bluuebunny/crossref_metadata_2025 huggingface dataset:
Further Search for Small Molecular Inactivants Capable of Eliciting Respiratory Mucosal Immunogenicity by Modifying Sendai Virus Core RNA
Authors: Miyamae Takeo
Published: November 2013

Five groups of 32 chemicals were examined regarding their immunological functions as modifier inactivants to make inactivated Sendai nasal vaccine using a contact exposure experiment, direct immunofluorescent method, and serum HI titer. (1) Five of the nine reactive groups of reactive dyes (2‐chloropyridine, 2, 4, 6‐trichloropyrimidine, vinylsulfonic acid, epichlorohydrin and beta‐chloroethylamine) induced complete or almost complete defense in the entire respiratory tract, and the four other vaccines brought about slight infection in the respiratory tracts. There was no marked rise in serum HI titers post‐exposure, despite uneven development. (2) Of the four sizable substituted AS naphthol vaccines, naphthol AS‐IRG and AS‐G vaccines elicited nearly complete defense, but the two other vaccines, inactivated with more elongated molecules, invited rare and successive infections. The three immune groups produced invariably high serum HI titers. (3) Of the six naphthalene derivative vaccines, two (3‐hydroxy‐2‐naphthoic acid methylester and 2‐naphthol‐6‐sulfonic acid) induced complete or almost complete protection. But two vaccines brought about less protection, and the remaining two vaccines caused heavy infections. (4) Of the six benzene derivative vaccines, both m‐nitrobenzenesulfonic acid and isatoic anhydride induced complete protection. Three vaccines permitted slight infections but 2, 4, 6‐trinitrobenzenesulfonic acid vaccine caused severe infection. (5) Of the seven food dye vaccines, only orange I induced complete or nearly complete defense, while the other dye vaccines were inferior. In effect, twelve inactivated Sendai nasal vaccines modified the ribose and/or phosphate groups of the virus core RNA through five groups of small‐sized molecules with specially fixed side chains, and elicited complete or almost complete respiratory mucosal defense. The viral stabilization requiring the least alteration of the configuration will be involved in the chemical modification.
Nosocomial Diarrhoea in the Elderly Due to Enterotoxigenic Clostridium perfringens
Authors: Wada Akihito , Masuda Yoshishige , Fukayama Makiko , Hatakeyama Tsutomu , Yanagawa Yoshitoki , Watanabe Haruo , Inamatsu Takashi
Published: November 2013

To diagnose sporadic diarrhoea due to Clostridium perfringens infection, faecal specimens from elderly patients were examined directly for C. perfringens enterotoxin using reverse passive latex agglutination assay, and then cultured for this organism. C. perfringens isolates from those samples were grouped by slide agglutination and by pulsed‐field gel electrophoresis (PFGE). Fifty of the 60 isolates agglutinated with newly raised antiserum WX2 and 38 shared the same genomic PFGE pattern. Characteristics of the epidemics and experimental data suggest that the diarrhoea was caused by a nosocomial spread of C. perfringens, and not by a food‐borne outbreak.
Investigation of IFN Type‐I Receptor and IFN Regulatory Factor Expression Relating to Induction of 2′, 5′‐Oligoadenylate Synthetase in Cells Persistently Infected with the Mumps Virus
Authors: Fujii Nobuhiro , Yokosawa Noriko , Ishida Setsuko , Shirakawa Sachiko , Kubota Toru , Indoh Tomokazu , Fujinaga Kei , Yashiki Teruo
Published: November 2013

Poor induction of interferon‐induced 2′, 5′‐oligoadenylate synthetase (2–5AS) activity has been demonstrated in cells persistently infected with the mumps virus or human T‐lymphotropic virus type‐I (HTLV‐I). The suppression of 2–5AS induction is the result of the repression of 2–5AS gene expression at the transcription level. In a general way, after the binding of interferon‐α (IFN‐α) to cell surface‐specific receptors, expression of 2–5AS gene is thought to be regulated by some transacting factors, IFN‐regulatory factors (IRF‐1 and IRF‐2) and the IFN‐stimulated gene factor (ISGF‐3, a complex consisting of STAT‐1α, STAT‐2 and p48). To clarify the cause of the suppression mechanism(s), fluctuation in the number of IFN receptors and the levels of mRNAs in both IRF‐1 and IRF‐2 were examined in cells persistently infected with the mumps virus (FLMT and KBMT). There were few defferences in the number of IFN receptors and the level of IRF‐2 mRNA between persistently infected cells and uninfected control cells. After the treatment of cells with IFN, a slight reduction of IRF‐1 mRNA was found in persistently infected cells as compared with that of the uninfected control cells.
Sequence Variation of the Hypervariable Region in HCV Carriers with Normal ALT Levels: A Comparison with Symptomatic Carriers
Authors: Murashima Shiro , Sata Michio , Suzuki Hiroshi , Noguchi Seiji , Tanikawa Kyuichi
Published: November 2013

The clinical significance of the hypervariable region (HVR) in the N‐terminus of the E2/NS1 region, which encodes the putative envelope glycoprotein (gp 70) of HCV, has not yet been elucidated. We studied the relation between HVR changes and elevation of the alanine aminotransaminase (ALT) level due to liver cell injury as well as the persistence of HCV infection. Three patients (carrier group) who were HCV RNA positive and had normal ALT levels for as long as five years and three patients with high ALT levels were studied. None of the six patients had a history of treatment. HCV RNA was extracted from serum obtained from each patient in 1990 and 1995. The E2/NS1 region, including HVR‐1 and HVR‐2, was amplified using the RT‐PCR method. PCR products were cloned and nucleotide sequences were determined using the dideoxynucleotide chain termination method. No clear correlation was found between the ALT levels and the number of nucleotide substitutions in HVR‐1. The number of nucleotide substitutions in HVR‐1 during the five years was greater than in other regions. Furthermore, more nucleotide substitutions occurred in the 1st and 2nd codon positions of HVR‐1 than in the control region, even in the carrier group. In conclusion, HVR‐1 changes are probably a more important factor in persistent viral infection than liver cell injury.
Analysis of the Epitopes Recognized by Mouse Monoclonal Antibodies Directed to Yersinia enterocolitica Heat‐Shock Protein 60
Authors: Yamaguchi Hiroyuki , Miura Hitoshi , Ohsumi Kazuoki , Osaki Takako , Taguchi Haruhiko , Yamamoto Tomoko , Hanawa Tomoko , Ogata Sachio , Kamiya Shigeru
Published: November 2013

