bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Immunodeficiency Virus Vpx Is Required for the Early Phase of Replication in Peripheral Blood Mononuclear Cells
Authors: Kawamura Meiko , Sakai Hiroyuki , Adachi Akio
Published: November 2013

Functional importance of Vpx protein of human immunodeficiency virus type 2 was evaluated in various types of cells. In 8 lymphocytic or monocytic cell lines tested, vpx mutant virus grew as well as wild‐type virus. Only in primary peripheral blood mononuclear cell cultures, severely retarded growth of mutant virus was observed. No replication of vpx‐minus virus was detected in primary macrophage cells. A highly sensitive single‐round replication assay system was used to determine the defective replication phase in primary mononuclear cells of vpx mutant virus. In all cell lines examined, vpx mutant displayed no abnormality. In contrast, the vpx mutant was demonstrated to be defective at an early stage of the infection cycle in primary cell cultures. No evidence of a replication‐defect at a late phase in primary cells of the vpx mutant was obtained by a transfection‐coculture method. These results indicate that the virion‐associated Vpx protein is essential for early viral replication process in natural target cells such as primary macrophages.
Inhibition of T‐Cell Proliferation Induced by a Cell‐Free Salmonella typhimurium Extract Does Not Involve a Nitric Oxide‐Mediated Mechanism
Authors: Matsui Katsuhiko , Arai Toshihiko
Published: November 2013

In a previous study, we observed that a cell‐free Salmonella typhimurium extract induced suppression of mitogen‐induced T‐cell proliferation and that this suppression involved non‐responsiveness of T‐cells to interleukin‐2 (IL‐2) and augmentation of IL‐2 receptor (IL‐2R) expression. In this study, we found that inhibition of phytohemagglutinin (PHA)‐stimulated murine spleen cell proliferation induced by a cell‐free S. typhimurium extract was reversed by treatment with an anti‐interferon‐γ monoclonal antibody (anti‐IFN‐γ Ab), but not by interleukin‐4 or NG‐monomethyl‐l‐arginine, which is known to inhibit nitric oxide (NO)‐secretion from spleen cells in culture. However, IL‐2R expression was augmented by treatment with the extract, although this was independent of an NO‐mediated mechanism. Only anti‐IFN‐γ Ab treatment reduced the augmented IL‐2R expression to a normal level. These results suggest that the suppression of T‐cell proliferation induced by the Salmonella cell‐free extract is associated with augmentation of IL‐2R expression in an NO production‐independent manner.
Inducement of Respiratory Mucosal Immunogenicity for Nasal Vaccine Due to Amine‐ and Amide‐Inactivation of Parainfluenza Virus Type 1, Sendai
Authors: Miyamae Takeo
Published: November 2013

The protective effects in mice by nasal vaccination of amine‐ and amide‐inactivated Sendai viruses were investigated by a contact exposure experiment, immunofluorescent examination of the entire respiratory tract, and checking the serum HI antibody development. Of 10 monoamines, ethanolamine and 2‐methoxyethylamine vaccines induced complete protection, and methylamine, ethylamine, n‐propylamine, n‐butylamine, 2‐ethoxyethylamine, diethylamine and triethylamine vaccines brought about almost complete protection or lower respiratory infection. The methoxyamine‐treated mouse conferred the least protection. Of 5 diamines, 1, 3‐diaminopropane vaccine inhibited completely the infection, but hydrazine, ethylenediamine, putrescine, and cadaverine vaccines produced regional infection. Two polyamines, spermine and spermidine, did not inactivate the virus. Of 4 amides, only semicarbazide vaccine conferred complete mucosal defense, while acetamide, propionamide, and isonicotinic acid hydrazide vaccines lead to regional infection. Serum HI titers developed by vaccination were low on the whole, following their slight rise, fall or maintenance postexposure. In effect, the 4 vaccines inactivated by a best‐suited interstrand crosslink between phosphate groups in helix of viral RNA brought about the strongest protection, and showed the necessity of a definite length of molecules for inactivants.
Tumor Necrosis Factor Receptor Expression in HIV‐1‐Infected CD4+ T Cells
Authors: Hober Didier , de Groote Donat , Vanpouille Nathalie , Dehart Isabelle , Shen Lu , Wattré Pierre , Maniez‐Montreuil Michèle
Published: November 2013

We investigated whether HIV‐1 can regulate tumor necrosis factor receptor (TNFR) expression in SupT‐1, a CD4 + T‐cell line. The cells were infected with HIV‐1 containing 1,000 cpm RT activity, as early as day 3 after infection and all along the culture the supernatant level of core protein p24 was >250 pg/ml, and on days 6 and 9 after infection, p24 was found in 10 % of the cells as determined by indirect immunofluorescence assay. The cells were growing without loss of viability. The study of TNFR expression was based on a microassay for measurement of binding of 125I‐TNFα to cells, in which free and cell‐bound ligand separation was performed by centrifugation through oil. Scatchard analysis of TNFα binding on days 6 and 9 after infection revealed a 90 % increase in the expression of high‐affinity membrane receptors in HIV + SupT‐1 culture compared with uninfected cells (mean +/‐S.D. = 501 +/‐148.5 vs. 263 +/‐77.8 receptors/cell, n = 9, P< 0.001) with no change in dissociation constants (mean +/− S.D. = 4.36 +/−1.06 vs. 4.00 +/−1.12 × 10−10 m).
Serological Studies of the Antigenic Similarity between Typhus Group Rickettsiae and Weil‐Felix Test Antigens
Authors: Amano Ken‐ichi , Kyohno Kaori , Aoki Sumie , Suto Tsunehisa
Published: November 2013

