Effect of the Modified Live Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Vaccine on European and North American PRRSV Shedding in Semen from Infected Boars

Han, Kiwon,Seo, Hwi Won,Shin, Jeoung Hwa,Oh, Yeonsu,Kang, Ikjae,Park, Changhoon,Chae, Chanhee American Society for Microbiology 2011 CLINICAL AND VACCINE IMMUNOLOGY Vol.18 No.10

<B>ABSTRACT</B><P>The objective of the present study was to compare the effects of the modified live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine (Ingelvac PRRS MLV; Boehringer Ingelheim Animal Health, St. Joseph, MO) on European and North American PRRSV shedding in the semen of experimentally infected boars. The boars were randomly divided into six groups. Vaccinated boars shed the North American PRRSV at the rate of 10<SUP>0.1</SUP>to 10<SUP>1.0</SUP>viral genome copies per ml and 3.63 to 10<SUP>1.1</SUP>50% tissue culture infective doses (TCID50)/ml, respectively, in semen, whereas nonvaccinated boars shed the North American PRRSV at the rate of 10<SUP>0.2</SUP>to 10<SUP>4.7</SUP>viral genome copies per ml and 1.14 to 10<SUP>3.07</SUP>TCID50/ml, respectively, in semen. Vaccinated boars shed the European PRRSV at the rate of 10<SUP>0.1</SUP>to 10<SUP>4.57</SUP>viral genome copies per ml and 1.66 to 10<SUP>3.10</SUP>TCID50/ml, respectively, in semen, whereas nonvaccinated boars shed the European PRRSV at the rate of 10<SUP>0.3</SUP>to 10<SUP>5.14</SUP>viral genome copies per ml and 1.69 to 10<SUP>3.17</SUP>TCID50/ml, respectively, in semen. The number of genomic copies of the European PRRSV in semen samples was not significantly different between vaccinated and nonvaccinated challenged European PRRSV boars. The present study demonstrated that boar vaccination using commercial modified live PRRSV vaccine was able to decrease subsequent shedding of North American PRRSV in semen after challenge but was unable to decrease shedding of European PRRSV in semen after challenge.</P>

A Short Isoform of Human Cytomegalovirus US3 Functions as a Dominant Negative Inhibitor of the Full-Length Form

Shin, Jinwook,Park, Boyoun,Lee, Sungwook,Kim, Youngkyun,Biegalke, Bonita J.,Kang, Seongman,Ahn, Kwangseog American Society for Microbiology 2006 Journal of virology Vol.80 No.11

<B>ABSTRACT</B><P>Human cytomegalovirus encodes four unique short (US) region proteins, each of which is independently sufficient for causing the down-regulation of major histocompatibility complex (MHC) class I molecules on the cell surface. This down-regulation enables infected cells to evade recognition by cytotoxic T lymphocytes (CTLs) but makes them vulnerable to lysis by natural killer (NK) cells, which lyse those cells that lack MHC class I molecules. The 22-kDa US3 glycoprotein is able to down-regulate the surface expression of MHC class I molecules by dual mechanisms: direct endoplasmic reticulum retention by physical association and/or tapasin inhibition. The alternative splicing of the <I>US3</I> gene generates two additional products, including 17-kDa and 3.5-kDa truncated isoforms; however, the functional significance of these isoforms during viral infection is unknown. Here, we describe a novel mode of self-regulation of US3 function that uses the endogenously produced truncated isoform. The truncated isoform itself neither binds to MHC class I molecules nor prevents the full-length US3 from interacting with MHC class I molecules. Instead, the truncated isoform associates with tapasin and competes with full-length US3 for binding to tapasin; thus, it suppresses the action of US3 that causes the disruption of the function of tapasin. Our results indicate that the truncated isoform of the <I>US3</I> locus acts as a dominant negative regulator of full-length US3 activity. These data reflect the manner in which the virus has developed temporal survival strategies during viral infection against immune surveillance involving both CTLs and NK cells.</P>

Mouse emi1Has an Essential Function in Mitotic Progression during Early Embryogenesis

Lee, Ho,Lee, Dong Jun,Oh, Sang Phil,Park, Hee Dong,Nam, Hyun Hee,Kim, Jin Man,Lim, Dae-Sik American Society for Microbiology 2006 Molecular and cellular biology Vol.26 No.14

