bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of Common Flagella Antigen in Bacillus cereus by Monoclonal Antibody
Authors: Mikami Takeshi , Hiraoka Kenji , Murakami Tomonori , Boon‐Long Jotica , Matsumoto Tatsuji , Suzuki Masuko
Published: November 2013

Bacillus cereus has been classified into 23 types by immunochemical analyses of flagella antigen, but a common antigenic determinant of flagella had not been determined. When the immunochemical method of classification had changed from “agglutination method” to “enzyme‐linked immunosorbent assay (ELISA),” the cross‐reactivity between each flagella type was increased. These results indicated that the application of ELISA method would enable detection of the common antigenic determinant of B. cereus. Therefore, we attempted to make monoclonal antibody against common flagella antigen that could be detected by ELISA. Monoclonal antibody provided was specific for B. cereus (H‐1 to H‐23) and did not show the cross‐reactivity with Escherichia coli NIH and Bacillus subtilis.
A Nosocomial Outbreak of Epidemic Keratoconjunctivitis due to Adenovirus Type 37
Authors: Takeuchi Rie , Nomura Yasuhiro , Kojima Motoyoshi , Uchio Eiichi , Kobayashi Nobuyoshi , Matumoto Minoru
Published: November 2013

An outbreak of epidemic keratoconjunctivitis occurred at the department of ophthalmology of a hospital in Yokohama, involving 14 inpatients, 12 outpatients, and 2 doctors. Adenovirus type 37 (Ad‐37) was isolated from the conjunctival swab in 12 of 18 cases. In neutralization tests, the isolates showed some cross‐reaction with adenovirus type 19 (Ad‐19). The Ad‐37 isolates were indistinguishable from each other and from the prototype Ad‐37, but distinct from the prototype Ad‐19 in the restriction endonuclease analysis of viral DNA.
Pili of an Aeromonas hydrophila Strain as a Possible Colonization Factor
Authors: Hokama Akira , Honma Yasuko , Nakasone Noboru
Published: November 2013

Aeromonas hydrophila Ae6 had 2 morphologically distinctive kinds of pili. One appeared rigid and straight with a diameter of 9 nm (R‐pili). The other appeared wavy and flexible with a diameter of 7 nm (W‐pili). W‐pili were very few on the cell as compared with R‐pili. In this study, W‐pili were purified and characterized. The pili consisted of a subunit protein with a molecular weight of 21 kDa as estimated by SDS‐PAGE. There was no immunological cross‐reaction between W‐pili and other cellular components. The strain Ae6 and its purified W‐pili adhered to human and rabbit intestine and agglutinated human and rabbit erythrocytes. Organisms pretreated with the Fab fraction of anti‐pilus antibody failed to adhere to the intestine. Pretreatment of intestine with purified W‐pili blocked adherence of the organisms to the intestine. These results suggest that the W‐pili are the colonization factor of A. hydrophila Ae6.
Electron Microscopic Observation of Branhamella catarrhalis
Authors: Ahmed Kamruddin , Rikitomi Naoto , Nagatake Tsuyoshi , Matsumoto Keizo
Published: November 2013

The hemagglutination (HA) test was done on 85 strains of Branhamella catarrhalis, isolated from sputum of patients with respiratory infections; 53% were HA positive strains. Three HA positive and three HA negative strains were selected and were observed under the electron microscope. The bacterial cell wall appeared to be lobulated and its total thickness was about 38 nm. The nuclear region consisted of whorls or fibrils and dense bodies. Five strains were fimbriated and one strain was nonfimbriated. The size of fimbriae was about 68 nm in length and 4.5 nm in width. The fimbriae of Branhamella catarrhalis were densely arranged and peritrichous in distribution. There was no change of fimbriation between broth and agar cultures.
Studies on the Relationship between Serum Colloidal Reactions (ZTT and TTT) and IgG Subclasses, Especially IgG1 and IgG2
Authors: Miyagawa Nobuyuki , Okamoto Yasuyuki , Morita Keiko , Dohi Kazuhiro
Published: November 2013

The relationship between zinc sulfate turbidity test (ZTT) and thymol turbidity test (TTT), and IgG subclasses, especially IgG1 and IgG2, was studied. Serum colloidal reactions of specimens from patients with IgG‐myeloma usually show abnormally high values in ZTT and low values in TTT. But in some cases. values in ZTT and TTT are both low, and in a few cases both values are abnormally high. In order to see the reason why IgG‐myelomas are classified into these three groups according to serum colloidal reactions, IgG subclasses of monoclonal proteins (MPs) isolated by zone electrophoresis were determined by immunodiffusion with anti IgG‐subclasses. Results revealed that IgG1‐myeloma showed high ZTT and low TTT and IgG2‐myeloma showed low ZTT and TTT. Only two out of our 29 cases with IgG‐myeloma showed high ZTT and TTT. MP from one of them belonged to IgG1‐k and that from the other to IgG2‐λ. We checked the relationship between turbidity tests and electrophoretic mobility of MP and also that between these tests and L‐chains' type of MP, but did not find any relationship between them. These findings suggest that IgG1 innately reacts with ZTT but not with TTT, while IgG2 does not react with any of these tests. MP from cases showing high values in both ZTT and TTT may have a special idiotype.
Production of H2O2 by Alveolar Macrophages in Experimental Allergic Alveolitis
Authors: Tremblay Guy , Thibault Sylvie , Cormier Yvon
Published: November 2013

