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Well-defined 1,2,4-triazolium-based polymers were synthesized by reversible addition–fragmentation chain transfer (RAFT) polymerization of N-vinyl-1,2,4-triazolium salts, i.e., N-vinyl-4-ethyl-1,2,4-triazolium bromide (NVETri-Br) and N-vinyl-(4-ethoxyethyl)-1,2,4-triazolium bromide (NVEtOETri-Br). Reasonable control of the polymerization of these monomers was attained using a trithiocarbonate-type chain transfer agent (CTA), producing poly(N-vinyl-1,2,4-triazolium bromide)s with controlled molecular weights (Mn,SEC > 20000) and low Ð values (Mw/Mn

Die attach material for LED package plays a significant role in performance and reliability of LED device. There are two common materials used for die attach in the LED package and each material has its own characteristics. Epoxy material which is well balanced industrial material and used for a wide variety of applications, offers high die share strength. However, epoxy material cannot used in high temperature because it turns yellow and shows low transmittance at high temperature. By comparison to epoxy, silicone material shows high heat resistance. Because of higher heat resistance, silicone material is the first choice for high brightness LEDs for now. However, as the high brightness of the light elements in recent years, the die attach material exposure to high heat from the light for a long time, failures such as cracks and delamination has occurred. Therefore, development of the material can be obtained with high die share strength and high heat resistance has been desired.In this paper, we propose new concept of the die attach material that offers high die shear strength and high heat resistance. In order to achieve these properties, we use the polysilsesquioxane (PSQ) as the base compound. PSQ is a new organic-inorganic hybrid material and it has high flexibility since the inorganic structure shows excellent transparence and heat resistance, and the organic functional group gives good reactivity. To increase the die shear strength and to enhance anti-delamination performance, we add unique additives such as silane coupling agents and silicone powders into the base compound of PSQ. As a result of evaluation, our newly developed material shows equal heat resistance performance as silicone material, and also shows high die shear strength, more than twice as high as silicone material.

Bartonella henselae is the causative agent of cat scratch disease (CSD). To clarify the population structure and relationship between human and cat strains of B. henselae, 55 specimens isolated in Japan, including 24 B. henselae DNA-positive clinical samples from CSD patients and 31 B. henselae isolates from domestic cats, were characterized by multilocus sequence typing (MLST) and the 16S-23S tRNA-Ala/tRNA-Ile intergenic spacer (S1) sequence, which were used previously for strain typing of B. henselae. Three different sequence types (STs) were identified by MLST, one of which was novel. Fifty-two strains (94.5%), including all strains detected in CSD patients, were assigned to ST-1. Eight S1 genotypes were observed, three of which were novel. The 52 ST-1 strains were classified into seven S1 genotypes, two of which were predominant in both human and cat strains. In addition, 5.5% of the strains (3/55) contained two different intergenic spacer S1 copies. These results indicate that the predominant B. henselae MLST ST-1 in Japan is a significantly genetically diverse population on the basis of the sequence diversity of intergenic spacer S1, and that highly prevalent S1 genotypes among cats are often involved in human infections.

Bartonella henselae is a causative agent of cat scratch disease. We preliminarily tested four media for the bacterial growth, including agar plates with sheep, horse or rabbit blood, and chocolate agar. Of these media, rabbit blood and chocolate agar plate were found to be more excellent for the growth than the medium with sheep or horse blood. Blood samples from 60 domestic cats in Yamaguchi Prefecture were then cultured using 7% rabbit blood agar plates and BACTEC9050 (BD), automated blood culture microbial detection system. B. henselae was isolated from six of the 60 (10%) blood samples. Tiny colonies of B. henselae were visible on the agar medium after one week of culture at 35 degrees C in the 5% CO2 atmosphere. BACTEC 9050 detected B. henselae in one of the 10 blood samples and it took two weeks to detect the bacteria automatically, though gram stain failed to show organisms in the blood culture bottle. In conclusion, rabbit blood or chocolate agar and incubation of agar media more than one week and of BACTEC more than two weeks are recommended for the detection of B. henselae.

In cytological examination, human cytomegalovirus (HCMV) infection can not be implied unless typical HCMV-infected cells like owl's-eye cells are present. However, such cells are not always observed in HCMV-infection cases. The aim of our study is to establish the cytopathological features induced by HCMV. In vitro transfection and fluorescence in situ hybridization (FISH) were performed on human embryo lung (HEL) cells. Marked cellular aggregation was observed at 6-hr postinfection (hpi). Multinucleated cells, giant cells, and, particularly, small vacuoles were present in the nuclei or cytoplasm before the appearance of inclusion bodies. However, molding and ground glass in nuclei were absent. Cell clusters displayed round cytoplasm, dispersed later, and showed anisocytosis. All features occurred before 48 hpi when the owl's-eye cell appeared. In FISH, the positive signal highlighted viral particles that became predominant and localized in nuclei. These cytological aspects are dependent on viral replication and contribute to the cytological detection of HCMV infection.

