bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and Characterization of the pswP Gene Required for the Parallel Production of Prodigiosin and Serrawettin W1 in Serratia marcescens
Authors: Sunaga Shinyu , Li Hong , Sato Yohei , Nakagawa Yoji , Matsuyama Tohey
Published: November 2013

Serratia marcescens mutants defective in production of the red pigment prodigiosin and the biosurfactant serrawettin W1 in parallel were isolated by transposon mutagenesis of strain 274. Cloning of the DNA fragment required for production of these secondary metabolites with different chemical structures pointed out a novel open reading frame (ORF) named pswP. The putative product PswP (230 aa) has the distinct signature sequence consensus among members of phosphopantetheinyl transferase (PPTase) which phosphopantetheinylates peptidyl carrier protein (PCP) mostly integrated in the nonribosomal peptide synthetases (NRPSs) system. Since serrawettin W1 belongs to the cyclodepsipeptides, which are biosynthesized through the NRPSs system, and one pyrrole ring in prodigiosin has been reported as a derivative of L‐proline tethered to phosphopantetheinylated PCP, the mutation in the single gene pswP seems responsible for parallel failure in production of prodigiosin and serrawettin W1.
Microarray Analysis of Temperature‐Induced Transcriptome of Yersinia pestis
Authors: Han Yanping , Zhou Dongsheng , Pang Xin , Song Yajun , Zhang Ling , Bao Jingyue , Tong Zongzhong , Wang Jin , Guo Zhaobiao , Zhai Junhui , Du Zongmin , Wang Xiaoyi , Zhang Xiuqing , Wang Jian , Huang Peitang , Yang Ruifu
Published: November 2013

Yersinia pestis, the etiologic agent of plague, must acclimatize itself to temperature shifts between the temperature (26 C) for flea blockage and the body temperature (37 C) of warm‐blooded hosts during its life cycle. Here a whole‐genome DNA microarray was used to investigate transcriptional regulation upon the upshift of growth temperature from 26 to 37 C in a chemically defined medium. Four hundred and one genes were regulated differentially under the two temperatures. About 39% of these genes were up‐regulated at 37 C, whereas 61% were down‐regulated. Temperature‐induced changes occurred at the level of transcription of genes encoding proven or predicted virulence factors, regulators, metabolism‐associated proteins, prophages, and hypothetical proteins. Strikingly, many gene clusters displayed a co‐transcription pattern in response to temperature upshift. Our data provided a genome‐wide profile of gene transcription induced by temperature shift and should shed light on the pathogenicity and host‐microbe interaction of this deadly pathogen.
Antibodies to Human‐Related H3 Influenza A Virus in Baikal Seals (Phoca sibirica) and Ringed Seals (Phoca hispida) in Russia
Authors: Ohishi Kazue , Kishida Noriko , Ninomiya Ai , Kida Hiroshi , Takada Yoshitake , Miyazaki Nobuyuki , Boltunov Andrei N. , Maruyama Tadashi
Published: November 2013

Antibodies to influenza A virus were detected using enzyme‐linked immunosorbent assay (ELISA) in the sera from two of seven Baikal seals (Phoca sibrica) and from five of six ringed seals (Phoca hispida) in Russia. In a hemagglutination‐inhibition test using H1–H15 reference influenza A viruses, ELISA‐positive sera from one Baikal seal and four ringed seals reacted to A/Aichi/2/68 (H3N2) and A/Bangkok/1/79 (H3N2) strains. One ringed seal serum sample reacted to A/seal/Massachusetts/1/80 (H7N7). The present results suggested that human‐related H3 viruses were prevalent in Baikal seals and ringed seals inhabiting the central Russian Arctic.
Epizootiological and Epidemiological Study of Hantavirus Infection in Japan
Authors: Lokugamage Nandadeva , Kariwa Hiroaki , Lokugamage Kumari , Iwasa Masahiro A. , Hagiya Tomohiro , Yoshii Kentaro , Tachi Atsushi , Ando Shuji , Fukushima Hiroshi , Tsuchiya Kimiyuki , Iwasaki Takuya , Araki Koichi , Yoshimatsu Kumiko , Arikawa Jiro , Mizutani Tetsuya , Osawa Kazutaka , Sato Hiroshi , Takashima Ikuo
Published: November 2013

Epizootiological surveys on hantavirus infections in rodents were carried out in various areas of Japan, including the four major islands of Hokkaido, Honshu, Shikoku, and Kyushu from 2000 to 2003. A total of 1,221 rodents and insectivores were captured. Seropositive animals were found in Apodemus (A.) speciosus (5/482, 1.0%), Rattus (R.) norvegicus (4/364, 1.1%), R. rattus (3/45, 6.7%), and Clethrionomys (C.) rufocanus (7/197, 3.6%). The partial S segment was amplified from one seropositive R. rattus captured at Hakodate. The nucleotide sequence showed 96% identity with the Seoul virus (SEOV) prototype strain SR‐11. In addition, we conducted an epidemiological survey on human hantavirus infection in a high‐risk population, the personnel of the Japan Ground Self‐defense Force on Hokkaido. One out of 207 human blood samples was positive for anti‐hantavirus antibody by IFA, ELISA, and WB analysis. The result of the serotype specific ELISA indicates that this individual acquired SEOV infection. This study indicates that A. speciosus, R. norvegicus, R. rattus, and C. rufocanus carry hantaviruses as the reservoir animals in Japan. Infected R. rattus and R. norvegicus in port areas could be the sources of human SEOV infection and a threat to travelers and individuals working in seaports.
Quantum Dots Targeted to the Assigned Organelle in Living Cells
Authors: Hoshino Akiyoshi , Fujioka Kouki , Oku Taisuke , Nakamura Shun , Suga Masakazu , Yamaguchi Yukio , Suzuki Kazuo , Yasuhara Masato , Yamamoto Kenji
Published: November 2013

