bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of Candida albicans Growth by Murine Peritoneal Neutrophils and Augmentation of the Inhibitory Activity by Bacterial Lipopolysaccharide and Cytokines
Authors: Tansho Shigeru , Abe Shigeru , Yamaguchi Hideyo
Published: November 2013

Anti‐Candida activity of murine neutrophils and its regulation by immunomodulators were studied in vitro. Murine neutrophils which were prepared from peritoneal‐exudated cells inhibited the growth of Candida albicans at an effector: target (E/T) ratio of 30/1 or above. This anti‐Candida activity of neutrophils was augmented by lipopolysaccharide from Escherichia coli, murine tumor necrosis factor (TNF), murine interferon‐γ (IFN‐γ) and murine granulocyte macrophage colony‐stimulating factor (GM‐CSF) but not by granulocyte colony‐stimulating factor (G‐CSF) added to the incubation medium. Greater extent of augmentation was obtained when TNF plus GM‐CSF or INF‐γ plus GM‐CSF were used in combination. These results indicate that anti‐Candida activity of murine neutrophils is regulated similarly to that of the human neutrophils reported previously. Therefore murine peritoneal neutrophils can be used as a favorable substitute for human neutrophils in studies on protective machinery against C. albicans infection.
A Rapid Colorimetric Assay for Determination of Leukocyte‐Mediated Inhibition of Mycelial Growth of Candida albicans
Authors: Abe Shigeru , Satoh Tamae , Tokuda Yoshiko , Tansho Shigeru , Yamaguchi Hideyo
Published: November 2013

A staining method with crystal violet (CV) was demonstrated to be useful for a simple, quick and objective assessment of in vitro growth inhibitory activity of leukocytes against Candida albicans cells. Candida cells incubated with murine neutrophils or macrophages for 14 hr in microwells were stained with CV and, after washing with 0.25% sodium dodecyl sulfate (SDS), treated with isopropanol containing HCl (0.04 N) to extract Candida cell‐bound CV. Then the absorbance at 590 nm of the isopropanol extract was photometrically measured. The results showed that the photometrical absorbance was proportional to the amount of3H‐glucose taken up by C. albicans cells, which reflected the number of viable Candida cells.
Comparative Studies on Borrelia afzelii Isolated from a Patient of Lyme Disease, Ixodes persulcatus Ticks, and Apodemus speciosus Rodents in Japan
Authors: Nakao Minoru , Miyamoto Kenji , Fukunaga Masahito , Hashimoto Yoshio , Takahashi Hidetoshi
Published: November 2013

The genospecies Borrelia afzelii was isolated from a patient of Lyme disease in Hokkaido, Japan, for the first time, by culturing the minced erythema lesion in BSK II medium. Two analytical methods, rRNA gene restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR) using the specific primer set to amplify the 16S rRNA gene, revealed that this clinical isolate belongs to the group of B. afzelii. In our culture collection of spirochetes, part of the isolates from Ixodes persulcatus ticks, and from Apodemus speciosus rodents, were also classified as B. afzelii. These results strongly suggest that the agent pathogenic to humans is maintained in “rodent‐tick” transmission cycle.
Human T Cell Leukemia Virus Type 1 (HTLV‐1) Antibodies in Healthy Populations and Renal Transplanted Patients in the North‐East of Brazil
Authors: Linhares Maria Ieda Siqueira , Eizuru Yoshito , de Andrade Gildete Patriota , Fonseca Ivailda Barbosa , Carvalho Luiz Bezerra , Moreira Ivanildo Tenorio , Minamishima Yoichi
Published: November 2013

The seroprevalence of human T cell leukemia virus type 1 (HTLV‐1) infection was investigated in Brazilians (570): native inhabitants (298) and descendants from Japanese (272) living in Recife and its neighborhoods—North‐east of Brazil. Furthermore, polytransfused renal transplanted patients (54) were also examined for the serological status to this virus. The seropositivity to HTLV‐1, screened by enzyme‐linked immunosorbent assay (ELISA), was low: 1.34% for the local population and 0.73% for the descendants from Japanese. However, the seropositivity for the renal transplanted patients was found to be 11.1%. This higher value suggests that this retrovirus infection seems to be of importance in this clinical condition.
Sero‐Epidemiology of Lyme Disease in an Endemic Area in China
Authors: Ai Cheng‐xu , Zhang Wei‐fen , Zhao Jien‐hua
Published: November 2013

A sero‐epidemiological investigation on Lyme disease was carried out in a forestry center of Hailin County, Heilongjiang Province, China. A total of 381 participants including forestry workers and their dependents completed questionnaires and had blood samples taken for detection of antibody against Borrelia burgdorferi by indirect immunofluorescent assay. Of 381 participants, 250 (65.6%) had a history of tick bites between May and July, 1987, and 379 (99.5%) at some time in the past, 56 (14.7%) developed erythema migrans at the site of tick attachment, 138 (36.2%) had late manifestations of Lyme disease, and 101 (26.2%) were seropositive. There was a significant difference in the seropositive rate between the persons with tick bites and those without tick bites in 1987 (P<0.01). The titers in the older age group (over 30) were slightly higher than those of the younger age group (under 30). No relationship between the prevalence of Lyme manifestations by tick bites and the result of serological test was observed. Further investigations are necessary to assess the actual prevalence and incidence of infections using antigen of Lyme disease borreliae isolated in China.
Detection of Bacteria in Phagocyte‐Smears from Septicemia‐Suspected Blood by In Situ Hybridization Using Biotinylated Probes
Authors: Matsuhisa Akio , Saito Yoshihiro , Sakamoto Yoshimasa , Keshi Hiroyuki , Ueyama Hiroshi , Aikawa Youko , Kishi Youichiro , Ohno Tsuneya
Published: November 2013