The sera from two patients with murine typhus reacted with whole cells of Rickettsia prowazekii, R. typhi, and Proteus vulgaris OX19, and with lipopolysaccharides (LPS) from the spotted fever group rickettsia strain TT‐118 and P. vulgaris OX19 in the enzyme‐linked immunosorbent assay. Sera from these patients reacted with ladder‐like bands of LPS from R. prowazekii and R. typhi in the immunoblot, whereas the reactivity of these sera with LPS from P. vulgaris OX19 differed from each other. These results indicate that LPS from the typhus group rickettsiae and P. vulgaris OX19 contain similar epitopes.
Influence of Exogenous Interleukin‐2 Concentration on the Isolation of Human Immunodeficiency Virus
Authors: Zöllner Bernhard , Feucht Heinz‐hubert , Hamann Alf , Laufs Rainer
Published: November 2013

A significant increase (P=0.015) in the HIV isolation rate from plasma samples was achieved by use of 10 U/ml exogenous interleukin‐2 compared to 20 U/ml. The sensitivity rose from 0% to 29% on patients negative for p24 core antigen (P=0.031) and from 71% to 86% in patients positive for p24 core antigen in plasma (P>0.05). Titration of infectious HIV revealed that 10 U/ml interleukin‐2 is the optimal concentration to isolate low numbers of infectious particles of HIV.
C3d and Epstein‐Barr Virus (CR2/CD21 Ligands) Stimulate Cells of an HTLV‐I Line, MT‐2
Authors: Kuraya Mikio , Sato Tetsuo , Fujita Teizo
Published: November 2013

We studied the physiological role of complement receptor type II (CR2, C3d/EBV receptor) expressed on T cells using MT‐2 cells. First, we confirmed CR2 expression on MT‐2 cells by flow cytometry and found that the MW of CR2 molecules on these cells and Raji B cells were the same by SDS‐PAGE analysis. When MT‐2 lysates were incubated with anti‐CR2 mAb HB5 and thereafter with 32P‐labeled ATP, 52‐ and 74‐kDa proteins were phosphorylated, suggesting the activation of MT‐2 cells through the complex of CR2 with these proteins. In this respect, we measured lymphotoxin production by MT‐2 cells when incubated with C3d or EBV. The cytotoxicity of the MT‐2 supernatant against L929 cells was elevated in a dose‐ and time‐dependent manner. Next, we confirmed EBNA expression on EBV‐infected MT‐2 cells and attempted to establish an EBV‐positive MT‐2 clone by in vitro EBV infection. However, these clones disappeared during cloning. To clarify this mechanism, we examined the EBV genome in MT‐2 cells. By Southern blot analysis, BamHI digestion of DNA extracts from MT‐2 cells 3 days after EBV treatment gave a 3.0‐kb signal which comigrated with the EBV BamHI‐W probe. The 3.0‐kb signal of genomic EBV‐DNA was detected at 1, 2, 3, 5, and 7 days after EBV treatment, but could not be detected at 14 days. Thus, natural ligands of CR2 stimulate CR2‐positive MT‐2 cells through their functionally active CR2 molecules and in vitro EBV infection of MT‐2 cells might be transient.
Conspicuous Growth of Intravenously Inoculated Staphylococcus aureus in Subcutaneously Established Ehrlich Ascites Tumor Tissue of Mice
Authors: Murai Miyo , Seki Keiko , Sakurada Junji , Usui Akemi , Masuda Shogo
Published: November 2013

Intratumoral growth of Staphylococcus aureus was compared with the intrarenal growth, to examine the usefulness of the method as a marker of its pathogenicity. When 5 × 107 CFU/mouse of three derivatives from S. aureus Cowan I with different intrarenal growth were intravenously injected into Ehrlich tumor‐bearing mice, they lodged in the tumor tissue at approximately 103 CFU/0.1 g by 30 min after infection, and grew in the range of 106 CFU/0.1 g to 108 CFU/0.1 g by day 4, regardless of their intrarenal growth capacity. In contrast, S. saprophyticus lodged in both tissues to the same degree as S. aureus, but did not grow at all. The time course of the staphylococcal growth was different between tumor tissue and kidney, suggesting differences in the local responses against S. aureus.
Nutrient Starvation Induces Cross Protection against Heat, Osmotic, or H2O2 Challenge in Vibrio parahaemolyticus
Authors: Koga Tetsuro , Takumi Kenji
Published: November 2013

Glucose‐starved cells of Vibrio parahaemolyticus were compared with non‐starved counterparts with respects to heat, osmotic, and oxidative challenges. The starved cells demonstrated greater thermal and oxidative resistance than did the non‐starved cells. The starved cells also showed greater resistance against low osmotic challenge than did the non‐starved cells although both cells showed a comparable resistance against high osmotic challenge.
Differentiation of Capsular Polysaccharides from Acetobacter diazotrophicus Strains Isolated from Sugarcane
Authors: Stephan MaríLia Penteado , Fontaine Thierry , Previato José Osvaldo , Mendonça‐Previato Lúcia
Published: November 2013

Capsular polysaccharides (CPSs) from six representative strains of Acetobacter diazotrophicus were isolated and fractionated by gel filtration and anion‐exchange chromatography. Purified CPSs obtained in the non‐adsorbed fraction of a DEAE‐Sephadex A‐25 column were qualitatively and quantitatively analyzed for sugar composition. Uronic acid and amino sugars were not detected in all purified CPSs. Basically the CPSs of A. diazotrophicus are composed of rhamnose, mannose, galactose and glucose. The presence of fucose was only observed in the CPS of strains PR2 and PAL3. Based on these results, the six strains of A. diazotrophicus could be divided into four groups according to the sugar content of their capsules: (i) fucose‐containing capsules (PR2 and PAL3, localized in roots), (ii) mannose‐rich capsule (PAL5, localized in root), (iii) capsules with a high ratio of hexose to rhamnose (PR4 and PR20, localized in stems) and (iv) capsules with a low ratio of hexose to rhamnose (PR14, localized in rhizosphere). For all CPSs, sodium dodecy sulfate‐polyacrylamide gel electrophoresis showed diffuse bands of slow mobility in silver‐stained gels. The different CPS migration patterns could not be correlated with sugar composition. The purified CPS of strain PAL3 was found to be immunogenic and immunochemically similar to the CPS of strain PR2. The serological specificity to CPS of strains PAL3 and PR2 correlated well with the presence of fucose, indicating that this deoxyhexose is immunodominant. These findings demonstrated the feasibility of preparing specific antibodies to fucose‐containing CPS of A. diazotrophicus, indicating the possibility of utilization of this antiserum for future taxonomic studies or to select strains with chemically related capsular polysaccharides from their natural habitat.
Proliferative Response of Human Peripheral Blood Mononuclear Cells to Japanese Encephalitis Virus
Authors: Desai Anita , Ravi Vasanthapuram , Chandramuki Akkepati , Gourie‐Devi Mandaville
Published: November 2013