Fluorescent nanocrystal quantum dots (QDs) have the potential to be applied to bioimaging since QDs emit higher and far longer fluorescence than conventional organic probes. Here we show that QDs conjugated with signal peptide obey the order to transport the assigned organelle in living cells. We designed the supermolecule of luminescent QDs conjugated with nuclear‐ and mitochondria‐targeting ligands. When QDs with nuclear‐localizing signal peptides were added to the culture media, we can visualize the movements of the QDs being delivered into the nuclear compartment of the cells with 15 min incubation. In addition, mitochondrial signal peptide can also transport QDs to the mitochondria in living cells. In conclusion, these techniques have the possibility that QDs can reveal the transduction of proteins and peptides into specific subcellular compartments as a powerful tool for studying intracellular analysis in vitro and even in vivo.
Growth Competition of Macrolide‐Resistant and ‐Susceptible Helicobacter pylori Strains
Authors: Kanai Kyoko , Shibayama Keigo , Suzuki Satowa , Wachino Jun‐ichi , Arakawa Yoshichika
Published: November 2013

We examined population dynamics in a mixed culture of clonally related macrolide‐resistant and ‐susceptible Helicobacter pylori strains isolated from a single patient. The resistant strain had a macrolide resistance‐conferring A2143G mutation in the 23S rRNA gene. The growth rate of these two strains did not apparently differ when cultured separately. On the other hand, by conducting sequential passage of a mixed culture of the resistant and the susceptible strains, the ratio of the resistant strain to the susceptible strain in the culture typically decreased per passage, indicating that the resistance imposed a significant disadvantage on bacterial fitness in the population.
A Hemolysin of Vibrio mimicus (VMH) Stimulates Cells to Produce ATP and Cyclic AMP Which Appear to Be Secretory Mediators
Authors: Li Yunshan , Okamoto Keinosuke , Takahashi Eizo , Miyoshi Shin‐ichi , Shinoda Sumio , Tsuji Takao , Fujii Yoshio
Published: November 2013

The hemolysin of Vibrio mimicus (VMH) is a pore‐forming toxin with both enterotoxic and hemolytic activity. The hemolysis by VMH is induced by creation of pores in the membrane of erythrocyte; however, the mechanism for the enterotoxic action of VMH has remained unclear. In order to clarify the mechanism, we incubated T84 cells (a human colon carcinoma cell line) with VMH and found that the levels of ATP and cyclic AMP of culture medium increased after exposure of the cells to VMH. Subsequently, we found that the fluid accumulating activity of VMH in a mouse internal loop assay was reduced by administration of glibenclamide, an inhibitor of cyclic AMP‐dependent chloride channels, into the intestinal loop. These results suggest that the stimulation of cells to produce nucleotides by VMH is linked to the enterotoxic activity of the toxin.
A Calcium Binding Protein, S100A4, Mediates T Cell Dependent Cytotoxicity as a Transformation‐Associated Antigen
Authors: Kondo Nobuhiko , Ichimiya Shingo , Tamura Yasuaki , Tonooka Akiko , Koshiba Shigeru , Torigoe Toshihiko , Kamiguchi Kenjiro , Takenaga Keizo , Sato9 Noriyuki
Published: November 2013

The nature of the target molecule of TCRγδ T cell‐mediated lysis remains to be determined. As we previously reported, #067 monoclonal antibody (mAb) recognizes one of the transformation‐associated antigens, designated as #067 antigen. This antigen is expressed on the cell surface of rat fibrosarcoma W31 cells, which are established by transformation of fetal fibroblastic WFB cells with H‐ras oncogene. It has been suggested that the #067 antigen is a target molecule for TCRγδ T cells since #067 mAb inhibited TCRγδ T cell‐mediated lysis against #067 positive cells. In this study we attempted to identify the protein sequence of the #067 antigen. By using molecular cloning techniques, we demonstrated that a calcium binding protein, S100A4, was possibly one and the same molecule as the #067 antigen. It was shown that the expression of S100A4 was higher in W31 cells than in WFB cells at transcription and protein level. Flow cytometry and immunocytochemical studies showed that #067 antigen partially co‐localized with S100A4 on the cell surface as well as the cytoplasm of W31 cells. Moreover, rabbit anti‐S100A4 polyclonal antibodies (pAb) inhibited TCRγδ T cell‐mediated lysis against #067 positive cells. Our results indicated that S100A4 may play a role as a possible target molecule for TCRγδ T cell‐mediated lysis although how S100A4 is involved in TCRγδ T cell‐mediated lysis remains to be determined.
Differentiation of Anaplasmataceae through Partial groEL Gene Analysis
Authors: Park Hyo‐Soon , Lee Jung‐Hee , Jeong Eun‐Ju , Park Tae‐Kyu , Kim Tae‐Yung , Chae Joon‐Seok , Park Jin‐Ho , Klein Terry A. , Jang Won‐Jong , Park Kyung‐Hee , Lee Seung‐Hyun
Published: November 2013

The nucleotide sequences (287 bp) of the partial groEL gene from 14 reference strains of Anaplasmataceae were determined and compared. A partial groEL gene is useful for the identification and characterization of Anaplasmataceae, in spite of its short nucleotide sequences.
Intermedilysin Is Essential for the Invasion of Hepatoma HepG2 Cells by Streptococcus intermedius
Authors: Sukeno Akiko , Nagamune Hideaki , Whiley Robert A. , Jafar Syed I. , Aduse‐Opoku Joseph , Ohkura Kazuto , Maeda Takuya , Hirota Katsuhiko , Miyake Yoichiro , Kourai Hiroki
Published: November 2013