To determine amino acid sequences of the epitopes recognized by monoclonal antibodies (mAbs) 3C8 and 5C3 directed against Yersinia enterocolitica heat‐shock protein (HSP60), a dot blot analysis was perfomed using synthesized peptides of Y. enterocolitica HSP60 such as peptides p316‐342, p327‐359, p340‐366, p316‐326, p316‐321, p319‐323, and p321‐326 which represent positions of amino acids in Y. enterocolitica HSP60. The dot blot analysis revealed that 5C3 mAb reacted with p316‐342, p316‐326 and p321‐326, and 3C8 mAb p316‐342 and p316‐326. These results indicate that the epitopes recognized by the mAbs were associated with eleven amino acids, Asp Leu Gly Gln Ala Lys Arg Val Val Ile Asn, of p316‐326. The sequence homology between p316‐326 of Y. enterocolitica HSP60 and the rest of the HSP60 family suggests that the five amino acids of Lys, Arg, Val, Ile and Asn, which are highly conserved in the HSP60 family, might be related with the epitope recognized by 3C8. In contrast, it was also demonstrated that three amino acids of Leu, Gly and Val, which are not well conserved in the HSP60 family, might be related to the epitope recognized by 5C3.
Polymerase Chain Reaction for the Detection of Chlamydia psittaci in the Feces of Budgerigars
Authors: Takashima Ikuo , Imai Yumi , Itoh Norihiko , Kariwa Hiroaki , Hashimoto Nobuo
Published: November 2013

The polymerase chain reaction (PCR) targeting the ompA gene of Chlamydia psittaci was evaluated for its ability to detect chlamydiae in fecal specimens of budgerigars as compared with isolation procedures using cell culture and embryonated egg inoculations. Several procedures for PCR template DNA preparation were compared so as to determine their detection levels for chlamydiae propagated in cell culture in the presence of fecal materials. Tween‐20 and proteinase K treatments followed by centrifugation of the template DNA were found to be an appropriate procedure for DNA preparation for primary PCR. Subsequent nested PCR was shown to detect 4.8 IFU/ml or 84 particles/ml of chlamydiae. Chlamydiae in 50 fecal specimens from apparently healthy budgerigars were examined by nested PCR and several other methods. Nested PCR detected chlamydiae at a higher rate (12/50, 24%) than the isolation procedure in embryonated eggs (6/50, 12%). Primary PCR combined with the isolation procedure in cell culture gave a detection rate (5/50, 10%) similar to that of isolation from embryonated eggs. Detection rates by primary PCR (1/50, 2%) and in cell culture (0%) were inferior to the other procedures.
Colorimetric Assay System for Screening Antiviral Compounds against Hepatitis B Virus
Authors: Sudo Kenji , Konno Kenji , Shigeta Shiro , Yokota Tomoyuki
Published: November 2013

A highly sensitive, rapid, and accurate assay system was developed for the in vitro evaluation of anti‐hepatitis B virus (anti‐HBV) agents. Chronic HBV‐producing HB611 cells were used in combination with immunoaffinity purification, polymerase chain reaction (PCR), and hybrid capture detection. HB611 cells were incubated with putative anti‐HBV agents for 7 days in 96‐well microtiter plates. HBV was purified from HB611 cell culture media using immunoaffinity purification. The HBV DNA was extracted, amplified with PCR, and assayed using a hybrid capture colorimetric method. This assay provided quantitative detection of extracellular HBV DNA from 25 μl of cell culture media. Using the colorimetric method, we found that 50% effective concentration levels of several known anti‐HBV agents (HPMPA, PMEDAP, PMEA and others) were similar to those reported in studies using Southern blot analysis. These results demonstrate that this new and easily automated colorimetric assay system can be used for the rapid and accurate assessment of anti‐HBV compound selectivity.
The trpF Nucleotide Sequence and Its Promoter Analysis in Pseudomonas aeruginosa
Authors: Murata Takahiro
Published: November 2013

The nucleotide sequence of the gene for the phosphoribosyl anthranilate isomerase (PRAI) (trpF) in Pseudomonas aeruginosa was determined. The gene encoded a polypeptide of 211 amino acid residues and its initiation codon was UUG. Northern hybridization analysis showed only one transcript for trpF (about 700 nucleotides). Primer extension analysis revealed three transcription start sites which were at positions 52, 55 and 57 bases upstream of the initiation codon. A highly GC‐rich 9‐base‐pair inverted repeat with 5‐base‐pair spacing was found upstream of a putative Shine‐Dalgarno sequence. This inverted repeat could form a 23‐nucleotide stem‐loop structure in the transcript. Deletion of the inverted repeat slightly decreased the promoter activity of trpF. The presence of tryptophan had no effect on transcription of trpF.
The Prevalence of Listeria monocytogenes in the Environment of Dairy Farms
Authors: Ueno Hiroshi , Yokota Keiichi , Arai Takanori , Muramatsu Yasukazu , Taniyama Hiroyuki , Iida Takashi , Morita Chiharu
Published: November 2013

The prevalence of Listeria monocytogenes in the environment of dairy farms was surveyed from December 1993 to June 1994 in one city of Hokkaido. L. monocytogenes was isolated from 3 out of 5 farms investigated. Serovar 4b organism was isolated from the brain stem of a cow from one farm which was clinically diagnosed as having listeriosis. The same serovar of L. monocytogenes was also isolated from the rectal contents of a healthy cow, straw on the floor, straw in the barn, and silage scattered around the silo from the same farm. At another farm, with no reported cases of bovine listeriosis, serovar 1/2 organism was isolated from the same types of samples as the above mentioned farm except from straw on the floor. The difference in the isolation rates of the organism from straw on the floor between the two farms (22%: 5/23 vs 0%: 0/24) is considered to be caused by the different feeding methods of silage between the two farms.
Kinetics of Anti‐Mannan Antibodies Useful in Confirming Invasive Candidiasis in Immunocompromised Patients
Authors: van Deventer A. Johanna M. , Goessens Wil H.F. , van Zeijl Jan H. , Mouton Jan W. , Michel Marc F. , Verbrugh Henri A.
Published: November 2013

