bluuebunny/crossref_metadata_2025 huggingface dataset:
Further Studies on Guinea Pig Z‐1 Antigen That Is Involved in Phagocytosis of Zymosan by Macrophages: Cell Type Distribution of the Antigen and Cross‐Reactivity of Anti‐Z‐1 with Human CR3
Authors: Tamoto Koichi , Nochi Hiromi , Tada Masahito , Kakinuma Toshihiko , Mori Yoki
Published: November 2013

We have previously identified a heterodimer molecule, Z‐1, on guinea pig peritoneal macrophages (Møs) by the newly prepared monoclonal antibody, anti‐Z‐1, and Z‐1 has been assumed to be the complement receptor type three (CR3) in this species. To clarify this assumption, the cell type distribution of the antigen in guinea pig and the cross‐reactivity of anti‐Z‐1 with other species were analyzed. It was demonstrated that Z‐1 was expressed on peritoneal Møs, pulmonary Møs, peritoneal polymorphonuclear leukocytes (PMN), peripheral neutrophils, and some subpopulations of spleen cells and of bone marrow cells, but not on erythrocytes, circulating lymphocytes, and lymphocytes in both spleen and bone marrow in detectable amounts. Thus the expression of Z‐1 seems to be restricted to phagocytes as is CR3 of other species. Furthermore, it was found that anti‐Z‐1 bound with peripheral neutrophils from human, horse and goat and HL‐60 cells differentiated into monocytes. Any cross‐reactivity of the antibody was not detected with neutrophils from rabbit, cow, sheep and dog and nondifferentiated HL‐60 cells. Human Z‐1 was indistinguishable from human CR3, since both were the heterodimer consisting of α chain of 170 kDa (pI = 6.6‐7.2) noncovalently associated with β chain of 100 kDa (pI = 5.6‐6.7). In addition, human CR3 in detergent‐lysate of neutrophils was completely adsorbed with anti‐Z‐1 F(ab')2‐Sepharose. These findings indicate that guinea pig Z‐1 shares an antigenic determinant with human CR3. It thus seems to be possible that Z‐1 may function as CR3 in guinea pigs.
Diversity in Molecular Mass of the Common EDTA‐Soluble Antigens of Clostridium chauvoei and Clostridium septicum
Authors: Hamaoka Takafumi , Terakado Nobuyuki , Nakamura Shinichi
Published: November 2013

Common EDTA‐soluble antigens of Clostridium chauvoei and C. septicum were examined by indirect‐immunofluorescence (IFA) and immunoblot analysis. The monoclonal antibodies (mAbs) specific for the 35 kDa antigen of C. chauvoei strain ATCC 10092 were used. These mAbs reacted with all 11 strains, 6 of C. chauvoei and 5 of C. septicum, in IFA. In immunoblot analysis with the mAbs, the bands at molecular mass of 35 kDa were found in all C. chauvoei strains, while the bands at 36 kDa were found in 4 of 5 strains of C. septicum. These results indicate that the 35 kDa antigen of C. chauvoei and the 36 kDa antigen of C. septicum possess a similar epitope recognized by the mAb.
Degradation of a Polymerase Chain Reaction (PCR) Product by Heat‐Stable Deoxyribonuclease (DNase) Produced from Yersinia enterocolitica
Authors: Nakajima Hiroshi , Itoh Ken‐ichiro , Arakawa Eiji , Inoue Masanao , Mori Tadashige , Watanabe Haruo
Published: November 2013

When crude deoxyribonucleic acid (DNA) preparations by boiling were used for the polymerase chain reaction (PCR) from pathogenic and non‐pathogenic Yersinia enterocolitica strains, the amplified products were degraded after their storage at 4 C. The degradation of products was prevented by the addition of ethylenediaminetetraacetate (EDTA) or treatment with proteinase K. These findings indicate that Y. enterocolitica produced heat‐stable deoxyribonuclease (DNase). Proteinase K treatment would be recommended to prevent heat‐stable DNase contamination in the DNA preparations for PCR from Y. enterocolitica strains.
Stimulation of the Synthesis of Histamine and Putrescine in Mice by a Peptidoglycan of Gram‐Positive Bacteria
Authors: Ando Takanobu , Endo Yasuo , Abe Masataka , Kumagai Katsuo
Published: November 2013

To clarify the base of in vivo biological activities of peptidoglycans of Gram‐positive bacteria, the effects of a polysaccharide peptide of Staphylococcus epidermidis peptidoglycan (SEPS) on the synthesis of histamine and putrescine in BALB/c mice were examined and compared with those of a lipopolysaccharide (LPS or endotoxin) of Gram‐negative bacteria. Within a few hours after its injection into BALB/c mice, SEPS induced histidine decarboxylase (HDC), the enzyme forming histamine, in the liver, lung, spleen and bone marrow, and ornithine decarboxylase (ODC), the enzyme forming putrescine, in the tissues except for the lung. SEPS induced HDC activity even in mast cell‐deficient mice and in nude mice. These effects of SEPS were essentially the same as those of LPS. However, the dosage of SEPS capable of inducing HDC and ODC was much higher (100 to 1,000 times) than that of LPS. We have reported that C3H/HeN mice are resistant to SEPS in producing acute arthritis, and their productions of IL‐1 and prostaglandin E2 are less than BALB/c mice sensitive to producing acute arthritis. In the present study, it was also found that C3H/HeN mice were markedly resistant to SEPS in inducing HDC activity.
Quantitative PCR for Detection of Femtogram Quantities of Interleukin‐8 mRNA Expression
Authors: Izutani Ryo , Ohyanagi Harumasa , MacDermott Richard P.
Published: November 2013