<B>ABSTRACT</B><P>For successful mitotic entry and spindle assembly, mitosis-promoting factors are activated at the G2/M transition stage, followed by stimulation of the anaphase-promoting complex (APC), an E3 ubiquitin ligase, to direct the ordered destruction of several critical mitotic regulators. Given that inhibition of APC activity is important for preventing premature or improper ubiquitination and destruction of substrates, several modulators and their regulation mechanisms have been studied. Emi1, an early mitotic inhibitor, is one of these regulatory factors. Here we show, by analyzing Emi1-deficient embryos, that Emi1 is essential for precise mitotic progression during early embryogenesis. Emi1<SUP>−/−</SUP> embryos were found to be lethal due to a defect in preimplantation development. Cell proliferation appeared to be normal, but mitotic progression was severely defective during embryonic cleavage. Moreover, multipolar spindles and misaligned chromosomes were frequently observed in Emi1 mutant cells, possibly due to premature APC activation. Our results collectively suggest that the late prophase checkpoint function of Emi1 is essential for accurate mitotic progression and embryonic viability.</P>

Characterization of a Thermoacidophilic l-Arabinose Isomerase from Alicyclobacillus acidocaldarius: Role of Lys-269 in pH Optimum

Lee, Sang-Jae,Lee, Dong-Woo,Choe, Eun-Ah,Hong, Young-Ho,Kim, Seong-Bo,Kim, Byoung-Chan,Pyun, Yu-Ryang American Society for Microbiology 2005 Applied and environmental microbiology Vol.71 No.12

<B>ABSTRACT</B><P>The <I>araA</I> gene encoding l-arabinose isomerase (AI) from the thermoacidophilic bacterium <I>Alicyclobacillus acidocaldarius</I> was cloned, sequenced, and expressed in <I>Escherichia coli</I>. Analysis of the sequence revealed that the open reading frame of the <I>araA</I> gene consists of 1,491 bp that encodes a protein of 497 amino acid residues with a calculated molecular mass of 56,043 Da. Comparison of the deduced amino acid sequence of <I>A. acidocaldarius</I> AI (AAAI) with other AIs demonstrated that AAAI has 97% and 66% identities (99% and 83% similarities) to <I>Geobacillus stearothermophilus</I> AI (GSAI) and <I>Bacillus halodurans</I> AI (BHAI), respectively. The recombinant AAAI was purified to homogeneity by heat treatment, ion-exchange chromatography, and gel filtration. The purified enzyme showed maximal activity at pH 6.0 to 6.5 and 65°C under the assay conditions used, and it required divalent cations such as Mn<SUP>2+</SUP>, Co<SUP>2+</SUP>, and Mg<SUP>2+</SUP> for its activity. The isoelectric point (pI) of the enzyme was about 5.0 (calculated pI of 5.5). The apparent <I>Km</I> values of the recombinant AAAI for l-arabinose and d-galactose were 48.0 mM (<I>V</I>max, 35.5 U/mg) and 129 mM (<I>V</I>max, 7.5 U/mg), respectively, at pH 6 and 65°C. Interestingly, although the biochemical properties of AAAI are quite similar to those of GSAI and BHAI, the three AIs from <I>A. acidocaldarius</I> (pH 6), <I>G. stearothermophilus</I> (pH 7), and <I>B. halodurans</I> (pH 8) exhibited different pH activity profiles. Based on alignment of the amino acid sequences of these homologous AIs, we propose that the Lys-269 residue of AAAI may be responsible for the ability of the enzyme to act at low pH. To verify the role of Lys-269, we prepared the mutants AAAI-K269E and BHAI-E268K by site-directed mutagenesis and compared their kinetic parameters with those of wild-type AIs at various pHs. The pH optima of both AAAI-K269E and BHAI-E268K were rendered by 1.0 units (pH 6 to 7 and 8 to 7, respectively) compared to the wild-type enzymes. In addition, the catalytic efficiency (<I>k</I>cat/<I>Km</I>) of each mutant at different pHs was significantly affected by an increase or decrease in <I>V</I>max. From these results, we propose that the position corresponding to the Lys-269 residue of AAAI could play an important role in the determination of the pH optima of homologous AIs.</P>

The Zinc-Responsive Regulator Zur Controls a Zinc Uptake System and Some Ribosomal Proteins in Streptomyces coelicolor A3(2)

Shin, Jung-Ho,Oh, So-Young,Kim, Soon-Jong,Roe, Jung-Hye American Society for Microbiology 2007 Journal of Bacteriology Vol.189 No.11