In studying the anti‐mannan antibodies longitudinally in serial serum samples of three immunocompromised patients, it was observed that anti‐mannan antibodies started to increase shortly after the moment that cultures of deep‐tissue sites became positive with Candida albicans. The mean anti‐mannan antibody titers determined in a group of 36 immunocompromised patients with invasive candidiasis increased within two weeks after the probable onset of invasive candidiasis. In contrast, anti‐mannan antibody levels in serial serum samples of 14 immunocompromised patients who were only colonized with C. albicans remained stable or decreased over time. The HA test measuring the anti‐mannan antibodies was 64% sensitive and 89% specific in determining invasive candidiasis. In contrast, antibodies specific for candidal cytoplasmic antigens or enolase alone were of little value in confirming invasive candidiasis in these immunocompromised patients.
Threonine‐74 Is a Key Site for the Activity of Clostridium perfringens Alpha‐Toxin
Authors: Nagahama Masahiro , Sakurai Jun
Published: November 2013

A mutant toxin (MT) that abolished almost 99% of the hemolytic activity of alpha‐toxin was isolated by random polymerase chain reaction (PCR) mutagenesis of the gene for Clostridium perfringens alpha‐toxin. In the mutant toxin, the amino acids at Tyr (Y)‐62, Thr (T)‐74 and Ile (I)‐345 were substituted with His, Ile and Met, respectively. Replacement of T‐74 with Ile by site‐directed mutagenesis resulted in the loss of hemolytic, phospholipase C and sphingomyelinase activities by 1/250‐fold of that of the wild‐type. The replacement of Y‐62 with Ile or I‐345 with Met alone did not affect the activities of the toxin. T74I mutant bound to sheep erythrocyte membranes and specifically bound [65Zn]2+ in Tris‐buffered saline, in the same manner as the wild‐type, and contained 2 mol of zinc ions per mol of protein. These results suggest that the T‐74 residue plays a key role in these biological activities of C. perfringens alpha‐toxin.
Detection of the Disease Severity‐Related Molecular Differences among New Thai Dengue‐2 Isolates in 1993, Based on Their Structural Proteins and Major Non‐Structural Protein NS1 Sequences
Authors: Thant Kyaw‐Zin‐ , Morita Kouichi , Igarashi Akira
Published: November 2013

We determined the nucleotide sequences of the whole structural protein gene of four new dengue‐2 viruses by the primer extension dideoxy chain termination method, using multiple cDNA clones for six overlapping gene regions. The nucleotide sequences of the major non‐structural protein NS1 gene of these viruses were also determined by direct sequencing of the reverse‐transcription polymerase chain reaction products. These viruses were isolated from dengue patients with different clinical severities in Nakhon Phanom, Northeastern Thailand in 1993. The results were compared with the sequences of prototype New Guinea C strain and other reference strains. All four viruses revealed highest homology to New Guinea C strain. The homology between each of the four strains and New Guinea C strain varies from 95.09% to 95.29% in its nucleotide sequences, and from 97.24% to 97.78% in its amino acid sequences covering all structural proteins and NS1 protein. The PreM region shows the highest divergence (6.59% to 7.32%) in its nucleotide sequence, whereas C protein is most highly conserved (only 1.75% to 2.63% divergence). Our data showed that there are certain molecular differences in the genomic structure of these four new isolates, which indicate the possibility that these changes are related with the virulence of the virus strains.
Fgr Expression Restricted to Subpopulation of Monocyte/Macrophage Lineage in Resting Conditions Is Induced in Various Hematopoietic Cells after Activation or Transformation
Authors: Hatakeyama Shigetsugu , Iwabuchi Kazuya , Ato Manabu , Iwabuchi Chikako , Kajino Kiichi , Takami Kimitaka , Katoh Masahito , Ogasawara Kazumasa , Good Robert A. , Onoé Kazunori
Published: November 2013

The c‐fgr gene product (Fgr) is a member of the src‐family of protein tyrosine kinases. We have established a monoclonal antibody (2H2) which recognizes the unique N‐terminal domain of the murine Fgr. In the present study, using immunohistochemical analysis and immune complex kinase assay with the 2H2, we investigated expression of Fgr in various cell populations and tissues in a murine system. In resting conditions, Fgr expression was confined to subsets of a monocyte/macrophage lineage. Thus, Fgr+ cells were detected in paracortical areas and medullas of lymph nodes, but seen only in marginal zones of the spleen and the medulla of the thymus. No Fgr+ macrophage was detected in other tissues, Peyer's patches, brain, heart, lung, liver, pancreas, kidney and peritoneal cavity. However, immune complex kinase assay revealed that, upon stimulation, T and B cells as well as peritoneal macrophages expressed significant levels of Fgr molecules. Transformed cell lines of lymphoid origin, EL‐4 and LK35.2, which are T and B lineage lymphomas, respectively, also expressed Fgr molecules. Thus, various cells of hematopoietic origin appeared to possess a potentiality to express Fgr following activation or transformation. The present findings may help elucidate the functional significance of Fgr in immunologically committed cells in either activated or non‐activated conditions.
Immunochemical Characteristics and Localization on Cells of Protective Antigen (PAg) Prepared from Leptospira interrogans Serovar lai
Authors: Masuzawa Toshiyuki , Nakamura Ryuta , Beppu Yoshihito , Yanagihara Yasutake
Published: November 2013