The cell envelope and cytoplasmic architecture of the Mycobacterium leprae Thai-53 strain were examined using the freeze-substitution technique of electron microscopy and compared with those of the M. tuberculosis H37Rv strain. Both strains had similarly multilayered envelope architectures composed of an electron-translucent layer, a peptidoglycan layer and the plasma membrane, from outside to inside. A comparison of the structures of these two mycobacteria revealed that the M. leprae cell was smaller in size and had a thinner peptidoglycan layer than the M. tuberculosis cell. The cell widths measured on electron micrographs were 0.44 microm for M. tuberculosis and 0.38 microm for M. leprae. The peptidoglycan layer of M. leprae was 4-5 nm, while the corresponding layer of M. tuberculosis was 10-15 nm.

To investigate basic characteristics of 10 virulent phages active on silage-making lactobacilli, morphological properties, host ranges, protein composition and genome characterization were separated into five groups based on host ranges and basic properties. The seven phages of groups I, II and V were active on Lactobacillus plantarum and Lactobacillus pentosus . Phage φPY4 (group III) infected both L. casei and Lactobacillus rhamnosus . Phage φPY5 (group IV) specifically infected Lactobacillus casei . Morphologically, three phages of groups I belonged to the Myoviridae family, while seven other phages of groups II, III and V belonged to the Siphoviridae family. SDS-PAGE profiles, restriction analysis, G+C contents of DNA and Dot blot hybridization revealed a high degree of homology in each group. Clustering derived from host range analysis was closely related to results of DNA and protein analyses. These phages may be applicable to phage typing for silage-making lactobacilli.

Summary Uncoating of clathrin-coated vesicles in neuronal cells requires hsc70 in concert with the cofactor auxilin which contains a J-domain as well as a domain with homology to dual specific phosphatases and tensin, known as PTEN. The question of whether an analogous factor operates in other cell types has until now remained unanswered. Here we show that it is the recently discovered and widely expressed cyclin G-associated protein kinase which fulfils the function of neuronal auxilin in hsc70-mediated clathrin coat dissociation. GAK possesses a J-domain, which stimulates the hsc70 ATPase, it competes with auxilin for clathrin binding and at sufficiently high concentrations acts as a clathrin assembly protein. Moreover, GAK binds to the γ- and α-appendage domains of the adaptor proteins AP-1 and AP-2 in vitro and phosphorylates their medium chains. Cells that transiently overexpress GAK are impaired in respect of receptor-mediated endocytosis. In transfected cells clathrin is dislodged from coated pits/vesicles and co-localizes with GFP-GAK in the form of large aggregates. The cellular distribution of membrane-associated adaptors was unaffected by overexpression of GAK. Our results point to a hsc70/auxilin-based uncoating system as a ubiquitous feature of eukaryotic cells.

The bursa of Fabricius is a gut-associated lymphoid organ that is essential for the generation of a diversified B cell repertoire in the chicken. We describe here a novel gene preferentially expressed in bursal B cells. The gene encodes an 85-kDa protein, designated BASH (B cell adaptor containing SH2 domain), that contains N-terminal acidic domains with SH2 domain-binding phosphotyrosine-based motifs, a proline-rich domain, and a C-terminal SH2 domain. BASH shows a substantial sequence similarity to SLP-76, an adaptor protein functioning in TCR-signal transduction. BASH becomes tyrosine-phosphorylated with the B cell Ag receptor (BCR) cross-link or by coexpression with Syk and Lyn and associates with signaling molecules including Syk and a putative chicken Shc homologue. Overexpression of BASH results in suppression of the NF-AT activation induced by BCR-cross-linking. These findings suggest that BASH is involved in BCR-mediated signal transduction and could play a critical role in B cell development in the bursa.

Bacterial images can be obtained rather easily with an atomic-force microscope (AFM) in the magnification range of 5,000 to 30,000 times without any pretreatment of the specimens for such observations as chemical fixation, dehydration or staining. The bacterial shapes or the presence of flagella can be clearly recognized in these magnification ranges. In addition, we were also able to distinguish between gram-negative and gram-positive bacteria based on the specific wavy surface appearance of the former. AFM could thus be a useful tool for the identification of bacteria in the resolution range between electron and light microscopy.

The fine structure of the cell walls of Gram-positive and -negative bacteria were determined by electron microscopy with the new technique of freeze substitution method, and analysed the cell wall structure of Staphylococcus aureus in detail. The surface of Staphylococcal cell wall was covered with a fuzzy coat consisting of fine fibers or electron-dence mass. This coat was completely removed after extraction of teichoic acid from the cell wall with trichloroacetic acid treatment, but was not affected by sodium dodecyl sulfate or trypsin treatment. It was suggested that many amount of teichoic acid was located on the surface of the cell wall and less inside the cell wall. The capsule of strain Smith diffuse was assumed to play the role as the barrier protected from the penetration of antibody against teichoic acid.

