Microbiology and Immunology

Yokoyama, Hideaki; Ikedo, Masanari; Kohbata, Shunro; Ezaki, Takayuki; Yabuuchi, Eiko

doi: 10.1111/j.1348-0421.1987.tb03063.xpmid: 2438540

Scanning electron micrograph of HeLa S3 monolayered cells, inoculated with viable bacteria of a Salmonella typhi strain GIFU 10007, revealed that the extended microvilli tangled the bacteria within 10 min after inoculation. The micrographs of HeLa cells, at 1 hr after inoculation, indicate the following: shortening of bacterium‐attached microvilli, subsiding of tangled bacteria into microvilli bush, and then attachment of bacterial soma to cell surface making the cell membrane depressed. The transmission electron micrographs, at 1 hr after inoculation, demonstrated the findings of interaction between HeLa cell and S. typhi 10007, similar to those observed on scanning electron micrographs. Hair‐like fine structures from the soma of challenge organisms were also observed. They were in contact with HeLa cell microvilli and cell membrane. The bacteria were first partially and then totally surrounded by the HeLa cell plasma membrane. One, two, or several bacteria with intact outer membrane were enclosed in intracytoplasmic membrane‐bound vacuoles. Fragmented vacuolar membrane was still visible around the intracellularly accumulated bacteria at 24 hr after inoculation. The viable cells of S. typhi 10007 are regarded as internalizing into HeLa cells by a process of endocytosis and to multiply within the membrane‐bound vacuoles.

Miyoshi, Noriko; Shimizu, Chie; Miyoshi, Shin‐Ichi; Shinoda, Sumio

doi: 10.1111/j.1348-0421.1987.tb03064.xpmid: 3295490

A protease was purified from a strain of Vibrio vulnificus isolated from the blood of a septicemic human. The vibrio was cultured in bacto peptone‐yeast extract medium, and the protease was purified by a purification procedure including ultrafiltration of the culture supernatant with an Amicon YM 5 membrane, diethylaminoethyl‐Sephacel column chromatography, Sephacryl S‐200 column chromatography and fast protein liquid chromatography on Mono Q column. The protease preparation revealed homogeneity on polyacrylamide gel electrophoresis and about 30,000‐fold purification was achieved, with a yield of about 30%. The isoelectric point of the purified V. vulnificus protease was about 5.80 and its molecular weight was ca. 45,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH of the protease activity was 8.0. The V. vulnificus protease was inhibited by a metalloprotease inhibitor and zinc ion and/or ferrous ion were essential for its enzyme activity. No cysteine residue was detected in the V. vulnificus protease. The protease had caseinolytic, elastolytic and collagenolytic activities.

El‐Belbasi, Hussein I.; Imagawa, Masayoshi; Oku, Yuichi; Nishihara, Tsutomu; Kondo, Masaomi

doi: 10.1111/j.1348-0421.1987.tb03065.xpmid: 3108631

Spore coat proteins obtained by extraction with sodium dodecylsulfate/dithiothreitol from six Bacillus spores were compared by immunoblot analysis using antibodies to spore coat proteins from two strains of B. megaterium. Although the extract from spores of each strain had heterogenous proteins with various molecular weights, there were some bands which cross‐reacted with specific antibodies from B. megaterium spores. Specific antibody to 48K protein from B. megaterium ATCC 12872 cross‐reacted with 17K protein from B. megaterium ATCC 19213, 13K protein from B. cereus and 50K protein from B. subtilis 60015 and B. subtilis NRRL B558. Also, specific antibody to 22K protein from the same strain cross‐reacted with 22K and 17K proteins from B. megaterium ATCC 19213 and 13K protein from B. cereus T. Specific antibody to 17K protein from B. megaterium ATCC 19213 reacted with 22K and 19K proteins in addition to 17K protein of own strain, and it was cross‐reactive with 16K protein from B. megaterium ATCC 12872, 19K and 27K proteins from B. thiaminolyticus, 13K protein from B. cereus.

Nakasone, Noboru; Kawata, Tomio; Masuda, Kuniyoshi

doi: 10.1111/j.1348-0421.1987.tb03066.xpmid: 3587087

Mesosomes of Staphylococcus aureus 209P were observed to be extruded as tubules upon protoplast formation by electron microscopy and isolated under hypertonic conditions to maintain their structural integrity by differential centrifugation followed by sucrose density gradient centrifugation. Isolated mesosomes were composed of long, branched tubules of irregular sizes and they were shortened during purification. Thin sections of isolated mesosomes showed that the mesosomal tubule was surrounded by a triple‐layered membrane and contained ribosome‐like particles in diameter of about 15 to 20 nm. These particles were isolated from purified mesosomal preparation by disrupting the mesosomal tubule with deoxycholate and Triton X‐100 under hypotonic conditions followed by a linear sucrose density gradient centrifugation. Negatively stained preparations of the isolated particles revealed the same appearance as those of the ribosomes isolated from the cytoplasm. The mesosomal particles sedimented at 70S in sucrose gradients in the presence of 10 mm Mg2+, but they were dissociated into two subparticles, 50S and 30S subunits, upon lowering the Mg2+ concentration to 1 mm. These findings indicate that the mesosomal tubule is packed with ribosomes.