A quantitative polymerase chain reaction (PCR) assay for mRNA expression of interleukin‐8 (IL‐8), a neutrophil chemotactant and activator, was developed to examine the expression of this cytokine by colonic mucosa. A synthetic IL‐8 RNA deleted in size of native IL‐8 mRNA was used as an external control. The synthetic IL‐8 RNA was mixed with total RNA from cells and converted to cDNA and amplified by PCR simultaneously. The lower limit of sensitivity for the assay was found to be more than 1 femtogram of IL‐8 mRNA. The assay determined IL‐8 mRNA expression when the RNA was isolated from either human histiocytic lymphoma cell line U937 cells or human colonic mucosa obtained from colitis patients and healthy controls. The development of the rapid and sensitive assay should provide a means to more fully evaluate the role of this cytokine in diverse disease states with small scale.
Comparison of Antibody Titers against Borrelial Strains Isolated in Japan by the Microcapsule Agglutination Test for Serological Studies of Early Lyme Disease
Authors: Arimitsu Yoshiko , Seki Masaaki , Nakao Minoru , Miyamoto Kenji
Published: November 2013

Results of a passive microcapsule agglutination test using 2 Japanese strains, Borrelia garinii and Borrelia afzelii, were comparable to that of the passive microcapsule agglutination test using a Swiss strain Borrelia burgdorferi IRS in detecting antibodies of an early stage of Lyme disease patients in Japan. MCAT with microcapsule sensitized strain IRS seems likely to become one of the important tools for the early diagnosis of Lyme disease in the world. The highest titer of MCAT was detected in the serum samples of patients which were taken around 2 weeks after erythema chronicum migrans. The test is very simple and can be employed easily in diagnostic laboratories.
Successful Application of Enzyme‐Labeled Oligonucleotide Probe for Rapid and Accurate Cholera Diagnosis in a Clinical Laboratory
Authors: Miyagi Kazufumi , Matsumoto Yasukazu , Hayashi Kazu , Yoh Myonsun , Yamamoto Koichiro , Honda Takeshi
Published: November 2013

Two cholera cases were diagnosed using an enzyme‐labeled oligonucleotide probe (ELONP) hybridization test for detection of cholera toxin gene (ctx) in a clinical laboratory at Osaka Airport Quarantine Station. The ELONP test with suspicious colonies of Vibrio cholerae O1 grown on TCBS or Vibrio agar plates gave positive result for ctx within 3 hr. We also tried to apply the ELONP test for direct detection of ctx in their stool and their non‐selective culture. Specimens from Case #1, which contained 5.9 × 105 CFU/g of V. cholerae O1 in the stool, cultured for 7–8 hr or longer in alkaline peptone water or Marine broth at 37C, became positive for ctx. On the other hand, specimens from Case #2, which contained 8.7 × 108 CFU/ml (of V. cholerae O1 in the stool), gave positive result in this stool itself without any further culture. These data suggest that the ELONP test provides successfully a more rapid and accurate means of identifying “toxigenic” V. cholerae O1 in a clinical laboratory.
Gene Expression of Tumor Necrosis Factorα and Interferonγ in the Lungs of Mycoplasma pulmonis‐Infected Mice
Authors: Nishimoto Mitsunobu , Akashi Akira , Kuwano Koichi , Tseng Cheng‐chuang , Ohizumi Kotaro , Arai Sumio
Published: November 2013

ICR mice were infected intranasally with Mycoplasma pulmonis isolated freshly from the lungs of a rat with pneumonia. We demonstrated with high reproducibility the expressions of messenger RNAs of cytokines, tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ) in the lung tissue of M. pulmonis‐infected mice by the reverse transcriptase‐polymerase chain reaction and confirmed specific mRNA of the cytokines by restriction endonuclease digestion. Both the viable population of M. pulmonis in the lung tissue and the titers of the neutralizing antibody in the serum increased between 7 and 21 days, and reached their maximum 35 days after infection. The pneumonia in mice progresses with the development of lung lesions after 7 days of infection. The early lesions are characterized primarily by neutrophils and edema in the alveolar spaces. mRNAs prepared from the lung tissue of M. pulmonis‐infected and ‐uninfected mice were also tested for the presence of messages specific to TNFα and IFNγ by the reverse transcriptase‐polymerase chain reaction. The expression of the genes encoding TNFα and IFNγ was constitutively demonstrated from 24 hr through 35 days after the intranasal inoculation of M. pulmonis. Furthermore, cells of two types, adherent and nonadherent cells, in bronchoalveolar lavage fluids obtained from the mice 3 weeks after inoculation of M. pulmonis were also found to express the genes of TNFα and IFNγ respectively. These data suggest that these cytokines would play a role in both stimulation in the development of pathological changes in mycoplasmal infection, affecting the inflammatory responses.
Immunological Recognition of Fibronectin‐Binding Proteins of Staphylococcus aureus and Staphylococcus capitis, Strain LK499
Authors: Sakata Noriaki , Rozalska Barbara , Wadström Torkel
Published: November 2013

Antibodies to fibronectin‐binding proteins (FnBPs) of Staphylococcus aureus, including binding domain of FnBPA, the D region, or the A‐C regions of FnBPB were produced in rabbits and mice. These antibodies were used to characterize cell‐associated FnBPs of S. aureus strain Cowan I, S. aureus strain U320 and a coagulase‐negative Staphylococcus capitis strain LK499 as well as extracellular FnBPs in culture supernatants of the strain U320. FnBPs of S. aureus were predominantly FnBPA, while FnBPB was hardly detected on the cells or in culture supernatant of these S. aureus strains. Moreover, S. capitis strain LK499 possessed different FnBP(s) compared to S. aureus because the antibodies to S. aureus FnBPs did not recognize FnBP(s) on S. capitis.
Characterization of Monoclonal Antibodies for Identification of Borrelia japonica, Isolates from Ixodes ovatus
Authors: Masuzawa Toshiyuki , Kawabata Hiroki , Beppu Yoshihito , Miyamoto Kenji , Nakao Minoru , Sato Nanao , Muramatsu Koichi , Sato Nobutake , Johnson Russell C. , Yanagihara Yasutake
Published: November 2013