Experimental extrinsic allergic alveolitis (EAA) was induced in guinea pigs with Saccharopolyspora rectivirgula. Bronchoalveolar lavages were performed before inducing EAA (day 1, BAL 1), on day 23 (BAL 2), and on day 48 (BAL 3). The number of cells/ml in lavage fluid was increased at BAL 2 (4.79 × 106) and BAL 3 (4.29 × 106) compared with BAL 1 (0.56 × 106). The number of major cell types increased simultaneously, neutrophil becoming the predominant cell type over alveolar macrophages (AM). The production of H2O2 by AM was measured at the different phases of EAA. Adherent AM were either non‐stimulated or triggered with phorbol myristate acetate (PMA), zymosan, S. rectivirgula opsonized with normal guinea pig serum (SRNS), or S. rectivirgula opsonized with guinea pig anti‐S. rectivirgula serum (SRAS). Stimulated AM produced larger quantities of H2O2 than unstimulated cells, PMA being the most potent stimulus. At day 1, AM stimulated with S. rectivirgula and zymosan produced similar quantities of H2O2. After the induction of the disease, AM stimulated with S. rectivirgula produced larger quantities of H2O2 than with zymosan. Production of H2O2 by AM stimulated with S. rectivirgula or PMA, respectively, stayed the same at day 1 and 23, but increased sharply for both stimuli at day 48. There was no difference between H2O2 production by AM triggered with SRNS or with SRAS. This study shows that: 1) H2O2 production is enhanced, especially by the offending agent, in the course of the disease; 2) the enhanced production of H2O2 by AM is preceded by the development of alveolitis; and, 3) the opsonization with specific antibodies does not enhance the avidity of AM for the antigen.
Mycolic Acid‐Containing Glycolipid as a Possible Virulence Factor of Rhodococcus equi for Mice
Authors: Gotoh Kohkichi , Mitsuyama Masao , Imaizumi Sadao , Kawamura Ikuo , Yano Ikuya
Published: November 2013

By the use of various Rhodococcus equi strains differing in the length of carbon chains of glycolipid, we examined whether the glycolipid, glucose mono‐mycolate, was contributing to the virulence of R. equi for mice. R. equi strains with longer carbon chain mycolic acid showed a higher virulence as determined by lethality and granuloma formation in mice than those with shorter ones. When purified glycolipid was injected into mice, granuloma formation and liver damage were most prominent with the glycolipid having longer carbon chain mycolic acid. Only a representative strain with longer carbon chain mycolic acid persisted in the spleen of mice after intravenous injection, while a strain with shorter carbon chain mycolic acid was readily eliminated. These results suggested that glycolipid was at least one of the virulence factors of R. equi and that the carbon chain length of mycolic acid might be critical in the expression of virulence.
Factors Influencing the in vitro Growth of Mycobacterium leprae: Effect of Sulfhydryl Compounds
Authors: Dhople Arvind Madhav , Lamoureux Laurie Christine
Published: November 2013

In an attempt to determine the factors that influence the in vitro growth of Mycobacterium leprae in DH medium, the effects of sulfhydryl compounds were studied. Growth of M. leprae was monitored using two biochemical indicators. Only the sulfhydryl compounds, in reduced form, containing carboxyl group could support the growth of M. leprae. Higher cell yields were obtained when these sulfhydryl compounds were supplemented with dithiothreitol, presumable to keep the monothiols in reduced state during long incubation periods. Ascorbic acid could not replace dithiothreitol for this purpose. It is suggested that these carboxylated sulfhydryl compounds play a role in the metabolic activity of M. leprae along with maintaining low redox potential of the medium.
Establishment of a New Perchloric Acid Treatment Method to Allow Determination of the Total Endotoxin Content in Human Plasma by the Limulus Test and Clinical Application
Authors: Inada Katsuya , Endo Shigeatsu , Takahashi Kazuhiko , Suzuki Miyuki , Narita Tomofumi , Yoshida Toshimi , Suda Hidetoshi , Komuro Tetsuo , Yoshida Masao
Published: November 2013

We established a new method of plasma treatment for the removal of interfering factors in the plasma to allow detection of endotoxin by limulus test. The limulus test used was an endotoxin‐specific chromogenic test, the Endospecy test. Perchloric acid (PCA) treatment and centrifugation (PCA method) is usually used to remove interfering factors from plasma, with the precipitate being discarded and the supernatant used to detect endotoxin. As the solubilized precipitates of endotoxin‐spiked plasma and some patient plasma were found to contain the Endospecy activity, we have devised a new method assaying endotoxin in both the supernatant and precipitate. This study confirmed that the solubilized precipitate of endotoxin‐spiked plasma had Endospecy activity and found that the precipitate had other endotoxin activities, such as lethality in galactosamine‐sensitized mice and pyrogenicity in rabbits. We also confirmed that interfering factors were completely removed from plasma samples by this new method. The endotoxin level after the new PCA method was found to be about 8 times higher than that determined after PCA treatment and the new PCA method surpasses the conventional PCA method with regard to the positive rate of endotoxin contents in clinical samples. These results indicate that the new PCA method is superior to the PCA method as a plasma pretreatment method for limulus test.
The Analysis of the Defense Mechanism against Indigenous Bacterial Translocation in X‐Irradiated Mice
Authors: Kobayashi Toshiya , Ohmori Toshihiro , Yanai Minoru , Kawanishi Gosei , Mitsuyama Masao , Nomoto Kikuo
Published: November 2013

The defense mechanism against indigenous bacterial translocation was studied using a model of endogenous infection in X‐irradiated mice. All mice irradiated with 9 Gy died from day 8 to day 15 after irradiation. The death of mice was observed in parallel with the appearance of bacteria from day 7 in various organs, and the causative agent was identified to be Escherichia coli, an indigenous bacterium translocating from the intestine. Decrease in the number of blood leukocytes, peritoneal cells and lymphocytes in Peyer's patches or mesenteric lymph nodes was observed as early as 1 day after irradiation with 6 or 9 Gy. The mitogenic response of lymphocytes from various lymphoid tissues was severely affected as well. The impairment of these parameters for host defense reached the peak 3 days after irradiation and there was no recovery. However, in vivo bactericidal activity of Kupffer cells in mice irradiated with 9 Gy was maintained in a normal level for a longer period. It was suggested that Kupffer cells play an important role in the defense against indigenous bacteria translocating from the intestine in mice.
Sensitivity of Bacteria to NaNO2 and to L‐Arginine‐Dependent System in Murine Macrophages
Authors: Saito Shinji , Onozuka Kazuyasu , Shinomiya Hiroto , Nakano Masayasu
Published: November 2013