A negatively stained electron micrograph of regularly arranged porin proteins of Campylobacter jejuni on the isolated outer membrane of bacteria was analyzed in detail by the correlation averaging method using a computer-assisted program. The results showed that the porin of C. jejuni had a trimeric structure separated by about 10.4 +/- 0.15 nm. In addition, the pores in the trimers were also separated by about 4.3 +/- 0.1 nm.

The surfaces of the disrupted-cell surfaces of the Campylobacter jejuni strains FUM158432 and M1 were examined using the negative-staining technique and electron microscopy. The surfaces of the whole cells and the outer membranes were covered with small dark dots which, in some areas, were arranged in hexagonal patterns. The hexagonal arrangement was more clearly seen in extracted outer membrane. The size of each structure was measured based on a center-to-center distance with the adjacent structure, and was determined to be 9.9 +/- 0.9 nm. A profile of the proteins in the outer membrane by SDS-PAGE, performed in 0.1% SDS and at 100 C, showed 42 kDa proteins to comprise the major outer membrane protein of this bacterium. Digestion of the outer membrane materials with proteinase reduced this protein band in the SDS-PAGE, and the amount of dark dots on the electron micrograph indicated the structure to be the major outer membrane protein (porin) of this bacterium. The power spectrogram of a computer-assisted Fourier transformation of the hexagonally arranged porin proteins suggests that the porin has a trimeric structure rather than a monomeric one.

走査プローブ顕微鏡の生物学的応用の現状について, 主として原子間力顕微鏡での応用研究について総説的に解説した。現在生物試料として, DNA線維, コレラ毒素分子, バクテリオファージ, 外膜タンパク分子, S-layer タンパク分子などが, 溶液中, あるいは大気中で様々な試料作成法の検討をふまえて観察されつつあり, かなりの成果を上げつつある。全く新しい発想のもとに設計された顕微鏡として, 電子顕微鏡と光学顕微鏡の不備を補完する役割を果たしうるか否かは, これからの応用技術の開発にかかっていると言える。

From the nares of 11 healthy adults, 253 strains of coagulase negative staphylococcus were isolated and 88% of them were identified as Staphylococcus epidermidis using the API STAPH system. Chromosomal DNA fingerprinting of the isolated strains revealed that each person carried multiple types of S. epidermidis in his or her nares. The colonization of the strains was not stable; the types of the isolates changed in the first and the second examinations 5 months apart. The results contrasted with previous findings in which only one strain of S. aureus colonized persistently in the nares of healthy adults.

Strain Eri-1T was isolated from a water sample on the campus of Kyushu University, Fukuoka, Japan. The motility and morphology of the isolate were similar to those of members of the genus Leptospira , but the spiral structure of the isolate was sharper under dark-field microscopy. Cells were 10.6±1.3 µm long and 0.2 µm in diameter, with a wavelength of 0.9 µm and an amplitude of 0.4 µm. Strain Eri-1T grew in Korthof’s medium at both 13 and 30 °C, and also in the presence of 8-azaguanine. 16S rRNA gene-based phylogenetic analysis placed strain Eri-1T within the radiation of the genus Leptospira where it formed a unique lineage within the clade of the known saprophytic species of the genus Leptospira . The strain was not pathogenic to hamsters. Strain Eri-1T exhibited low levels (11.2–12.6 %) of similarity by DNA–DNA hybridization to the three most closely related species of the genus Leptospira . The DNA G+C content of the genome of strain Eri-1T was 42.5±0.1 mol%. These results suggest that strain Eri-1T represents a novel species of the genus Leptospira , for which the name Leptospira idonii sp. nov. is proposed. The type strain is Eri-1T ( = DSM 26084T = JCM 18486T).

Summary The surface structures of the cell envelopes of 16 clinical isolates of Pseudomonas aeruginosa were examined by electronmicroscopy with the new fixation technique of freezesubstitution. Two types of structures were observed among the organisms. In one group of strains, mostly isolated from blood, a dense fibrous layer c. 30 nm thick was found around the outer-membrane surface, whereas no such structure was observed in the other group of isolates, most of which were from sputum. Lipopolysaccharides extracted from the isolates with a dense fibrous layer were found by SDS-PAGE to have long O-polysaccharide chains, whereas strains without such a layer mostly had lipopolysaccharides that lacked high mol. wt. O-polysaccharide chains.

Alterations of the p53 tumor suppressor gene have been observed in a variety of human and mouse tumors. For investigation of the role of this gene in tumors in cats, feline p53 cDNA was molecularly cloned by PCR amplifications using primers based on the sequences conserved among several species. The cloned cDNA appeared to cover approximately 90% of the open reading frame of the feline p53 gene and had characteristic structures in common with the p53 genes of several other species. The amino acid sequence similarities of the feline p53 with the human, mouse, rat and chicken counterparts were 82.9%, 75.6%, 76.5% and 57.2% respectively. Moreover, using a panel of feline x rodent somatic cell hybrids, the feline p53 gene was assigned to feline chromosome E1. These data will be useful for determining the role of the p53 tumor suppressor gene in feline tumors.