Cell‐mediated immune response to Japanese encephalitis virus (JEV) and its purified envelope (E) protein was measured in 45 laboratory confirmed JE patients using a proliferation assay of peripheral blood mononuclear cells (PBMC). In parallel, JEV‐specific IgM antibodies were measured by ELISA. No correlation was observed between the antibody response and results of the lymphocyte proliferation assay. Only 11 of the 42 patients positive in the antibody test were positive in the proliferation assay, and PBMC from 14/45 (31%) patients did not respond to either phytohemagglutinin and to JEV and/or its purified E protein antigen. No correlation was observed between the cell‐mediated immune response and the final clinical outcome (fatality vs. recovery).
Development of a New Selective Plating Medium for Rhodococcus equi
Authors: von Graevenitz Alexander , Pünter‐Streit Verena
Published: November 2013

A new selective plating medium for Rhodococcus equi containing ceftazidime (20 mg/l) and novobiocin (25 mg/l) on a Mueller‐Hinton agar basis is described. It proved to be less inhibitory for R. equi than selective plating media devised earlier and grew only very few other nocardioform bacteria.
Rapid Identification by Polymerase Chain Reaction of Staphylococcal Exfoliative Toxin Serotype A and B Genes
Authors: Sakurai Susumu , Suzuki Hitoshi , Machida Katsuhiko
Published: November 2013

A new system was designed to detect staphylococcal exfoliative toxin A (ETA) and B (ETB) genes by the polymerase chain reaction (PCR). The primer pairs for the ETA gene (eta) were 20 and 20‐mer, and its PCR product was a 741‐bp eta fragment, while the primer pairs for the ETB gene (etb) were also 20 and 20‐mer, and its PCR product was a 629‐bp etb fragment. When these primers were simultaneously used in the PCR, the two types of ET were clearly detected as two bands in an ETA and ETB double‐producer using only one colony within 3 hr. We examined 66 strains of Staphylococcus aureus isolated from patients with staphylococcal scalded skin syndrome (SSSS) and compared the results obtained by ELISA and PCR. The same results were obtained for 56 of the strains, i.e., 30 strains were ETA producers, 20 strains were ETB producers, and 6 strains were double‐producers. However, positive results were obtained for 5 of the 10 non‐ET‐producing strains. Two of these strains were judged by PCR as ETA producers and three as ETB producers. Thus, PCR is very sensitive and rapid in detecting ETA and ETB gene fragments in colonies isolated from patients with SSSS.
Characterization of the Dextranase Gene (dex) of Streptococcus mutans and Its Recombinant Product in an Escherichia coli Host
Authors: Igarashi Takeshi , Yamamoto Ayako , Goto Nobuichi
Published: November 2013

The gene (dex), which encodes the Streptococcus mutans dextranase (Dex), was cloned in Escherichia coli. The E. coli host harboring a recombinant plasmid (pSD2) containing an 8‐kb BamHI insert produced a Dex protein of 133 kDa as well as smaller enzymes of 118, 104, and 88 kDa. The Dex produced by the recombinant E. coli was apparently located in the cytoplasmic fraction, not in the periplasmic nor the extracellular fractions. Subcloning and deletion analysis of pSD2 showed that the structural gene of Dex was encoded by a 4‐kb BamHI‐SalI fragment. The fragment also contained the dex promoter which was effective in the E. coli cell.
Suppression of Anti‐Candida Activity of Murine Neutrophils by Progesterone In Vitro: A Possible Mechanism in Pregnant Women's Vulnerability to Vaginal Candidiasis
Authors: Nohmi Takao , Abe Shigeru , Dobashi Kazuyoshi , Tansho Shigeru , Yamaguchi Hideyo
Published: November 2013

Sex steroid hormones were examined for their effect on mycelial growth of Candida albicans, and the inhibitory activity of casein‐induced murine peritoneal neutrophils against mycelial growth of C. albicans was examined in vitro using a crystal violet staining method or a [3H]glucose incorporation method. Four steroid hormones, danazol, estradiol, estriol and testosterone had no effect on mycelial growth of C. albicans, but progesterone appeared to convert the growth form of C. albicans from hyphal to yeast. Danazol (10–6 m) and progesterone (10–5 m) suppressed anti‐Candida activity of neutrophils of non‐treated mice, while testosterone, estradiol, and estriol did not. The anti‐Candida activity of neutrophils of estradiol‐pretreated mice was clearly suppressed by progesterone even at 10–6 m which corresponded to its plasma concentration in pregnant women in the third trimester. The physiological significance of this suppressive effect of progesterone was discussed in relation to the vulnerability of pregnant women to vaginal candidiasis.
Biomaterial‐Associated Infection with Candida albicans in Mice
Authors: Różalska Barbara , Ljungh Asa , Burow Aleksandra , Rudnicka Wiesława
Published: November 2013