Streptococcus intermedius causes endogenous infections leading to abscesses. This species produces intermedilysin (ILY), a human‐specific cytolysin. Because of the significant correlation between higher ILY production levels by S. intermedius and deep‐seated abscesses, we constructed ily knockout mutant UNS38 B3 and complementation strain UNS38 B3R1 in order to investigate the role of ILY in deep‐seated infections. Strain UNS38 reduced the viability of human liver cell line HepG2 at infection but not of rat liver cell line BRL3A. Isogenic mutant strain UNS38 B3 was not cytotoxic in either cell line. Quantification of S. intermedius revealed that in infected HepG2 cells UNS38 but not UNS38 B3 increased intracellularly concomitantly with increasing cell damage. This difference between UNS38 and UNS38 B3 was not observed with UNS38 B3R1. Invasion and proliferation in BRL3A cells was not observed. Masking UNS38 or UNS38 B3R1 with ILY antibody drastically decreased adherence and invasion of HepG2. Moreover, coating strain UNS38 B3 with ILY partially restored adherence to HepG2 but without subsequent bacterial growth. At 1 day post‐infection, many intact UNS38 were detected in the damaged phagosomes of HepG2 with bacterial proliferation observed in the cytoplasm of dead HepG2 after an additional 2 day incubation. These results indicate that surface‐bound ILY on S. intermedius is an important factor for invasion of human cells by this bacterium and that secretion of ILY within host cells is essential for subsequent host cell death. These data strongly implicate ILY as an important factor in the pathogenesis of abscesses in vivo by this streptococcus.
Establishment of Highly Specific and Quantitative Immunoassay Systems for Staphylococcal Enterotoxin A, B, and C Using Newly‐Developed Monoclonal Antibodies
Authors: Sasaki Takanori , Terano Yoshitake , Shibata Tadayoshi , Kawamoto Hiroyoshi , Kuzuguchi Tsuyoshi , Kohyama Erina , Watanabe Toshihiro , Ohyama Tohru , Gemba Munekazu
Published: November 2013

Staphylococcal enterotoxin (SE) activities remain after boiling or treating with proteases. The main symptoms such as vomiting and diarrhea, are caused by the ingestion of SEs. Among SEs, SEA has been reported to be the major and most toxic protein. A highly specific and simple assay system is required to diagnose staphylococcal food poisoning. Therefore, the development of a suitable assay system is strongly anticipated. In this study, we have established a highly specific and sensitive avidin‐biotin sandwich ELISA (ABS‐ELISA) system for SEA, SEB, and SEC1 using newly‐developed monoclonal antibodies. The linearity of these systems obtained was in the range of 0.78–25 ng/ml for each SE, and furthermore, the lower concentrations of SEs could also be detected. The recoveries of SEs from murine serum, skim milk solution, and raw milk were found to be over 90%, suggesting that our systems could detect SEs without any interventions, such as these from milk or serum proteins. We were also able to quantify SEs in 22 specimens of culture supernatants of S. aureus isolated in past occurrences. Our established system should be very useful not only in the clinical field but also in various fields of investigation because of its quantification and simplicity in detecting SEs.
Promoter Analysis of Cytolethal Distending Toxin Genes (cdtA, B, and C) and Effect of a luxS Mutation on CDT Production in Campylobacter jejuni
Authors: Jeon Byeonghwa , Itoh Kikuji , Ryu Sangryeol
Published: November 2013

Quorum sensing is known to be related to regulation of virulence factors in some pathogens. We report transcriptional analysis of cdt genes (cdtA, B, and C) encoding cytolethal distending toxin (CDT) in Campylobacter jejuni and demonstrate that a luxS mutation affects the cdt transcription in C. jejuni. The reverse transcriptase‐polymerase chain reaction (RT‐PCR) showed that cdtA, cdtB, and cdtC genes constitute a polycistronic operon in C. jejuni. A putative transcriptional start site was found at 81‐bp upstream from the start codon of cdtA by the primer extension assay. A decrease in cdt transcription was observed in the luxS null mutant, which is defective in AI‐2 production. The level of cdt transcription in the luxS mutant was approximately 61% of that in the wild‐type. Flow cytometric analysis showed that the number of HeLa cells arrested in the G2/M phase, a common biological feature of CDT activity, was reduced by treatment with the cell‐free supernatant of the luxS mutant as compared to that of the wild‐type. Our observations suggest that the function of luxS is related to the regulation of cdt in C. jejuni
Involvement of Reactive Oxygen Intermediate in the Enhanced Expression of Virulence‐Associated Genes of Listeria monocytogenes inside Activated Macrophages
Authors: Makino Masato , Kawai Mariko , Kawamura Ikuo , Fujita Masashi , Gejo Fumitake , Mitsuyama Masao
Published: November 2013

Listeriolysin O encoded by 1,587 bp hly is the essential virulence factor of Listeria monocytogenes that replicates in the cytosolic space after escaping from phagosome of macrophages. By using murine macrophage‐like J774.1 cells with or without activation by IFN‐γ plus LPS, the expression of both hly and its positive regulator prfA was monitored by means of RT‐PCR. In activated J774.1 cells, the level of hly expression was enhanced although the multiplication of bacteria was significantly suppressed. The elevated expression of hly inside activated macrophage was abolished by addition of SOD and catalase, suggesting that reactive oxygen intermediates contribute to the upregulation of prfA and hly transcriptions. Moreover, we found that exposure of L. monocytogenes to H2O2 dramatically enhanced the expression of both prfA and hly mRNAs. Spontaneous ONOO– generator, SIN‐1, also promoted the transcription to a certain level. These results suggested that oxygen radicals generated in activated macrophages provide a positive signal for up‐regulation of virulence genes in L. monocytogenes.
Physiological Roles of Three Na+/H+ Antiporters in the Halophilic Bacterium Vibrio parahaemolyticus
Authors: Kuroda Teruo , Mizushima Tohru , Tsuchiya Tomofusa
Published: November 2013