Bartonella henselae is the causative agent of cat scratch disease (CSD). To clarify the population structure and relationship between human and cat strains of B. henselae, 55 specimens isolated in Japan, including 24 B. henselae DNA-positive clinical samples from CSD patients and 31 B. henselae isolates from domestic cats, were characterized by multilocus sequence typing (MLST) and the 16S-23S tRNA-Ala/tRNA-Ile intergenic spacer (S1) sequence, which were used previously for strain typing of B. henselae. Three different sequence types (STs) were identified by MLST, one of which was novel. Fifty-two strains (94.5%), including all strains detected in CSD patients, were assigned to ST-1. Eight S1 genotypes were observed, three of which were novel. The 52 ST-1 strains were classified into seven S1 genotypes, two of which were predominant in both human and cat strains. In addition, 5.5% of the strains (3/55) contained two different intergenic spacer S1 copies. These results indicate that the predominant B. henselae MLST ST-1 in Japan is a significantly genetically diverse population on the basis of the sequence diversity of intergenic spacer S1, and that highly prevalent S1 genotypes among cats are often involved in human infections.

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.

Many electron-dense granules were found in the nucleoid area of Pseudomonas aeruginosa strain K by electron microscopy with the technique of the freeze-substitution method. These granules contained phosphorus and calcium as determined by X-ray microanalysis. The size and the numbers of the granules decreased when the bacteria was cultured in the medium from which phosphate-containing compounds were depleted. From these observations we concluded that the granule was a phosphate-containing granule and possibly a polyphosphate granule. The excellent preservation of the fine structures by the freeze-substitution technique enables us to show very small polyphosphate granules in the nucleoid area of the bacterial cells which cannot be revealed by the conventional chemical fixation method. As we could not see the granules in other bacteria cultured in nutrient medium such as Serratia, Escherichia, Bacillus and Vibrios, the accumulation of the phosphate granules in Ps. aeruginosa might be a unique character of this bacteria and might be related to the growing capability of this bacteria in extremely low nutrient supply.

Helicobacter pylori divides in the human stomach resulting in persistent infections and causing various disorders. Bacterial cell division is precisely coordinated by many molecules, including FtsZ and Min proteins. However, the role of Min proteins in H. pylori division is poorly understood. We investigated the functional characteristics of Min proteins in wild-type HPK5 and five HPK5-derivative mutants using morphological and genetic approaches. All mutants showed a filamentous shape. However, the bacterial cell growth and viability of three single-gene mutants (minC, minD, minE) were similar to that of the wild-type. The coccoid form number was lowest in the minE-disruptant, indicating that MinE contributes to the coccoid form conversion during the stationary phase. Immunofluorescence microscopic observations showed that FtsZ was dispersedly distributed throughout the bacterial cell irrespective of nucleoid position in only minD-disruptants, indicating that MinD is involved in the nucleoid occlusion system. A chase assay demonstrated that MinC loss suppressed FtsZ-degradation, indicating that FtsZ degrades in a MinC-dependent manner. Molecular interactions between FtsZ and Min proteins were confirmed by immunoprecipitation (IP)-western blotting (WB), suggesting the functional cooperation of these molecules during bacterial cell division. This study describes the intrinsic characteristics of Min proteins and provides new insights into H. pylori cell division.

Strain ST67P of Staphylococcus hyicus was capsular type I (++)/II(+) of S. epidermidis as determined by the method of Ichiman. To mice immunized with heat-killed vaccine of strain ST67P, homologous strain and strain ATCC 31432 (capsular type I), SE-360 (capsular type II) and SE-10 (capsular type III) of S. epidermidis were injected intraperitoneally into mice, then, the viable cell number of the organisms in the peritoneal cavity were enumerated of 30 minutes and 20 hours after the injection. Results showed that the viable cell number of the homologous strain and strain ATCC 31432 was remarkably decreased at 20 hours after the injection, however, the cells were increased with strain SE-360 and SE-10. Passive protective activity of rabbit anti-ATCC 31432 serum was absorbed either with homologous strain or strain ST67P in the mouse, however, protective activity of anti-SE-360 strain serum and anti-SE-10 strain serum was not absorbed with these organisms although the activity was absorbed with homologous organisms. With an ultrathin section preparation of strain ST67P conjugated with ferritin-labelled rabbit anti-homologous strain serum, numerous ferritin granules surrounding the outermost layer of large capsule were electronmicro-scopically demonstrated. In the same organisms treated with ferritin-labelled anti-ATCC31432 strain serum, the all walls were surrounded by a relatively thinner capsule and a number of ferritin granules were located in the outermost layer of the capsule. However, with the organisms treated with ferritin-labelled anti-SE-360 strain serum only a number of ferritin granules were shown on the surface of the cell walls, and neither capsule nor ferritin granules were exhibited in the organisms treated with ferritin-labelled anti-SE-10 strain serum.

