bluuebunny/crossref_metadata_2025 huggingface dataset:
Concentrations of Immunoglobulins and Protein in Piglet Feces
Authors: Inoue Takeshi , Yamada Yasuko , Saito Toshio
Published: November 2013

The concentrations of immunoglobulins and protein in piglet feces at one day to ten weeks of age were assayed by the single radial immunodiffusion and by the Lowry method, respectively.

The concentrations of IgG, IgA, IgM and total immunoglobulin were found to be significantly higher at one day and two days of age than those at one week to ten weeks of age. The concentrations of IgA, IgM and total immunoglobulin at two days of age were the highest and those at one day of age were the next highest. However, in case of IgG, there seemed to be no such difference in concentrations.

There was almost no significant difference in concentrations of IgG, IgA and total immunoglobulin in feces at one week to ten weeks of age. On the other hand, the concentration of IgM at eight weeks of age seemed to be the least from one day to ten weeks of age.

The concentration of protein at two days of age was the highest and that at one day of age was the next highest. Those at one week to three weeks of age were the least among those at one day to ten weeks of age.
Effects of Cyclophosphamide on the Expression and Induction of Delayed Hypersensitivity
Authors: Himeno Kunisuke , Nomoto Kikuo , Takeya Kenji
Published: November 2013

Erythematous delayed reactions without induration, presumably assigned to Jones‐Mote type, were characterized by the resistance to treatment with cyclophosphamide (CY) before elicitation or immunization in guinea pigs immunized with BGG in IFA or CFA. CY‐treatment before elicitation converted delayed erythematous reactions from negative to positive at late intervals after immunization with BGG in IFA. Such a treatment augmented erythematous delayed reactions in animals immunized with BGG in CFA, but abolished induration at the reaction sites. CY‐treatment before elicitation or immunization reduced the numbers of basophils at the reaction sites, although erythematous delayed reactions were augmented. Effector T cells responsible for delayed erythematous reaction without induration appear to persist for a long period of time after immunization in the presence of antibody production or tuberculin hypersensitivity and the expression of their function may be inhibited by suppressive mechanisms.
Replication of Mouse Cytomegalovirus in Thymidine Kinase‐Deficient Mouse Cells
Authors: Eizuru Yoshito , Minamishima Yoichi , Hirano Akiko , Kurimura Takashi
Published: November 2013

In an attempt to determine whether mouse cytomegalovirus (MCMV) requires thymidine kinase (TK) for replication and whether it induces TK, TK‐deficient mouse cells were isolated and used as host cells for MCMV. Mutant cells resistant to 200 μg/ml of 5‐Bromodeoxyuridine (BUdR) were selected from SV40‐transformed mouse cells, mks‐A TU‐7, by propagating the cells in the presence of varying concentrations of BUdR graded by serial 2‐fold increments. The mutant cells, designated as TU‐7 BU, showed a very low TK activity (less than 1/20 that of mks‐A TU‐7). Herpes simplex virus type 1 (HSV‐1) replicated in starved as well as in unstarved TU‐7 BU, whereas MCMV could replicate only in growing TU‐7 BU and could not form plaques in monolayers of mks‐A TU‐7 or TU‐7 BU. HSV‐1 infection enhanced TK activity equally in both mks‐A TU‐7 and TU‐7 BU. In contrast, TK activity of MCMV‐infected mks‐A TU‐7 was lower than that of uninfected cells or cells inoculated with UV‐inactivated virus. In addition, TK activity of the MCMV‐infected TU‐7 BU remained minimal without showing any increase. The replication of HSV‐1 was completely inhibited in the presence of BUdR (10 μg/ml), whereas MCMV could replicate even in the presence of 50 μg/ml of BUdR. The results indicate that MCMV neither requires TK nor induces TK activity in the infected cells.
Isolation and Characterization of Defective Interfering Particle of Newcastle Disease Virus
Authors: Maeda Akitoshi , Suzuki Yasuo , Matsumoto Makoto
Published: November 2013

Newcastle disease virus grown in embryonated eggs was separated and purified by sucrose density gradient centrifugation into two distinct types of particles, B and T, the former being normal virus particles with high activities of hemagglutination, hemolysis, neuraminidase and infectivity, the latter being non‐infectious virus particles with low activities of hemolysis and neuraminidase but high hemagglutination activity. B and T particles were shown to share a common antigen by immunodiffusion test. T particles were deficient in viral RNA, since they contained only 13s RNA in a small amount, whereas B particles possessed a large amount of 57s RNA and a small amount of 13s RNA. T particles interfered with the multiplication of normal Newcastle disease virus in primary cultures of chick embryo cells.
Studies on Delayed Hypersensitivity to Protein Antigen: Antigen‐specific Suppression of Delayed Hypersensitivity by Chemically Modified Antigen
Authors: Nakashima Shoichi , Sakai Yasuo , Umeda Yukio , Takatsu Kiyoshi
Published: November 2013