Sensitivity of bacteria to NO2‐/NO and to L‐arginine‐dependent system in murine macrophages was tested. The growth of all bacteria tested, Salmonella typhimurium, Pseudomonas aeruginosa and Staphylococcus epidermidis, was significantly inhibited by NaNO2 at the concentration of 5 to 10 mM. However, L‐arginine‐dependent effector mechanism of LPS‐activated murine macrophages was ineffective in killing these bacteria. It seems that the concentration of NO2‐/NO in phagolysosome in the activated macrophages is insufficient to kill the bacteria in situ. We concluded that L‐arginine‐dependent effector mechanism can hardly work on bacteria killing in activated macrophages.
Bacterial ADP‐Ribosylating Toxins: Molecular Structures and Signal Transducing Functions
Authors: Kato Iwao
Published: November 2013

Mono‐ADP‐ribosylation is a posttranslational modification of proteins employed by a variety of bacterial ADP‐ribosylating toxins to modify the metabolism of target cells. The ADP‐ribosyltransferases of bacterial toxins, in general, use NAD as a substrate for covalent modification by ADP‐ribose to certain GTP‐binding proteins (G proteins) as signal transducers resulting in altered enzymatic activity of the membrane enzymes as effectors. Such a mechanism has the potential of being of importance in the physiological regulation of cellular metabolism, particularly if the process is reversible. These ADP‐ribosylating toxins are characterized in Table 1.
The Herald Waves of Influenza Virus Infections Detected in Sendai and Yamagata Cities in 1985–1990
Authors: Moriuchi Hiroyuki , Oshima Takeko , Komatsu Shigeo , Katsushima Noriko , Kitame Fumio , Nakamura Kiyoto , Numazaki Yoshio
Published: November 2013

The community surveillance of respiratory virus infections performed during 1985–1987 in Sendai and 1988–1990 in Yamagata has identified a total of five herald waves of influenza virus infections: A/H3N2 virus infections in 1985 and 1989, A/H1N1 virus infections in 1986 and 1988, and type B virus infections in 1989. To investigate the antigenic and genetic relationships between the herald wave and epidemic strains, influenza A/H1N1 viruses isolated during the 1986 and 1988 herald waves were compared with those isolated during the 1986–1987 and 1988–1989 epidemic seasons, respectively, utilizing hemagglutination inhibition tests with anti‐hemagglutinin monoclonal antibodies and oligonucleotide mapping of total viral RNAs. The results showed that multiple variants differing in antigenic and genetic properties were cocirculating during the 1986 herald wave as well as during each of the two epidemics (only one strain was isolated in the 1988 herald wave). It was also observed that viruses which had the antigenic and/or genetic characteristics closely similar to those of the viruses circulating in the 1986 and 1988 herald waves, were isolated during the winters of 1986–1987 and 1988–1989, respectively.
Characterization of Two Bacteriocins Produced by Clostridium perfringens
Authors: Higa Akiko , Yoshida Etsuko , Miyoshi Yasushi
Published: November 2013

Two types of bacteriocins were shown to be produced in succession by a strain of Clostridium perfringens SN‐17. They were separated by diethylaminoethyl cellulose (DEAE) column chromatography at pH 8.5 with a linear concentration gradient of NaCl. One type of bacteriocin (named SN‐a) was eluted at 0.07 M and the other type (named SN‐b) was at 0.12 M. Each of these was partially purified in a series of column chromatographies: DEAE, Sephadex G‐200 (or Bio Gel P‐150), and hydroxyapatite. Specific activities of SN‐a and SN‐b after the last chromatography were at most 30‐ to 50‐fold that of culture filtrate of the organisms. Chromatographed SN‐a migrated as a single zone in polyacrylamide gel electrophoresis (PAGE) and the zone showed high biological activity. On the other hand, PAGE pattern of SN‐b revealed the presence of a few contamination materials. The activity of SN‐b after the last chromatography was hardly recovered from the gel but inactivated SN‐b was identified in the gel by examining bacteriocin activity of the DEAE fractions recovered from the gel. The molecular weight of the SN‐a and SN‐b was determined to be about 70,000 and 100,000, respectively, by molecular sieve chromatography. These bacteriocins were very sensitive to protease but insensitive to DNase and RNase. Bacteriocins were both completely inactivated at 55 C and they were more stable in alkaline pH than in acidic pH. SN‐a and SN‐b were adsorbed in different ways on the surface of the producer and insensitive strains. Several differences and similarities between these 2 bacteriocins are discussed with special reference to the relationship between them.
A Synthetic Peptide Homologous to Retroviral Transmembrane Envelope Proteins Depresses Protein Kinase C Mediated Lymphocyte Proliferation and Directly Inactivated Protein Kinase C: A Potential Mechanism for Immunosuppression
Authors: Kadota Jun‐ichi , Ciangiolo George J. , Snyderman Ralph
Published: November 2013