We report herein the detection of intracellular bacteria in phagocyte‐smears obtained from septicemia‐suspected blood samples by in situ hybridization. This was obtained by using nick‐translated biotin‐11‐dUTP‐labeled DNA probes and streptavidin‐alkaline phosphatase conjugates for visualization of the hybridized signals. The probes were made from random genomic DNA clones of bacteria which are frequently the causative agents of bacteremia, such as Staphylococcus spp., Pseudomonas aeruginosa, Enterococcus faecalis, Escherichia coli, Klebsiella spp. and Enterobacter spp. When our in situ hybridization method was compared with conventional culture protocols for the ability to detect bacteria from the blood of patients suspected of having septicemia, 30 positive results were obtained in 50 specimens by in situ hybridization methods. In contrast, only 7 positive results were obtained by blood cultures. Thus, even if bacteria cannot be detected by conventional blood cultures and histology, our in situ hybridization method allows for direct observation of bacterial foci in circulating phagocytes and identification of the bacteria. Our investigations suggest that in septicemia, circulating polymorphonuclear neutrophils carry some surviving bacteria as well as metabolized bacterial DNA and RNA for a considerable period of time. Thus, our in situ hybridization method using the phagocyte‐smears have diagnostic value for detecting most bacteria which cause septicemia.
Role of Breast Milk in Acquisition of Cytomegalovirus Infection
Authors: Minamishima Ikuko , Ueda Kohji , Minematsu Toshio , Minamishima Yoichi , Umemoto Masakazu , Take Hiromichi , Kuraya Kazue
Published: November 2013

The prevalence of IgG antibody against cytomegalovirus (CMV) was compared between the age‐matched (0 month to 2 years of age) groups of 212 breast‐fed children and 223 bottle‐fed children to examine the role of breast milk for acquisition of CMV. Mothers of both groups of children were also examined for CMV IgG antibodies. Both the breast‐fed and bottle‐fed children groups showed high seropositivity for CMV at 0 to 2 months of age, which gradually decreased and bottomed at 6 to 8 months of age. Thereafter, in the breast‐fed children group, the seropositivity rate increased up to 70% by 1 year of age. In contrast, in the bottle‐fed children group, the seropositivity rate remained at the bottom level of lower than 30%, without showing any apparent increases. The serological data of the children whose mothers were confirmed to be seropositive, revealed that mother‐to‐child transmission of CMV occurred in 11 of 17 (64.7%) of the breast‐fed children and in 24 of 87 (27.6%) of the bottle‐fed children. All the bottle‐fed children born to seronegative mothers remained seronegative for CMV up to 1 year of age. The bottle‐fed children showed significantly lower seropositivity than the breast‐fed children, although most of both groups of children were born to seropositive mothers. The results strongly suggested that about 40% of the breast‐fed children acquire CMV via breast milk and breast‐feeding has certain protective effects on congenital CMV disease in the offspring.
Prevalence of Human Herpesvirus 6 Variants A and B in Patients with Chronic Fatigue Syndrome
Authors: Yalcin Safak , Kuratsune Hirohiko , Yamaguchi Koji , Kitani Teruo , Yamanishi Koichi
Published: November 2013

Peripheral blood mononuclear cells collected from 13 patients with chronic fatigue syndrome and 13 healthy controls were analyzed for the presence of human herpesvirus 6 (HHV‐6) DNA by variant‐specific polymerase chain reaction and dot blot hybridization. HHV‐6 DNA was detected in 7 of 13 (53%) patients, and of those 7 patients, 4 were positive for HHV‐6 variant A DNA and 3 were for variant B. No HHV‐6 DNA was detected in the controls. Serum antibody titers to the late antigen and antibody prevalence to the early antigen of HHV‐6 were significantly higher in the patient group. These results suggest active replication of HHV‐6 in patients with chronic fatigue syndrome.
The Role of MRSA (Methicillin‐Resistant Staphylococcus aureus) Adherence and Colonization in the Upper Respiratory Tract of Geriatric Patients in Nosocomial Pulmonary Infections
Authors: Rikitomi Naoto , Nagatake Tsuyoshi , Sakamoto Tasuku , Matsumoto Keizo
Published: November 2013

The mechanism of nosocomial respiratory infections caused by MRSA (methicillin‐resistant Staphylococcus aureus) in geriatric patients was investigated. Seriously ill patients (SIP) undergoing naso‐gastric tube feeding or intravenous hyperalimentation and moderately ill patients (MIP) who were orally fed, were examined for their colonization and infection by Staphylococcus aureus (S. aureus) in the respiratory tract. Colonization of MRSA in the upper respiratory tract in SIP was from six to ten times higher than that in MIP and was associated with a high incidence of MRSA pulmonary infections. In vitro S. aureus adherence to nasal or oropharyngeal cells demonstrated that bacteria binding to nasal cells was higher, which probably can be interpreted as an elevated occurrence of S. aureus colonization in the nasal cavity than in the throat. The binding activity of MRSA was not superior to that of MSSA (methicillin‐sensitive S. aureus). Though MRSA binding to the nasal cells from SIP was not higher than those from MIP, MRSA colonization in the upper respiratory tract was more frequently seen in SIP (P < 0.01). A higher incidence of total infectious episodes (P < 0.02‐0.001) and more frequent use of antibiotics (P < 0.02‐0.001), which were potent against MSSA might be the basis for selection of MRSA in these patients. In fact, the rate of MRSA colonization on the skin (pressure sores) was also higher in SIP (P < 0.01). A low nutritional state in SIP (P < 0.01‐0.02) might also be associated with MRSA colonization. The present results indicate that the high frequency of infections, antibiotic administration, MRSA skin colonization and low nutritional condition, are enhancing factors of MRSA acquisition in the respiratory tract for SIP undergoing artificial feeding, in a geriatric hospital.
Crystallization and Analyses of Crystals of Various Chemotypes of R‐Form Lipopolysaccharides from Salmonella Spp.
Authors: Kato Nobuo , Arakawa Yoshichika , Sugiyama Tsuyoshi , Ito Hideo , Naito Setsuko , Kido Nobuo , Ohta Michio , Sasaki Kyoyu
Published: November 2013