Candida yeasts are frequently isolated from patients with continuous ambulatory peritoneal dialysis peritonitis or other biomaterial‐associated infections. The mouse model of candidal peritonitis was used to study the interaction of Candida cells with end‐point attached heparinized polyethylene (H‐PE) and with polymorphonuclear leukocytes (PMNs) or macrophages (Mφ). Two Candida strains differing in cell surface hydrophobicity and in expression of fibronectin (Fn) binding were used for the study. Cells of both Candida strains adhered at higher numbers to H‐PE surfaces preadsorbed with Fn or with human dialysis fluid (HDF) than to non‐modified H‐PE, supporting a role of Fn in mediating adhesion. C. albicans 4016 cells expressing low hydrophobicity and low binding of soluble Fn demonstrated stronger adhesion to PMNs than the more hydrophobic C. albicans 3248 yeasts, which express high binding of soluble Fn. However, C. albicans 4016 cells were more resistant to phagocytic killing and were hardly eradicated in intraperitoneally infected mice. The animals depleted in PMNs by treatment with CY were neither able to eradicate C. albicans 3248 (rapidly eliminated by normal mice) nor C. albicans 4016 yeasts (with a tendency to persist in the tissues of normal mice).
Sequential Involvement of NK Cells and CD8+ T Cells in Granuloma Formation of Rhodococcus aurantiacus‐Infected Mice
Authors: Asano Misako , Nakane Akio , Kohanawa Masashi , Minagawa Tomonori
Published: November 2013

We investigated the effect of in vivo administration of antibodies against T‐cell subsets and natural killer (NK) cells on endogenous gamma interferon (IFN‐γ) production and granuloma formation in Rhodococcus aurantiacus‐infected mice. High titers of endogenous IFN‐γ were detected in the extracts of the livers and spleens during 24 hr of the infection, reaching the peak at 8 hr, and the IFN‐γ production was reduced by in vivo administration of anti‐NK 1.1 monoclonal antibody (MAb) or antibody against asialo GM1+ cells. Endogenous IFN‐γ declined until 2 days of the infection, then reappeared from 1 week and peaked at 3 weeks. Endogenous IFN‐γ at 1 and 3 weeks was reduced by in vivo administration of anti‐CD8 MAb, but not by anti‐CD4 MAb or anti‐NK 1.1 MAb. Granulomatous lesions in the livers and spleens began to appear from 1 week of the infection and developed in 3 weeks. In vivo administration of rat anti‐IFN‐γ MAb reduced the development of granulomas. In addition, granuloma formation was reduced by depletion of NK cells prior to the infection or depletion of CD8+ T cells at 1 week of the infection. Based on these findings, it is presumed that the biphasic production of IFN‐γ is attributable to NK cells in the early phase of the infection and CD8+ T cells in the phase of granuloma formation, and that granuloma formation is regulated by NK cells and CD8+ T cells through the secretion of endogenous IFN‐γ.
Effect of Porphyromonas gingivalis ATCC 33277 Vesicle on Adherence of Streptococcus mutans OMZ 70 to the Experimental Pellicle
Authors: Kamaguchi Arihide , Baba Hisae , Hoshi Masaaki , Inomata Koshiro
Published: November 2013

The vesicles of Porphyromonas gingivalis ATCC 33277 strongly aggregated Streptococcus cricetus, S. rattus, and S. mutans, but poorly aggregated S. sobrinus. The adherence of S. mutans OMZ 70 to hydroxyapatite (HA) coated with whole saliva was increased in parallel with the quantity of the vesicles. The significant increase of adherence of S. mutans OMZ 70 by the vesicles was also observed on the HA coated with parotid saliva, submandibular saliva, serum, and type I collagen. These findings suggest that the vesicles may act as a bridge between mutans streptococcus and the tooth surface.
Further Characterization of Chlamydia pneumoniae Specific Monoclonal Antibodies
Authors: Puolakkainen Mirja , Parker Julia , Kuo Cho‐chou , Grayston J. Thomas , Campbell Lee Ann
Published: November 2013

Studies using monoclonal antibodies have demonstrated species‐specific reactivities with Chlamydia pneumoniae. In this study, further characterization of C. pneumoniae specific monoclonal antibodies TT‐205 and RR‐402 and description of C. pneumoniae specific antibodies prepared against other isolates are presented. TT‐205 and RR‐402 were shown to neutralize infectivity. Neutralization in cell culture was specific and enhanced by complement. Attempts to characterize the reactive antigen by immunoblotting, immunoaffinity chromatography and radioimmunoprecipitation were unsuccessful, probably due to difficulties in solubilizing the immunoreactive epitope without denaturing it. Recognition of the determinant by the monoclonal antibodies is labile to physical and chemical treatments suggesting that the reactive epitope is conformational.
Sequences of E/NS1 Gene Junction from Four Dengue‐2 Viruses of Northeastern Thailand and Their Evolutionary Relationships with Other Dengue‐2 Viruses
Authors: Thant Kyaw Zin , Morita Kouichi , Igarashi Akira
Published: November 2013

We determined the 240‐nueleotide sequences of the E/NS1 gene junction of four dengue‐2 viruses by the primer extension dideoxy chain termination method. These viruses were isolated from dengue patients with different clinical severities in Nakhon Phanom, Northeastern Thailand in 1993. The results were compared with the 52 published dengue‐2 sequences of the same gene region. Sequence divergence of four new isolates varied from 4.17% to 5.42% compared with dengue‐2 prototype New Guinea C strain whereas it varied from 5.42% to 6.67% and from 6.67% to 7.09% when compared with Jamaica 1409 strain and PR159/S1 strain, respectively. All nucleotide substitutions were found at the 3rd position of the codons which were silent mutations. All 56 isolates studied were classified into five genotypic groups by constructing the dendrogram. The results indicated that four new isolates from Northeastern Thailand belong to genotype II of dengue virus serotype 2, and were most closely related to prototype New Guinea C strain. We also observed the variation in nucleotide and amino acid sequences among clusters of isolates (Thailand‐1980, Malaysia‐1989 and Thailand‐1993) which were obtained from the dengue patients with different clinical severities. The significance of these genetic differences have been discussed in terms of the possible correlation between genetic variability and virulence.
Antibody Responses Raised against a Conformational V3 Loop Peptide of HIV‐1
Authors: Bukawa Hiroki , Fukushima Jun , Hamajima Kenji , Kimura Mari , Tsuji Takashi , Xin Ke‐qin , Okuda Kenji
Published: November 2013