CKS‐17, an immunosuppressive peptide homologous to certain retroviral transmembrane envelope protein, has been shown to inhibit lymphocyte proliferation in response to mitogens or alloantigens when covalently attached to bovine serum albumin (CKS‐17‐BSA). To define its site of action, we determined if CKS‐17 conjugated to human serum albumin (CKS‐17‐HSA) could block the direct activation of lymphocytes by phorbol‐12‐myristate‐13‐acetate (PMA) or by a synthetic diacylglycerol, dioctanoylglycerol (DiCs). CKS‐17‐HSA inhibited lymphocyte proliferation in response to PMA and ionomycin in a dose‐dependent manner with up to 88% inhibition occurring with 15μM CKS‐17‐HSA. The conjugated peptide also inhibited the proliferation of lymphocytes in response to DiC8 and ionomycin by up to 57% at 15 μM CKS‐17‐HSA. Based on these findings we investigated the effect of CKS‐17‐HSA on the activity of protein kinase C (PKC), an enzyme directly activated by PMA and DiC8. PKC was isolated chromatographically from the cytosol of human neutrophils or the human lymphoblastoid cell line Jurkat. CKS‐17‐HSA caused a dose‐dependent enzyme inhibition with a concentration giving half‐maximal inhibition (IC50) of ca.3 μM and >95% inhibition at 15 μM CKS‐17‐HSA. Inhibition of PKC by the conjugated peptide was not reversed by increasing concentrations of Ca2+, Mg2+, phosphatidylserine, diolein, or adenosine triphosphate (ATP), indicating that the conjugated peptide did not function as a chelator or competitive inhibitor. In contrast to its effects on PKC, CKS‐17‐HSA did not inhibit the activity of adenosine 3′: 5′‐cyclic monophosphate (cyclic AMP)‐dependent protein kinase (PK‐A) nor the calcium and phospholipid‐independent form of PKC (PK‐M). Moreover the peptide inhibited in vivo PKC activity in cytosol of intact cells and in membrane of PMA‐stimulated cells. These results suggest that the inhibition of lymphocyte proliferation by CKS‐17‐HSA may be due to the direct inactivation of PKC.
S Layer Protein of Clostridium tetani: Purification and Properties
Authors: Takumi Kenji , Susami Yukie , Takeoka Aya , Oka Tatsuzo , Koga Tetsuro
Published: November 2013

S layer protein of Clostridium tetani strain AO 174, a nontoxigenic derivative of strain Harvard A 47, was prepared from the cell walls by 4 M urea extraction and purified by DEAE‐Sepharose CL‐6B chromatography followed by a combination of anion‐exchange chromatography and reverse‐phase chromatography using an HPLC system. The molecular weight of the S layer protein was estimated to be 140 kilodaltons (kDa) by SDS‐PAGE. The amino acid composition of the 140 kDa protein was very similar to those of S layer proteins from the other bacterial species: it was rich in acidic amino acid and lacked cysteine. Also, the protein was unique in its extremely low content of proline (0.02 to 0.03 mol%o). Multiple isoelectric forms ranging from pH 4.0 to 4.5 were observed in the purified preparation. Immunodiffusion analysis showed that the 140 kDa protein was a common antigen to the three strains of C. tetani tested.
Serological Evidence that the Q Fever Agent (Coxiella burnetii) Has Spread Widely among Dairy Cattle of Japan
Authors: Yoshiie Kiyotaka , Oda Hiroshi , Nagano Naoyuki , Matayoshi Seiken
Published: November 2013

Serological examination of bovine and human sera for antibodies against Coxiella burnetii was carried out by the immunofluorescence technique. Twenty to 30%o of the cows examined were antibody‐positive. Sera from two veterinarians also had antibody against C. burnetii. These results suggest an increase in the number of infected cows with C. burnetii in Japan since 1954, and also imply the possibility of the prevalence of acute Q fever in the human population, which had been underestimated and undiagnosed for the last three decades.
Separation of the Prodigiosin‐Localizing Crude Vesicles Which Retain the Activity of Protease and Nuclease in Serratia marcescens
Authors: Kobayashi Nobuharu , Ichikawa Yoichi
Published: November 2013

Crude vesicles in which prodigiosin is localized were separated from pigmented Serratia marcescens. The bacteria were grown on peptonegiycerol agar plate, suspended in saline, and fractionated into cells, vesicles, and supernatant by differential centrifugation. Electron microscopic observations showed that the fractionation was conducted properly and the separated vesicles were lysed in distilled water. The vesicles suspended in saline retained 100 kilodalton protein of which amount is correlated with prodigiosin level, but the 100 kDa protein was found in the supernatant when the vesicles were lysed in distilled water. The vesicle fraction retained few colony‐forming units and little detectable activity of NADH oxidase, but showed much higher activities of protease and nuclease than the cell fraction. The profiles of the activities of the protease and the nuclease in the fractions were different from each other, that is, the protease activity in the vesicle fraction was lower than that in the supernatant fraction, whereas the nuclease activity in the vesicle fraction was higher than that in the supernatant fraction, suggesting that the two extracellular enzymes were released from the pigmented bacteria by different mechanisms.
Examination of Host Defense Factors Responsible for Experimental Chronic Respiratory Tract Infection Caused by Klebsiella pneumoniae in Mice
Authors: Iizawa Yuji , Nishi Takeshi , Kondo Masahiro , Imada Akira
Published: November 2013

We have studied the host defense factors that operate during the course of chronic respiratory tract infection caused by Klebsiella pneumoniae 27 in CBA/J mice. A large number of polymorphonuclear leukocytes (PMNs) rapidly infiltrated the alveolar spaces after infection. Treatment with cyclophosphamide (CY) before infection greatly reduced the infiltration of PMNs and caused an increase in bacterial counts. CY treatment of mice in the chronic phase also caused bacterial proliferation in the lungs. The administration of a high titer immune serum efficiently reduced the bacterial counts in the lungs during the early phase but not during the chronic phase. The proliferation of bacteria induced by CY treatment was not suppressed by the administration of the immune serum in either phase. When the mice were exposed to an aerosol containing Pseudomonas aeruginosa P9 in the chronic phase, the organisms from the secondary infection were eliminated from the lungs in the same manner as in the case of primary infection with P, aeruginosa. Thus, PMNs seem to play an important role in the suppression of bacterial proliferation in the early and chronic phases, and the specific antibody might have a supplementary effect on the defensive action of PMNs in the chronic phase. It is also presumed that the bacteria in the chronic phase of infection are sequestered at sites hardly accessible to PMNs.
Coexistence of Fusion Receptors for Human T‐Cell Leukemia Virus Type‐I (HTLV‐I) and Human Immunodeficiency Virus Type‐1 (HIV‐1) on MOLT‐4 Cells
Authors: Kannagi Mari , Kuroda Marcelo J. , Maeda Yosuke , Harada Shinji
Published: November 2013