Bartonella henselae is a causative agent of cat scratch disease. We preliminarily tested four media for the bacterial growth, including agar plates with sheep, horse or rabbit blood, and chocolate agar. Of these media, rabbit blood and chocolate agar plate were found to be more excellent for the growth than the medium with sheep or horse blood. Blood samples from 60 domestic cats in Yamaguchi Prefecture were then cultured using 7% rabbit blood agar plates and BACTEC9050 (BD), automated blood culture microbial detection system. B. henselae was isolated from six of the 60 (10%) blood samples. Tiny colonies of B. henselae were visible on the agar medium after one week of culture at 35 degrees C in the 5% CO2 atmosphere. BACTEC 9050 detected B. henselae in one of the 10 blood samples and it took two weeks to detect the bacteria automatically, though gram stain failed to show organisms in the blood culture bottle. In conclusion, rabbit blood or chocolate agar and incubation of agar media more than one week and of BACTEC more than two weeks are recommended for the detection of B. henselae.

To clarify the clinical manifestations of cat scratch disease (CSD), we evaluated a total of 130 seropositive patients with CSD. The patients' ages ranged from 1 to 68 years; 103 (79.2%) were under 18 years of age. CSD occurred predominantly in the fall and winter months. Regional lymphadenopathy was noted in 110 (84.6%) of the cases, and the most common sites were the neck (33%), axillary (27%), and inguinal (18%) regions. One hundred of the patients (77%) had general symptoms, such as fever, headache, and malaise. The clinical manifestations of CSD showed a wide spectrum from typical or classical CSD, with regional lymphadenopathy, to atypical or systemic CSD. Of the 130 cases, 103 (79.2%) were typical CSD and 27 (20.8%) were atypical CSD. Atypical cases of CSD were commonly reported as fever of unknown origin (37.0%), neuroretinitis (22.2%), encephalopathy (14.8%), hepatosplenic granuloma (11.1%), and Parinaud's oculoglandular syndrome (7.4%). Fever of unknown origin or prolonged fever lasting more than 14 days was evident in 27 (20.8%) of the 130 cases in this study. Eleven of the 27 cases lacked lymphadenopathy. Our findings suggest that CSD is not a rare disease in Japan. The indirect fluorescent antibody (IFA) test to detect Bartonella species may provide a prompt diagnosis of CSD and facilitate appropriate therapy.

Objective: To evaluate the typing power of cluster analysis of antimicrobial susceptibility. Methods: Results of pulsed-field gel electrophoresis in 71 strains of meth icillin-resistant Staphylococcus aureus were compared with cluster analysis of the diameter of growth inhibition in 11 drugs. Subjects were a consecutive series of patients (n = 71) from the wards and outpatient units of a community teaching hospital. Results: The cluster analysis took 2 to 3 seconds once the data were entered into a computer. The sensitivity, specificity, and accuracy of the cluster analysis were 76.3%, 58.3%, and 73.2%, respectively, using genotyping as the reference. Conclusions: The cluster analysis offered real-time epidemiologic data at minimal cost and labor, warranting its cost-effective role.

We studied the serological cross-reactions among Bartonella henselae, Chlamydia pneumoniae and Coxiella burnetii by indirect fluorescence antibody (IFA) method, using sera from 8 patients with cat scratch disease (CSD), 13 patients with C. pneumoniae infection and 12 patients with acute Q fever. B. henselae IgG antibody was negative in 13 patients with C. pneumoniae infection, and was positive in 3 (titers being 1:64) of 12 patients with Q fever, whereas B. henselae IgM antibody was negative in all the patients with C. pneumoniae infection or Q fever. C. burnetii IgG antibody was removed by absorption of these 3 sera with C. burnetii antigens, whereas B. henselae IgG antibody did not change. C. pneumoniae IgG antibody was positive in 3 (titers being 1:125 in two, 1:32 in one) of 8 patients with CSD. Both C. pneumoniae and B. henselae IgG antibody titers were significantly reduced by absorption of these 3 sera with B. henselae antigens. C. burnetii IgG or IgM antibodies were negative in all patients with CSD. In conclusion, no serological cross-reaction between B. henselae and C. burnetii was observed. On the other hand. B. henselae IgG antibody cross-reacted to C. pneumoniae antigens, whereas C. pneumoniae IgG antibody did not cross-react to B. henselae antigens. Our findings suggest that determination of B. henselae IgG or IgM antibodies were not influenced by C. pneumoniae and C. burnetii antigens.

A total of 200 isolates of Haemophilus influenzae were analyzed by serotyping, biotyping, and pulsed-field gel electrophoresis (PFGE). A total of 178 epidemiologically unrelated strains of H. influenzae demonstrated a variety of genome patterns by PFGE, and 165 genotypes were thus obtained in this study. PFGE typing proved to have a much stronger discriminatory power than either serotyping or biotyping. Six serotype b strains were all classified into discrete genotypes. A PFGE analysis of 18 strains obtained from the nasopharynx, blood, and cerebrospinal fluid of patients with meningitis also supported the hypothesis that invasive H. influenzae disseminates from the nasopharynx to the bloodstream and then subsequently to other body sites. PFGE typing of 10 other strains isolated from household contacts of patients with H. influenzae infection revealed that the strain that caused the H. influenzae infection often colonized the nasopharynges of household contacts. Our findings suggest that PFGE analysis is useful for the epidemiological study of H. influenzae infection, even when the invasive disease is caused by serotype b strains.