The amino acid sequence of the principal neutralizing determinant (PND) of 224 cases of human immunodeficiency virus type 1 (HIV‐1) was determined and the most frequently occurring sequence was used as a peptide antigen for studying virus‐specific antibody responses. In our present study, a linear peptide of the most frequent PND was first synthesized and then oxidized to create a disulfide‐bridged loop conformation. Then, in order to construct a macromolecular structure for the purpose of increasing anti‐genicity, the synthetic peptide was conjugated to a core peptide. We compared the immunogenicity of the disulfide‐bridged loop PND peptide antigen (AG4) and the linear PND peptide antigen (AG5). After immunizing rabbits 5 and 6 times with both peptides, the results obtained using ELISA revealed that AG4 (conformational‐loop type) was more capable of inducing a high titer of antigen‐specific antibodies than was AG5 (linear type). Despite an amino acid sequence homology of 72%, a 1:8 dilution of serum raised against AG4 inhibited 81.9% of HIV‐1IIIB‐mediated cell fusion, suggesting that conformational V3 loop peptide is able to elicit an antibody response which is strongly HIV‐1‐specific.
The Purification of a GroEL‐Like Stress Protein from Aerobically Adapted Campylobacter jejuni
Authors: Takata Tohru , Wai Sun Nyunt , Takade Akemi , Sawae Yoshiro , Ono Junko , Amako Kazunobu
Published: November 2013

From plate cultures of Campylobacter jejuni grown in room air a particulate protein of 62 kDa was isolated by ion‐exchange chromatography. The protein had a square shape from the side view but when viewed from the top it had a star‐shaped structure. The molecular size of the whole particle determined by gel filtration was 850 kDa which suggested the presence of 14 subunits of 62 kDa in each particle. The N‐terminal 37 amino residues showed more than 80% homology with the sequence of these heat shock protein (HSP) 60 homologs of Chlamydia trachomatis, Helicobacter pylori, and Escherichia coli (GroEL). This protein is immunologically cross‐reactive with the antiserum for the 60‐kDa HSP of Yersinia enterocolitica. Production of the 62‐kDa protein increased under heat stress and growth in an aerobic atmospheric environment. From these observations we concluded that the 62‐kDa protein is a Campylobacter stress protein (Cj62) which belongs to the HSP 60 family.
Prevalence of Antibody to Hepatitis E Virus among Healthy Individuals in Southern Taiwan
Authors: Peng Chien‐Fang , Lin Meei‐Ru , Chue Pay‐Yu , Tsai Jung‐Fa , Shih Ching‐Hua , Chen I‐Lung , He Junkun , Carl Mitchell
Published: November 2013

The seroprevalence of hepatitis E virus (HEV) among 997 healthy individuals aged 6 to 84 years, collected between July 1993 and June 1994 at Kaohsiung‐Pingtung area in Southern Taiwan was studied. Of the study populations of vegetable farmers, elementary school children, volunteer blood donors and college students, the prevalence of IgG anti‐HEV ranged from 6.4% to 8.8%. In suburban elemantary school children of Mang‐Chou Village at Pingtung‐Hsien, the seroprevalence rate (9.6%) was significantly higher than the positive rate (1.5%) found in rural aboriginal elementary school of San‐Min Village at Kaohsiung‐Hsien. IgG anti‐HEV antibodies were widely distributed among all age groups, with a significantly higher percentage (13.1%) in the age group of 46‐55 years old.
Mapping and Characterization of a Sequential Epitope on the Rabies Virus Glycoprotein Which Is Recognized by a Neutralizing Monoclonal Antibody, RG719
Authors: Ni Yajin , Tominaga Yuichi , Honda Yoshikazu , Morimoto Kinjiro , Sakamoto Shin'ichi , Kawai Akihiko
Published: November 2013

We have established a murine hybridoma cell line RG719 which produces a rabies virus‐neutralizing IgM‐type monoclonal antibody (referred to as MAb RG719). Immunoblot analysis indicated that the antibody recognized a sequential epitope of G protein. Among four rabies virus strains tested, the antigenicity to MAb RG719 was absent from the Nishigahara strain, while the other three strains (HEP, ERA and CVS) reacted to the MAb. Studies with deletion mutants of the G protein indicated that the epitope was located in a middle region of the primary structure of G protein, ranging from position 242 to 300. By comparing the estimated amino acid sequence of the four strains, we found in this region two amino acids (at positions 263 and 291) which are common to three of those strains but are not shared by the Nishigahara strain. The site‐directed point mutagenesis revealed that replacement of phenylalanine‐263 by leucine destroyed the epitope of the HEP G protein, while the epitope was generated on the Nishigahara G protein whose leucine‐263 was replaced by phenylalanine. These observations suggest that phenylalanine‐263 is essential for constructing the epitope for MAb RG719. The synthetic 20‐mer peptide produced by mimicking the amino acid sequence (ranging from amino acid positions 249 to 268) of the presumed epitope region was shown to bind specifically to MAb RG719 and also to raise the virus‐neutralizing antibodies in rabbits. Vaccination with the HEP vaccine produced in Japan induced in humans and rabbits production of significant amounts of the antibodies which reacted with the 20‐mer peptide.
Differential Effects of Prostaglandin E1 and Prostaglandin E2 on Growth and Differentiation of Murine Myeloid Leukemic Cell Line, M1
Authors: Sawamura Hiroshi , Hayashi Hidetoshi , Onozaki Kikuo
Published: November 2013

