunized foals. Immunization in mice via intranasal (i.n.) conferred good protection. However, compared to the i.n. route, a greater degree of protection was obtained in foals following immunization via i.m. route. Despite variation in the degree of protection due to different routes of immunization in the two animal species, our results have established significant evidence that immunization with L-particles confers protection in the natural host. It is suggested that non-infectious L-particles should be used as immunizing agents for vaccination of horses against EHV-1 infection.

Transformations of 2,4,6-Trinitrotoluene in Various Conditions by Klebsiella sp. Strain C1 Isolated from Activated Sludge: Chong-Suk Chang , Hyoun-Young Kim , Yang-Mi Kang , Kyung Sook Bae , Hong-Gyu Song; J. Microbiol. 2002;40(3):193-198.

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Abstract: Several 2,4,6-Trinitrotoluene (TNT) degrading bacteria were isolated from an activated sludge by an enrichment culture technique, and their TNT removal activities were examined. Among the isolates, strain C1 showed the highest degrading capability, and completely removed 100 or 200 mg l^-1 of TNT within 6 hours of incubation. This bacterium was identified as Klebsiella sp. The effects of different carbon sources on the removal of the parent TNT by Klebsiella sp. C1 were negligible, but the transformation rates of TNT metabolites such as amino-dinitrotoluenes and diamino-nitrotoluenes were higher with fructose addition compared to glucose addition. When nitrate was used as the nitrogen source, the degradation rates of TNT and hydroxylamino-dinitrotoluenes were higher than those with the ammonium addition. Although the TNT removal rate of Klebsiella sp. C1 was slightly higher in anaerobic conditions, the further transformations of TNT metabolites were more favorable in aerobic conditions.

Nosema sp. isolated from Cabbage White Butterfly (Pieris rapae) Collected in Korea: Ji Young Choi , Jong Gill Kim , Young Cheol Choi , Tae Won Goo , Jin Hee Chang , Yeon Ho J e , Keun Young Kim; J. Microbiol. 2002;40(3):199-204.

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Abstract: A microsporidium, from cabbage white butterflies, Pieris rapae, collected in Korea, was purified and characterized according to its gene structure, spore morphology and pathogenicity. From the observation of the isolate by SEM and TEM, the endospores, exospores and nuclei, about 12 polar filament coils of the polar tube and posterior vacuoles were all identified. The nucleotide sequence was determined for a portion of genomic DNA which spans the V4 variable region of the small subunit rRNA gene. Comparison with the GenBank database for 15 other microsporidia species suggests that this isolate is most closely related to Nosema species. The pathogenicity against cabbage white butterflies was quantified by inoculating variable doses of spores to the second instar larvae. Peroral inoculation at a dosage of 10^8 spores/ml resulted in the death of all larvae prior to adult eclosion, but at lower spore dosages of 10^4 ?0^5 spores/ml, many adults successfully emerged. The median lethal dose (LD_50 ) was determined to be 4.6 X 10^6 spores/ml and the isolate also transmitted transovarially to the progeny eggs at a frequency of 92%.

P22-Based Challenge Phage Constructs to Study Protein-Protein Interactions between the [sigma]^54 -Dependent Promoter, dctA, and Its Transcriptional Regulators: Jeong Min Song , Eungbin Kim , Joon H. Lee; J. Microbiol. 2002;40(3):205-210.

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Abstract: To study interactions between C_4 -dicarboxylic acid transport protein D and E[sigma]^54 in the dctA promoter regulatory region, we used the challenge phage system. An ant'-'lac fusion was recombined onto the challenge phage, and this ant'-'lac fusion along with Pant and the R. meliloti dctA promoter regulatory region were cloned onto a plasmid. The plasmid bearing the ant'-'lac fusion was used as a reporter plasmid in a coupled transcription-translation system. Addition of purified [sigma]^54 to the coupled system specifically repressed transcription of the plasmid-borne ant'-'lac fusion. When DCTD was added along with [sigma]^54 to the coupled system, transcription of the ant'-'lac fusion was even further repressed, suggesting that DCTD may stabilize closed complexes between E[sigma]^54 and the dctA promoter.