Immuno‐electron microscopic methods revealed that the protective antigen (PAg) of Leptospira interrogans serovar lai exists on the outer envelope sheathing the leptospiral cell body. PAg lost its protective activity after treatment by hydrolysis with 2 M formic acid at 100 C for 2 hr, or oxidation with periodate at 4 C for 40 hr. The antigenic oligosaccharide fraction was further purified from the hydrolyzed PAg by immunoaffinity column coupled with protective monoclonal antibody, LW2, and by gel filtration of HPLC. The antigenic oligosaccharide fraction contained two unknown sugars and 4‐O‐methylmannose (molar ratio 3:5:1). These findings suggested that these sugars are components of an antigenic determinant contributing to the protective immunity against serovar lai infection.
Isolation and Characterization of Teichoic Acid‐Like Substance as an Adhesin of Staphylococcus aureus to HeLa Cells
Authors: Matsuura Takashi , Miyake Yoichiro , Nakashima Seiji , Komatsuzawa Hitoshi , Akagawa Yasumasa , Suginaka Hidekazu
Published: November 2013

A cell wall component that bound to HeLa cells (HeLa cell‐binding CWC) was isolated from a clinical isolate of Staphylococcus aureus. The HeLa cell‐binding CWC was resistant to heat (100 C, 1 hr) and proteases, did not stain with Coomassie Brilliant Blue R‐250 on SDS‐PAGE but stained as a broad band with antiserum against the strain on Western blots. These data suggest that the HeLa cell‐binding CWC is not a protein, and may be teichoic acid. Purified teichoic acid bound to HeLa cells, whereas fractions without teichoic acid did not. In Western blots, HeLa cell‐binding CWC appeared as a broad band of less than 35 kDa, similar to that of purified teichoic acid. These data suggest that the HeLa cell‐binding CWC obtained in this study is teichoic acid. Teichoic acid inhibited S. aureus adherence to HeLa cells and bound to the cells time and dose dependently, in a saturable and reversible manner, and therefore appears to be an adhesin of S. aureus to HeLa cells.
Analysis of the Phospholipase C Gene of Clostridium perfringens KZ1340 Isolated from Antarctic Soil
Authors: Kameyama Kohtaro , Matsushita Osamu , Katayama Seiichi , Minami Junzaburo , Maeda Masazumi , Nakamura Shinichi , Okabe Akinobu
Published: November 2013

Clostridium perfringens KZ1340 isolated from Antarctic soil was first classified as Clostridium plagarum and later as a lecithinase‐negative variant of C. perfringens. Although the strain produced no detectable lecithinase (phospholipase C, PLC) activity in the culture supernatant, it was shown by Southern blot hybridization to possess a PLC‐encoding gene (plc). To determine the cause of the PLC deficiency, we cloned and sequenced the plc gene from KZ1340. The deduced amino acid sequence consists of 398 amino acid residues, coinciding with those of the plc genes previously determined. Tyrosine was substituted for histidine at amino acid position 148, which is thought to bind a zinc ion essential for PLC activity. Northern blot analysis revealed that KZ1340 expressed the plc gene at an extremely low level. Furthermore, the plc gene cloned from C. perfringens strain 13 into a plasmid was expressed weakly in KZ1340, compared to that in strain 13. This indicates that the former strain represses plc gene expression in trans. When a phylogenetic tree of plc genes was constructed, the KZ1340 plc gene formed a monophyletic branch along with those of various other C. perfringens strains, supporting the classification of the strain as a variant of C. perfringens.
Evidence That Two Distinct Populations of Rabbit Anti‐Idiotypic Antibodies Are Induced by Three Monoclonal Antibodies Specific for Bluetongue Virus Core Protein VP7
Authors: Lin Min , Zhou En‐Min
Published: November 2013

Three groups of anti‐idiotypic antibodies (anti‐Id or Ab2), designated RAb2‐A, RAb2‐B1, and RAb2‐B2, were isolated from rabbit antiserum raised against three monoclonal antibodies (mAbs) (M1875, M1877, and M1886) specific for the bluetongue virus core protein, VP7. RAb2‐A was specific for the idiotype of M1875. RAb2‐B1 and RAb2‐B2, isolated through the M1877 and M1886 affinity columns, respectively, were directed against the common idiotype that is shared by M1877 and M1886 and therefore classified in the same population (RAb2‐B). Further characterization revealed that the two anti‐Id populations, RAb2‐A and RAb2‐B, were significantly different. RAb2‐A was an Ab2β type of anti‐Id since (i) its reaction with M1875 was inhibited by the antigen; (ii) it inhibited the M1875‐VP7 interaction; and (iii) it elicited anti‐VP7 antibody response in Balb/c mice. In contrast, RAb2‐B may represent an Ab2α type of anti‐Id since its reactions with M1877 or M1886 were not inhibited by the antigen, even though it inhibited mAbs from binding to the antigen. These results indicated that RAb2‐A and RAb2‐B represent two distinct populations of anti‐Ids to anti‐VP7 mAbs with similar epitope specificity.
Characterization of Glycoprotein H and L of Human Herpesvirus 7
Authors: Mukai Tetsu , Hata Atsuko , Isegawa Yuji , Yamanishi Koichi
Published: November 2013

The genes encoding the glycoproteins H (gH) and L (gL) of human herpesvirus 7 (HHV‐7) have been identified. The gH open reading frame (ORF) was 2,070 base pairs in length and encoded a predicted 690 amino‐acid protein. The gH contained characteristics of a transmembrane glycoprotein including 10 consensus N‐linked glycosylation sites, 12 cysteine residues, a potential amino‐terminal signal sequence and a predicted transmembrane segment located near the carboxyl terminus. The gL ORF was 738 base pairs in length and encoded a predicted 246 amino‐acid protein. Four possible N‐glycosylation sites and 6 cysteine residues existed within gL. The predicted amino‐acid sequences of the HHV‐7 gH and human herpesvirus 6 variant A (HHV‐6A) gH gene products exhibited 23.6% identity to each other, and those of the gL gene products had 26.0% identity. Upon in vitro translation of the gL gene, the addition of microsomal membranes resulted in two modified products with molecular weights of 32 kDa and 35 kDa from the unmodified initial translation product of 26 kDa. An amino‐terminal portion of gH and the full length of gL were expressed as glutathione S‐transferase fusion proteins, and these proteins were used to raise immune sera in mice. Lysates of cells infected with HHV‐7 were subjected to immunoprecipitation analysis. Approximate molecular weights of 33, 37, 80 and 90 kDa polypeptides were immunoprecipitated with antibodies against the gH protein. Antibodies against the gL protein polypeptides with the same molecular weights were also precipitated, and were observed with the antibodies against the gH protein. These results suggest that HHV‐7 gH and gL may form a heterodimeric complex with each other in HHV‐7 infected cells, as has been reported for other herpesviruses.
Suppressed Induction of 2‐5AS in Cells with HTLV‐I Is Not Associated with the Fluctuation of IFN‐Receptor, IFN‐Regulatory Factors (IRF‐1 and IRF‐2) or STAT‐1α
Authors: Yokosawa Noriko , Indoh Tomokazu , Kubota Toru , Yashiki Teruo , Fujii Nobuhiro
Published: November 2013