Effects of prostaglandins (PGs) of the E series on growth and differentiation of murine myeloid leukemic cell line M1 were studied. PGE1, but not PGE2, inhibited the growth of M1 cells. PGE2 neither inhibited nor augmented the antiproliferative effect of PGE1. PGE1 augmented the differentiation of M1 cells into macrophage‐like cells induced by interleukin 6. PGE2, however, did not exhibit any effect on the differentiation. PGE1 caused a marked increase in intracellular cAMP level in M1 cells, whereas PGE2 had no effect. These results indicate that M1 cells are able to respond only to PGE1. Radiolabeled PGE1 binding experiments, however, revealed that there was no specific binding in M1 cells, suggesting that the cells express low numbers of receptors or very low affinity receptors specific for PGE1. Stable agonists of PGI2, iloprost, cicaprost or carbacyclin, also potently inhibited the growth of M1 cells. These findings suggest that PGE1 as well as PGI2 may play a role in the differentiation of monocyte‐macrophage lineage cells.
Yersinia pseudotuberculosis in China
Authors: Zheng Xue Bin , Tsubokura Misao , Wang Yongkun , Xie Chun , Nagano Tetsuji , Someya Kazuhiko , Kiyohara Tomoko , Suzuki Kiyomi , Sanekata Takeshi
Published: November 2013

Thirty strains of Yersinia pseudotuberculosis were isolated from rabbits (17 strains), wild rats (9 strains) and house rats (4 strains) in China between 1990 and 1993. The biochemical properties of these isolates were identical with those of Y. pseudotuberculosis and no special characteristics were found in these strains. Serologically, serogroups 4b and 5b were identical to isolates found in Japan, and a new serogroup 1c and unclassified strains have also been detected. The existence of virulence‐associated properties were different among strains. The pYV plasmid was detected from 6 strains of 30 isolates. This report documents the presence of Y. pseudotuberculosis in China, providing important epidemiological information.
Demonstration of a Ferric Vibrioferrin‐Binding Protein in the Outer Membrane of Vibrio parahaemolyticus
Authors: Yamamoto Shigeo , Akiyama Toshihito , Okujo Noriyuki , Matsu‐ura Shiro , Shinoda Sumio
Published: November 2013

Under iron‐restricted conditions, Vibrio parahaemolyticus produces a siderophore, vibrioferrin, accompanying expression of two major outer membrane proteins of 78 and 83 kDa. Autoradiographic analysis of nondenaturing polyacrylamide gel electrophoregrams of outer membrane preparations previously incubated with [55Fe]ferric vibrioferrin revealed a single radiolabeled band, in which the 78‐kDa protein was detected predominantly by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The antiserum against the purified 78‐kDa protein partially inhibited Fe‐VF binding to isolated OMPs. The 78‐kDa protein was cleaved by the treatment of whole cells with proteinase K, indicating that a portion of this protein is exposed on the surface of the outer membrane. The treated cells lost most of their iron uptake activity mediated by vibrioferrin. These results suggest that the ferric vibrioferrin‐binding protein of 78 kDa may function as the receptor for ferric vibrioferrin involved in the initial step of vibrioferrin‐mediated iron uptake. Immunoblot analysis using the antiserum against the 78‐kDa protein demonstrated that the molecular mass and antigenic properties of the protein were highly conserved among V. parahaemolyticus strains examined. The antiserum also recognized an iron‐repressible outer membrane protein of 78 kDa from iron‐restricted V. alginolyticus strains, some of which appeared to produce vibrioferrin.
Three Antigenic Regions in p17 of Human Immunodeficiency Virus Type 1 (HIV‐1) Revealed by Mouse Monoclonal Antibodies and Human Antibodies in HIV‐1 Carrier Sera
Authors: Liu Xiaoliang , Ota Akemi , Watanabe Michiko , Ueda Shigeharu , Saitoh Atsushi , Shinagawa Hideo , Nakata Atsuo , Kurimura Takashi , Wang Xiaoui , Zhao Yu , Kondo Kiyoshi , Seki Jiro , Miyake Shinichi , Sakato Nobuo , Fujio Hajime
Published: November 2013

We investigated the murine antibody response to recombinant p17 (rp17) of human immunodeficiency virus type 1 (HIV‐1) and the human antibody response directed to p17 in HIV‐1 infection. Three large peptides covering residues 12‐29, 53‐87 and 87‐115 of p17 were synthesized. The cysteine residues 57 and 87 of peptide 53‐87 were reoxidized to form a disulfide bridge. Eighteen out of 19 murine monoclonal anti‐rp17 antibodies had relatively high affinities (KA = 1.9 × 105−1.4 × 108 M−1) with one of the 3 p17 peptides in the liquid phase. Each monoclonal antibody reacted only with one particular peptide and had no reactivity with the other 2 p17 peptides. All the monoclonal antibodies reacted with rp17 in the liquid phase with a reasonable degree of affinity (KA = 2.0 × 105−1.8 × 107 M−1). Four HIV‐1 carrier sera, which were positive in ELISA using rp17 as the antigen, reacted positively in an ELISA using 3 p17 peptides which were used to titrate murine monoclonal antibodies. Murine monoclonal antibodies having specificity for the 3 p17 peptides stained live HIV‐1‐infected cells by means of indirect membrane immunofluorescence, irrespective of their specificity. This suggests that the various portions of p17 (at least 3 regions of p17) were exposed on the surface of live infected cells, probably as short polypeptide chains.
A 38‐kDa Antigen of Mycobacterium tuberculosis Predominantly Induces the Secretion of Interleukin‐2, Interferon‐gamma and IgG2a Antibodies
Authors: Agrewala Javed N. , Mishra Gyan C.
Published: November 2013

In the present study, mice of 3 different haplotypes (H‐2d, H‐2k and H‐2b) were sensitized subcutaneously with heat‐killed H37Ra or 38‐kDa antigen of Mycobacterium tuberculosis. Lymphocytes obtained from immunized animals were challenged in vitro with 38‐kDa antigen in both cases. The dominant pattern of Th1‐like lymphokines (IL‐2 and IFN‐gamma) and preferential production of 38‐kDa specific IgG2a‐type antibody were observed. It was noted that 38‐kDa antigen was recognized permissively by all 3 strains of mice used in the present study. It was interesting to note that C3H/HeJ mice, which express BCG‐resistant alleles showed a higher level of proliferative as well as cytokine response as compared to BALB/c and C57BL/6 mice, which bear BCG‐susceptible alleles. These results suggest that not only in recall responses but also during the induction as well as expression phase of the immune response mediated by 38‐kDa antigen of M. tuberculosis the Th1‐like immune response predominates.
Transfer of Two Burkholderia and An Alcaligenes Species to Ralstonia Gen. Nov.
Authors: Yabuuchi Eiko , Kosako Yoshimasa , Yano Ikuya , Hotta Hisako , Nishiuchi Yukiko
Published: November 2013