Monoclonal antibodies for identification of Borrelia japonica isolated from tick, Ixodes ovatus and long‐tailed shrew, Sorex unguiculatus in Japan and Borrelia related to Lyme disease (Borrelia burgdorferi sensu lato) were prepared and characterized. All isolates belonging to B. japonica and isolates from I. dentatus and cottontail rabbit in North America reacted with MAb O1441b against flagellin which was prepared from immunized mice with strain HO14, type strain of B. japonica, but isolates from I. persulcatus, patient, and wood mouse, Apodemus speciosus ainu, in Japan, and isolates belonging to B. burgdorferi, B. garinii and B. afzelii from North America and Europe did not. Strains used in this study reacted with MAb P62 against common antigen which was prepared from immunized mice with strain NT24 isolated from I. persulcatus in Japan, but B. japonica did not. These MAbs are useful for identification and differentiation of B. japonica and B. burgdorferi sensu lato in Japan.
Biological Activities of Lipopolysaccharide Isolated from Vibrio cholerae O139, a New Epidemic Strain for Recent Cholera in Indian Subcontinent
Authors: Shimizu Tadayori , Yanagihara Yasutake , Isshiki Yasunori , Kawamata Yasuhisa , Kondo Seiichi , Hisatsune Kazuhito
Published: November 2013

Biological activities of lipopolysaccharide (LPS) isolated from Vibrio cholerae O139, a new causative agent for recent cholera epidemic in Indian subcontinent, were investigated in comparison with those of LPS from O1 V. cholerae. V. cholerae O139 LPS exerted mitogenic activity, lethal toxicity and Shwartzman reaction to the same extent as those observed for O1 V. cholerae LPS, although these activities except for lethal toxicity were obviously lower than those of Salmonella typhimurium LT‐2 LPS used as a reference. It was, therefore, suggested that O139 LPS does not contribute to the high infective and pathogenic potentials of the V. cholerae O139 strain as in the case of O1 V. cholerae.
The Presence of Low Molecular Weight Germinative Substances in Bacillus cereus T Spore Extract
Authors: Shibata Hirofumi , Odahara Yoshihiko , Sasaki Fumiko , Hirota Osamu , Nishida Mikio , Tani Isamu
Published: November 2013

An extract from intact spores of Bacillus cereus T having a germination‐inducing activity was studied. Two distinct germinative principles were found through dialysis of the extract. One was diffusible through the dialysis membrane and the other was non‐diffusible. The activity of the former fraction was inhibited by the addition of 1 mM glycoletherdiamine‐N, N, N', N'‐tetraacetic acid (GEDTA), whereas the latter fraction was inactive unless GEDTA was added to the assay system. The diffusible principle maintained the major portion of the activity found in the crude spore extract. By means of high‐performance liquid chromatography (HPLC) using a gel permeation chromatography column, 9 fractions were obtained from the deproteinized diffusible fraction. Of those fractions, two fractions (No. 1 and No. 8) were responsible for the germination‐inducing activity, but no reconstituted activity was observed unless both fractions No. 1 and No. 8 were added to the assay system. Amino acid analysis of fraction No. 1 revealed that the fraction was rich in free amino acids, especially in alanine. On the other hand, by the use of reverse‐phase HPLC and fast atom bombardment mass spectrometry, it was concluded that the effective substance in fraction No. 8 was inosine. Based on these findings, it was suggested that the active substances in fraction No. 1 might be a free amino acid such as L‐alanine and/or Ca2+ and a Ca2+‐binding substance.
Evaluation of Salmonella Porins as a Broad Spectrum Vaccine Candidate
Authors: Tabaraie Bahman , Sharma Bal Krishan , Sharma Praveen Rishi nee , Sehgal Rakesh , Ganguly Nirmal Kumar
Published: November 2013

Porins were prepared from smooth strain of Salmonella typhi 0–901 and chemotype of rough mutant of S. typhimurium Ra‐30. Mice were immunized with both the porin preparations in different groups and challenged with S. typhimurium LT2–71 and S. enteritidis SH‐1269. Porin immunized mice showed significant protection (P <0.01) against challenge with homologous as well as heterologous strains. Hence, the use of porins may be attempted in future to protect against salmonellosis.
Migration of Murine Epidermal Langerhans Cells to Regional Lymph Nodes: Engagement of Major Histocompatibility Complex Class II Antigens Induces Migration of Langerhans Cells
Authors: Yamashita Keizo , Yano Akihiko
Published: November 2013

Langerhans cells are resident dendritic cells in the epidermis. Once they are loaded with epicutaneously‐delivered antigens, they leave the epidermis and migrate to the regional lymph nodes where they initiate primary T cell responses as antigen‐presenting cells. However, the stimulus that initiates such migration remains unknown. Because major histocompatibility complex class II (Ia) antigens on B lymphocytes or monocytic cells have been shown to function as signal transducers, we evaluated the effect of the engagement of Ia antigens on the migration of murine epidermal Langerhans cells. The intradermal injection of an anti‐Ia monoclonal antibody (mAb) reduced the density of Langerhans cells in epidermis and produced a dose‐ and time‐dependent increase in the frequency of cells reactive with NLDC145 (Langerhans cell‐ and dendritic cell‐specific mAb) within the regional lymph nodes. Injection of a control mAb had no effect. The NLDC145+ cells that were induced to accumulate in the regional lymph nodes were Ia+, large dendritic cells, some of which were positive for both NLDC145 and F4/80, a phenotype corresponding to that of murine epidermal Langerhans cells. Thus, the engagement of Ia antigens on Langerhans cells by mAb induces the migration of Langerhans cells from the epidermis to the regional lymph nodes. Analysis of these changes in Langerhans cells in vitro may help to reveal the biochemical sequence of events involved in the activation and differentiation of Langerhans cells.
Infectivity and Early Antibody Response to Borrelia burgdorferi Sensu Lato Isolated in Japan in Outbred Mice
Authors: Masuzawa Toshiyuki , Kurita Toru , Kawabata Hiroki , Muramatsu Koichi , Uchikawa Kimito , Yanagihara Yasutake
Published: November 2013