<B>ABSTRACT</B><P>In various bacteria, Zur, a zinc-specific regulator of the Fur family, regulates genes for zinc transport systems to maintain zinc homeostasis. It has also been suggested that Zur controls zinc mobilization by regulating some ribosomal proteins. The antibiotic-producing soil bacterium <I>Streptomyces coelicolor</I> contains four genes for Fur family regulators, and one (named <I>zur</I>) is located downstream of the <I>znuACB</I> operon encoding a putative zinc uptake transporter. We found that zinc specifically repressed the level of <I>znuA</I> transcripts and that this level was derepressed in a Δ<I>zur</I> mutant. Purified Zur existing as homodimers bound to the <I>znuA</I> promoter region in the presence of zinc, confirming the role of Zur as a zinc-responsive repressor. We analyzed transcripts for paralogous forms of ribosomal proteins L31 (RpmE1 and RpmE2) and L33 (RpmG2 and RpmG3) for their dependence on Zur and found that RpmE2 and RpmG2 with no zinc-binding motif of conserved cysteines (C's) were negatively regulated by Zur. C-negative RpmG3 and C-positive RpmE1 were not regulated by Zur. Instead, they were regulated by the sigma factor σ<SUP>R</SUP> as predicted from their promoter sequences. The <I>rpmE1</I> and <I>rpmG3</I> genes were partially induced by EDTA in a manner dependent on σ<SUP>R</SUP>, suggesting that zinc depletion may stimulate the σ<SUP>R</SUP> regulatory system. This finding reflects a link between thiol-oxidizing stress and zinc depletion. We determined the Zur-binding sites within <I>znuA</I> and <I>rpmG2</I> promoter regions by footprinting analyses and identified a consensus inverted repeat sequence (TGaaAatgatTttCA, where uppercase letters represent the nucleotides common to all sites analyzed). This sequence closely matches that for mycobacterial Zur and allows the prediction of more genes in the Zur regulon.</P>

Rhizobium etli USDA9032 Engineered To Produce a Phenazine Antibiotic Inhibits the Growth of Fungal Pathogens but Is Impaired in Symbiotic Performance

Krishnan, Hari B.,Kang, Beom Ryong,Hari Krishnan, Ammulu,Kim, Kil Yong,Kim, Young Cheol American Society for Microbiology 2007 Applied and environmental microbiology Vol.73 No.1

<B>ABSTRACT</B><P>Phenazine production was engineered in <I>Rhizobium etli</I> USDA9032 by the introduction of the <I>phz</I> locus of <I>Pseudomonas chlororaphis</I> O6. Phenazine-producing <I>R. etli</I> was able to inhibit the growth of <I>Botrytis cinerea</I> and <I>Fusarium oxysporum</I> in vitro. Black bean inoculated with phenazine-producing <I>R. etli</I> produced brownish Fix<SUP>−</SUP> nodules.</P>

Development of a Biofilm Production-Deficient Escherichia coli Strain as a Host for Biotechnological Applications

Sung, Bong Hyun,Lee, Choong Hoon,Yu, Byung Jo,Lee, Jun Hyoung,Lee, Ju Young,Kim, Mi Sun,Blattner, Frederick R.,Kim, Sun Chang American Society for Microbiology 2006 Applied and environmental microbiology Vol.72 No.5

<B>ABSTRACT</B><P>Bacteria form biofilms by adhering to biotic or abiotic surfaces. This phenomenon causes several problems, including a reduction in the transport of mass and heat, an increase in resistance to antibiotics, and a shortening of the lifetimes of modules in bioindustrial fermentors. To overcome these difficulties, we created a biofilm production-deficient <I>Escherichia coli</I> strain, BD123, by deleting genes involved in curli biosynthesis and assembly, Δ(<I>csgG-csgC</I>); colanic acid biosynthesis and assembly, Δ(<I>wcaL-wza</I>); and type I pilus biosynthesis, Δ(<I>fimB-fimH</I>). <I>E. coli</I> BD123 remained mostly in the form of planktonic cells under the conditions tested and became more sensitive to the antibiotics streptomycin and rifampin than the wild-type <I>E. coli</I> MG1655: the growth of BD123 was inhibited by one-fourth of the concentrations needed to inhibit MG1655. In addition, the transformation efficiency of BD123 was about 20 times higher than that of MG1655, and the production and secretion of recombinant proteins were ∼16% and ∼25% greater, respectively, with BD123 than with MG1655. These results indicate that the newly created biofilm production-deficient strain of <I>E. coli</I> displays several key properties that substantially enhance its utility in the biotechnology arena.</P>

Glutathione Reductase and a Mitochondrial Thioredoxin Play Overlapping Roles in Maintaining Iron-Sulfur Enzymes in Fission Yeast

Song, Ji-Yoon,Cha, Joonseok,Lee, Joon,Roe, Jung-Hye AMERICAN SOCIETY OF MICROBIOLOGY 2006 EUKARYOTIC CELL Vol.5 No.11