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.
No Relationship between HTLV‐1 Infection and Filariasis—Serological Study on Patients with Filariasis in Recife, Brazil
Authors: Linhares Maria Ieda Siqueira , Malagueño Elizabeth , Carvalho Luiz Bezerra , de Oliveira Valdete Felix , Minamishima Yoichi
Published: November 2013

The seroprevalence of antibodies against human T‐cell leukemia virus was determined by ELISA in 68 patients with filarial infestation living in an endemic area. The total seropositivity was 2.9% and the HTLV‐1‐positive cases were detected in 2 microfilaremic patients 12 and 40 years old. This value is very close to that obtained for healthy individuals in the same region and age groups. This result suggests that there is no relationship between filariasis and HTLV‐1 infection as previously proposed.
Genetic Identification of Staphylococcus aureus by Polymerase Chain Reaction Using Single‐Base‐Pair Mismatch in 16S Ribosomal RNA Gene
Authors: Saruta Katsutoshi , Hoshina Sadayori , Machida Katsuhiko
Published: November 2013

Staphylococcus aureus is the most predominant and important pathogen in clinical microbiology. A DNA amplification assay using the polymerase chain reaction (PCR) was designed to identify S. aureus through a single‐base‐pair mismatch in the sequences of staphylococcal 16S ribosomal RNA (16S rRNA) genes. It was able to detect and identify S. aureus without requiring additional analytical techniques. Twenty‐eight staphylococcal and non‐staphylococcal strains were tested to verify the specificity of the assay, and only S. aureus strains gave a positive reaction. It may be possible to provide immediate and exact information for the identification of S. aureus.
Gene Expression of Perform and Granzyme A in the Liver in Chronic Hepatitis C: Comparison with Peripheral Blood Mononuclear Cells
Authors: Fukuda Ryo , Ishimura Norihisa , Nguyen Xuan‐Thanh , Chowdhury Aktaruzzarman , Ishihara Shunji , Sakai Sino , Akagi Shuji , Tokuda Alejandlo , Watanabe Makoto , Fukumoto Shiro
Published: November 2013

Perforin and granzyme A are the major effectors of cytotoxic T cells in cell‐mediated cytotoxicity. However, there has been no report on these effectors in chronic viral hepatitis. In the present study, the expression of perforin and granzyme A mRNA was investigated by the reverse transcription polymerase chain reaction method using liver biopsy specimens and peripheral mononuclear cells (PBMC) from 21 patients with chronic hepatitis C and 5 control cases. Perforin mRNA was detected only in the liver of chronic hepatitis patients but not in the control livers. Conversely, perforin mRNA was not expressed in PBMC of the patients with chronic hepatitis (P<0.01). Granzyme A mRNA was detected both in the liver and PBMC of all the cases including control cases. These results indicated that the perforin is an important effector molecule in the hepatocyte lysis in chronic viral hepatitis C.
The Complete Nucleotide Sequence of Odontoglossum Ringspot Virus (Cy‐1 Strain) Genomic RNA
Authors: Ikegami Masato , Isomura Yoshinori , Matumoto Yoshinori , Chatani Masaaki , Inouye Narinobu
Published: November 2013

The complete nucleotide sequence of the genomic RNA of odontoglossum ringspot virus Cy‐1 strain (ORSV Cy‐1) was determined using cloned cDNA. This sequence is 6611 nucleotides long containing four open reading frames, which correspond to 126 K, 183 K, 31 K, and 18 K proteins. Its genomic organization is similar to other tobamoviruses, TMV‐V(vulgare), TMV‐L (tomato strain), tobacco mild green mosaic virus (TMGMV) and cucumber green mottle mosaic virus (CGMMV). The 5′ non‐coding regions of ORSV Cy‐1 is 62 nucleotides. The ORFs encoded a 126 K polypeptide and a 183 K read‐through product in which helicase‐sequence and polymerase‐sequence motifs are found. The ORFs encoding the 126 K and 183 K proteins have 61% and 63% identities with those of TMV‐V. The third ORF encoded a 31 K protein homologous to TMV cell‐to‐cell movement protein. It has 63% identities with that of TMV‐V. The fourth ORF encoded an 18 K coat protein. The 5′ non‐coding region, which extends from base 1 to 62 has 2 G residues and a ribosome binding site (AUU). The 3′ non‐coding region, 414 nucleotides in length, is entirely different from that of other tobamoviruses.
Rapid and Sensitive PCR Detection of Vibrio trachuri Pathogenic to Japanese Horse Mackerel (Trachurus japonicus)
Authors: Iwamoto Yoshihisa , Suzuki Yusaku , Kurita Akinobu , Watanabe Yoko , Shimizu Tadayori , Ohgami Hirohisa , Yanagihara Yasutake
Published: November 2013

A polymerase chain reaction (PCR) method was performed for rapid and sensitive detection of pathogenic Vibrio trachuri isolated from cultured Japanese horse mackerel. A set of primers was selected from the base sequence of the Pst I fragment of T9210 chromosomal DNA and used for PCR detection of T9210. This PCR specifically amplified the DNAs from V. trachuri T9210, T9213, and T9216 but not of those other bacterial strains. PCR using a Pst I‐1 primer set made it possible to detect 100 fg of T9210 DNA. The PCR method reported here may be useful for detection and identification of V. trachuri pathogenic to Japanese horse mackerel.
Different Properties of Wild Type and Drug‐Resistant Mutants of Human Immunodeficiency Virus Type 1 Reverse Transcriptase In Vitro
Authors: Ijichi Katsushi , Fujiwara Masatoshi , Shigeta Shiro , Konno Kenji , Yokota Tomoyuki , Baba Masanori
Published: November 2013