Borrelia burgdorferi sensu lato isolated from Ixodes ovatus (B. japonica), I. persulcatus and patients with erythema migrans (EM) in Japan were determined on infectivity and arthritis induction‐activity in outbred mice. Infectivity of B. japonica was weak and did not induce the development of footpad swelling by subcutaneous (s.c.) inoculation into the footpad. Challenged strain, NO129‐M of B. japonica, to ddY mice were reinoculated to the mice at various cell numbers (1 × 10‐1 × 106 cells/mouse). The strain isolated from the mouse did not reinfect ddY mice and did not induce the production of specific antibody to the homologous strain. On the other hand, strains from I. persulcatus and patients with EM in Japan infected the mice and induced a serious inflammatory response in Borrelia‐inoculated footpad as well as strains belonging to the three genospecies, B. burgdorferi sensu stricto, B. garinii, and B. afzelii, related to Lyme disease, from North America and Europe. The mice were infected with 10 cells of strain HP1 isolated from I. persulcatus in Hokkaido and of strain 297 isolated from a patient in the U.S.A. by subcutaneous inoculation into the hind footpad, or by intradermal inoculation into the back. Antigens of ca. 20, 23–24 (Osp C), 29, 39, 41 (flagellin) and 45 kDa reacted with the pooled sera from mice inoculated with strains HP1 and 297, but Osp A and Osp B did not.
Possible Mechanism of Action of β‐Lactam‐Enhancing Factor on Methicillin‐Resistant Staphylococcus aureus
Authors: Tajima Yutaka , Nagasawa Zenzo , Tanabe Ichiro , Kusaba Koji , Tadano Jutaro
Published: November 2013

We have recently found a factor (Factor T) in aged mixtures of tungstate and phosphate which greatly enhances the antibacterial effects of β‐lactams on methicillin‐resistant strains of staphylococcal species such as methicillin‐resistant Staphylococcus aureus (MRSA), but shows only weak effects on methicillin‐susceptible S. aureus and bacterial strains other than staphylococci. Factor T alone did not strongly inhibit cell metabolism and bacterial growth unless an excess amount was added. When Factor T was added to the culture medium beforehand, the growth of MRSA cells was rapidly suppressed just after addition of oxacillin (MPIPC). However, the growth of the cells was inhibited gradually when these two reagents were added in reverse order. For full expression of the enhancing effect, it seemed necessary for cells of MRSA strains to be incubated with Factor T for at least 2–3 hr. When the cells were washed after being sensitized by incubating them for 5 hr with Factor T, it took approximately 1 hr for the cells to recover their resistance to MPIPC. Factor T reduced the amount of penicillin‐binding protein‐2′ (PBP 2′), and thus sensitized the MRSA strains to β‐lactams.
A Similar Expression Pattern of Adhesion Molecules between Intermediate TCR Cells in the Liver and Intraepithelial Lymphocytes in the Intestine
Authors: Ohtsuka Kazuo , Hasegawa Katsuhiko , Sato Kazunari , Arai Katsumitsu , Watanabe Hisami , Asakura Hitoshi , Abo Toru
Published: November 2013

Two major populations of extrathymically differentiated T cells exist in the liver and intestine. Such T cells in the liver have TCR of intermediate intensity (i.e., intermediate TCR cells) and constitutively express IL‐2 receptor β‐chain (IL‐2Rβ), whereas those in the intestine, especially intraepithelial lymphocytes, have TCR of bright intensity, consisting of a mixture of IL‐2Rβ+ and IL‐2Rβ–. All mature thymocytes and thymus‐derived T cells seen in the peripheral immune organs are TCR‐bright+IL‐2Rβ– under resting conditions. When the expression pattern of adhesion molecules, including CD44, L‐selectin, LFA‐1 and ICAM‐1, was compared among these T‐cell populations, they displayed quite unique patterns of expression. All extrathymic T cells in the liver, intestine, and even other organs were CD44+L‐selectin– LFA‐1++ICAM‐1+, whereas thymocytes and thymus‐derived T cells were CD44– L‐selectin+LFA‐1+ICAM‐1–. This inverted expression of adhesion molecules between extrathymic T cells and thymus‐derived T cells might be associated with their unique tissue‐localization.
Approach to the Involvement of Influenza B Neuraminidase in the Cleavage of HA by Host Cell Protease Using Low pH‐Induced Cell Fusion Reaction
Authors: Yamamoto‐Goshima Fumi , Maeno Koichiro
Published: November 2013

ts7, a temperature‐sensitive mutant defective in neuraminidase (NA) of influenza B/Kanagawa/73, lacks NA enzymatic activity at the nonpermissive temperature (37.5 C). When MDCK cells were infected with the mutant at the permissive temperature (32 C) and exposed to pH 5.2 medium, extensive cell fusion occurred. In contrast, at the nonpermissive temperature cells did not show cell fusion at all unless they were pretreated with trypsin, suggesting that at 37.5 C the hemagglutinin (HA) of ts7 is expressed at the cell surface in an uncleaved form. It was also found that the replacement of RNA segment 6 of ts7 with that of wild‐type B/Lee resulted in the emergence of low pH‐induced fusion activity as well as NA enzymatic activity at the incubation temperature of 37.5 C and that the addition of bacterial NA to the cultures infected with ts7 at 37.5 C early in infection brought about low pH‐induced cell fusion. We suggest that the removal of neuraminic acid from the carbohydrate moiety of HA by NA is essential for the cleavage of HA by cellular protease.
Interspecies Transmission of Animal Rotaviruses to Humans as Evidenced by Phylogenetic Analysis of the Hypervariable Region of the VP4 Protein
Authors: Shirane Koji , Nakagomi Osamu
Published: November 2013