Some viruses seem to be capable of suppressing interferon (IFN)‐induced 2′, 5′‐oligoadenylate synthetase (2‐5AS) induction. Cells infected with human T‐lymphotropic virus type‐I (HTLV‐I) show different natures for the constitutive production of IFN‐γ or sensitivity to IFN. Poor induction of 2‐5AS was found in IFN‐γ producer cells carrying HTLV‐I (MT‐1, MT‐2 and SMT‐1). On the other hand, in non‐ or low‐producing cell lines of IFN‐γ such as HUT102 and OKM‐2, significant activity of 2‐5AS was induced by treatment with IFN‐α. A satisfactory level of IFN receptor was detectable in SMT‐1 cells in spite of the poor induction of 2‐5AS. There were no differences in either the interferon regulatory factor‐2 (IRF‐2) mRNA transcript or the level of STAT‐1α between SMT‐1 and HUT102 cells. However, the transcription of IRF‐1 mRNA was slightly reduced in SMT‐1 cells as compared with that of HUT102 cells.
Adjuvanticity of an Ornithine‐Containing Lipid of Flavobacterium meningosepticum as a Candidate Vaccine Adjuvant
Authors: Kato Hiroshi , Goto Norihisa
Published: November 2013

Ornithine‐containing lipids (OrnL) extracted from Flavobacterium meningosepticum have been reported to have various biological activities such as B‐cell mitogenicity and macrophage activation to generate interleukin‐1 and prostaglandin E2. We, using ovalbumin (OVA) as an antigen, evaluated the adjuvant activity of OrnL as an immunological adjuvant in BALB/c mice. OrnL showed the function of forming liposome‐like vesicles retaining biological activities when prepared as either small unilamellar or dehydration‐rehydration vesicles. Although OrnL was not shown to have enough entrapping efficacy for use as a vaccine adjuvant, phosphatidylglycerol (PG) and cholesterol (CHOL) added to stabilize the vesicle membrane increased the entrapping efficacy to the same extent as that of conventional liposomes. Furthermore, the stabilized OrnL vesicles tolerated centrifugation to remove non‐entrapped antigens. Completely antigen‐entrapped OrnL vesicles including PG and CHOL induced a significantly greater enhancement of IgG antibody production than did aluminum hydroxide gel in BALB/c mice from week 6. These results indicate that OrnL can be utilized as an immunological adjuvant for vaccines.
Studies of Polymorphic DNA Fingerprinting and Lipid Pattern of Mycobacterium tuberculosis Patient Isolates in Japan
Authors: Chaicumpar Kunyaluk , Fujiwara Nagatoshi , Nishimura Osamu , Hotta Hisako , Pan Jiong Wei , Takahashi Mitsuyoshi , Abe Chiyoji , Yano Ikuya
Published: November 2013

Strain differentiation by DNA restriction fragment length polymorphism (RFLP) has been used mainly for the epidemiological purpose of Mycobacterium tuberculosis infection. In this study, we tried to connect the molecular and phenotypic characteristics of M. tuberculosis patient isolates by comparing the DNA fingerprints obtained by RFLP using IS6110 and lipid patterns using two‐dimensional thin‐layer chromatography (2‐D TLC) with silica gel, since M. tuberculosis has a lipid‐rich cell envelope which contributes to the virulence and immunomodulatory properties. We found that 66 isolates of M. tuberculosis from tuberculosis patients showed that the occurrence of IS6110 varied from 1 to 24 copies. The IS6110 patterns were highly variable among isolates. Fifty different RFLP patterns were observed, and 12 RFLP patterns were shared by two or more strains. By computerized analysis of the RFLP patterns of M. tuberculosis patient isolates, we found that 95% of the isolates fell into seven clusters, from A to G, with at least two isolates in each (> 30% similarity). Among the cellular lipids, the phospholipid composition did not differ by strain, whereas the glycolipid pattern differed markedly. Especially, the relative concentration of cord factor and sulfolipid, both of which were known as virulent factors, varied by strain. The fingerprints of some strains showed an association between the DNA and glycolipid patterns, even though some of the same DNA fingerprint strains showed differences in lipid patterns. Among the patient isolates, M. tuberculosis strain 249 possessed a specific glycolipid with 2‐O‐methyl‐L‐rhamnose and L‐rhamnose, which is rarely found in other strains. This glycolipid showed serological activity against the sera of tuberculosis patients, even if the reactivity was not as strong as trehalose dimycolate. It also showed the inhibition of phagosome‐lysosome fusion in macrophages, suggesting involvement with virulence. These results suggest that RFLP analysis using IS6110 is useful for clustering the human isolates of M. tuberculosis, however, for further strain differentiation on virulence, a lipid analysis provides more information.
Father‐to‐Mother‐to‐Infant Transmission of HIV‐1: Clonally Transmitted Isolate of Infant Mutates More Rapidly than That of the Mother and Rapidly Loses Reactivity with Neutralizing Antibody
Authors: Okamoto Yukari , Shiosaki Koichi , Eda Yasuyuki , Tokiyoshi Sachio , Yamaguchi Yumi , Gojobori Takashi , Hachimori Takashi , Yamazaki Shudo , Honda Mitsuo
Published: November 2013