Various chemotypes (Re, Rd2, Rd1P–, Rd1, RcP–, Rc, Rb3, Rb2, Rb1, and Ra) of R‐form lipopolysaccharides (LPSs) of Salmonella spp. were crystallized by treatment with 70% ethanol containing 250 mM MgCl2, and crystals of the LPSs were observed electron microscopically and analyzed by electron diffraction and synchrotron X‐ray diffraction. All the LPSs tested formed three‐dimensional crystals showing very similar shapes; hexagonal plate, solid column, discoid, square or rectangular plate, lozenge plate and truncated hexangular or rectangular pyramid forms. Electron diffraction patterns from the hexagonal plate crystals of all these LPSs obtained by electron irradiation from the direction perpendicular to the basal plane showed that they consist of hexagonal lattices with the lattice constant of 4.62 Å. The crystals of all the LPSs thus formed gave ring‐like X‐ray diffraction patterns because of their small sizes. The long‐axis values were calculated from the X‐ray diffraction patterns from crystals of all the LPSs in the low‐angle region and they corresponded roughly to the length of the proposed primary chemical structures of the R cores of the LPSs. The volume occupied by a single molecule of all the LPSs were calculated from the molecular weights based on the proposed structures and the crystallographic data obtained by electron diffraction, X‐ray diffraction, and density determination.
Siderophore‐Mediated Utilization of Iron Bound to Transferrin by Vibrio parahaemolyticus
Authors: Yamamoto Shigeo , Okujo Noriyuki , Matsuura Shyoko , Fujiwara Ikuko , Fujita Yasuhiro , Shinoda Sumio
Published: November 2013

Vibrio parahaemolyticus produces a structurally novel type of siderophore, termed vibrioferrin, in response to iron‐limitation. This study was performed to examine whether vibrioferrin can assimilate iron from human iron‐binding proteins for growth. Comparison of the growth rates between V. parahaemolyticus AQ 3354 and its spontaneously arising, vibrioferrin‐deficient mutant revealed that vibrioferrin was able to sequester iron from 30% iron‐saturated human transferrin for growth, but not from human lactoferrin even if fully saturated with iron. In both strains, iron limitation induced two high‐molecular‐weight outer membrane proteins with apparent molecular masses of approximately 78 and 83 kDa. Since only the outer membrane fraction including these proteins showed a binding capacity to ferric vibrioferrin complex, either of them may function as its cell surface receptor. These results suggested that the organism might utilize such a source of host iron through the action of vibrioferrin during in vivo survival and proliferation, although its importance in pathogenesis is unknown.
Pulsed Field Gel Electrophoresis Analysis of Borrelia burgdorferi Sensu Lato Isolated in Japan and Taxonomic Implications with Lyme Disease Spirochetes
Authors: Fukunaga Masahito , Takahashi Yukie
Published: November 2013

Genomic DNAs of Borrelia burgdorferi sensu lato isolates obtained in Japan sharing different rRNA gene ribotypes were digested with rare‐cutter restriction endonucleases and the fragments obtained were separated by pulsed field gel electrophoresis (PFGE). The sizes of large restriction cleavage bands with MluI endonuclease were quite similar among isolates in each ribotype group. On the other hand, the PFGE profiles obtained with the other enzymes (NruI, Sal I or SplI) were rather divergent, and Japanese isolates were distinguishable from the United States and European isolates. The Japanese isolates classified as ribotypes group II (Borrelia garinii) and III (B. afzelii) showed different PFGE patterns from that of European isolates. The isolates grouped into ribotype IV revealed distinctively different PFGE profiles. These results indicate that the Japanese isolates may be genetically divergent and distinct from the United States and European isolates.
Complement‐Mediated Killing of Borrelia garinii —Bactericidal Activity of Wild Deer Serum
Authors: Isogai Emiko , Kamewaka Yuri , Isogai Hiroshi , Kimura Koichi , Fujii Nobuhiro , Nishikawa Takeshi
Published: November 2013

The susceptibility of Borrelia garinii to fresh wild deer sera was determined by incubating strain SIKA2 at 10% serum concentration for 1 hr at 37 C in an in vitro bactericidal assay. Each serum showed bactericidal effects at various levels. The effect was dependent on the concentration of antibody to the spirochetes. Complement was essential in the bactericidal assay because the inactivated deer serum showed greatly decreased activity. Our results suggest that B. garinii is sensitive to deer serum, in the presence of antibody and the bactericidal effect is important for preventing Lyme disease in wild sika deer.
Isolation and Classification of Temperature‐Sensitive Mutants of Influenza B Virus
Authors: Yamamoto‐Goshima Fumi , Aoki Hiizu , Takada Toshihiko , Toda Shigeru , Yoshii Saiji , Maeno Koichiro
Published: November 2013

We isolated 25 temperature‐sensitive mutants of B/Kanagawa/73 strain generated by mutagenesis with 5‐fluorouracil and classified them into seven recombination groups by pair‐wise crosses. All mutants showed a ratio of plaquing efficiency at the nonpermissive temperature (37.5 C) to the permissive temperature (32 C) of 10–4 or less. At 37.5 C most of group I, II, and III mutants did not produce appreciable amounts of protein, but all other group mutants were protein synthesis‐positive. A group VII mutant produced active hemagglutinin (HA) and neuraminidase (NA) at the nonpermissive temperature, but Group V mutants produced only active NA and were defective in the HA molecule. The other group mutants, including group IV mutants with mutation only in the NA gene (8, 10), lacked both activities at the nonpermissive temperature. One of nine influenza B virus isolates in 1989 had EOP 37.5/32 of 1/3 × 10–2 and belonged to recombination group VII.
Expression of Fimbriae and Host Response in Branhamella catarrhalis Respiratory Infections
Authors: Ahmed Kamruddin , Masaki Hironori , Dai Iran Cong , Ichinose Akitoyo , Utsunomiya Yoshiaki , Tao Misao , Nagatake Tsuyoshi , Matsumoto Keizo
Published: November 2013