Vibrio parahaemolyticus mutants lacking three Na+/H+ antiporters (NhaA, NhaB, NhaD) were constructed. The AnhaA strains showed significantly higher sensitivity to LiCl regarding their growth compared to the parental strain. The AnhaA and AnhaB strains exhibited higher sensitivities to LiCl. The mutant XACabd lacking all of the three antiporters could not grow in the presence of 500 mM LiCl at pH 7.0, or 50 mM at pH 8.5. The XACabd mutant was also sensitive to 1.0 M NaCl at pH 8.5. These results suggest that Na+/H+ antiporters, especially NhaA, are responsible for resistance to LiCl and to high concentrations of NaCl. Reduced Na+/H+ and Li+/H+ antiport activities were observed with everted membrane vesicles of AnhaB strains. However, Li+/H+ antiport activities of AnhaB strains were two times higher than those of AnhaA strains when cells were cultured at pH 8.5. It seems that expression of nhaA and nhaB is dependent on medium pH to some extent. In addition, HQNO (2‐heptyl‐4‐hydroxyquinoline N‐oxide), which is a potent inhibitor of the respiratory Na+ pump, inhibited growth of XACabd, but not of the wild type strain. Moreover, survival rate of XACabd under hypoosmotic stress was lower than that of wild type strain. It is likely that the Na+/H+ antiporters are involved in osmoregulation under hypoosmotic stress. Based on these findings, we propose that the Na+/H+ antiporters cooperate with the respiratory Na+ pump in ionic homeostasis in V. parahaemolyticus
Proportion of Sporadic Gastroenteritis Cases Caused by Rotavirus, Norovirus, Adenovirus and Bacteria in Japan from January 2000 to December 2003
Authors: Sumi Ayako , Kobayashi Nobumichi , Ohtomo Norio
Published: November 2013

Many kinds of virus and bacterium have been identified as pathogens that cause sporadic gastroenteritis (SG). Among the pathogens, rotavirus (RV), norovirus (NV; previously known as Norwalk‐like virus), and adenovirus (AdV) types 40/41 have been considered as the prevalent viruses implicated in the aetiology of childhood gastroenteritis. In the present study, we attempted to estimate monthly proportions of SG cases caused by the viral agents (RV, NV, AdV and other viruses (OV)) and bacterial agents in the whole of Japan. The estimation was carried out by using time series data of the incidence of SG and the viral and bacterial agents of SG, which were collected by a nationwide surveillance system in Japan from January 2000 to December 2003. It was confirmed that, in the dominant period of RV and NV, the proportion of RV‐associated SG and that of NV‐associated SG indicate almost same level with each other: 46–69% during February–May for RV, and 41–75% during October–December for NV.
Tepidimonas arfidensis Sp. Nov., a Novel Gram‐Negative and Thermophilic Bacterium Isolated from the Bone Marrow of a Patient with Leukemia in Korea
Authors: Ko Kwan Soo , Lee Nam Yong , Oh Won Sup , Lee Jang Ho , Ki Hyun Kyun , Peck Kyong Ran , Song Jae‐Hoon
Published: November 2013

A Gram‐negative bacillus, SMC‐6271T, which was isolated from the bone marrow of a patient with leukemia but could not be identified by a conventional microbiologic method, was characterized by a genotypic analysis of 16S rRNA gene. Sequences of the 16S rRNA gene revealed that this bacterium was closely related to Tepidimonas ignava and other slightly thermophilic isolates but diverged distinctly from them. Analyses of cellular fatty acid composition and performance of biochemical tests confirmed that this bacterium is a distinct species from the other Tepidimonas species. Based on the evaluated phenotypic and genotypic characteristics, it is proposed that SMC‐6271T (=ABB 0301T =KCTC 12412T =JCM 13232T) should be classified as a new species, namely Tepidimonas arfidensis sp. nov.
A Novel Aerobactin Utilization Cluster in Vibrio vulnificus with a Gene Involved in the Transcription Regulation of the iutA Homologue
Authors: Tanabe Tomotaka , Naka Ayaka , Aso Hiroaki , Nakao Hiroshi , Narimatsu Shizuo , Inoue Yuji , Ono Tomomichi , Yamamoto Shigeo
Published: November 2013

We demonstrated that Vibrio vulnificus M2799 utilizes aerobactin for growth as an exogenous siderophore under iron‐limiting conditions, concomitant with enhanced production of the 76‐kDa iron‐repressible outer membrane protein. Subsequently, by applying the Fur titration assay method to the M2799 genomic libraries followed by further cloning of the regions surrounding the candidate genes, we identified the 8.4‐kb aerobactin utilization gene cluster which consists of five genes arranged in three distinct transcriptional units. It was confirmed by disruption of the corresponding genes that the first unit forming a three‐gene operon (vatCDB) and the third unit of a single gene (iutA) encode an ATP‐binding cassette transport component and the 76‐kDa ferric aerobactin receptor, respectively. The second unit of another single gene (iutR), encodes a homologue of the GntR family of transcriptional repressors. Although transcription of the first and third units was iron‐regulated, the iutR gene was transcribed regardless of iron status in the growth medium. Construction of an iutR disruptant coupled with genetic complementation experiments suggested that the gene encodes a transcriptional repressor for iutA. This is the first example of a regulator gene involved in aerobactin‐enhanced production of IutA.
Cell‐Specific Inhibition of Paramyxovirus Maturation by Proteasome Inhibitors
Authors: Watanabe Hitoshi , Tanaka Yoshikazu , Shimazu Yukie , Sugahara Fumihiro , Kuwayama Masaru , Hiramatsu Akira , Kiyotani Katsuhiro , Yoshida Tetsuya , Sakaguchi Takemasa
Published: November 2013

Effects of proteasome inhibitors on the replication of a paramyxovirus in comparison with the effects on replication of an orthomyxovirus and rhabdovirus were investigated. Treatment of Sendai virus (SeV)‐infected LLC‐MK2 cells with 50 μM MG132 reduced virus growth to ca. 1/10,000, and treatment with different concentrations of MG132 reduced virus growth in a dose‐dependent manner. Released amounts of viral proteins were reduced in correspondence with decrease in infectivity. The inhibition of virus maturation was confirmed by an SeV‐like particle formation system. Lactacystin also impaired SeV growth and zLL impaired the growth to a lesser extent, suggesting involvement of proteasomes in the restriction of virus growth. In the presence of MG132, localizations of the M protein and viral F and HN glycoproteins on the cell membrane appeared to be partly dissociated, although the viral glycoproteins were normally transported to the cell surface. These results suggest that an early step of SeV assembly was disturbed by proteasome inhibitors. The relationship of the results with ubiquitin is also discussed. SeV maturation was less susceptible and resistant to MG132 in CV1 cells and A549 cells, respectively, indicating cell specificity of the drug effect. Release of vesicular stomatitis virus also showed high susceptibility to MG132 and release of influenza virus A/WSN/33 was only mildly susceptible to the drug in LLC‐MK2 cells. Effects of proteasome inhibitors on virus maturation are thus highly cell‐specific and partly virus‐specific.
Survival of Virulent Mycobacterium tuberculosis Involves Preventing Apoptosis Induced by Bcl‐2 Upregulation and Release Resulting from Necrosis in J774 Macrophages
Authors: Zhang Jianling , Jiang Ru , Takayama Hisao , Tanaka Yoshinori
Published: November 2013