The swimming patterns of Campylobacter jejuni in environments of low and high viscosity were examined by a video tracking method. In media of low viscosity, C. jejuni swam with an average velocity of 39.3 microm/s with frequent changes in direction. The velocity of C. jejuni increased in a medium at a little higher viscosity than that of a low viscosity buffer. In addition to this, C. jejuni showed a second increase of velocity in media of a high viscosity of about 40 centipoise. The swimming patterns at these two velocity peaks were compared. In the second peak the wild-type C. jejuni exhibited repeated back and forth swimming patterns which were more like the swimming pattern of spirochaetes than that of monotrichous bacteria. Thus C. jejuni may presumably use a different swimming mode in media of high viscosity than the original swimming mode mediated by the propelling force of the flagella. The spiral shape of this bacterium like that of spirochaetes may strongly influence its swimming ability in media of high viscosity such as the mucous layer of the intestinal tract.

To investigate the effects of conjugated bile acid on bacterial growth and cholyglycine hydrolase activity, Clostridium perfingens from human feces was exposed to varying concentrations of taurine- or glycine-conjugated chenodeoxycholic acid. Extracellular enzyme activity was determined by deconjugation of radiolabeled taurocholic acid and viable cells were counted after anaerobic culture at 37°C for 24 h. Viable cells were decreased with more than 1.0 mg conjugated chenodeoxycholic acid per mL and there were no viable cells with 10.0 mg of bile acid per mL. Although enzyme activity was decreased according to the bile acid concentration, enzyme activity per bacterium was increased between 1.0 and 4.0 mg/mL. There were no statistically significant differences between the types of conjugation. It was concluded that conjugated bile acids may exert inhibitory effect on bacterial growth and extracellular cholyglycine hydrolase activity in Clostridium perfringens . However, under the physiologic condition in the human intestine, conjugated bile acid might induce production of extracellular cholyglycine hydrolase per bacterium. (Steroids 59 :485–489, 1994)

Heat-inactivated horse serum inhibited penicillin-induced L-form colony formation in Staphylococcus aureus when included in an osmotically stabilized culture medium. Most, perhaps all, L-form colonies that appeared with low frequencies on the serum-supplemented medium were of the penicillin-independent, stable type. This relationship must be taken into account when use of serum is considered for L-form cultivation.

Anti-peptidoglycan (PG) and anti-teichoic acid (TA) antibodies were prepared from sera of rabbits immunized with the cell wall fraction of Staphylococcus aureus Cowan I by the specific adsorption technique with purified teichoic acid or peptidoglycan. The anti-PG antibody recognized the trichloroacetic acid-treated walls (TCA wall) prepared from S. aureus, Bacillus subtilis, and Micrococcus luteus but did not react with teichoic acid or proteins extracted from the cell wall of Staphylococcus. The anti-TA antibody specifically reacted with cell wall teichoic acid of beta-type sugar configuration. The reaction sites of these antibodies on the cell wall of S. aureus Wood 46 were determined by immunoelectron microscopy using colloidal gold as a probe. The anti-TA antibody reacted mostly with the fibrous electron-dense mass on the cell surface. The reaction was also seen on the inner surface of the cell wall. The anti-PG antibody reacted with the fibrous structures and also directly on the cell wall surface. The distribution of the probes on the cell wall surface examined with the scanning electron microscope showed that there was no localized distribution in respect to the cell division. We knew from these observations that the external surface of the cell wall of Staphylococcus is covered with the fibrous mass which consists mostly of teichoic acid but partially of peptidoglycan.

The fine structure of the capsule of Staphylococcus aureus Smith diffuse was examined by the technique of freeze-substitution and immunoelectron microscopy. The cell surface was covered with a thick layer consisting of fine fibrous structures which were absent from an unencapsulated strain, Smith compact. Anti-teichoic acid antibody did not react with this surface layer but reacted with the surface of strain Smith compact. Anti-capsular antibody, made from the serum of a rabbit immunized with strain Smith diffuse and specific absorption with unencapsulated strain Wood 46, reacted with the fibrous layer of the Smith diffuse strain. Since the anti-teichoic acid antibody did not react with the encapsulated strain Smith diffuse, the capsular layer acts as a barrier to penetration of the anti-teichoic acid antibody through the capsular layer. A portion of a few cell surfaces of the encapsulated strain remained accessible to the anti-teichoic acid antibody. The capsular layer in this portion of the cell surface was thin, and this surface seemed to be a new cell wall surface created by the cell separation.