Human immunodeficiency virus type‐1 (HIV‐1) and human T‐cell leukemia virus type‐1 (HTLV‐I) have a similar tropism for target cell types, especially for CD4+ T cells. In this study, we provide evidence that receptors of these two viruses exist independently on the target cell. We established an HTLV‐I‐producing CD8+ T cell line (ILT‐8M2) with a remarkable cell fusion capacity. When cocultured with MOLT‐4 cells, ILT‐8M2 cells induced giant syncytia more efficiently than any other tested HTLV‐I‐producer cell lines. In contrast to other HTLV‐I‐producers, ILT‐8M2 cells were minimally susceptible to cytopathic effects of HIV‐1 due to very low expression of CD4, although they were able to be persistently infected by HIV‐1. The indicator MOLT‐4 cells are known to respond well to HIV‐1‐induced cell fusion, but they lose this ability if they become persistently infected with HIV‐1 because of the reduction of CD4 receptor expression. ILT‐8M2 was, however, still capable of inducing syncytia with the MOLT‐4 cells persistently infected by HIV‐1 (MOLT‐4/IIIB). This syncytium formation was dependent on the HTLV‐I‐envelope, as it was inhibited by HTLV‐I‐positive human sera or a monoclonal antibody to HTLV‐I gp46 but not by monoclonal antibodies to HIV‐1 gp120 or CD4. Moreover, ILT‐8M2 cells persistently infected by HIV‐1 (ILT‐8M2/IIIB) induced both HTLV‐I‐ and HIV‐1‐mediated syncytia with uninfected MOLT‐4 cells. These results suggest that HTLV‐I induces cell fusion utilizing receptors on the target cells independent of HIV‐1‐receptors.
Effects of Biocidal Treatments to Inhibit the Growth of Legionellae and Other Microorganisms in Cooling Towers
Authors: Yamamoto Hiroyuki , Ezaki Takayuki , Ikedo Masanari , Yabuuchi Eiko
Published: November 2013

The effects of biocidal treatments for cooling towers were examined through the use of chemicals and ultraviolet irradiation to inhibit the growth of legionellae and other microorganisms. In the water of cooling towers without continuous biocidal treatments, heterotrophic bacteria and bacterivorous protozoan first appeared, and then legionellae increased up to 104 CFU/100 ml. When a UV sterilizer was connected to the cooling tower, the legionellae count was 1/10 or 1/100 of that in the nontreated tower water. In the water of towers supplemented continuously with the biocidal chemicals, legionellae were not found during a 4‐month period. The biocidal treatments tested were proved to suppress the increase of legionellae in cooling‐tower water, and thus are useful in preventing the outbreak of legionellosis due to inhalation of contaminated aerosol from the cooling tower system.
Different Antiviral Potencies of BV‐araU and Related Nucleoside Analogues against Herpes Simplex Virus Type 1 in Human Cell Lines and Vero Cells
Authors: Machida Haruhiko , Nishitani Makiko , Suzutani Tatsuo , Hayashi Kozaburo
Published: November 2013

Antiviral potencies against herpes simplex virus type 1 (HSV‐1) of 1‐β‐D‐arabinofuranosyl‐E‐5‐(2‐bromovinyl)uracil (BV‐araU) and ten other nucleoside analogues in human embryonic lung fibroblast (HEL) cells were compared with those in Vero cells. 5‐Halogenovinylarabinosyluracils, highly active in HEL cells, were inactive against all three laboratory‐stocked strains of HSV‐1 but exerted moderate antiviral effects on three clinical isolates in Vero cells. The reduction in anti‐HSV‐1 potencies of other representative nucleoside analogues in Vero cells was much less than those of 5‐halogenovinylarabinosyluracils. However, significant antiviral potencies of BV‐araU against laboratory strains were observed in other human and monkey fibroblast cells including an immortalized cell line. Significant enhancement of antiviral activity of BV‐araU against a laboratory strain and a clinical isolate was demonstrated in Vero cells by the addition of 0.1 μM aminopterin or FUdR, an inhibitor of thymidylate synthesis. The potentiated anti‐HSV‐1 activity in Vero cells was comparable to the potency in HEL cells without the inhibitor. These results suggested that high activity of thymidylate synthesis and a large thymidylate pool size in Vero cells seem to be related to loss of anti‐HSV‐1 potency of BV‐araU. Original tissue type, species, and the immortality may not be responsible for the reduced antiviral activity of BV‐araU in Vero cells.
Vascular Permeability Enhancement by Vibrio mimicus Protease and the Mechanisms of Action
Authors: Chowdhury Mohammad Afzalur Rahim , Miyoshi Shin‐ichi , Shinoda Sumio
Published: November 2013