A phylogenetic tree constructed for the hypervariable region (aa 71–203) of the VP4 protein of 28 human and animal rotaviruses that were previously reported to belong to 13 distinct VP4 genotypes revealed unique positions of human rotavirus strains HCR3 and Ro1845, together with feline strain FRV64 and canine strains K9 and CU‐1, in the animal rotavirus lineages, lending strong support to the view that both HCR3 and Ro1845 were of animal rotavirus origin.
Thermal Stability of an Oral Killed‐Cholera‐Whole‐Cell Vaccine Containing Recombinant B‐Subunit of Cholera Toxin
Authors: Ahmed Zia Uddin , Hoque Mohammad Mainul , Rahman Abu Syed Mohammad Hamidur , Sack Richard Bradley
Published: November 2013

An oral killed cholera vaccine containing 1×1011 cells of Vibrio cholerae O1 (heat‐ or formalin‐killed) representing the Ogawa and Inaba biotypes and containing 1 mg of B‐subunit of cholera toxin (CTB) produced by recombinant DNA technology (the WC/rCTB vaccine) was subjected to temperatures of 4 C, 30 C or 42 C for up to 6 months time. Lipopolysaccharide antigen (LPS) and CTB content of the vaccine samples determined at various times remained unchanged during the study except for the CTB component which decreased by about 50% after 6 months of storage at 42 C. Immunogenicity determined by immunization of rabbits with the vaccine in Freund's complete adjuvant and measuring anti‐LPS and anti‐CTB antibody titers in the serum by an ELISA was also found to be unaltered. Lyophilization of the vaccine and storage at room temperature for 7 days also did not have any adverse effect on antigen content or immunogenicity as tested above. There was up to one log reduction in serum antibody titers after immunization without using any adjuvant or using Freund's incomplete adjuvant, and up to two logs following oral immunization. Immunization by oral feeding of the vaccine followed by RITARD challenge with a virulent V. cholerae O1 strain showed evidence of protection against severe or lethal diarrhea. The results suggest that the vaccine retains its antigen content and ability to induce antibodies unchanged when maintained at elevated temperatures for relatively long periods of time.
Nucleotide Sequence of Polymerase Chain Reaction Product Amplified from Rickettsia japonica DNA Using Rickettsia rickettsii 190‐Kilodalton Surface Antigen Gene Primers
Authors: Yan Yansheng , Uchiyama Tsuneo , Uchida Takahiro
Published: November 2013

The PCR product amplified from Rickettsia japonica with the primer pair Rr 190.70p and Rr 190.602n of R. rickettsii 190‐kDa antigen gene was cloned into M13mp19 RF DNA at the EcoRI site and sequenced by chemiluminescent DNA sequencing. The sequence revealed a molecular size of 533 base pairs (bp). The primer‐flanking region of 491 bp, an open reading frame, was compared with the corresponding region of R. rickettsii, demonstrating 35 nucleotide substitutions in R. japonica. The sequence of primer portions in R. japonica DNA was also analyzed, revealing one nucleotide substitution in the Rr 190.70p and two in the Rr 190.602n portion. The homology in the overall sequence of PCR‐amplified regions between R. japonica and R. rickettsii was 93% in nucleotide and 85% in putative amino acid structure. The sequence contains no cleavage site for the restriction endonuclease AfaI but two PstI sites giving three fragments of 121, 159, and 253 bp, which differentiated R. japonica from other spotted fever group rickettsiae in addition to R. rickettsii. The cleavage sites for endonucleases AluI, HinfI, and MunI that disappeared or appeared in the sequence by nucleotide substitution differentiated R. japonica from others, as did PstI. The estimation of molecular size of DNA fragments on polyacrylamide gel electrophoresis is discussed.
Restriction Fragment Length Polymorphism (RFLP) of Genomic DNA of Moraxella (Branhamella) catarrhalis Isolates in a Hospital
Authors: Kawakami Yoshiyuki , Ueno Ichiro , Katsuyama Tsutomu , Furihata Ken'ichi , Matsumoto Hideki
Published: November 2013

Epidemiological typing, based on restriction fragment length polymorphism (RFLP) by pulsed‐field gel electrophoresis (PFGE), was attempted for the 38 clinical isolates of Moraxella catarrhalis obtained at Shinshu University Hospital during the years 1987 and 1993. Digestion with SmaI or NotI generated well separable, 12 to 5 genomic DNA fragments ranging from 1,000 kb to 30 kb and the strains could be classified into 14 or 13 types, respectively. The electrophoretic profile differed with the strain in most of them and was hence useful to distinguish the each strain. Investigation for their RFLP have, however, suggested that majority of them, including the type strain ATCC25238, may have derived from a common ancestor.
Growth Medium Containing Cyclodextrin and Low Concentration of Horse Serum for Cultivation of Helicobacter pylori
Authors: Morshed Muhammad Golam , Karita Mikio , Konishi Hisanori , Okita Kiwamu , Nakazawa Teruko
Published: November 2013

The growth of Helicobacter pylori, a Gram‐negative microaerophilic bacterium, is often difficult and requires complex media with the supplementation of 5% to 10% blood or blood derivatives. We have found that Brucella broth supplemented with 1% heated horse serum and 0.1% β‐cyclodextrin supports the good growth of H. pylori. The degree of growth and production of urease and vacuolating cytotoxin in this medium were equal to those in the medium supplemented with 5% horse serum. This medium was found to be suitable for both the routine laboratory culture and primary isolation of H. pylori from biopsy samples.
Strand‐Specific Detection of Hantaan Virus RNA Sequences by In Vitro DNA Amplification
Authors: Isegawa Yuji , Ohshima Atsushi , Sokawa Yoshihiro , Yamanishi Koichi
Published: November 2013