Induction of tumor necrosis factor-alpha (TNF-alpha) by Staphylococcus aureus L-form was investigated. The supernatant of a macrophage culture mixed with S. aureus L-form showed a potent cytotoxic activity to L cells. Addition of anti TNF-alpha antibody inhibited completely the cytotoxic activity of the supernatant, indicating that the activity might be due mostly to TNF-alpha. To investigate localization of TNF-alpha production, the membranes of hypotonicity treated L-form were layered on a step-gradient composed of an upper and lower layers of 35% and 50% sucrose, respectively. The membranes were banded at the interface of 35% and 50% of sucrose. The activity of TNF-alpha production of the membrane fraction was 10-times higher than that of the soluble fraction.

Aims: To identify an extreme thermophile, strain TMY, isolated from silica scale from the geothermal electric power plant and to examine microdiversity of Thermus thermophilus strains. Materials and Results: The isolated strain TMY was identified by morphological, biochemical and physiological tests. Phylogenetic comparison of the strain and other Thermus strains with 16S rDNA analysis, RAPD and ERIC-PCR fingerprinting were performed. Strain TMY was closely related to strain which was isolated from a hot spring in New Zealand and shown to belong to the Japanese Thermus cluster. However, there were considerable genetic differences between strain TMY and other Thermus species using DNA fingerprinting. Conclusions: Based on morphological, physiological and genetic properties, strain TMY could be a strain of T. thermophilus. The distinct properties of strain TMY suggest that microdiversity of T. thermophilus strains should be considered. Significance and Impact of the Study: The results of this study have demonstrated genetic diversity within T. thermophilus strains, which were previously masked by an almost identical 16S rDNA sequence. RAPD and ERIC-PCR could be potential methods for distinguishing between Thermus strains.

An outbreak of nosocomial Campylobacter fetus meningitis occurred in a neonatal intensive care unit (NICU). Eight C. fetus strains were isolated from 4 infants with meningitis, the mother of the index patient and 2 infants who were asymptomatic intestinal carriers. The pulsed-field gel electrophoresis (PFGE) pattern with the restriction endonucleases Smal and Sall were found to be identical for the nosocomial C. fetus isolates, but the patterns were different from those of sporadic strains. These nosocomial strains were strongly suspected to be a single strain. The finding revealed that the index patient was infected by the mother, and that the outbreak developed from this patient by cross-infection. This is the first confirmed nosocomial C. fetus meningitis outbreak spread by cross-infection in a NICU.

Pseudomonas aeruginosa is an important pathogen in the infection of compromised host. As pathogenic factors the roles of some cell associated structures like flagella, cell surface layer and the production of slime are reported. Therefore for the understanding of the mechanisms of the infection of this bacteria, the exact data on the morphological observations are indispensable. In this paper we described our recent observations on the fine structures of P. aeruginosa determined by the mild fixation method, the freezesubstitution method.

The fine structure of the Staphylococcus aureus cell wall was determined by electron microscopy with the new technique of rapid freezing and substitution fixation. The surface of the cell wall was covered with a fuzzy coat which consisted of fine fibers or an electron-dense mass. Morphological examination of the cell wall, which was treated sequentially with sodium dodecyl sulfate, trypsin, and trichloroacetic acid, revealed that this coat was partially removed by trypsin digestion and was completely removed by trichloroacetic acid extraction but was not affected by sodium dodecyl sulfate treatment, suggesting that the fuzzy coat consists mostly of a complex of teichoic acids and proteins. This was confirmed by the application of the concanavalin A-ferritin technique for teichoic acid and antiferritin immunoglobulin G technique for protein A.

Morphological aspects of the outgrowth of Bacillus subtilis strain w23 from heat-activated spores were examined by scanning electron microscopy. After 60 min incubation at 37 °C on agar medium, cracks appeared on the spore shells at their equatorial position and vegetative bacteria emerged by breaking open the cracks. In the following 180 min, the bacteria continued to grow without cell separation or the formation of flagella, so that, at 240 min, long filamentous forms of the vegetative bacteria were seen. Cell separation occurred at 300 min and simultaneously the development of flagella was observed. In liquid medium, the vegetative bacteria showed cell separation at an earlier stage (120 min) of outgrowth and flagella were visible at this stage. These results suggested that cell separation and flagella formation were closely related events. The role of autolytic enzymes in cell separation and flagella formation in relation to spore outgrowth is discussed.

The process of cell separation and arrangement of Staphylococcus was investigated using a scanning electron microscope. After two cycles of cell division, the Staphylococcal cells cultured on an agar medium were generally observed to be arranged in three morphological types: linear, square, and crooked arrangements. Results of the examination of cell surface structure revealed that separations had occurred in these clustered cells following two patterns. One type of second separation occurred parallel to the transversal axis of the preceding pair of the parental cells (X-type) and the other occurred tangential to it (Y-type). In the former type, the four daughter cells were usually arranged tetragonally after the separations, and in the latter type they were arranged either linearly or crookedly depending on the direction of the second separation. The final pattern of the cell arrangement was thus determined by the type of septal wall formation and the direction of cell separation. After several cycles of cell divisions, the cells were finally arranged in an irregular grape-like cluster, even though the cross walls were formed regularly at the rectangular face of the preceding cross walls.