Vibrio mimicus, a causative agent of gastroenteritis, has also been reported to attribute to extraintestinal infections. Recently we have purified a metalloprotease produced by the pathogen: however, the role of the protease in V. mimicus infection has not been documented. The V. mimicus protease (VMP) was found to enhance vascular permeability and form edema when injected into the dorsal skin of guinea pig and rat. The permeability enhancement by VMP was observed in a dose‐dependent manner in both guinea pig and rat skin. In guinea pig, an inhibitor of the angiotensin‐converting enzyme was found to augment the permeability enhancement reaction. The permeability enhancement was significantly blocked by soybean trypsin inhibitor (SBTI), an inhibitor of plasma kallikrein reaction. In vitro conversion of plasma prekallikrein to kallikrein by VMP was also noted. In rat skin, the permeability enhancement reaction was not blocked by antihistamine or SBTI. However, the reaction was partially blocked when a mixture of antihistamine and SBTI was administered with VMP. It is apparent from the study that in guinea pig skin, VMP enhances vascular permeability through activation of plasma kallikrein‐kinin system which generates bradykinin, whereas in addition to the activation of plasma kallikrein‐kinin cascade in the case of rat, stimulation of histamine release from mast cells and other unknown mechanism seem to be also a cause of the permeability enhancement reaction. These results suggest that VMP may play a role in extraintestinal infections with edema caused by the pathogen.
Monoclonal Antibody to a Structure Expressed on a Subpopulation of Rat CD8 T Cell Subsets
Authors: Nagoya Satoshi , Kikuchi Kokichi , Uede Toshimitsu
Published: November 2013

We have developed a monoclonal antibody, RTS‐1, which can divide a rat CD8 (+) peripheral T cell population into two functionally distinct subsets. The cell‐surface structure defined by this antibody is a glycoprotein with a molecular weight of 220 kDa found to be a high molecular isoform of rat CD45 antigen. CD4 (+) T cells were not stained by RTS‐1 antibody. The cytotoxic T cell‐enriched population did not express RTS‐1 epitope on the cell surface. CD8 (+) spleen cells as well as RTS‐1(+)CD8(+)T cells exhibited strong inhibition on mitogen‐induced immunoglobulin G production by rat B cells. Furthermore, RTS‐1 antibody, but not the control antibody, abolished CD8(+)T cell‐mediated inhibition of immunoglobulin G production by rat B cells. These data suggest that RTS‐1 antibody recognizes a unique determinant of rat CD45 antigen that is expressed on a fraction of CD8(+) cells.
A Monoclonal Antibody Specific for Conidia and Mycelium Wall Layer of Penicillium and Aspergillus
Authors: Fuhrmann B. , Lebreton V. , Van Hoegaerden M. , Kamphuis H.J. , Strosberg A.D.
Published: November 2013

A monoclonal antibody was obtained from BALB/c mice immunized with Penicillium frequentans mycelium. The specificity of the antibody was evaluated by enzyme‐linked immunosorbent and indirect immunofluorescence assays against the same mycelium. This IgM antibody cross‐reacted with various strains of the Penicillium and Aspergillus genera. By indirect immunofluorescence assays, the antibody was able to stain about 10% of Penicillium and Aspergillus conidia, but major part of conidia did not absorb the fluorescence‐labeled antibody before swelling. During germination of P. frequentans conidia, the germ tube wall which constitutes a continuation of an inner wall layer was also stained. During germination of P. griseofulvum, the protrusion of the germ tube wall was not always recognized by the antibody because the germ tube wall was constituted by a continuation of an outer spore wall layer. The study of the staining patterns of the spores and the protrusions suggests that the antibody specifically recognizes an antigen of the inner spore wall layer. The monoclonal antibody reacts with extracelluar galactomannans produced by genera Aspergillus and Penicillium but is not directed against β‐(1,5)‐linked galactofuranose units.
Clostridium botulinum Type C in Healthy Swine in Japan
Authors: Yamakawa Kiyotaka , Kamiya Shigeru , Yoshimura Kiyoto , Nakamura Shinichi
Published: November 2013

Healthy cattle and swine bred in a district of Japan were examined for the presence of Clostridium botulinum in their liver. Liver specimens were cultivated in chopped meat‐glucose medium and the cultures were examined for botulinum toxin. In cattle, none of the cultures of 100 liver specimens yielded the toxin. In swine, however, C1 or C2 toxin was demonstrated in 8 of 100 liver specimens from 36 farms. One of the five farms where the carrier‐state swine were present was surveyed for about 2 years to determine whether the carrier‐state was transient or resident. C. botulinum type C was found in swine livers and feces, and environmental specimens at extremely high rates during the surveillances, with 76% of specimens yielding botulinum toxin following the culture. These data suggest that it is not uncommon for healthy swine to carry C. botulinum type C in the liver and that there is a close relationship between C. botulinum carrier‐state in swine and the presence of this organism in their raising environments. In 20 cattle and 20 swine suffering from parturient paresis of unknown etiology no evidence for involvement of C. botulinum type C was obtained.
Organization of the Ribosomal RNA Genes in Treponema phagedenis and Treponema pallidum
Authors: Fukunaga Masahito , Okuzako Noriko , Mifuchi Ichiji , Arimitsu Yoshiko , Seki Masaaki
Published: November 2013

The genomic DNA fragment which contains ribosomal RNA (rRNA) genes for Treponema phagedenis was cloned into bacteriophage vector lambda EMBL3. A restriction map of the fragment was constructed and the organization of the rRNA genes was determined. The fragment contained at least one copy of the 16S, 23S and 5S sequences and the genes are arranged in the order 16S‐23S‐5S. Southern hybridization using radiolabeled rRNA gene probes to genomic DNA from T. phagedenis strain Reiter and T, pallidum strain Nichols showed that these organisms have two radioactive fragments which hybridize to the probes in their genome. These results suggest that both pathogenic and non‐pathogenic strains of Treponema may carry at least two sets of rRNA genes on their chromosomes.
Geographic/Ethnic Differences in Human Herpesvirus‐6 Antibody Patterns
Authors: Levine Paul H. , Neequaye Janet , Yadav Manmohan , Connelly Roger
Published: November 2013