The sequences of the V3 loop and surrounding regions of human immunodeficiency virus type‐1 from a father‐to‐mother‐to‐infant trimmer were studied and the horizontal and vertical transmissions compared. The father's virus was variable for reactivity with neutralizing antibody and sequences of the V3 loop central core sequence. In contrast, the mother's viral sequences were much less diverse and reacted with a virus neutralizing antibody. The infant's viral sequences were also less diverse than those of the father, and N‐glycosylation sites were conserved. By phylogenetic analysis, the major clone, of which V3‐peptide reacted with the neutralizing antibody, was found to be transmitted from the mother to her infant; however, the mutated minor clones did not bind to the antibody. These findings suggest that both horizontal and vertical virus transmission were selective, and that the clonally transmitted virus in infants mutates more rapidly than viruses in the mother, to whom the virus was horizontally transmitted.
Two Distinct Populations of Primary Cytotoxic Cells Infiltrating into Allografted Tumor Rejection Sites: Infiltration of Macrophages Cytotoxic against Allografted Tumor Precedes That of Multiple Sets of Cytotoxic T Lymphocytes with Distinct Specificity to Alloantigens
Authors: Yoshida Ryotaro , Matsuura Akihiro , Einaga Kuniko , Ushio Yumiko , Yamamoto Naoki , Yoneda Yukio
Published: November 2013

It has been reported that the rejection of tumor allografts is mainly mediated by cytotoxic T lymphocytes (CTLs). Here, we characterized the cytotoxic effector cells of C57BL/6 (B6; H‐2b) mice infiltrating into the rejection site of the i.p. allografted Meth A fibrosarcoma (or P815 mastocytoma) cells of H‐2d origin. Two types of cytotoxic cells (i.e., CD8+ CTLs and macrophages (Mφs)) were identified by flow cytometric fractionation of the infiltrates or by specific in vitro elimination of cells either with antibody (Ab)‐coated beads or with an Ab‐plus complement. Of particular interest, these effector cells showed distinct and unique target specificities. First, the CTLs were inactive against transplanted tumor (e.g., Meth A) cells, whereas they were cytotoxic against donor‐related concanavalin A (Con A) blasts as well as CTLL‐2 (H‐2b) cells transfected with a class I gene of H‐2d origin. A cold target competition assay suggested that the CTLs were composed of multiple sets of T cells, each of which specifically recognized different allo‐antigens. Second, the Mφs lysed the allografted tumor cells but were inert toward the Con A blasts and the CTLL‐2 transfectants. Unexpectedly, the infiltration of Mφs preceded the infiltration of CTLs by several days during the course of rejection. These results indicate that two distinct populations of unique cytotoxic cells (i.e., CTLs and Mφs) are induced in the allografted tumor rejection site, and that the infiltration of cytotoxic Mφs responsible for rejection precedes that of the CTLs cytotoxic against cells expressing donor‐related allo‐antigens.
Seasonal Variations in the Presence of Antibodies against Coxiella burnetii in Dairy Cattle in Hokkaido, Japan
Authors: Yanase Tsuyoshi , Muramatsu Yasukazu , Ueno Hiroshi , Morita Chiharu
Published: November 2013

The prevalence and seasonal variations of infection by Coxiella burnetii in cattle were investigated seroepidemiologically on a farm in Hokkaido, Japan, by an immunofluorescent antibody test. A total of 364 serum samples from 28 cows were collected from August 1993 to October 1995 in two barns on the farm. It was found that the number of antibody‐positive cows and their antibody titers were significantly elevated in winter and decreased in summer. In addition, antibodies were detectable in seroconverted cows for about five months.
Consensus Sequence on the Genes Encoding the Major Outer Surface Proteins (OspA and OspB) of Borrelia garinii Isolate
Authors: Jianhui Wang , Masuzawa Toshiyuki , Komikado Tetsuro , Yanagihara Yasutake
Published: November 2013

Japanese Lyme borrelias classified as ribotype IV is predominant among isolates derived from clinical specimens, reservoir rodents and Ixodes persulcatus ticks, and has been characterized as Borrelia garinii. These B. garinii isolates have antigenic and genetic features apparently different from North American, European and other Asian isolates, especially in major outer surface proteins A (OspA) and B (OspB). In this study, we cloned and sequenced the genes encoding OspA and OspB from B. garinii strain FujiP2 (ribotype IV strain) isolated from I. persulcatus in Shizuoka, Japan. A sequence analysis revealed significant differences to the previously published sequences of ospA and ospB of B. burgdorferi sensu lato. The open reading frames of ospA and ospB consist of 822 and 888 nucleotides corresponding to the proteins of 273 and 295 amino acids, with molecular weights of 29,643 and 31,786 daltons, respectively. The most interesting finding is that the two osp genes share a consensus 282 bp sequence in their carboxy‐terminal portions and that the ospB gene is flanked by a 282 bp‐long direct repeat sequence. The deduced amino‐acid (aa) sequences of OspA and OspB of strain FujiP2 showed 60.1% homology, and have overall similarities of 70.5%, 70.3% and 75.6% to OspAB proteins of B. burgdorferi sensu stricto strain B31, Borrelia afzelii strain ACA1 and Borrelia garinii strain Ip90, respectively.
Characteristics and Pathogenicity of Non‐Melibiose‐Fermenting Strains of Yersinia pseudotuberculosis O3
Authors: Nagano Tetsuji , Ichimura Kazuhisa , Haji Norihiko , Nagao Kazuya , Someya Kazuhiko , Kiyohara Tomoko , Suzuki Kiyomi , Tsubokura Misao , Otsuki Koichi
Published: November 2013