Sputum during the acute exacerbation of chronic respiratory diseases were observed under the electron microscope, to determine the in vivo expression of surface structures of Branhamella catarrhalis (B. catarrhalis), the polymorphonuclear neutrophil (PMN) response to B. catarrhalis infections, and the composition of sputum. It was found that during infection fimbriae are expressed in B. catarrhalis. However, there were sparsely to densely fimbriated bacteria in each sputum sample. The length of the fimbriae were from 50 to 76 nm. In the sparsely fimbriated B. catarrhalis, external to the cell wall, a thin, granular, electron‐dense layer was observed. Due to the presence of fimbriae, this layer was not seen in densely fimbriated B. catarrhalis. Blebs were also found in B. catarrhalis. PMNs were found to phagocytose both B. catarrhalis and debris. Evidence was found that debris were formed mainly by the destruction of PMNs. Bacteria as well as debris were phagocytosed by PMNs.
Use of the Recombinant 38‐kDa Antigen of Mycobacterium tuberculosis as an Immunogen for Specific Antisera Production
Authors: D'Souza Celine D. , Kadival Gururaj V. , Samuel Aban M.
Published: November 2013

The Mycobacterium tuberculosis 38‐kDa protein antigen is one of the secreted immunodominant antigens showing high immunogenicity at B‐cell and T‐cell levels. Although monoclonal antibodies to this antigen have been produced, specific polyclonal antisera is required for standardization of specific immunodiagnostic assays. This protein has been overexpressed and purified from recombinant Escherichia coli using an inducible vector system. During each stage of expression and purification, the recombinant protein was used to immunize mice and rabbits by several methods: 1) as overexpressed protein present as inclusion bodies in recombinant E. coli; 2) embedded in a polyacrylamide gel; 3) fixed to a solid‐phase nitrocellulose membrane and 4) emulsified with an adjuvant. All strategies yielded specific antisera as determined by enzyme‐linked immunosorbent assay (ELISA) and immunoblot analyses. The results obtained, both quantitative (ELISA) and qualitative (immunoblot) demonstrate that the purified recombinant antigen retains its antigenicity and immunogenicity throughout the various steps in the process of expression and purification and serves as a potent antigen for production of specific antisera to be used in immunoassays.
Borrelia japonica in Nature: Genotypic Identification of Spirochetes Isolated from Japanese Small Mammals
Authors: Nakao Minoru , Miyamoto Kenji , Fukunaga Masahito
Published: November 2013

Spirochetes were isolated from earlobe tissues of shrews (Sorex unguiculatus, Sorex caecutiens, and Crocidura dsinezumi), voles (Clethrionomys rufocanus), and mice (Apodemus argenteus and Apodemus speciosus) captured in various localities in Japan. The isolates were identified as Borrelia japonica by rRNA gene restriction fragment length polymorphism analysis. The data suggest that these small mammals are candidates of reservoir hosts for B. japonica.
Comparative Analyses of Thymocyte and Thymic Low‐Density Adherent Cell Functions
Authors: Wambua Peter P. , Iwabuchi Kazuya , Iwabuchi Chikako , Ogasawara Kazumasa , Itoh Yasushi , Arase Hisashi , Kajiwara Masaharu , Gotohda Toshihiko , Kajino Ki‐ichi , Good Robert A. , Onoé Kazunori
Published: November 2013

Thymocytes which have developed in the C3H thymus showed depressed proliferative responses to stimulation with anti‐CD3 antibody as compared with those which have developed in the thymus of other strains of mice (i.e. AKR). The present study was conducted to analyze immunological functions of the thymic stromal cell population (low‐density adherent cells, LDAC) in the C3H mice using allogeneic bone marrow (BM) chimeras established by BM transplantation in the reciprocal combination of AKR and C3H mice as donor or recipient. The thymic LDAC from C3H mice or the [AKR(donor)→C3H(recipient)] chimeras contained a high proportion of Mac‐1+ cells as compared to AKR mice or the [C3H→AKR] chimeras. The proportion of Mac‐1+ cells paralleled the IL‐1‐ and PGE2‐secreting ability of the LDAC cultured either in the presence or absence of LPS and also paralleled the antigen‐presenting cell functions of the LDAC. Furthermore, after anti‐CD3 stimulation the PGE2 inhibited more profoundly proliferative responses of [AKR→C3H] or normal C3H thymocytes than those of the [C3H→AKR] chimera or normal AKR thymocytes. A PGE2 inhibitor, indomethacin, reversed the depressed responses of the thymocytes which had developed in the C3H thymus. These findings suggest that the lower responsiveness of thymocytes from [AKR→C3H] chimeras to anti‐CD3 stimulation may be attributable to large amounts of PGE2 secreted by LDAC and/or to increased sensitivity of thymocytes themselves to PGE2.
Increased Phosphodiesters in Lipopolysaccharide Prepared from the Polymyxin B‐Resistant Isolate of Klebsiella pneumoniae
Authors: Yokochi Takashi , Inoue Yoshiko , Jiang Guo‐zhi , Kato Yutaka , Sugiyama Tsuyoshi , Kawai Makoto , Fukada Masako , Takahashi Kazuko
Published: November 2013

A polymyxin B (PXB)‐resistant mutant of Klebsiella pneumoniae O3 was isolated. Lipopolysaccharide (LPS) extracted from the PXB‐resistant isolate bound little PXB, although LPS from the parental strain did. The 31P nuclear magnetic resonance (NMR) spectrum of PXB‐resistant type LPS showed that it contained much less of the phosphomonoesters and the pyrophosphate esters, and an increased amount of the phosphodiesters, compared to the parental type LPS. The decrease in the binding of PXB might be due to altered phosphate groups on the PXB‐resistant type LPS, suggesting that it might explain the PXB‐resistance of the mutant.
Isolation and Characterization of Three Porin‐Like Proteins from Vibrio vulnificus: Effect of Different Growth Media on Their Production
Authors: Koga Tetsuro , Takumi Kenji
Published: November 2013