Serum samples from healthy adults in four geographic/ethnic groups (Ghanaian Blacks, Malaysian Chinese, Malaysian Indians and United States Caucasians) were tested under code for antibodies to human herpesvirus‐6 (HHV‐6). The prevalence and titer of HHV‐6 antibody in the healthy Ghanaians were significantly higher than in the Malaysian Chinese; United States Caucasians and Malaysian Indians had intermediate prevalence and titer of antibodies. Thus far, no specific differences in HHV‐6‐associated diseases have been noted between geographic/ethnic groups with these marked variations in antibody patterns.
Purification and Characterization of Heterologous Component IIs of Botulinum C2 Toxin
Authors: Ohishi Iwao , Hama Yasuo
Published: November 2013

Botulinum C2 toxin (C2T) elaborated by certain strains of Clostridium botulinum types C and D is composed of separate and dissimilar two proteins, components I and II. Previous studies have shown that these two components of C2T produced by type C and D strains were immunologically heterologous and that C2T‐producers were classified into three groups depending on the difference in molecular characteristics of the components I and II. In the present study, the heterologous component IIs of C2T were purified from three representative strains of the groups and the molecular characteristics of the components were compared. Immunological analyses by agar gel double immunodiffusion test showed that the component IIs purified from the three strains have the specific epitope(s) in addition to the common one(s). The biological activity of C2Ts reconstituted with component I purified from a fixed strain and component II each from the three strains differed depending on the source of the component II. These results indicate that the component II of C2T produced by C. botulinum types C and D differs in molecular structure, which reflects on the difference in the biological activity of the toxin. The present study suggests that the pathophysiological activity of C2T, which possibly causes a necrotic enteritis, is dependent on the C2T‐producing bacteria infected.
Gelatin Particle Agglutination Test for Early Serodiagnosis of Japanese Spotted Fever
Authors: Fujita Hiromi , Mahara Fumihiko , Watanabe Yuriko , Sato Tadashi , Ohara Yoshiro , Homma Morio
Published: November 2013

A gelatin particle agglutination (PA) test for Japanese spotted fever has been developed. Gelatin particles were sensitized with a sonicated causative rickettsia and used as antigens. The antibodies by PA test were detected as early as days 4–7 after the onset, whereas those by indirect immunoperoxidase (IP) test were detected after days 8–11. In addition, PA titers were higher than IP titers before days 20–23. The agglutinins detected by PA test were proven to be IgM because they were all sensitive to dithiothreitol. PA test was, however, less specific than IP test, giving a little nonspecific reaction to the sera from patients with scrub typhus and from individuals unrelated to those two rickettsioses. Nevertheless, PA test, which is simple, rapid, and easy to interpret the results, is useful for the early serodiagnosis of Japanese spotted fever.
Bactericidal Activities of Rat Defensins and Synthetic Rabbit Defensins on Staphylococci, Klebsiella pneumoniae (Chedid, 277, and 8N3), Pseudomonas aeruginosa (Mucoid and Nonmucoid Strains), Salmonella typhimurium (Ra, Rc, Rd, and Re of LPS Mutants) and Escherichia coli
Authors: Kohashi Osamu , Ono Tamiko , Ohki Kazunori , Soejima Toshinori , Moriya Tetsuhiro , Umeda Akiko , Meno Yuko , Amako Kazunobu , Funakosi Susumu , Masuda Masao , Fujii Nobutaka
Published: November 2013

Rat defensins were purified and tested for in vitro bactericidal assay against gram‐positive and gram‐negative bacteria. Staphylococcus aureus (209P, Cowan I, Smith diffuse and Smith compact) were resistant to defensins, whereas Staphylococcus epidermidis, Staphylococcus saprophyticus, Micrococcus lysodeikticus and Bacillus subtilis were less sensitive. Gram‐negative bacteria, such as Pseudomonas aeruginosa (mucoid and K) and Klebsiella pneumoniae (Chedid, 277, and 8N3 which were heavily capsulated, moderately capsulated and noncapsulated, respectively) were all very sensitive to defensins and killed within 20 min. Escherichia coli was moderately sensitive and the rough mutants of lipopolysaccharide (LPS) of Salmonella typhimurium LT2, such as Ra, Rc, Rd, and Re were equally sensitive to defensins, being killed within 40 min. Lysozyme did not show any bactericidal activity except against M. lysodeikticus and B. subtilis, whereas it enhanced the bactericidal activity of defensins against P. aeruginosa, E. coli, and K. pneumoniae and suppressed the killing activity of defensins against S. typhimurium and S. aureus. With regard to the three synthetic rabbit defensins, NP1, NP4, and NP5, NP1 showed strong bactericidal activity against K. pneumoniae 277, comparable to that of rat defensins. Neither NP4 nor NP5 showed any bactericidal activity, while NP5 rather enhanced the bactericidal activity of NP1 against K. pneumoniae 277.
Antiviral Activity of a Bile Pigment, Biliverdin, against Human Herpesvirus 6 (HHV‐6) In Vitro
Authors: Nakagami Tatsuyoshi , Taji Shiro , Takahashi Michiaki , Yamanishi Koichi
Published: November 2013

Biliverdin (BV), a bile pigment, was examined for its antiviral activity against human herpesvirus‐6 (HHV‐6) in vitro. BV (10 μg/ml) markedly inhibited HHV‐6 replication in MT‐4 cells when the cells were treated during a virus adsorption period. Its antiviral effect was weakened when cells were treated after adsorption. Treatment of cells with BV (40 μg/ml) 3 hr after virus infection had no inhibitory effect on virus replication. Virus replication was also significantly inhibited by treatment of MT‐4 cells with BV (10 μg/ml) before infection, while the virions were not inactivated by BV (20 μg/ml). Bilirubin and urobilin, metabolic derivatives of BV, showed slight inhibitory effects on virus replication in the cells. On the other hand, BV had no potent inhibitory activity in the replication of herpes simplex virus‐1 or human cytomegalovirus. These observations suggest that BV could interact with MT‐4 cells to inhibit an early stage of HHV‐6 infection in a virus‐specific manner.
Intracellular Localization of Staphylococcus aureus within Primary Cultured Mouse Kidney Cells
Authors: Murai Miyo , Usui Akemi , Seki Keiko , Sakurada Junji , Masuda Shogo
Published: November 2013