The biological properties of non‐melibiose‐fermenting (NMF) strains of Yersinia pseudotuberculosis 03 were investigated. These strains were clearly distinguished from representative melibiose‐fermenting (MF) strains of Y. pseudotuberculosis 03 by their pathogenicity in mice, sensitivity to some phages, production of catalase, restriction endonuclease analysis of virulence plasmid DNA with BamHI, detection of specific yersinia outer‐membrane proteins with SDS‐PAGE, antigenicity of the outer‐membrane proteins and neutrophil resistance to phagocytosis. The pathogenicity of NMF strains was clearly less than that of MF strains. In addition, the resistance of NMF strains to phagocytosis and catalase activity was evidently weaker than that of MF strains. These results suggested that the difference of pathogenicity was due to the ability of catalase production. Although the relationship between the above characteristics and melibiose‐fermentation was not analysed, the pathogenicity of Y. pseudotuberculosis 03 strains can probably be predicted by testing melibiose‐fermentation and catalase production.
Production of a T‐Cell Clone Which Reacts with Membrane Proteins of Acholeplasma laidlawii
Authors: Kuwano Koichi , Ono Saori , Akashi Akira , Ohishi Masahiro , Shigematsu Hideki , Arai Sumio
Published: November 2013

The role of cellular immunity in mycoplasma infection is not completely understood. In this study, we established mycoplasma‐specific T‐cell clones to evaluate cellular immunity in mycoplasma infection. We developed a T‐cell clone (G‐10) which was stimulated with Acholeplasma laidlawii. The T‐cell clone G‐10, CD4+ and T‐cell receptor (TCR) αβ+ recognized the 42‐ and 65‐kilodalton (kDa) membrane proteins of A. laidlawii and responded to A. hippikon. Hence, the application of mycoplasma‐specific T cells such as G‐10 in animal models may allow the assessment of cellular immune response to mycoplasma infection.
Normal Human Fibroblasts Immortalized by Introduction of Human Papillomavirus Type 16 (HPV‐16) E6‐E7 Genes
Authors: Shiga Takashi , Shirasawa Hiroshi , Shimizu Kumiko , Dezawa Mari , Masuda Yoshiaki , Simizu Bunsiti
Published: November 2013

This report demonstrates that normal human fibroblasts can be immortalized by the introduction of HPV‐16 E6‐E7 genes. We designed zinc‐inducible expression plasmids with HPV‐16 E6, E7 or both. Each plasmid was introduced into normal human fibroblasts (TIG‐3 cells) using lipofection methods. Only trans‐fectants with the HPV‐16 E6‐E7 zinc‐inducible expression plasmid, which were cultured in medium supplemented with 100 μM ZnSO4, overcame crisis and could be cultured over 200 population doubling levels (PDLs). These cell lines showed the reactivation of telomerase after crisis, and morphological alterations were also observed.
Identification of Murine T Cells Reactive with the Bacterial Superantigen Yersinia pseudotuberculosis‐Derived Mitogen (YPM) and Factors Involved in YPM‐Induced Toxicity in Mice
Authors: Miyoshi‐Akiyama Tohru , Fujimaki Wakae , Yan Xao‐Jie , Yagi Junji , Imanishi Ken'ichi , Kato Hidehito , Tomonari Kyuhei , Uchiyama Takehiko
Published: November 2013

We previously reported that Yersinia pseudotuberculosis‐derived mitogen (YPM) acts as a superantigen to human T cells. In this study, we assessed the superantigenicity and toxicity of YPM using murine experimental models. YPM activated T cells to produce interleukin‐2 in a major histocompatibility complex class II molecule‐dependent manner. The T‐cell blasts induced by YPM expressed T‐cell receptor (TCR) β‐chain variable region (Vβ)7, VβS.1, Vβ8.2 and Vβ8.3. The injection of YPM into mice pre‐sensitized with D‐galactosamine induced lethal shock. This shock was blocked by the injection of monoclonal antibodies (mAbs) to CD4, TCR Vβ7 plus Vβ8, tumor necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ), but not by injection to CD8 or unrelated Vβ. These results indicate that YPM‐induced shock requires the presence of CD4+ T cells bearing TCR Vβ7 and Vβ8, and that endogenous TNF‐a and IFN‐γ mediate the lethal effects.
An ATP Bioluminescence Assay Applicable to Rapid Fluconazole Susceptibility Testing of Dermatophytes
Authors: Yoshida Tatsumi , Uchida Katsuhisa , Yamaguchi Hideyo
Published: November 2013

An ATP bioluminescence assay as a rapid reference method for fluconazole (FLCZ) susceptibility testing of dermatophytes, as well as yeasts, was developed and evaluated by comparing it with viability, turbidity and fungal protein content‐based conventional methods. FLCZ susceptibility results obtained with strains of Candida albicans and dermatophytes by the bioluminescence method in high‐resolution medium were well correlated with those obtained by conventional methods currently used in clinical microbiology laboratories or reported previously, including a broth dilution method by the National Committee for Clinical Laboratory Standards (NCCLS). Thus, ATP bioluminescence assay can be used to monitor fungal growth in liquid culture media. The procedure has considerable potential for the rapid testing of FLCZ susceptibility of dermatophytes and other fungi.
Genetic Control of Immune Responses to HIV‐1 env DNA Vaccine
Authors: Ishii Norihisa , Sugita Yasuyuki , Nakajima Hiroshi , Bukawa Hiroki , Asakura Yusuke , Okuda Kenji
Published: November 2013

We investigated the genetic control of immunoglobulin production and the delayed‐type hypersensitivity (DTH) response produced by an HIV‐specific DNA vaccine using several strains of mice. Murine antigen‐specific immunoglobulin production was determined by ELISA. The DTH response was assessed in terms of the footpad swelling reaction. All strains of mice, except for B10.RIII and B10.T(6R), exhibited strong immunoglobulin production and footpad swelling in response to the DNA vaccine. In vitro treatment of lymphoid cells with monoclonal antibodies showed that the footpad swelling response was mediated by CD4+8− and Ia— T cells. However, CD8+ T cells did not suppress footpad swelling. There was no difference in the induction of HIV‐specific immunoglobulin production or DTH response induced by the DNA vaccine among the strains, suggesting that HIV‐specific DNA vaccine is useful for immunizing various populations against HIV‐1.
Levels of Endogenous lnterleukin‐1, Interleukin‐6, and Tumor Necrosis Factor in Congenic Mice Infected with Borrelia garinii
Authors: Isogai Hiroshi , Kimura Koichi , Hayashi Shunji , Kubota Toru , Fujii Nobuhiro , Nishikawa Takeshi , Kotake Satoshi , Isogai Emiko
Published: November 2013