The effect of the culture media on the composition of the outer membrane protein of Vibrio vulnificus strain 393 from human blood was examined. Only one major outer membrane protein, with an apparent molecular weight of 37,000 (37K protein) and 34,000 (34K protein), was formed in the cells grown in 3% NaCl‐BHI broth and chemically defined medium, respectively. The production of one major outer membrane protein was also observed in other isolates from humans and asari clam when they were grown in 3% NaCl‐BHI broth. On the other hand, three major outer membrane proteins, with apparent molecular weights of 48,000 (48K protein), 37,000 (37K protein), and 34,000 (34K protein), were produced in the cells grown in 3% NaCl‐nutrient broth. Three proteins, 48K, 37K, and 34K from strain 393, were purified and the amino acid compositions were determined. Although there was a little difference in the composition of amino acid among three proteins, the amino acid compositions of the three porin‐like proteins showed characteristic properties of the porins of Escherichia coli and Salmonella typhimurium. Immunoblot analysis of the outer membrane proteins from four vibrios, E. coli, and S. typhimurium using monospecific antisera against these three porin‐like proteins showed that only the antiserum against 37K protein cross‐reacted with the outer membrane proteins from all the strains tested.
Suppression of Anti‐Candida Activity of Murine and Human Neutrophils by Glucocorticoids
Authors: Nohmi Takao , Abe Shigeru , Tansho Shigeru , Yamaguchi Hideyo
Published: November 2013

Effects of glucocorticoid (GC) compounds on inhibitory activity of neutrophils to mycelial growth of Candida albicans were examined by in vitro crystal violet staining method with 14 hr co‐culture. Both GC hormones (hydrocortisone ≥6 × 10–7 m and corticosterone ≥10–6 m) and anti‐inflammatory GC agents (prednisolone ≥10–7 m and dexamethasone ≥10–8 m) significantly suppressed anti‐Candida activity of murine casein‐induced neutrophils. Anti‐Candida activity of human neutrophils prepared from peripheral blood was also suppressed by hydrocortisone (≥6 × 10–7 m). These GC compounds did not affect the Candida growth in the absence of neutrophils. Steroidal compounds without anti‐inflammatory activity, cholesterol, cholic acid, aldosterone did not suppress neutrophil activity. These results suggest that GCs at their physiological or clinical concentration may suppress anti‐Candida activity of neutrophils in vivo.
Isolation and Characterization of Staphylococcus aureus Mutants Which Form Altered Cell Clusters
Authors: Sugar Motoyuki , Komatsuzawa Hitoshi , Ooku‐Inomata Kaoru , Miyake Yoichiro , Ishida Eisaku , Suginaka Hidekazu
Published: November 2013

Staphylococcus aureus FDA 209P produces two extracellular bacteriolytic enzymes, 51‐kDa endo‐β‐N‐acetylglucosaminidase (GL) and 62‐kDa N‐acetylmuramyl‐l‐alanine amidase (AM), both of which can disperse cell clusters. To characterize the physiological roles of these enzymes in vivo, mutants with altered autolysin activity were isolated, and their degree of cluster formation in broth culture was assessed. Bacteriolytic activities of GL and AM, produced and secreted from these mutants into the culture fluid and detected with activity gels, coincided well with the degree of cluster formation of the mutants. The mutants with little or no enzyme activity grew in clusters, whereas those with high activity grew as well‐separated cocci, suggesting that these enzymes are involved in cell separation of S. aureus in vivo.
Seroepidemiological Survey of Coxiella burnetii in Domestic Cats in Japan
Authors: Morita Chiharu , Katsuyama Jun , Yanase Tsuyoshi , Ueno Hiroshi , Muramatsu Yasukazu , Hohdatsu Tsutomu , Koyama Hiroyuki
Published: November 2013

Cats are assumed to be one of the most important reservoirs of causative agent of human Q fever especially in urban areas. There is no evidence of Coxiella burnetii infection in cats in Japan prior to this. Sera from 100 cats, collected in various parts of Japan, were examined for antibody against C. burnetii. Sixteen out of the 100 samples contained antibodies against C. burnetii. The prevalence of the antibody decreased from the northeastern to the southwestern part of Japan. A high prevalence of the antibodies was observed in sera from cats of more than four years of age. It is difficult to deny that cats would be one of the important sources of human Q fever in Japan.
Purification and Characterization of Metallo‐β‐Lactamase from Serratia marcescens
Authors: Marumo Kenji , Takeda Atsushi , Nakamura Yoshiko , Nakaya Kazuyasu
Published: November 2013