<B>ABSTRACT</B><P>In the fission yeast <I>Schizosaccharomyces pombe</I>, the <I>pgr1</I><SUP><I>+</I></SUP> gene encoding glutathione (GSH) reductase (GR) is essentially required for cell survival. Depletion of GR caused proliferation arrest at the G1 phase of the cell cycle under aerobic conditions. Multicopy suppressors that restore growth were screened, and one effective suppressor was found to be the <I>trx2</I><SUP><I>+</I></SUP> gene, encoding a mitochondrial thioredoxin. This suggests that GR is critically required for some mitochondrial function(s). We found that GR resides in both cytosolic and organellar fractions of the cell. Depletion of GR lowered the respiration rate and the activity of oxidation-labile Fe-S enzymes such as mitochondrial aconitase and cytosolic sulfite reductase. Trx2 did not reverse the high ratio of oxidized glutathione to GSH or the low respiration rate observed in GR-depleted cells. However, it brought the activity of oxidation-labile Fe-S enzymes to a normal level, suggesting that the maintenance of Fe-S enzymes is a critical factor in the survival of <I>S. pombe</I>. The activity of succinate dehydrogenase, an oxidation-insensitive Fe-S enzyme, however, was not affected by GR depletion, suggesting that GR is not required for the biogenesis of the Fe-S cluster. The total iron content was greatly increased by GR depletion and was brought to a nearly normal level by Trx2. These results indicate that the essentiality of GR in the aerobic growth of <I>S. pombe</I> is derived from its role in maintaining oxidation-labile Fe-S enzymes and iron homeostasis.</P>

Distinct Antimicrobial Resistance Patterns and Antimicrobial Resistance-Harboring Genes According to Genomic Species of Acinetobacter Isolates

Lim, Y. M.,Shin, K. S.,Kim, J. American Society for Microbiology 2007 Journal of clinical microbiology Vol.45 No.3

The Naphthalene Catabolic (nag) Genes of Polaromonas naphthalenivorans CJ2: Evolutionary Implications for Two Gene Clusters and Novel Regulatory Control

Jeon, Che Ok,Park, Minjeong,Ro, Hyun-Su,Park, Woojun,Madsen, Eugene L. American Society for Microbiology 2006 Applied and environmental microbiology Vol.72 No.2

<B>ABSTRACT</B><P><I>Polaromonas naphthalenivorans</I> CJ2, found to be responsible for the degradation of naphthalene in situ at a coal tar waste-contaminated site (C.-O. Jeon et al., Proc. Natl. Acad. Sci. USA 100:13591-13596, 2003), is able to grow on mineral salts agar media with naphthalene as the sole carbon source. Beginning from a 484-bp <I>nagAc</I>-like region, we used a genome walking strategy to sequence genes encoding the entire naphthalene degradation pathway andadditional flanking regions. We found that the naphthalene catabolic genes in <I>P. naphthalenivorans</I> CJ2 were divided into one large and one small gene cluster, separated by an unknown distance. The large gene cluster (<I>nagRAaGHAbAcAdBFCQEDJI′ORF1tnpA</I>) is bounded by a LysR-type regulator (<I>nagR</I>). The small cluster (<I>nagR2ORF2I'KL</I>) is bounded by a MarR-type regulator (<I>nagR2</I>). The catabolic genes of <I>P. naphthalenivorans</I> CJ2 were homologous to many of those of <I>Ralstonia</I> U2, which uses the gentisate pathway to convert naphthalene to central metabolites. However, three open reading frames (<I>nagY</I>, <I>nagM</I>, and <I>nagN</I>), present in <I>Ralstonia</I> U2, were absent. Also, <I>P. naphthalenivorans</I> carries two copies of gentisate dioxygenase (<I>nagI</I>) with 77.4% DNA sequence identity to one another and 82% amino acid identity to their homologue in <I>Ralstonia</I> sp. strain U2. Investigation of the operons using reverse transcription PCR showed that each cluster was controlled independently by its respective promoter. Insertional inactivation and lacZ reporter assays showed that <I>nagR2</I> is a negative regulator and that expression of the small cluster is not induced by naphthalene, salicylate, or gentisate. Association of two putative <I>Azoarcus</I>-related transposases with the large cluster and one <I>Azoarcus</I>-related putative salicylate 5-hydroxylase gene (<I>ORF2</I>) in the small cluster suggests that mobile genetic elements were likely involved in creating the novel arrangement of catabolic and regulatory genes in <I>P. naphthalenivorans</I>.</P>