Molecular Cloning and Analysis of the Gene for P-450 Hydroxylase from Pseudonocardia autotrophica IFO 12743: Jung-Mee Kim , Younmie Jin , Chang-Gu Hyun , Jong-Hee Kim , Hong-Sub Lee , Dae-Kyung Kang , Dae-Jung Kang , Tae-Yong Kim , Joo-Won; J. Microbiol. 2002;40(3):211-218.

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Abstract: A 4.8-kb DNA fragment encoding the P-450 type hydroxylase and ferredoxin genes was cloned from Pseudonocardia autotrophica IFO 12743 that can convert vitamin D_3 into its hydroxylated active forms. In order to isolate the P-450 gene cluster in this organism, we designed PCR primers on the basis of the regions of an oxygen binding site and a heme ligand pocket that are general characteristics of the P-450 hydroxylase. Sequencing analysis of the BamHI fragment revealed the presence of four complete and one incomplete ORFs, named PauA, PauB, PauC, and PauD, respectively. As a result of computer-based analyses, PauA and PauB have homology with enoyl-CoA hydratase from several organisms and the positive regulators belonging to the tetR family, respectively. PauC and PauD show similarity with SuaB/C proteins and ferredoxins, respectively, which are composed of P-450 monooxygenase systems for metabolizing two sulfonylurea herbicides in Streptomyces griseolus PauC shows the highest similarity with another CytP-450_Sca2 protein that is responsible for production of a specific HMG-CoA reductase inhibitor, pravastatin, in S. carbophilus. Cultures of Streptomyces lividans transformant, containing the P-450 gene cluster on the pWHM3 plasmid, was unable to convert vitamin D_3 to its hydroxylated forms.

Characterization of Cell Wall Proteins from the soo1-1/ret1-1 Mutant of Saccharomyces cerevisiae: Dong-Won Lee , Ki-Hyun Kim , Se-Chul Chun , Hee-Moon Park; J. Microbiol. 2002;40(3):219-223.

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Abstract: In order to investigate the function of Soo1p/[alpha]-COP during post-translational modification and intracellular transport of cell wall proteins in Saccharomyces cerevisiae, cell wall proteins from the soo1-1/ret1-1 mutant cells were analyzed. SDS-PAGE analysis of biotin labeled cell wall proteins suggested that the soo1-1 mutation impairs post-translational modification of cell wall proteins, such as N- and/or O-glycosylation. Analysis of cell wall proteins with antibodies against [beta]-1,3-glucan and [beta]-1,6-glucan revealed alteration of the linkage between cell wall proteins and [beta]-glucans in the soo1-1 mutant cells. Compositional sugar analysis of the cell wall proteins also suggested that the soo1-1 mutation impairs glycosylation of cell wall protein in the ER, which is crucial for the maintenance of cell wall integrity.

Induction of Apoptosis in Human Monocytes by Human Cytomegalovirus is Related with Calcium Increase: Myung Sook Moon , Gyu Cheol Lee , Chan H. Lee; J. Microbiol. 2002;40(3):224-229.

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Abstract: The effect of human cytomegalovirus (HCMV) on three human monocyte cell lines at different stages of differentiation was investigated. While the viability of HL-60 cells or U-937 cells was not significantly affected by HCMV infection, the viability of THP-1 cells was reduced. Acridine orange/ethidium bromide staining revealed that the reduction of THP-1 cell viability was due to increased apoptotic death following HCMV infection. Apoptosis in HL-60 cells was not affected by HCMV infection, and induction of apoptosis of U-937 cells by HCMV was intermediate between HL-60 and THP-1 cells. Since HL-60 cells are the least differentiated and THP-1 cells are the most differentiated, the induction of apoptosis of human monocytes appears to be related to the degree of cell differentiation. Flow cytometric and confocal microscopic studies using fluorescent calcium indicator Fluo-3 suggested a significant increase in intracellular free calcium concentration ([Ca^2+ ]_i ) in THP-1 cells undergoing apoptosis by HCMV infection. Again [Ca^2+ ]_i in HCMV-infected HL-60 cells was not critically altered, and that in HCMV-infected U-937 cells was intermediate between THP-1 cells and HL-60 cells. Calcium influx blockers such as verapamil and nifedipine partially reversed HCMV-induced apoptosis in THP-1 cells.

Promotion of Asexual Development and Inhibition of Sexual Development of Aspergillus nidulans by Short-Chain Primary Amines: Myung Hoon Song , Kuppusamy Selvam , Chang-Jun Choi , Kwang-Yeop Jahng , Dong-Min Han , Keon-Sang Chae; J. Microbiol. 2002;40(3):230-233.