Macrophage apoptosis plays a role in mycobacterial infection. To define the mechanism by which virulent Mycobacterium tuberculosis escapes apoptosis and killing in macrophages, J774 macrophages were infected with virulent M. tuberculosis H37Rv and attenuated H37Ra strains. H37Rv induced less apoptosis than H37Ra, and caspase 3 was activated in H37Ra‐ and H37Rv‐infected macrophages. Intracellular H37Rv bacilli were released at a higher rate into the supernatant than were H37Ra by the sixth day of infection, and this was simultaneously accompanied by the increased necrosis of infected cells showing lactate dehydrogenase (LDH) release. Fas mRNA expression was downregulated and FasL was upregulated in H37Ra‐ and H37Rv‐infected macrophages, while Bcl‐2 was upregulated in H37Rv‐infected macrophages but downregulated in H37Ra‐infected macrophages as seen by real‐time PCR. These results indicate that M. tuberculosis H37Ra and H37Rv proliferate in macrophages by preventing them from inducing apoptosis during the early phase of infection, and that M. tuberculosis H37Rv‐infected macrophages are found to express Bcl‐2 mRNA, which leads to anti‐apoptotic activity, and that relatively distinct necrosis might occur during the later phase of infection.
Curli Fibers Are Required for Development of Biofilm Architecture in Escherichia coli K‐12 and Enhance Bacterial Adherence to Human Uroepithelial Cells
Authors: Kikuchi Tatsuya , Mizunoe Yoshimitsu , Takade Akemi , Naito Seiji , Yoshida Shin‐ichi
Published: November 2013

Sessile bacteria show phenotypical, biochemical, and morphological differences from their planktonic counterparts. Curli, extracellular structures important for biofilm formation, are only produced at temperatures below 30 C in Escherichia coli K‐12 strains. In this report, we show that E. coli K‐12 can produce curli at 37 C when grown as a biofilm community. The curli‐expressing strain formed more biofilms on polyurethane sheets than the curli‐deficient strain under growth temperatures of both 25 C and 37 C. Curli are required for the formation of a three‐dimensional mature biofilm, with characteristic water channels and pillars of bacteria. Observations by electron microscopy revealed the presence at the surfaces of the curli‐deficient mutant in biofilm of flagella and type I pili. A wild‐type curli‐expressing E. coli strain significantly adhered to several lines of human uroepithelial cells, more so than an isogenic curli‐deficient strain. The finding that curli are expressed at 37 C in biofilm and enhance bacterial adherence to mammalian host cells suggests an important role for curli in pathogenesis.
Immune Response to the Rhodococcus equi Infection in High and Low Antibody‐Producing Mice (Selection IV‐A)
Authors: Pedrini Sílvia C.B. , Acorci Michele J. , Pinto João G.G. , Silveira Liciana V.A. , Oliveira Silvio L.
Published: November 2013

Rhodococcus equi is a Gram‐positive, facultative intracellular bacterium which infects macrophages and causes rhodococcal pneumonia and enteritis in foals. Recently, this agent has been recognized as an opportunistic pathogen for immunocompromised humans. Several murine experimental models have been used to study R. equi infection. High (HIV‐A) and Low (LIV‐A) antibody (Ab)‐producers mice were obtained by bi‐directional genetic selections for their ability to produce antibodies against sheep and human erythrocytes (Selection IV‐A). These lines maintain their phenotypes of high and low responders also for other antigens than those of selection (multispecific effect). A higher macrophage activity in LIV‐A mice has been described for several intracellular infectious agents, which could be responsible for their intense macrophage antigens (Ag)‐handling and low Ab production. Due to these differences, LIV‐A mice were found to exhibit a better performance to trigger an effective immune response towards intracellular pathogens. The objective of this work was to characterize the immune response of Selection IV‐A against R. equi. HIV‐A and LIV‐A mice were infected with 2.0 ×106 CFU of ATCC 33701+R. equi by intravenous route. With regards to bacterial clearance and survival assays, LIV‐A mice were more resistant than HIV‐A mice to virulent R. equi. LIV‐A mice presented a higher hydrogen peroxide (H2O2) and nitric oxide (NO) endogenous production by splenic macrophages than HIV‐A mice. LIV‐A expressed the most intense cellular response, available by the Delayed‐Type Hypersensitivity (DTH) reaction, which activated macrophages and produced more H2O2 and NO. The three times higher specific antibodies titres in HIV‐A indicated that Selection IV‐A maintained the multispecific effect and the polygenic control of humoral and cellular responses also to R. equi.
Mutation in IRO1 Tightly Linked with URA3 Gene Reduces Virulence of Candida albicans
Authors: Chibana Hiroji , Uno Jun , Cho Tamaki , Mikami Yuzuru
Published: November 2013

Gene deletion in the pathogenic fungus Candida albicans has relied heavily on a URA3 cassette and a recipient Aura3 strain CAI4. The IRO1 gene adjacent to URA3 was inadvertently deleted during construction of CAI4. We report here that a mutation in IRO1 reduces virulence of C. albicans.
stx2vha Is the Dominant Genotype of Shiga Toxin‐Producing Escherichia coli O157:H7 Isolated from Patients and Domestic Animals in Three Regions of China
Authors: Zheng Han , Jing Huaiqi , Wang Hua , Xia Shengli , Hu Wangfu , Cui Shuyu , Bi Zhenqiang , Yang Jinchuan , Pang Bo , Zhao Guangfa , Zhang Jin , Li Hongwei , Xu Jianguo
Published: November 2013