Okada, Masanobu; Minamishima, Yoichi

doi: 10.1111/j.1348-0421.1987.tb03067.xpmid: 3035349

The host‐mediated antiviral effect of two biological response modifiers (BRM), OK‐432 and PS‐K, against murine cytomegalovirus (MCMV) was evaluated in normal and immunologically deficient mice of the same litters. In normal littermate mice, BALB/c (nu/+) or C57BL/6 (bg/+), the BRM‐induced resistance against MCMV infection was evidenced by increase in fifty percent lethal doses, decrease in titers of viruses replicated in the target organs and augmentation of natural killer (NK) cell activity of the spleen cells. In T cell‐deficient, athymic nude mice, BALB/c (nu/nu), the protective effect was manifested by prolongation of the survival, decrease in the virus titers, and increase in the NK‐cell activity, but without decrease in mortality. In NK cell‐deficient, beige mutant mice, C57BL/6 (bg/bg), the BRM‐induced protection was nullified or minimized, and there was little difference in those parameters between BRM‐treated and untreated mice. However, with higher doses of OK‐432, but not PS‐K, or with sublethal doses of MCMV, the NK cell activity was slightly augmented in the beige mutant mice. Thus both NK cell and T cell activity are essential for mice to overcome acute MCMV infection and it is likely that the protective effect of BRM manifests itself fully, at least in immunologically intact mice.

Iseki, Rieko; Koide, Yukio; Ueda, Ryuzo; Kondo, Nobuo; Hamuro, Junji; Yoshida, Takato O.

doi: 10.1111/j.1348-0421.1987.tb03068.xpmid: 2438541

Several lines of evidence suggest that subsets of resting lymphocytes naturally express interleukin‐2 receptors (IL‐2·R). Recombinant IL‐2 (rIL‐2) induced the enhancement of natural killer (NK) activity, the generation of activated killer (AK) cells, the proliferation of resting lymphocytes, and the production of interferon‐γ (IFN‐γ) in lymphocyte cultures. The subsets of lymphocytes mediating these responses appeared to be heterogeneous, but reside predominantly in nylon wool‐passed non‐T, non‐B cells (“lnull cells” or T3‐ cells); in response to rIL‐2, only Leul 1+T3‐ cells showed enhanced NK activity, and both Leul 1+T3‐ and Leul 1‐T3‐ cells showed predominantly AK activity, proliferation and production of IFN‐γ. These findings suggest that the T3‐ fraction (null cell fraction) contains predominantly cells expressing IL‐2·R at the resting state. Unlike the case with activated T cells, however, none of these responses was blocked by any of three monoclonal antibodies to IL‐2·R, including anti‐Tac antibody at any dilution. These results indicate that IL‐2·R on the resting T3‐ cells possess unique biological features compared to those on activated T or B cells. A most likely explanation is that T3‐ cells possess higher affinity IL‐2·R than activated T or B cells. Other possibilities are also discussed.

Ohshio, Gakuji; Furukawa, Fukumi; Yoshioka, Hideyuki; Taki, Yoshiro; Manabe, Tadao; Ozawa, Kazue; Tobe, Takayoshi; Hamashima, Yoshihiro

doi: 10.1111/j.1348-0421.1987.tb03069.xpmid: 3587088

We developed an easy enzyme‐linked immunosorbent assay (ELISA) for anti‐mitochondrial antibodies, and detected high levels of anti‐mitochondrial antibodies in MRL/Mp‐lpr/lpr (MRL 1/1) mice. The influence of the presence of anti‐DNA antibodies in the tested sera on this assay was also evaluated. The monoclonal anti‐DNA antibodies, which were made from non‐immunized MRL 1/1 mice, did not react with the mitochondrial antigens adhering to polystyrene plates. Absorption with DNA had little effect on the levels of mitochondrial antibodies in MRL 1/1 sera. Our assay for mitochondria‐binding antibody was very easy and sensitive, although it could not differentiate among heterogeneous anti‐mitochondrial antibodies.

Watanabe, Hiroshi; Kelly, Heath; Dawkins, Roger L.

doi: 10.1111/j.1348-0421.1987.tb03070.xpmid: 3495717

Binding of d‐Penicillamine (d‐Pen) to human monocytes was examined by flow cytometry with fluorescent d‐Pen conjugate. Cells from HLA DR1‐positive healthy females bound significantly more d‐Pen than cells from DR1‐negative healthy females (P=0.015), and DR1 was associated with the highest binding among HLA DR antigens. In contrast, DR4 was associated with the lowest binding in healthy females. A difference in d‐Pen binding between healthy females who were DR1‐positive, DR4‐negative and those who were DR1‐negative, DR4‐positive was statistically significant (P=0.026). Neither healthy females nor healthy males showed significant associations of d‐Pen binding with HLA A, B, or C antigens, nor did healthy males show an association between strength of d‐Pen binding and any DR antigens.

Goto, Norihisa; Ueno, Goro; Iwasa, Saburo

doi: 10.1111/j.1348-0421.1987.tb03071.xpmid: 3495718

Tochikubo, Kunio; Yasuda, Yoko; Kozuka, Satoshi

doi: 10.1111/j.1348-0421.1987.tb03072.xpmid: 3108632