Hantaan virus often causes a fatal human disease, hemorrhagic fever with renal syndrome (HFRS). An assay for strand‐specific detection and quantitation of Hantaan virus RNA was developed based on the polymerase chain reaction (PCR). A protocol for the efficient detection of both genomic RNA and its transcript is presented, in which a reverse transcriptase reaction (RTR) followed by PCR with two common primers was used to distinguish between positive‐ and negative‐stranded RNA. Using this method, the growth characteristics of Hantaan virus, strain 76‐118, were studied in Vero E6 cells. Positive‐stranded RNA was detected on the next day after infection, and the amount increased remarkably beginning from 3 days after infection. The negative‐stranded RNA (genomic), was detected at 2 days after infection which predominated over the transcript RNA. Three days after infection, the amount of viral RNA attained 80% of the maximum amount which was detected by 6 days after infection, while, 4 days after infection the amount of the positive‐stranded RNA became over 80% of the maximum amount of 6 days after infection and reached the amount of viral RNA. Both RNAs reached plateaus at 6 days after infection and after that the amounts of synthesized viral RNA and its transcripts were constant.
Failure of Mouse Peritoneal Macrophage Activation by the Purified Excreted/Secreted Antigens of Toxoplasma gondii
Authors: Cazabonne Patrick , Bessières Marie‐Hélène , Pipy Bernard , Séguéla Jean‐paul
Published: November 2013

This study was carried out to evaluate the effect of excreted/secreted antigens on macrophages infected by Toxoplasma gondii. Six proteins 28, 30, 45, 58, 63 and 145 kDa were separated by different chromatographic techniques. Mouse peritoneal macrophages were treated in vitro and in vivo with these purified fractions. Penetration and proliferation assays of T. gondii in the macrophages were performed in vitro. The different antigens used did not change the rate of penetration and proliferation of the parasites. Therefore, the secreted products, which are capable of provoking an immune response, could not directly activate the macrophages. Furthermore, the secreted products were not cytotoxic and neither did they possess a visible phospholipasic activity which would have increased penetration.
Thy.1lowCD3− Cells Sorted from Nylon Wool‐Passed Bone Marrow Cells Can Augment the H‐2 Identical but Not Non‐Identical Cytotoxic T Lymphocyte Precursors in Mixed Lymphocyte Cultures
Authors: Mukouyama Hideki , Tanabe Masao J.
Published: November 2013

Thy. 1lowCD3– cells obtained from nylon wool‐passed murine bone marrow (NW‐BM) cells by cell sorting did not express CD4, CD8, or T cell receptor‐α/β and ‐γ/δ on their cell surfaces. An extremely limited number of B10.BR (H‐2k) responder lymph node (LN) cells were stimulated with B10. D2 (H‐2d) stimulator spleen cells in cultures containing the minimum required dose of rat T cell growth factor (TCGF). In these cultures, the generation of cytotoxic T lymphocytes (CTL) was very low. B10.BR Thy.1lowCD3– NW‐BM cells, added to these cultures, could augment the CTL generation vigorously, but neither B10 (H‐2b) nor B10.D2 cells could. When B10 LN cells were used as responder cells in these cultures, B10 Thy. 1lowCD3– NW‐BM cells could augment the CTL generation, but neither B10.BR nor B10.D2 cells could. Similar findings were obtained when Lyt‐2+ cells or Thy.1+ L3T4– (CTL precursor) cells sorted from spleen cells were used as responder cells. Both elements, rat‐TCGF and Thy.1low CD3– NW‐BM cells, were essential for this augmentation of the CTL generation in this culture system because neither one alone could augment generation, and rat‐TCGF could be replaced by Thy.1+ Lyt‐2– helper T (Th) cells sorted from spleen cells. These findings showed that NW‐BM cells could augment CTL precursors in a self‐major histocompatibility complex (self‐MHC)‐antigen restricted manner, and further that both NW‐BM cells and Th cells had different and independent functions to induce CTL.
Signal Transmission through MHC Class II Molecules in a Human B Lymphoid Progenitor Cell Line: Different Signaling Pathways Depending on the Maturational Stages of B Cells
Authors: Naitoh Keiko , Ichigi Yasuhisa , Miyake Kensuke , Muraguchi Atsushi , Kimoto Masao
Published: November 2013

The function of MHC class II HLA‐DR molecules expressed on a human B lymphoid progenitor cell line FL8.2.4.4 (abbreviated as FL4.4) was examined. FL4.4 cells expressed HLA‐DR molecules and stimulation of the DR molecules by anti‐DR mAb or by superantigen TSST‐1 induced strong augmentation of homocytic aggregation and protein tyrosine phosphorylation in FL4.4 cells. Induced homocytic aggregation in FL4.4 consists both of LFA‐1/ICAM‐1‐dependent and ‐independent pathways as revealed by mAb blocking experiments. Metabolic inhibitors, NaN3 and cytochalasin B, blocked the induced homocytic aggregation of FL4.4. Early mature Daudi B cell lines also showed a similar type of homocytic aggregation by stimulation with anti‐DR mAb. Daudi cells are more sensitive to protein kinase inhibitors herbimycin A and H7 than FL4.4 cells in their blocking of induced homocytic aggregation, while W7 showed stronger inhibitory effects on FL4.4 cells than on Daudi cells. Western blotting analysis revealed that the stimulation of DR molecules induced protein tyrosine phosphorylation of 100‐kDa, 90‐kDa, 60‐kDa and 55‐kDa proteins in FL4.4 cells, while, in Daudi cells 110‐kDa, 100‐kDa and 80‐kDa proteins were phosphorylated. These results suggest that different signaling pathways through class II molecules are employed depending on the maturational stage of B‐cell differentiation.
Characterization of the Mode of Hantaan Virus Infection in Adult Mice Using a Nested Reverse Transcriptase Polymerase Chain Reaction: Transient Virus Replication in Adult Mice
Authors: Kariwa Hiroaki , Kamimura Mizuho , Arikawa Jiro , Yoshimatsu Kumiko , Takashima Ikuo , Hashimoto Nobuo
Published: November 2013