Shiga toxin‐producing Escherichia coli (STEC) O157:H7 strains were isolated from domestic animals and patients from Xuzhou City, Jiangsu Province, China and the bordering Anhui and Henan Provinces and were examined for the stx genotype. Of 390 strains, 277 were identified as genotype stx2vha; 41, stx2; 51, stx2‐stx1; 1, stx2‐stx2vha‐stx1; 5, stx2‐stx2vha; and 15 were un‐typeable. Of the 277 stx2vha‐bearing isolates, 116 were isolated from goats; 42, cattle; 38, hens, and 35 from pigs. The study shows stx2vha is the dominant genotype and goats are an important reservoir.
Evaluation of the Biofilm‐Forming Ability and Genetic Typing for Clinical Isolates of Pseudomonas aeruginosa by Enterobacterial Repetitive Intergenic Consensus‐Based PCR
Authors: Yang Weiqing , Shi Lei , Jia Wen‐xiang , Yin Xiaolin , Su Jian‐yu , Kou Yali , Yi Xie , Shinoda Sumio , Miyoshi Shin‐ichi
Published: November 2013

Biofilm formation is an important phenotype associated with chronic Pseudomonas aeruginosa infections. In the present study, a total of 48 P. aeruginosa strains isolated from clinical specimens were examined for their biofilm‐forming ability using a microtiter plate method. The different biofilm‐forming abilities were demonstrated among the strains; however, most strains formed a larger biofilm than strain PAO1, a reference strain. The genetic typing was also carried out by enterobacterial repetitive intergenic consensus‐based polymerase chain reaction. Although they were divided into five groups (A to E), most of the strains showing the higher biofilm‐forming ability were found to be in groups D and E, suggesting a significant relationship between the biofilm‐forming ability and the genetic group.
Protective Efficacy of Nonpathogenic Nef‐Deleted SHIV Vaccination Combined with Recombinant IFN‐γ Administration against a Pathogenic SHIV Challenge in Rhesus Monkeys
Authors: Kaneyasu Kentaro , Kita Masakazu , Ohkura Sadayuki , Yamamoto Toshiro , Ibuki Kentaro , Enose Yoshimi , Sato Akihiko , Kodama Makoto , Miura Tomoyuki , Hayami Masanori
Published: November 2013

We previously reported that a nef‐deleted SHIV (SHIV‐NI) is nonpathogenic and gave macaques protection from challenge infection with pathogenic SHIV‐C2/1. To investigate whether IFN‐γ augments the immune response induced by this vaccination, we examined the antiviral and adjuvant effect of recombinant human IFN‐γ (rIFN‐γ) in vaccinated and unvaccinated monkeys. Nine monkeys were vaccinated with nef‐deleted nonpathogenic SHIV‐NI. Four of them were administered with rIFN‐γ and the other five monkeys were administered with placebo. After the challenge with pathogenic SHIV‐C2/1, CD4+ T‐cell counts were maintained similarly in monkeys of both groups, while those of the unvaccinated monkeys decreased dramatically at 2 weeks after challenge. However, the peaks of plasma viral load were reduced to 100‐fold in SHIV‐NI vaccinated monkeys combined with rIFN‐γ compared with those in SHIV‐NI vaccinated monkeys without rIFN‐γ. The peaks of plasma viral load were inversely correlated with the number of SIV Gag‐specific IFN‐γ‐producing cells. In SHIV‐NI‐vaccinated monkeys with rIFN‐γ, the number of SIV Gag‐specific IFN‐γ‐producing cells of PBMCs increased 2‐fold compared with those in SHIV‐NI‐vaccinated monkeys without rIFN‐γ, and the NK activity and MIP‐1α production of PBMCs were also enhanced. Thus, vaccination of SHIV‐NI in combination with rIFN‐γ was more effective in modulating the antiviral immune system into a Th1 type response than SHIV‐NI vaccination alone. These results suggest that IFN‐γ augmented the anti‐viral effect by enhancing innate immunity and shifting the immune response to Th1.
Multicenter Evaluation of Commercial Frozen Plates for Microdilution Broth Antifungal Susceptibility Testing of Yeasts and Comparison of MIC Limits Recommended in NCCLS M27–A2
Authors: Makimura Koichi , Oguri Toyoko , Mikami Yuzuru , Kume Hikaru , Hanazawa Ryo , Abe Michiko , Ikeda Reiko , Shinoda Takako
Published: November 2013

A commercial kit, Frozen Plate for Antifungal Susceptibility Testing of Yeasts, Eiken (Eiken Chemical Co., Ltd., Tokyo), was tested in a multi‐institute study to evaluate the agreement between inter‐institute MICs and National Committee for Clinical Laboratory Standards (NCCLS) M27–A2 recommendation limits of MIC value. The kit was reported as a method equivalent to the standardized guidelines for antifungal susceptibility testing by the Committee for Clinical Laboratory Standards—1994, the Japanese Society for Medical Mycology, and which is widely used in Japan for amphotericin B, flucytosine, fluconazole, miconazole, and itraconazole. The degrees of inter‐institute and NCCLS agreements were good to excellent especially with 48‐hr incubation for all antifungal agents. However, the percent agreements to NCCLS recommendations against itraconazole were poor. Overall, MIC values obtained using the frozen plate antifungal susceptibility testing kit, with 48‐hr incubation, were thought to be reliable and convenient alternatives to the data obtained by the NCCLS M27–A2 reference macrodilution and microdilution method. This kit will allow matching of results between international laboratories. However, the MIC value for itraconazole requires careful interpretation.
Nasal Peptide Vaccination Elicits CD8 Responses and Reduces Viral Burden after Challenge with Virulent Murine Cytomegalovirus
Authors: Gopal Indulekha N. , Quinn Anita , Henry Stanley C. , Hamilton John D. , Staats Herman F. , Frothingham Richard
Published: November 2013