Carbapenem‐hydrolyzing β‐lactamase from Serratia marcescens FHSM4055 was purified 926‐fold by means of carboxylmethyl Sephadex C‐50, Sephacryl S‐200, and Mono S column chromatography. The molecular weight was 30,000 by SDS‐PAGE and the isoelectric point was 8.7. The enzyme activity was inhibited by EDTA, and restored by adding zinc (II) or manganese (II). It was inhibited by p‐chloromercuribenzoate and iodine as well as the heavy metals, Hg (II), Fe (II), Fe (III), and Cu (II). These results indicate that the enzyme is a metallo‐β‐lactamase and that the SH‐group of only one cysteine residue probably binds to the metal ion, thus contributing to the stability of the enzyme active center. The specific constant (kcat/Km) showed that the enzyme hydrolyzed various β‐lactam antibiotics such as carbapenems, cephalosporins, moxalactam, cephamycins, and penicillins other than monobactams. Ampicillin and piperacillin with respective amino‐ and imino‐groups, ceftazidime with a carboxypropyloxyimino‐group, and cefclidin with a carbamoylquinuclidine‐group were poor substrates among the β‐lactam antibiotics other than the monobactams tested. The plots of the turnover number (kcat) against pH for the hydrolysis of cephaloridine gave an asymmetrical curve with the ‘tail’ on the acid side (pK1, 5.9; pK2, 9.0; pK3, 10.8), whereas those of kcat/Km gave a bell‐shaped curve (pK1, 5.8; pK2, 9.8). Both results suggest that two ionic forms of an intermediate yield the same product at different rates and that the enzyme is stable under alkaline conditions. Since the N‐terminal amino acid sequence of 27 residues determined was consistent with that of the metalloenzyme (Antimicrob. Agents Chemother., 1994, 38: 71‐78), the above enzymatic characteristics seem to coincide.
Analysis of Cellular Response and Gamma Interferon Synthesis in Bronchoalveolar Lavage Fluid and Lung Homogenate of Mice Infected with Pneumocystis carinii
Authors: Ishimine Tomohiko , Kawakami Kazuyoshi , Nakamoto Atsushi , Saito Atsushi
Published: November 2013

The cellular and cytokine responses in the lungs of mice infected with Pneumocystis carinii were examined on both lung homogenates and bronchoalveolar lavage (BAL) fluids. In the lungs of infected mice, the number of P. carinii cysts rapidly decreased by day 7, then started to increase with a peak on day 14, and thereafter decreased gradually. When the presence of P. carinii was examined at the DNA level by dot blot hybridization, a similar clearance curve was obtained, and the organisms were shown to be completely eliminated on day 28. In the late phase of infection, leukocytes, mainly lymphocytes, increased in number when analyzed on lung homogenates, while no significant increase of inflammatory cells was observed in BAL fluids. An accumulation of both CD4+ and CD8+ T cells and an increase of activated T cells expressing IL‐2Rα were observed in lung homogenates of the infected mice. In addition, a considerable amount of IFN‐γ was detected in lung homogenates, but not in BAL fluids. These data indicate that lung homogenates are more suitable than BAL fluids for the analysis of cellular and cytokine responses in the lungs of mice infected with P. carinii. To define the involvement of IFN‐γ in host defense against P. carinii, the effect of this cytokine on the killing activity of macrophages against P. carinii was examined in vitro. IFN‐γ was found to augment this activity by increasing nitric oxide synthesis of the macrophages. Thus, it is suggested that IFN‐γ plays an important role in the protection of mice from P. carinii infection.
Production of Antigenically Related Exocellular Elastolytic Proteases Mediating Hemagglutination by Vibrios
Authors: Alam Munirul , Miyoshi Shin‐ichi , Shinoda Sumio
Published: November 2013

Exocellular proteases produced by Vibrio fluvialis, V. furnissii, V. metschnikovii and V. campbellii were characterized and compared to those of V. mimicus protease (VMP) and V. vulnificus protease (VVP). These proteases possessed both elastolytic and hemagglutinating abilities and were identified, except that of V. metschnikovii, as metalloprotease. Conversely, V. metschnikovii protease failed to exhibit some of the salient features for metalloproteases suggesting the existence of protease(s) other than metalloprotease. However, antibodies against VVP cross‐reacted to these proteases and to VMP indicating antigenic relatedness amongst vibrio proteases. This study, thus, demonstrated the prevalent distributions of antigenically related proteases both in pathogenic and non‐pathogenic vibrios, bringing their status as a virulence determinant into question.
Experimental Dental Caries on Gnotobiotic Inbred Mice
Authors: Maeda Nobuko , Arai Satoshi , Ozaki Akira , Oowada Tsutomu , Takahashi Akiko , Fujita Hiroshi , Mizutani Takeo
Published: November 2013

The purpose of this study in mono‐infected gnotobiotic BALB/cA and C3H/HeN mice was to evaluate the cariogenicity of Enterococcus faecalis. The caries incidence and mean caries score in the BALB/cA mice were significantly higher than those in the C3H/HeN. In both of the mouse strains, the mean number of E. faecalis isolated from the cecum content was almost the same, however, the mean number of E. faecalis from the maxilla of BALB/cA was significantly higher than that of C3H/HeN. These results indicate that C3H/HeN has some factors that prevent E. faecalis from attaching to the tooth surfaces.
Different Sensitivity of Complement to Salmonella typhimurium Accounts for the Difference in Natural Resistance to Murine Typhoid between A/J and C57BL/6 Mice
Authors: Nakano Akira , Kita Eiji , Kashiba Shuzo
Published: November 2013

The difference in natural resistance to Salmonella typhimurium between S. typhimurium‐resistant A/J mice and S. typhimurium‐susceptible C57BL/6 mice was analyzed. In both strains, the growth of S. typhimurium was controlled in the spleen until 48 hr of infection, while serum C3b levels were increased in A/J mice immediately after infection but not in C57BL/6 mice. Incubation of A/J mouse serum with S. typhimurium or its lipopolysaccharide (LPS) generated sufficient amounts of C3b, but that of C57BL/6 mouse serum with them did not. A/J macrophages had higher intracellular killing activity in vitro than did C57BL/6 cells against S. typhimurium pre‐opsonized with each corresponding fresh serum. However, the cells from both mice exhibited a similar level of killing activity against S. typhimurium pre‐opsonized with fresh A/J serum or rabbit complement. The resistance of C57BL/6 mice was significantly increased by opsonizing S. typhimurium with fresh A/J serum or rabbit complement before inoculation. The serum level of interferon‐γ (IFN‐γ) in A/J mice was 2.7 times as high as in C57BL/6 mice at 48 hr post‐infection. Recombinant murine IFN‐γ enhanced the intracellular killing activity of macrophages from both mice when S. typhimurium was pre‐opsonized with fresh A/J serum but not with fresh C57BL/6 serum. These findings suggest that A/J macrophages exhibit maximal killing activity against A/J serum‐opsonized S. typhimurium in vivo when the cells are activated with IFN‐γ. Therefore, the rapid and sufficient activation of complement by Salmonella LPS may render A/J mice more resistant against murine typhoid.
Cellular Lipid and Fatty Acid Compositions of Burkholderia pseudomallei Strains Isolated from Human and Environment in Viet Nam
Authors: Van Phung Le , Chi Tran Thi Bich , Hotta Hisako , Yabuuchi Eiko , Yano Ikuya
Published: November 2013