Summary: The colonization of the intestinal tract of suckling mice by Campylobacter jejuni was examined by orally challenging the mice with a wild-type strain and several nonmotile mutant strains which were isolated after treating the wild-type strain with mutagens. The wild-type strain had colonized the lower portion of the small intestine, the caecum and the colon 2 d after inoculation. Two nonmotile strains, one of which (M8) had lost all the flagellar structure including the filament, the hook and the basal structure, and the other (M1) which had lost only the filament region, were both cleared from the intestinal tract 2 d after challenge. Another nonmotile strain (M14), which had a complete flagellar structure like that of the wild-type strain, did not colonize and was cleared from the intestinal tract like the other nonmotile and nonflagellated strains. One atypically motile strain (M5), which had a shorter flagellar filament than that of the wild-type strain, colonized the intestinal tract only when mice were challenged with a large inoculum. None of the mice challenged with either the wild-type or any of the mutant strains showed signs of illness. We concluded that motility is an important factor in the colonization of the intestinal tract of suckling mice by C. jejuni.

The anti-staphylococcal IgE and IgG antibody levels of patients with hyper-IgE syndrome (HIE) were measured by microtiter solid phase radioimmunoassay (MSPRIA) and enzyme-linked immunosorbent assay (ELISA) using solubilized cell wall proteins (SCWP) and teichoic acid extracted from Staphylococcus aureus. Using MSPRIA, specific IgE antibodies to SCWP were detected at significant levels in all 5 patients with HIE, but not in 23 patients with atopic allergy or Wiskott-Aldrich syndrome with or without staphylococcal infections. IgG antibodies to SCWP and teichoic acid were lower in the patients with HIE than those in non-allergic infectious controls. These findings suggest an abnormal immunologic response to Staphylococcus aureus in patients with HIE.

The surface of Staphylococcus was examined in the field emission source scanning electron microscope and the following observations were obtained. The cell surface consisted of three areas, the smooth, the less smooth and the rough. Two cells were joined together facing each other at the smooth area. Many such pairs of cells were found in the cluster of cells. Among these pairs of cells the oval-shaped cells with rifts were occasionally detected. From this we discovered that in Staphylococcus cell separation proceeded asymmetrically along their septal wall from one end to the other and finally ceased at the end of septum, forming a pair of cells. We also found that newly exposed surface was smooth and it gradually became rough.

Surface structure and growth of the surface of Corynebacterium diphtheriae mitis strain were investigated by scanning electron microscopy and the immunofluorescence technique. The surface of the cell revealed by the scanning electron microscope showed a few elevated circular zones which encompassed the cell. The cell diameter increased at this zone and this gave the club-shaped appearance to this species. The cell surface labeled with specific antibodies against the whole bacterial cell and tagged with ferritin remained at a constant length during cell division cycles and the new cell surface emerged from the polar ends of the cell. This new wall surface was completely devoid of the ferritin particles indicating that the cell wall component on the old preexistent wall was completely conserved. A similar finding was obtained by immunofluorescence microscopy. C. diphtheriae, unlike Bacillus spp., showed apical growth as has been observed in fungal cells.

Lipid-peptide interaction has been investigated using cationic amphiphilic alpha-helical peptides and systematically varying their hydrophobic-hydrophilic balance (HHB). The influence of the peptides on neutral and acidic liposomes was examined by 1) Trp fluorescence quenched by brominated phospholipid, 2) membrane-clearing ability, 3) size determination of liposomes by dynamic light scattering, 4) morphological observation by electron microscopy, and 5) ability to form planar lipid bilayers from channels. The peptides examined consist of hydrophobic Leu and hydrophilic Lys residues with ratios 13:5, 11:7, 9:9, 7:11, and 5:13 (abbreviated as Hels 13-5, 11-7, 9-9, 7-11, and 5-13, respectively; Kiyota, T., S. Lee, and G. Sugihara. 1996. Biochemistry. 35:13196-13204). The most hydrophobic peptide (Hel 13-5) induced a twisted ribbon-like fibril structure for egg PC liposomes. In a 3/1 (egg PC/egg PG) lipid mixture, Hel 13-5 addition caused fusion of the liposomes. Hel 13-5 formed ion channels in neutral lipid bilayer (egg PE/egg PC = 7/3) at low peptide concentrations, but not in an acidic bilayer (egg PE/brain PS = 7/3). The peptides with hydrophobicity less than Hel 13-5 (Hels 11-7 and Hel 9-9) were able to partially immerse their hydrophobic part of the amphiphilic helix in lipid bilayers and fragment liposome to small bicelles or micelles, and then the bicelles aggregated to form a larger assembly. Peptides Hel 11-7 and Hel 9-9 each formed strong ion channels. Peptides (Hel 7-11 and Hel 5-13) with a more hydrophilic HHB interacted with an acidic lipid bilayer by charge interaction, in which the former immerses the hydrophobic part in lipid bilayer, and the latter did not immerse, and formed large assemblies by aggregation of original liposomes. The present study clearly showed that hydrophobic-hydrophilic balance of a peptide is a crucial factor in understanding lipid-peptide interactions.