Infection of BALB/c mice with murine cytomegalovirus (MCMV) leads to CD8 cell responses to an immunodominant epitope YPHFMPTNL. We presented this epitope as a nasal peptide vaccine in combination with cholera toxin adjuvant, and evaluated immune responses and protection from MCMV challenge. Vaccination of naïve mice generated elevated numbers of peptide‐specific interferon‐7‐secreting splenocytes (median 80/million, range 60 to 490), compared to control mice (median 2/million, range —4.5 to 8; P=0.008, Mann‐Whitney test). Twelve days after challenge with virulent MCMV, vaccinated mice had a 1.1 log10 reduction in salivary gland viral titer compared to unvaccinated controls (5.36±0.24 vs. 6.42±0.12, mean±SD log10 plaque‐forming‐units; P<0.001, t‐test). Mice with chronic MCMV infection had consistent responses to the peptide (183±24/million interferon‐γ‐secreting splenocytes). Nasal peptide vaccination during chronic infection boosted peptide‐specific responses in two of four mice to >900/million interferon‐γ‐secreting splenocytes. Nasal peptide vaccination was immunogenic in naïve and MCMV‐infected mice, and reduced viral burden in naïve mice after virulent MCMV challenge. The nasal route may be useful for peptide presentation by novel human vaccines.
Interleukin‐4 Up‐Regulates T‐Tropic Human Immunodeficiency Virus Type 1 Transcription in Primary CD4+ CD38+ T‐Lymphocyte Subset
Authors: Li Yong‐Gang , Iwabu Yukie , Warachit Jiranan , Kinomoto Masanobu , Ibrahim Madiha S. , Tsuji Shoutaro , Mukai Tetsu , Kameoka Masanori , Tokunaga Kenzo , Sata Tetsutaro , Ikuta Kazuyoshi
Published: November 2013

The capacity of human immunodeficiency virus type 1 (HIV‐1) to infect resting cells and to produce progeny particles may contribute significantly to its pathogenicity in vivo. We previously reported that primary culture of resting CD4+ CD38+ T‐lymphocyte subset had higher production rate of CXCR4‐using (X4) HIV‐1 than CD4+ CD38− subset. Interleukin (IL)‐4 highly contributed to the up‐regulation of the X4 virus production in the CD38+ subset. Here, we show evidences that IL‐4 treatment of both resting CD38+ and CD38− subsets allowed the adsorption, entry, and integration of X4 virus at similar rates, while the following viral transcription rate was significantly lower in the CD38− than CD38+ subset. Treatment of the CD38 subsets with IL‐4 or phytohemagglutinin revealed no association of X4 virus replication ability in the subsets with classic T‐cell activation or proliferation. Interestingly, the activator protein (AP)‐1 was significantly activated in the CD38+ subset after IL‐4 treatment, while both nuclear factor (NF)‐κB and signal transducers and activator of transcription (STAT)‐6 were activated in the IL‐4‐treated CD38+ and CD38− subsets at similar levels. Thus, IL‐4‐dependent X4 HIV‐1 transcription occurs efficiently in the CD38+ but not CD38− subset of CD4+ population and AP‐1 could play a significant role on viral transcription, leading to the up‐regulated X4 virus production in the CD38+ subset.
Experimental Candidosis and Recovery of Candida albicans from the Oral Cavity of Ovariectomized Rats
Authors: Junqueira Juliana Campos , Colombo Carlos Eduardo Dias , Martins Joyce da Silva , Ito Cristiane Yumi Koga , Carvalho Yasmin Rodarte , Jorge Antonio Olavo Cardoso
Published: November 2013

The aim of this study was to analyze the development of candidosis and the recovery of C. albicans from the oral cavity of ovariectomized and sham‐ovariectomized rats. One hundred and twenty‐four rats originally negative for Candida spp. in the oral cavity were divided into two groups: ovariectomized and sham‐ovariectomized. Fifty‐eight ovariectomized and the same quantity of sham‐ovariectomized rats were inoculated with C. albicans for the study of candidosis development and recovery of yeast. Four animals from each group were not inoculated with yeast suspension and were submitted to tongue dorsum morphologic analysis by optical and scanning electron microscopy. The development of candidosis in the tongue dorsum was observed by optical and scanning electron microscopy in the periods of 6 hr, 24 hr, 7 days and 15 days after the last inoculation. Recovery of C. albicans was performed by oral samples plating on Sabouraud agar after 1, 2, 5 and 7 days and progressively at each 15‐day interval until negative cultures for yeasts were obtained. The results were analyzed by Mann‐Whitney and Student's t tests. The tongue dorsum of sham‐ovariectomized and ovariectomized rats, not infected by Candida, presented normal aspect. Among the infected rats, the ovariectomized group showed less occurrence of candidosis lesions and lower recovery of C. albicans from the oral cavity in relation to the sham‐ovariectomized group. It could be concluded that candidosis was less frequent from the oral cavities of ovariectomized rats in relation to sham‐ovariectomized.
Remarkable Synergies between Baicalein and Tetracycline, and Baicalein and β‐Lactams against Methicillin‐Resistant Staphylococcus aureus
Authors: Fujita Mai , Shiota Sumiko , Kuroda Teruo , Hatano Tsutomu , Yoshida Takashi , Mizushima Tohru , Tsuchiya Tomofusa
Published: November 2013