Viet nam is known as an endemic area of melioidosis but its etiologic agent originated in Viet nam was not extensively studied. For the first time, we analyzed the cellular lipid and fatty acid compositions of 15 Vietnamese isolates of Burkholderia pseudomallei, 10 from humans and 5 from the environment. Cellular lipid compositions were analyzed by two‐dimensional thin‐layer chromatography on silica gel G plates. Cellular fatty acid methyl esters were analyzed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The major lipids in all the isolates were phosphatidylglycerol (PG), two forms of phosphatidylethanolamine (PE‐1 and PE‐2), and two forms of ornithine‐containing lipid (OL‐1 and OL‐2). PE‐1 contained non‐hydroxy fatty acids at both sn‐1 and −2 positions, while PE‐2 possessed 2‐hydroxy fatty acids and non‐hydroxy fatty acids in a ratio of 1: 1. Since snake venom phospholipase A2 digestion of PE‐2 liberated 2‐hydroxy fatty acids, it was confirmed that these acids are at the sn‐2 position of glycerol moiety. In both OL‐1 and OL‐2, amide‐linked fatty acid was 3‐hydroxy palmitic acid (3‐OH‐C16: 0), while ester‐linked fatty acids were non‐hydroxy acids in OL‐1 and 2‐hydroxy acids in OL‐2. The total cellular fatty acid compositions of the test strains were characterized by the presence of 2‐hydroxy palmitic (2‐OH‐C16: 0), 2‐hydroxy hexadecenoic (2‐OH‐C16: 1), 2‐hydroxy octadecenoic (2‐OH‐C18: 1), 2‐hydroxy methylene octadecanoic (2‐OH‐C19CPA), 3‐hydroxy myristic (3‐OH‐C14: 0) and 3‐hydroxy palmitic (3‐OH‐C16: 0) acids. There were significant differences in the concentration of hexadecenoic (C16: 1), methylene hexadecanoic (C17CPA), octadecenoic (C18: 1) and methylene octadecanoic (C19CPA) acids among the Vietnamese isolates of B. pseudomallei. However, no significant difference was observed in cellular lipid and fatty acid components between strains of human and environmental origins.
T Cell‐Dependent Activation of Macrophages and Enhancement of Their Phagocytic Activity in the Lungs of Mice Inoculated with Heat‐Killed Cryptococcus neoformans: Involvement of IFN‐γ and Its Protective Effect against Cryptococcal Infection
Authors: Kawakami Kazuyoshi , Kohno Shigeru , Kadota Jun‐ichi , Tohyama Masaki , Teruya Katsuji , Kudeken Norifumi , Saito Atsushi , Hara Kohei
Published: November 2013

Previous investigations have demonstrated that macrophages play a critical role in the first‐line cellular defense mechanism against infection with Cryptococcus neoformans. In the present study, to elucidate the way in which anticryptococcal activity of macrophages is regulated at the site of infection, pulmonary intraparenchymal macrophages were directly analyzed for expression of their surface molecules and their phagocytic activities against the organism, and the effects of depletion of T cells and endogenous IFN‐γ in vivo on these parameters were examined. In the lungs of mice intratracheally inoculated with heat‐killed C. neoformans, macrophages were activated, as indicated by augmented expression of MHC class II, intercellular adhesion molecule‐1 (ICAM‐1) and Fc receptor (FcR), and about two‐thirds of macrophages were found to have ingested an average of 3.77 ± 0.12 yeast cells per macrophage. In mice depleted of both CD4+ and CD8+ T cells by injecting the specific monoclonal antibodies (mAbs) or anti‐IFN‐γ mAb, not only augmentation of the expression of macrophage activation markers but also phagocytosis of C. neoformans was significantly reduced. These results suggest that anticryptococcal activity of macrophages is regulated by IFN‐γ endogenously produced by T cells. Additionally, treatment with IFN‐γ were shown to significantly prolong the survival time of mice infected with viable C. neoformans. Additionally, preimmunization with heat‐killed C. neoformans significantly prolonged the survival time of mice which received the following infection.
Evaluation of Two Assay Kits for Thermostable Direct Hemolysin (TDH) as an Indicator of TDH‐Related Hemolysin (TRH) Produced by Vibrio parahaemolyticus
Authors: Yoh Myonsun , Kawakami Nobuhiro , Funakoshi Yasunobu , Okada Keishi , Honda Takeshi
Published: November 2013

Reversed passive latex agglutination (RPLA) or enzyme‐linked immunosorbent assay kits with beads (Bead‐ELISA) are commercially available in Japan to detect the thermostable direct hemolysin (TDH) produced by Vibrio parahaemolyticus isolates. We evaluated whether these kits can be used to assay the pathogenic toxin, TDH‐related hemolysin (TRH), produced by some so‐called Kanagawa phenomenon‐negative V. parahaemolyticus strains isolated from patients with diarrhea. Our results showed that the two kits, RPLA and Bead‐ELISA, can detect TRH, although they were originally developed for detection of TDH. This may be due to the use of polyclonal anti‐TDH antisera that cross react with TRH. Although the sensitivity for TDH detection by RPLA and Bead‐ELISA differed tenfold, that for TRH detection was essentially equal. The minimum concentration of TRH required for detection by the two assay kits was about 10 ng/ml.
In Vivo Production of Various Cytokines in Splenocytes of Sheep Erythrocyte‐Immunized Mice after Intravenous Administration of Bacterial Lipid A
Authors: Mashimo Jun‐ichi , Mita Akira
Published: November 2013