A polymerase chain reaction (PCR) for the detection of hantavirus genome was established and applied to analyze the mode of infection of Hantaan virus in adult ICR mice. The cDNA for the S genome segment of Hantaan virus was reverse‐transcribed from the total RNA of organs of the infected mice. The sequence in the S genome segment of Hantaan virus was successfully amplified by reverse transcriptase (RT)‐PCR followed by nested PCR. In 5‐week‐old ICR mice inoculated intraperitoneally with Hantaan virus, strain 76‐118 (1.3 × 105 FFU/mouse), the virus was detected in clots and lungs from 3 to 10 days post‐inoculation (p.i.) by nested PCR and virus‐isolation techniques. No virus was detected in any specimens collected on 1 day and after 28 days p.i., and in spleens and brains through the observation period by both methods. The antibody which was measured by indirect immunofluorescence antibody assay (IFA) appeared at 7 days p.i. and the geometric mean titer was elevated to its maximum level of 1: 203 at 10 days p.i., maintaining the same level until 35 days p.i. These results suggest that adult mice are transiently infected with Hantaan virus.
Comparison of a Reversed Passive Latex Agglutination and a Polymerase Chain Reaction for Identification of Cholera Toxin Producing Vibrio cholerae O1
Authors: Honma Yasuko , Higa Naomi , Tsuji Takao , Iwanaga Masaaki
Published: November 2013

Production of cholera toxin (CT) in AKI medium and conservation of CT gene (ctx) of 49 strains of Vibrio cholerae O1 were compared by reversed passive latex agglutination (RPLA) and polymerase chain reaction (PCR). The production of CT agreed with conservation of the ctx in 48 out of the 49 strains. Ten strains were positive, and 38 strains were negative by both methods. Only one strain was negative in RPLA and positive in PCR. This suggested that the combination of AKI‐SW and RPLA is comparable to PCR to identify CT‐producing V. cholerae O1.
Carbohydrate‐Dependent Biological Activities of Glycosylated Human Interferon‐β on Human Hepatoblastoma Cells In Vitro
Authors: Utsumi Jun , Matsuo‐Ogawa Etsuko , Nagahata Takemitsu , Kasama Kyoko , Kagawa Yasuhiro , Satoh Yu‐ichiro
Published: November 2013

To evaluate the relationship between the sugar chain structure and biological activity, fibroblast‐derived glycosylated human interferon‐β, Chinese hamster ovary cell‐derived glycosylated recombinant human interferon‐β and Escherichia coli‐derived unglycosylated recombinant human interferon‐β were evaluated using human hepatoblastoma cells in vitro. Native fibroblast interferon‐β expressed more cell‐growth inhibitory action, 2′5′‐oligoadenylate synthetase induction, and the inhibition of hepatitis B virus DNA replication than its asialoform and two recombinant interferon‐βs. These results showed that the sugar chain structure of human interferon‐β affects its biological activity on human hepatoblastoma cells.
Characterization of an Enterotoxin Produced by Vibrio cholerae O139
Authors: Nakashima Kazutoshi , Eguchi Yukinori , Nakasone Noboru
Published: November 2013

A cholera‐like enterotoxin was purified from Vibrio cholerae O139 strain AI‐1841 isolated from a diarrheal patient in Bangladesh. Its characteristics were compared with that of cholera toxins (CTs) of classical strain 569B and El Tor strain KT25. Al‐1841 produced as much toxin as O1 strains. The toxins were indistinguishable in terms of their migration profiles in conventional polyacrylamide gel disc electrophoresis, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and isoelectrofocusing as well as their affinity for hydroxyapatite. The skin permeability factor activity and the fluid accumulation induced in rabbit ileal loops of the toxin of AI‐1841 were identical to those of the CTs. Three toxins equally reacted against anti‐569B CT antiserum in Western blotting, and their B subunits formed a precipitin line against any anti‐B subunit antiserum by double gel immunodiffusion. Anti‐569B CTB antibody neutralized the three toxins in their PF activities and enterotoxicities. The amino acid sequence of 1841 toxin B subunit was identical with that of KT25 CTB, corresponding to the DNA sequence of ctxB from El Tor strains of the seventh pandemic. We concluded 1841 toxin was identical to CT of the seventh pandemic El Tor vibrios.
CD3+/TCR‐αβ− Cells Are Important in Protecting Spinal Cord Tissues against Theiler's Virus Strain GD VII Infection
Authors: Kohanawa Masashi , Nakane Akio , Asano Misako , Minagawa Tomonori
Published: November 2013

Intravenous infection with Theiler's virus strain GD VII causes acute encephalomyelitis in mice. Endogenous IFN‐γ produced in the spinal cord is important to protect the tissue in mice infected with this virus. Neither CD4+ cells nor CD8+ cells infiltrated the spinal cords of infected mice until Day 9 postinfection. However, the number of CD3+/TCR‐γδ+ cells increased in the spinal cords of mice infected with the virus. These cells resided in the spinal cords of normal mice, and produced IFN‐γ as a result of stimulation by immobilized anti‐CD3 mAb. Elimination of CD3+ cells by the administration of a specific mAb augmented viral replication and suppressed production of endogenous IFN‐γ. Depletion of TCR‐αβ+ cells and ASGM1+ cells did not affect the viral replication, and did not alter the production of IFN‐γ. Therefore, CD3+/TCR‐αβ– cells producing IFN‐γ play an important role in the protection of the spinal cord against Theiler's virus infection. These results suggest that CD3+/TCR‐αβ– cells might be identical to TCR‐γδ+ cells.
Effect of Substitution for Arginine Residues near Position 146 of the A Subunit of Escherichia coli Heat‐Labile Enterotoxin on the Holotoxin Assembly
Authors: Okamoto Keinosuke , Takatori Rie , Okamoto Kyoko
Published: November 2013