Staphylococcus aureus Cowan I was incubated with monolayers of cells derived from several portions of mouse kidney, and found to be ingested by all types of the renal cells. Intracellular localization of S. aureus was determined by resistance of intracellular cocci against lysostaphin digestion and confirmed by electron microscopy. From renal medulla, three morphological variants of the hyperosmolarity‐tolerant (HOT) cells were obtained. The rate of cocci‐ingesting cells varied from 16.9% to 93.4% among those of the HOT cells at the end of 3‐hr incubation. From renal cortex, three morphological variants of epithelial cells grew in medium RK‐1. Among them, only the cells on the edge of colony ingested Cowan I, while the epithelial cells on the center of colony ingested few cocci. Transferred from medium RK‐1 to MEM supplemented with 10% FBS, part of the cortical cells changed into fibroblast‐like appearance and obtained the capacity to ingest Cowan I. This result may indicate the correlation between ingesting capacity and cellular morphology. From a glomerulus, epithelial (GE) cells and fibroblast‐like (GF) cells were obtained. The GE cells ingested not only S. aureus Cowan I but Staphylococcus epidermidis and Staphylococcus saprophyticus after 30‐min incubation, while the GF cells, like both of the HOT cells and the cortical cells, ingested only S. aureus. These results suggest a possibility that S. aureus is located within nonprofessional phagocytes during its infection and intracellular coccus plays an important role in its pathogenicity.
Analysis of Borderline‐Resistant Strains of Methicillin‐Resistant Staphylococcus aureus Using Polymerase Chain Reaction
Authors: Hiramatsu Keiichi , Kihara Hidetoshi , Yokota Takeshi
Published: November 2013

Identification of methicillin‐resistant Staphylococcus aureus by drug‐susceptibility tests alone poses a serious problem, because a considerable number of clinical S. aureus isolates are borderline resistant to methicillin. To circumvent this problem, we have developed a quick and sensitive method of PCR amplification for the detection of mecA gene, which, coding for PBP2′, is the specific genetic element of methicillin‐resistant Staphylococcus aureus. This method made it possible to identify MRSA strains in a short time using as few as 30 cells as a starting material for template DNA. Using this method, we found that the strains of borderline methicillin‐resistance could be accurately identified. We also found one S. aureus clinical strain, T3, which lacked mecA gene in spite of its resistance to methicillin.
Definition and Application of a Histopathological Scoring Scheme for an Animal Model of Acute Mycoplasma pneumoniae Pulmonary Infection
Authors: Cimolai Nevio , Taylor Glenn P. , Mah Diana , Morrison Brenda J.
Published: November 2013

A histopathological scoring system was developed to assess the pathology of acute Mycoplasma pneumoniae pulmonary infection in a hamster model. A final score per animal (ranging 0–26) is obtained by averaging scores from each lung which have been accumulated by the addition of subscores from the assessments of quantity and quality of peribronchiolar and peribronchial infiltrates, luminal exudates, perivascular infiltrates, and parenchymal pneumonia. The scoring scheme was then applied to test the ability of a heat‐killed inoculum to induce pulmonary pathology and to the trial of a 43 kDa protein‐associated antigen as a vaccine immunogen. A heat‐killed inoculum delivered by both intratracheal and intranasal routes did not induce pulmonary pathology compared to a live inoculum (respective mean scores 0.1, 6.7; P<0.01). Animals prevaccinated with the 43 kDa antigen developed an accentuated pathological response after live challenge compared to those unvaccinated (respective mean scores 16.8, 5.8; P=0.00007). Hypersensitization to growth medium components may, however, have contributed to the accentuated disease since the lungs of vaccinated animals challenged with culture‐negative media also were affected (mean score 5.4). Reproducibility of the scoring system was measured by duplicate reading of histology slides which were randomized to the observer upon the second reading (r=0.93; P=0.000009). The scoring system has the ability to differentiate disease severity in small groups of animals.
IgM Neutralizing Antibody Responses to Human Herpesvirus‐6 in Patients with Exanthem Subitum or Organ Transplantation
Authors: Suga Sadao , Yoshikawa Tetsushi , Asano Yoshizo , Nakashima Toshihiko , Yazaki Takehiko , Fukuda Minoru , Kojima Seiji , Matsuyama Takaharu , Ono Yoshinari , Oshima Shinichi
Published: November 2013

The assay for detecting IgM neutralizing (NT) antibody activity to human herpesvirus‐6 (HHV‐6) was developed by using pretreatment of blood sample with staphylococcal protein A. The activity was mostly present in IgM fractions of serum but not in IgA fractions separated by ultracentrifugation. The assay was used for seroepidemiological studies for HHV‐6 infection. In primary HHV‐6 infection, IgM NT antibodies appeared 5 to 7 days after onset of exanthem subitum, reached maximum titers at 2 to 3 weeks, and tended to decline to undetectable levels after 2 months. In contrast, reactivation of HHV‐6 observed in organ transplants showed somewhat greater degree of IgM NT antibody responses that persisted for 2 to 3 months and became undetectable 5 to 6 months after transplantation. The level and persistence of NT antibody titers measured by the conventional method was generally greater than those of the IgM titers. The prevalence of the IgM NT antibodies was examined in healthy individuals. The antibody was first detected at 4 to 7 months of age (5%), reached maximum level at 8 to 11 months (40%), and was detectable by 4 to 6 years (17%). A few (4 to 5%) of adolescents and adults were positive for the antibody.