The effects of an intravenous administration of lipid A from Salmonella minnesota R595 lipopolysaccharide on the in vivo production of interleukin‐2 (IL‐2), IL‐4, IL‐5 and IL‐6 in the spleens of mice intravenously immunized with sheep red blood cell (SRBC) antigen were investigated. The increased number of antigen‐specific IgM antibody‐producing cells and the titer of the IgM serum antibody were measured using the plaque‐forming cell (PFC) assay and an enzyme‐linked immunosorbent assay (ELISA). Simultaneous injections of SRBC antigen and lipid A adjuvant enhanced IgM‐PFC number on days 3 and 4 and the serum IgM titer on days 4 and 5 after the immunization. We found that the enhanced IL‐4 and IL‐5 levels correlated with the PFC number and IgM titer. When lipid A was injected intravenously 2 days after immunization with SRBC, the PFC number in lipid A‐treated groups were similar to those in controls 3 and 4 days after the immunization. However, it was found that a twofold increase in the IgM titer in serum was induced by lipid A 5 days after immunization. In relation to this increase, lipid A stimulated the production of only IL‐5 among the cytokines tested.
Nucleotide Sequence of the SrRNA Gene of Entamoeba gingivalis: Applications for Construction of a Species‐Specific DNA Probe and Phylogenetic Analysis
Authors: Yamamoto Ayako , Kikuta Noriko , Hashimoto Tetsuo , Oyaizu Hiroshi , Goto Nobuichi
Published: November 2013

The small subunit ribosomal RNA (SrRNA) gene of Entamoeba gingivalis was amplified by PCR and the product of 1.9‐kbp sequence was cloned into a plasmid vector pUC18. Four clones were isolated and sequenced. The insert DNAs were 1918‐ to 1921‐bp long and A+T rich (65.5%). The four SrRNA sequences of E. gingivalis were found to be aligned with those of nine related protozoans while searching for E. gingivalis‐specific sequences. A sequence of 28 oligonucleotides was chosen, chemically synthesized, and labeled with digoxigenin for use as a DNA probe. The probe thus constructed was shown to hybridize only with either the SrRNA‐coding DNAs or the cells of the two E. gingivalis strains and not with those of other protozoans or oral fungi tested. A representative SrRNA‐sequence was analyzed and a phylogenetic tree was constructed by the neighbor‐joining (NJ) method. Among the protists examined, E. gingivalis was placed next to Entamoeba histolytica as expected from the traditional taxonomy.
Malaria Antigens Stimulate Neopterin Secretion by PBMC and U937 Cells
Authors: Facer Christine A.
Published: November 2013

Lysates of Plasmodium falciparum parasitized human erythrocytes stimulate U937 cells to secrete neopterin during a 48 hr co‐culture period. Neopterin secretion by U937 cells was enhanced by the addition of human interferon gamma (IFN‐γ). Several P. falciparum antigens, ‘FC27’ (a synthetic ‘S’ antigen), Ag16 (a recombinant ‘S’ antigen) and Ag44/RHOP3 (a recombinant merozoite rhoptry protein), also activated U937 cells to neopterin secretion and produced a similar effect on peripheral blood mononuclear cells (PBMC) from 2 of 3 normal healthy donors cultured with the antigens for 7 days. Plasma from six Nigerian malaria patients contained high neopterin concentrations ranging from 5.06 to 14.17 ng/ml. This preliminary pilot study lends support for further investigation incorporating a larger number of malaria patients and further culture experiments with U937 cells and PBMC with the aim of defining the cause and source of the large quantities of plasma neopterin produced in this infection.
Analysis of the NH2‐Terminal 83rd Amino Acid of Escherichia coli GyrA in Quinolone‐Resistance
Authors: Yonezawa Minoru , Takahata Masahiro , Banzawa Naoko , Matsubara Nobuyuki , Watanabe Yasuo , Narita Hirokazu
Published: November 2013

Artificial mutations of Gyrase A protein (GyrA) in Escherichia coli by site‐directed mutagenesis were generated to analyze quinolone‐resistant mechanisms. By genetic analysis of gyrA genes in a gyrA temperature sensitive (Ts) background, exchange of Ser at the NH2‐terminal 83rd position of GyrA to Trp, Leu, Phe, Tyr, Ala, Val, and Ile caused bacterial resistance to the quinolones, while exchange to Gly, Asn, Lys, Arg and Asp did not confer resistance. These results indicate that it is the most important for the 83rd amino acid residue to be hydrophobic in expressing the phenotype of resistance to the quinolones. These findings also suggest that the hydroxyl group of Ser would not play a major role in the quinolone‐gyrase interaction and Ser83 would not interact directly with other amino acid residues.
Chromatographic Study of Spirosin by Use of Monoclonal Antibodies to Spirosin of Yersinia enterocolitica
Authors: Yamato Masayuki , Tomotake Hiroyuki , Shiomoto Yasuhisa , Ota Fusao
Published: November 2013

Two monoclonal antibodies (MAbs) reacting with spirosins from Enterobacteriaceae were obtained in a course of screening MAbs to spirosin from Yersinia enterocolitica SYT‐11‐72 (YE72). The antibodies were designated MAbs‐S44 and S50. They were IgG2b and IgG2a, respectively, both with k light chains. On Western blotting after limited proteolysis of YE72 spirosin with Staphylococcus aureus V8 protease, they reacted markedly with peptide fragments of 27 and 35 kDa, suggesting the presence of an antigenic determinant on the fragments. When supernatant cell lysate from Escherichia coli K12 was chromatographed on DEAE‐cellulose and Sepharose CL‐6B columns successively, a 96‐kDa protein with alcohol dehydrogenase (ADH) activity was always associated with reactivity to MAb‐S50. These findings combined with N‐terminal amino acid sequences clearly indicate the identity of spirosin to ADH in E. coli.