During the screening of compounds that potentiate the effect of antimicrobial agents against methicillin‐resistant Staphylococcus aureus (MRSA), we found that an extract of thyme (Thymus vulgaris L) leaves greatly reduced the minimum inhibitory concentration (MIC) of tetracycline against MRSA. We isolated the effective compound and identified it as baicalein (5, 6, 7‐trihydroxyflavone). One of the clinically isolated MRSA strains possessed tetK, a gene encoding active efflux pump for tetracycline. We examined the effect of baicalein on the efflux of tetracycline, using Escherichia coli KAM32/pTZ1252 carrying the tetK. The E. coli KAM32/pTZ1252 showed 8 to 16 times higher MIC than E. coli KAM32. We observed strong inhibition of transport of tetracycline by baicalein with membrane vesicles prepared from E. coli KAM32/pTZ1252. Baicalein also showed synergy with tetracycline in a MRSA strain that doesn't possess tetK, or with β‐lactams. Thus, mechanisms of the synergies seem to be versatile.
Serratia marcescens Gene Required for Surfactant Serrawettin W1 Production Encodes Putative Aminolipid Synthetase Belonging to Nonribosomal Peptide Synthetase Family
Authors: Li Hong , Tanikawa Taichiro , Sato Yohei , Nakagawa Yoji , Matsuyama Tohey
Published: November 2013

Serrawettin W1 produced by Serratia marcescens is a surface active exolipid having various functions supporting behaviors of bacteria on surface environments. Through the genetic analyses of serrawettin‐less mutants of S. marcescens 274, the swrW gene encoding putative serrawettin W1 synthetase was identified. Homology analysis of the putative SwrW demonstrated the presence of condensation, adenylation, thiolation, and thioesterase domains which are characteristic for nonribosomal peptide synthetase (NRPS). NRPSs have been known as multi‐modular enzymes. Linear alignment of these modules specifying respective amino acids will enable peptide bond formation resulting in a specific amino acid sequence. Putative SwrW was uni‐modular NRPS specifying only L‐serine. Possible steps in this simple uni‐modular NRPS for biosynthesis of serrawettin W1 [cyclo‐(D‐3‐hydroxydecanoyl‐L‐seryl)2] were predicted by referring to the ingenious enzymatic activity of gramicidin S synthetase (multi‐modular NRPS) of Brevibacillus brevis.
Construction of Human Fab Library and Isolation of Monoclonal Fabs with Rabies Virus‐Neutralizing Ability
Authors: Ando Tadasuke , Yamashiro Tetsu , Takita‐Sonoda Yoshiko , Mannen Kazuaki , Nishizono Akira
Published: November 2013

A combinatorial human Fab library was constructed using RNAs from peripheral blood lymphocytes of 6 rabies vaccine‐boosted volunteers using pComb3X phagemid vector. The size of the constructed library was approximately 7.0×107 Escherichia coli transformants. The library was selected against purified rabies virus (RV) virion or purified RV glycoprotein for isolation of phages displaying RV‐neutralizing human Fab antibody. Among 132 selected clones, two Fab preparations revealed neutralizing activities against RV strain CVS when assayed in the rapid fluorescent focus inhibition test (RFFIT). The Fab preparation EP5G3 exhibited neutralizing activity with an infected cell count reduction of 76% at a dilution of 1:2, and of 20% at a dilution of 1:4. The Fab preparation GD2D12 also exhibited neutralizing activity with a 57% reduction at 1:2 and 41% reduction at 1:4. In the co‐immunoprecipitation using strain CVS, the RV glycoprotein was precipitated in reactions with both Fab preparations. The RV neutralizing ability of the Fab preparations described in the study were not directly correlated with their binding specificity for RV antigens detected by ELISA.
Experimental Abscess Formation Caused by Human Dental Plaque
Authors: Okayama Hidehito , Nagata Emi , Ito Hiro‐O , Oho Takahiko , Inoue Masakazu
Published: November 2013

Human dental plaque consists of a wide variety of microorganisms, some of which are believed to cause systemic infections, including abscesses, at various sites in the body. To confirm this hypothesis experimentally, we examined the abscess‐forming ability of native dental plaque in mice, the microbial features of the infectious locus produced by the plaque, and the anti‐phagocytic property of microbial isolates. Aliquots of a suspension of supragingival dental plaque containing 6 × 106 colony‐forming unit of bacteria were injected subcutaneously into the dorsa of mice. Abscess formation was induced in 76 of 85 mice using ten different plaque samples. Thirteen microorganisms were isolated from pus samples aspirated from abscess lesions. The microbial composition of pus, examined in 17 of 76 abscesses, was very simple compared to that of the plaque sample that had induced the abscess. The majority of the isolates belonged to the Streptococcus anginosus group, normally a minor component of plaque samples. S. anginosus was the most frequently detected organism and the most prevalent in seven abscesses, and Streptococcus intermedius and Streptococcus constellatus were predominant in one and three abscess samples, respectively. Each isolate of S. anginosus group produced abscesses in mice, and heat‐treated supragingival dental plaque influenced the abscess‐forming ability of S. anginosus isolate. These isolates possessed a high anti‐phagocytic capacity against human polymorphonuclear leukocytes. Our results suggest that human supragingival dental plaque itself is a source of the infectious pathogens that cause abscess formation.
Development of a Monoclonal Antibody against Epstein‐Barr Virus Nuclear Antigen Leader Protein (EBNA‐LP) That Can Detect EBNA‐LP Expressed in P3HR1 Cells
Authors: Shaku Fumio , Matsuda Go , Furuya Rie , Kamagata Chinatsu , Igarashi Mie , Tanaka Michiko , Kanamori Mikiko , Nishiyama Yukihiro , Yamamoto Naoki , Kawaguchi Yasushi
Published: November 2013

A mouse monoclonal antibody, LP4D3, was raised against purified Epstein‐Barr virus nuclear antigen leader protein (EBNA‐LP) fused to glutathione‐S‐transferase. The antibody detected endogenous and exogenous EBNA‐LP in immunoblotting, immunofluorescence and immunoprecipitation assays, and the epitope of the antibody was mapped in the W2 domain of EBNA‐LP. While another monoclonal antibody to EBNA‐LP, JF186, which is widely used for analyses of the viral protein, did not react with truncated forms of EBNA‐LP expressed in P3HR1 cells, as reported earlier, the LP4D3 antibody did. The LP4D3 antibody will be a useful tool for further studies of EBNA‐LP, especially investigations into the phenotypes of mutant EBNA‐LP expressed in P3HR1 cells.