Escherichia coli heat‐labile enterotoxin (LT) is a holotoxin which consists of one A and five B subunits. Although B subunit monomers released into periplasm can associate into pentameric structures in the absence of the A subunit, the A subunit accelerates the assembly. To express the function, A subunit constructs the proper spatial structure. However, the regions involved in the construction are unknown. To identify the regions, we substituted arginine residues near position 146 of the A subunit with glycine by oligonucleotide‐directed site‐specific mutagenesis and obtained the mutants expressing LT(R141G), LT(R143G), LT(R146G), LT(R143G, R146G), LT(R141G, R143G, R146G) and LT(R143G, R146G, R148G). We purified these mutant LTs by using an immobilized d‐galactose column and analyzed the purified mutant LTs by SDS‐PAGE to examine the amount of A subunit associated with B‐subunit oligomer. The substitution of an arginine residue at any position did not induce a significant alteration in the amount of A subunit associated with B‐subunit oligomer. However, the substitution of more than two arginine residues induced a significant decrease in the amount of A subunits associated with the B‐subunit oligomer. Subsequently, we measured the level of the intracellular B‐subunit oligomer of these mutant strains. The measurement revealed that the amount of B‐subunit oligomer in cells decreased as the number of substituted arginine residues increased. These results show that all arginine residues near position 146 are important for the construction of the functional A subunit, and thus for holotoxin formation, although each individual arginine residue is not an absolute requirement.
Comparison of the Selectivity of Anti‐Varicella‐Zoster Virus Nucleoside Analogues
Authors: Machida Haruhiko , Nishitani Makiko , Watanabe Yohko , Yoshimura Yuichi , Kano Fumitaka , Sakata Shinji
Published: November 2013

We compared the selectivity of six anti‐varicella‐zoster virus (VZV) drugs, which are clinically available or of which clinical efficacy for the treatment of VZV infections has been reported. Sorivudine (BV‐araU) had the most potent anti‐VZV effect in the plaque inhibition assay, followed by brivudine (BVDU) and 5‐propynyl‐arabinofuranosyluracil (Pry‐araU). All test compounds, except vidarabine (AraA), had only a very weak effect on human embryonic lung cell growth. The selectivity indexes (ID50 for cell growth/ED50 for VZV plaque inhibition) of BV‐araU, BVDU, and Pry‐araU were > 1,000,000, 20,000, and > 10,000, respectively, while those of acyclovir and penciclovir ranged from 600 to 800. AraA was much less selective than any of the other drugs tested. We measured the amount of pH] thymidine incorporated into the acid‐insoluble fraction of VZV‐infected cells to determine the ability of these drugs to selectively inhibit viral DNA synthesis. [3H]Thymidine incorporation was markedly inhibited by all anti‐VZV compounds, except BVDU. Treatment of infected cells with drugs from 32 to 38 hr after infection inhibited the DNA synthesis to the same extent as VZV plaque formation, except that AraA inhibited the DNA synthesis at a lower dose than for VZV plaque formation. DNA synthesis in non‐infected growing cells was inhibited to the same extent as cell growth. A particularly high selectivity index for the inhibition of DNA synthesis was noted for BV‐araU, which was defined as the ratio of inhibitions of DNA synthesis in VZV‐infected and non‐infected. The highest selectivity indexes were recorded for BV‐araU > Pry‐araU > acyclovir ≥ penciclovir > AraA.
Inhibition of Varicella‐Zoster Virus Glycoprotein Expression by Peripheral Blood Mononuclear Cells
Authors: Mizutani Ken‐ichi , Ito Masahiro , Kamiya Hitoshi , Sakurai Minoru
Published: November 2013

The effect of peripheral blood mononuclear cells (PBMC) on expression of varicella‐zoster virus (VZV) glycoproteins (Gps) was analyzed by flow cytometry. PBMC from VZV seropositive and seronegative donors and supernatant of PBMC co‐cultured with VZV‐infected human embryonic fibroblasts reduced VZV Gp expression. Neutralization of supernatant fluid with mixture of anti‐interferons (IFN)‐α, ‐β, ‐γ, and tumor necrosis factor (TNF)‐α partially reduced inhibitory activity of supernatant on VZV Gp expression. Deletion of natural killer (NK) cells and adherent cells from PBMC reduced inhibitory activity of PBMC on VZV Gp expression. These results suggest that IFN‐α, ‐β, ‐γ, TNF‐α and other soluble factors released from NK cells and monocytes by co‐cultivation with VZV‐infected fibroblasts inhibit VZV Gp expression.
Differentiation of Clostridium difficile, Clostridium bifermentans, Clostridium sordellii, and Clostridium perfringens from Diarrheal Stool by API ZYM and API LRA Oxidase Test
Authors: Fontana Carla , Jezzi Teresa , Testore Gian Piero , Dainelli Benedetto
Published: November 2013

A simple, rapid and reliable outline for identification of clostridia isolates from human infections was developed. It consists of a combination of API ZYM and API LRA Oxidase tests. The enzymatic activities were performed with strains sub‐cultured onto carbohydrate‐free medium (Columbia blood agar). Fifty‐five strains of Clostridium difficile, C. bifermentans, C. sordellii, and C. perfringens from clinical specimens and eight reference standard strains representing different species of the same genus were analyzed. The accuracy of the new method was evaluated by comparison with the results obtained by DNA/DNA analysis.
Frequent Detection of Hepatitis C Virus Subtype 3a (HCV‐3a) Isolates in Thailand by PCR Using Subtype‐Specific Primers
Authors: Apichartpiyakul Chatchawann , Miyajima Hirofumi , Doi Hisaya , Mizokami Masashi , Homma Morio , Hotta Hak
Published: November 2013

By means of a polymerase chain reaction (PCR) method using subtype‐specific primers for hepatitis C virus (HCV) subtypes 1a, 1b, 2a, 2b and 3a, the prevalence of each subtype among HCV isolates in Chiang Mai, Thailand, was determined. HCV‐3a appeared to be the most common subtype in blood donors, and was also frequently found in patients with liver disease. HCV‐1b, but not HCV‐2a or −2b, was also commonly found in this area, while a considerable percentage of the total HCV isolates still remained unclassifiable by the above methods. Serotype analysis of the HCV isolates using C14‐1 and C14‐2 recombinant peptides revealed that HCV‐3a was likely to carry an antigenic determinant(s) different from those of the major types 1 (HCV‐1a and −1b) and 2 (HCV‐2a and −2b).