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Abstract: Effects of short-chain primary amines on Aspergillus nidulans development were analyzed. Propylamine induced asexual development and inhibited sexual development. Even on medium containing lactose as the sole carbon source, on which little conidial heads are formed and sexual structures are formed preferentially, or when sexual development was induced, propylamine induced asexual development and inhibited sexual development. These effects of propylamine seemed to be due to accumulation of mRNA of the brlA gene, which has been identified as a positive regulator of asexual development, and due to the reduction of the veA mRNA level. The veA gene has been identified as an activator of sexual development and also as an inhibitor of asexual development. Other primary amines, methylamine and ethylamine, showed identical effects on development where short-chain primary amine also promoted asexual development and inhibited sexual development.

Bacterial Color Response to Hexavalent Chromium, Cr^6+: Ka Hong Cheung , Ji-Dong Gu; J. Microbiol. 2002;40(3):234-236.

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Abstract: A blue pigment-producing bacterium, Vogesella indigofera, was isolated and quantified for the relationship between its synthesis of a blue pigment and exposure concentrations of Cr^6+ . The concentration of Cr^6+ and the percentage of blue colonies on agar plates was negatively correlated (r^2 = -0.8683). Critical concentrations inhibiting bacterial pigment production were found to be between 100-150 ug Cr^6+ /ml on agar plates and 200-300 ug Cr^6+ /ml in liquid culture. As the blue color is characteristic and easily observable, the bacterium Vogesella indigofera may have potential applications in the detection and monitoring of environmental pollution.

Reclassification of a Carboxydobacterium, Acinetobacter sp. Strain JC1 DSM3803, as Mycobacterium sp. Strain JC1 DSM 3803: Taeksun Song , Hyeyoung Lee , Yong-Ha Park , Eungbin Kim , Young Ta e Ro , Si Wouk Kim , Young Min Kim; J. Microbiol. 2002;40(3):237-240.

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Abstract: A carboxydotrophic bacterium, isolated from a soil sample in Seoul, was classified initially as Acinetobacter sp. strain JC1 DSM 3803. Chemotaxanomic properties, analysis of the 16s rDNA sequence, fatty acid content, and molecular phylogenetic analysis based on rpoB gene, however, suggested that this bacterium belongs to the genus, Mycobacterium. On the basis of this evidence, it is proposed that Acinetobacter sp. strain JC1 DSM 3803 be reclassified as Mycobacterium sp. strain JC1 DSM 3803.

Comparative Enzyme Production by Fungi from Diverse Lignocellulosic Substrates: Marie K. W. Sin , Kevin D. Hyde , Stephen B. Pointing; J. Microbiol. 2002;40(3):241-244.

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Abstract: Fungi commonly encountered on monocotyledonous substrates were evaluated for their in vitro ability to produce enzymes involved in lignocellulose breakdown. Most were capable of structural polysaccharide utilization, but few produced enzymes associated with lignin breakdown. None of the monocotyledon-inhabiting fungi produced reactions as strongly as wood decay fungi.

Cloning of Genomic DNAs of Trametes versicolor Acting as Autonomously Replicating Sequences in Saccharomyces cerevisiae: Sora An , Kyoung Phil Park , Hyoung-Tae Choi , Kyu-Joong Kim , Kyunghoon Kim; J. Microbiol. 2002;40(3):245-247.

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Abstract: A genomic DNA library of the fungus Trametes versicolor was constructed in a yeast integration vector which contains the URA3 gene of the budding yeast Saccharomyces cerevisiae and the gene responsible for hygromycin B resistance, and fragments acting as autonomously replicating sequences (ARSes) in the budding yeast were identified from the genomic DNA library. Sixteen recombinant plasmids from the library transformed the budding yeast Saccharomyces cerevisiae to Ura^+ at high frequencies. They were maintained stably under selective conditions, but were gradually lost from yeast cells at different rates under nonselective conditions, indicating that they contain eukaryotic origins of DNA replication and exist as extrachromosomal plasmids. Base sequences of four ARS DNAs among the 16 cloned fragments revealed that all of the four contain at least one 11 bp [(A/T)TTTA(T/C)(A/G)TTT(A/T)] consensus sequence of the budding yeast ARS.

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