Drug‐resistant mutants of human immunodeficiency virus type 1 (HIV‐1) emerge during treatment with various reverse transcriptase (RT) inhibitors in vitro and in vivo. However, the virological nature and pathogenic importance of these mutants have not been fully elucidated. In this study, we have examined HIV‐1 mutants resistant to 3′‐azido‐3′‐deoxythymidine (AZT) and nonnucleoside RT inhibitors (NNRTIs) for their infectivity, RT activity, and replication in MT‐4 cells. Although the infectivity of AZT‐ and NNRTI‐resistant mutants was similar, the RT activity of AZT‐resistant mutants was much higher than that of NNRTI‐resistant mutants and their wild types. Furthermore, the RT activity of NNRTI‐resistant mutants was significantly lower than that of the wild types. In contrast, the replication of NNRTI‐resistant mutants was found to be greater than that of AZT‐resistant mutants and the wild types. When HIV‐1 proviral DNA (cDNA) synthesis was examined by PCR in MT‐4 cells infected with the wild type, AZT‐resistant mutant, or NNRTI‐resistant mutant, the PCR signal of the NNRTI‐resistant mutant was found to be much higher than those of the wild type and AZT‐resistant mutant. These results suggest that the drug‐resistant mutants differ from their corresponding wild types not only in drug sensitivity but also in other virological properties.
A Microassay for Determination of the Cytopathogenicity of Human Immunodeficiency Virus Type‐1 Isolates
Authors: Benyoucef Samira , Hober Didier , Shen Lu , Ajana Faïza , Gérard Yann , Bocket‐Mouton Laurence , Mouton Yves , Wattré Pierre
Published: November 2013

Correlations between the in vitro biological properties of HIV strains isolated from patients and the prognosis of their disease have been reported. We developed a technique to study the phenotype of HIV strains isolated from patients. We used the P4 cell line, derived from HeLa cells, which has been transfected with receptor CD4 gene. HIV laboratory strain (HIVLAI) and peripheral blood leukocytes (PBLs) from donors infected with HIVLAI induce syncytium in P4 cell cultures in vitro. The presence of reporter gene (LacZ gene) under the control of the HIV‐1 long terminal repeat (LTR) in these cells allows colorimetric visualization of syncytia in the cytoplasm using a β‐galactosidase (βgal) assay in the presence of X‐gal. We cocultivated 1 × 106 patient PBLs with 2 × 106 normal PHA‐activated normal PBLs for 4 days in the presence of IL‐2 in 24‐well plates. Half of the medium was replaced twice a week and PHA‐activated normal PBLs were added every 7 days. HIV‐1 was isolated from cocultured PBLs of 18 patients with advanced‐stage HIV infection as assessed by the production of HIV p24 detected with a commercially available HIV‐1 p24 ELISA. Supernatant and 105 cells were collected twice a week from cocultured PBLs and were added to P4 cells in 96‐well microtiter plates. The cultures were observed every day for 3 days and then the βgal assay was performed. We did not observe any effect with cells and supernatant from 8 patients, harvested from cultures incubated for as long as 28 days. The phenotype of these isolates was called NC (noncytopathic). In cells from 2 patients, we obtained blue multinucleated giant cells; the phenotype of these strains was called SI (syncytium inducing). In cultures from 8 other patients, we obtained the death of P4 cells without syncytium formation, and the phenotype of these strains was called CI (cell‐killing inducing). In every case, the cytopathic effect of HIV‐1 isolates could be detected with cocultured PBLs collected as early as day 4 of culture. Cocultured PBLs from 13 healthy controls did not alter the P4 cells. We displayed the replication of CI strains of HIV‐1, but not the one of NC strains in P4 cell line. Our micromethod allowed the detection of cytopathic effects of HIV isolates. Further investigations should define the clinical applications of this method.
Functional Characteristics of Enhanced Fc Receptor Expression of β2Integrin‐Deficient Bovine Mononuclear Phagocytes
Authors: Nagahata Hajime , Higuchi Hidetoshi , Goji Noriko , Noda Hiroshi , Kuwabara Mikinori
Published: November 2013

Fc receptor expression, cytoplasmic Ca2+signaling, chemiluminescent (CL) response, and electron spin resonance (ESR) combined with spin trapping of blood mononuclear phagocytes from control heifers and a heifer with leukocyte adhesion deficiency (LAD) were evaluated to elucidate the relationships between complement receptor type 3 (CR3) and Fc receptor expression and their functional responses. The mean fluorescence intensity of fluorescein isothiocyanate (FITC)‐conjugated anti‐bovine IgG bound to mononuclear phagocytes from the heifer with LAD was 1.8‐fold higher than that of control heifers. The mean increments of cytoplasmic Ca2+concentrations of mononuclear phagocytes from the heifer with LAD stimulated with OPZ, Agg‐IgG, and PMA were 39.4 (P<0.05), 118, and 71.6% compared with those of control heifers. A 1.27‐fold increase in the CL response relative to control heifers was detected when mononuclear phagocytes from the heifer with LAD were stimulated with Agg‐IgG. The OPZ‐induced CL response of mononuclear phagocytes from the heifer with LAD was significantly (P<0.05) decreased, whereas the PMA‐induced CL response was similar to that of control heifers. The ESR spectrum of mononuclear phagocytes from the heifer with LAD was increased when stimulated with Agg‐IgG, and was impaired when stimulated by OPZ compared with that of control heifers. The ESR spectrum of mononuclear phagocytes stimulated with PMA was similar in control heifers and the heifer with LAD. Fc receptors on mononuclear phagocytes from the heifer with LAD were enhanced, and their cytoplasmic Ca2+signaling, CL response, and ESR‐spin trapping when stimulated with Agg‐IgG and OPZ appeared to be associated with enhanced Fc receptors.