This study describes the levels of interleukin‐1 alpha (IL‐1α), tumor necrosis factor alpha (TNFα) and interleukin‐6 (IL‐6) in the sera and parenchymal organs of various congenic mouse strains infected with Borrelia garinii. A significant elevation of inflammatory cytokine levels was found in the organs of C3H/HeN (H‐2k) and B10.BR (H‐2k) mice but not in those of BALB/c mice (H‐2d). Focally produced cytokines can contribute to antimicrobial defense against these organisms. High levels of IL‐1α were observed in the sera of C3H/HeN, B10.BR and B10 (H‐2b) mice infected with B. garinii and they were associated with the presence of spirochetes in the skin. Thus, susceptible mice demonstrated a stronger cytokine response than resistant mice. This study presents in vivo evidence that B. garinii infection affects the immunopathogenesis of Lyme disease.
Replication of Feline Syncytial Virus in Feline T‐Lymphoblastoid Cells and Induction of Apoptosis in the Cells
Authors: Ikeda Yasuhiro , Itagaki Shin‐lchi , Tsutsui Shigeki , Inoshima Yasuo , Fukasawa Masashi , Tomonaga Keizo , Tohya Yukinobu , Maeda Ken , Mochizuki Masami , Miyazawa Takayuki , Mikami Takeshi
Published: November 2013

Feline syncytial virus (FSV) was isolated from feline peripheral blood mononuclear cells of FSV‐seropositive cats. When the susceptibility of feline T‐lymphocytes to FSV was examined using three strains of FSV, FSV antigens were detected in the FSV‐infected T‐lymphoblastoid cells. Further, a diversity of biological properties, including replication kinetics and syncytia formation, was noted among the strains, and condensation of chromatin and the fragmentation of cellular DNA were observed in the infected cells. From these data, we conclude that FSV is lymphotropic and can induce apoptosis in the lymphocytes.
RobA‐lnduced Multiple Antibiotic Resistance Largely Depends on the Activation of the AcrAB Efflux
Authors: Tanaka Toshihiko , Horii Toshinobu , Shibayama Keigo , Sato Kumiko , Ohsuka Shinji , Arakawa Yoshichika , Yamaki Ken‐ichi , Takagi Kenzo , Ohta Michio
Published: November 2013

RobA is a member of the XylS/AraC subfamily of DNA binding proteins, and when overexpressed, it induces multiple antibiotic resistance in Escherichia coli. In this study, we introduced a multicopy robA plasmid (pMEP1) and its derivative into OmpF mutants and an AcrAB‐deficient mutant. We found that a decrease in susceptibility to multiple antibiotics in these OmpF mutants when pMEP1 was introduced did not depend on OmpF porin expression. Interestingly, a ΔompF mutant (TK007) became more sensitive when pMEP1 was introduced. Moreover, no effect of RobA on the induction of multiple antibiotic resistance in an acrA1− mutant was observed. Therefore, we conclude that the multiple antibiotic resistance induced by the overexpression of RobA largely depends on the activation of the AcrAB efflux, as well as the activation of micF.
Successful Development of Air‐Dried Microplates (HP‐Plates) for Susceptibility Testing against Helicobacter pylori Isolates
Authors: Kawakami Yoshiyuki , Akahane Takayuki , Gotoh Akira , Okimura Yukie , Oana Kozue , Katsuyama Tsutomu
Published: November 2013

We have successfully developed and evaluated a new susceptibility testing procedure against Helicobacter pylori strains using air‐dried microplates “HP‐Plates” containing eight serially‐diluted anti‐H. pylori agents. HP‐Plate wells were reconstituted by the inoculation of 100 μl of H. pylori cell suspensions. After incubation at 37 C for 48 hr under humidified microaerophilic conditions, HP‐Plates were read visually with a circular mirror. We investigated the within‐day reproducibility tests of HP‐Plates using the six quality control (QC) strains we proposed. Of the 20 testings, determining the minimum inhibitory concentrations (MICs) of all the QC strains fell within ± 1 log2 dilution ranges. When 200 clinical isolates were tested with HP‐Plates and compared with the results obtained with the modified broth macrodilution method of NCCLS, more than 90% of the MICs also fell within ±1 log2 dilution ranges. We concluded that the HP‐Plate susceptibility test method is a practical and easily applicable alternative of susceptibility testing for clinical microbiology laboratories in determining the MICs of H. pylori isolates.
Triazine Dyes Inhibit HIV‐1 Entry by Binding to Envelope Glycoproteins
Authors: Hattori Toshio , Zhang Xiaoyan , Weiss Carol , Xu Younong , Kubo Toru , Sato Yoshimi , Nishikawa Shigemichi , Sakaida Hitoshi , Uchiyama Takashi
Published: November 2013

We have attempted to purify envelope (Env) glycoproteins of human immunodeficiency virus (HIV) from the culture supernatants of CHO‐Sec cells that secreted truncated 140‐kDa precursor and mature 120‐kDa Env glycoproteins. The concentrated culture supernatants were applied to a column coupled with cibacron blue 3GA (CB3GA) to separate albumin from the Env proteins because CB3GA, a triazine dye, has been known to have a high affinity to albumin. Unexpectedly, Env proteins as well as albumin bound to the column, and the bound Env proteins were eluted by increasing the ionic strength using KC1. Gp120 was eluted at 0.5–0.9 m of KC1, while a higher concentration (0.9–1.5 m) was necessary for the elution of gp140. The agarose gel coupled with reactive red 120 (RR120), another triazine dye with similar characteristics, also retained both Env proteins, and the bound Env proteins could be eluted in a similar manner. In addition, these agents inhibited syncytium formation caused by HTLV‐IIIB and HTLV‐IIMN. Inhibition was also seen when a virus‐free fusion assay between Env protein expressed in CHO cells and fluorescent labeled SupT1 cells were used. These findings indicate that triazine dyes bind to the functional regions of Env proteins of HIV‐1 that play important role(s) for HIV infection.