An extensively modified protein antigen (methylated bacterial α‐amylase, M‐BαA) which was neither reactive with anti‐BαA antibody nor able to induce a humoral anti‐BαA response, retained the ability to prime native BαA‐specific T cells which were responsible for the enhanced anti‐BαA response to subsequent immunization with BαA and delayed hypersensitivity (DH). The splenic T cell‐rich fraction from mice primed with M‐BαA collaborated with a native BαA‐primed B cell‐rich fraction to give a good adoptive IgG anti‐BαA response in syngeneic irradiated mice, whereas M‐BαA‐primed B cell fractions failed to cooperate with native BαA‐primed T cell fractions. Splenic T cells from mice given a subcutaneous (s.c.) injection of M‐BαA in complete Freund's adjuvant (CFA) exhibited DH in syngeneic cyclophosphamide‐treated mice. In the present study, native and methylated BαA were tested for their ability to generate suppressor T cells capable of inhibiting the development of DH.

An intraperitoneal (i.p.) injection of either native or methylated BαA in incomplete Freund's adjuvant (IFA) interferred with the development of DH to M‐BαA by an s.c. injection of the same antigen in CFA. Transfer of spleen cells from mice given an i.p. injection of either of these antigens 5 days previously, suppressed antigen‐specifically induction and expression of DH in the syngeneic recipient mice. The suppressive activity was sensitive to treatment with anti‐θ antiserum plus complement. These results indicate that the early phase of inhibition of DH after an i.p. injection is in part mediated by suppressor T cells and that M‐BαA cross‐reacts with native BαA at the suppressor T cell level as well as the level of effector T cells in DH.
Pathogenesis of Mouse Hepatitis Virus Infection: The Role of Nasal Epithelial Cells as a Primary Target of Low‐Virulence Virus, MHV‐S
Authors: Taguchi Fumihiro , Goto Yoshitaka , Aiuchi Masamine , Hayashi Toshiharu , Fujiwara Kôsaku
Published: November 2013

The pathogenesis of mouse hepatitis virus (MHV‐S) infection in suckling and weanling mice was comparatively studied after intranasal inoculation. In sucklings, infectious virus as well as specific antigen was first detected in the nasal mucosa at 12 hr, then in the nerve cells of the olfactory bulbs. At this stage viral particles were demonstrated both in the supporting cells and olfactory cells of the nasal mucosa. In the posterior part of the brain and spinal cord, virus was detected on days 3 to 4 postinoculation when viral growth was clearly demonstrable in the liver, spleen and intestines. In weanlings too, infection was first established in the nasal mucosa, shedding infectious virus in the nasal washing until day 6 postinoculation, and later infection spread to the brain and spinal cord. In weanling mice, however, neither infectious virus nor viral antigen was detected in the liver or other visceral organs, while serum neutralizing antibody became detectable on day 5 postinoculation, increasing in titer thereafter. Histopathologically degenerative and necrotic changes were observed in the nasal mucosa and central nervous system of both age groups of animals coincidentally with the presence of viral specific antigen, while inflammatory response was much less prominent in sucklings. In the liver, spleen and intestines, however, some lesions were observed only in sucklings.
Induction of Pre‐Early Nuclear Antigen(s) in HEL Cells Infected with Human Cytomegalovirus
Authors: Tanaka Shigeaki , Otsuka Masahiro , Ihara Seiji , Maeda Fumiko , Watanabe Yasushi
Published: November 2013

Human cytomegalovirus (HCMV)‐specific nuclear antigen could be detected within 1 hr after infection in human embryo lung cells by the anticomplement immunofluorescence (ACIF) test. This antigen has been named the pre‐early nuclear antigen (PENA) in this paper. Serum absorption tests suggested that PENA is immunologically different from the early antigen and the major nuclear inclusion antigens detected by the indirect immunofluorescence test before and after viral DNA replication, respectively. PENA‐forming ability of the virus corresponded to its plaque forming ability. PENA formation was not affected by phosphonoacetate but was inhibited by the addition of inhibitors of RNA and protein syntheses or by UV‐irradiation of infecting virus, suggesting that the formation of PENA depends on the expression of infecting virus gene functions. Virus‐specific proteins were isolated by indirect immunoprecipitation from HCMV‐infected cells exposed to 35S‐methionine. SDS‐polyacrylamide gel electrophoresis of the immunoprecipitate showed that at least two species of virus‐specific polypeptides with molecular weights of 70,000 and 30,000 were synthesized de novo within 3 hr after infection.
Chemical Composition of Streptococcus mutans Cell Walls and Their Susceptibility to Flavobacterium L‐11 Enzyme
Authors: Inoue Masakazu , Hamada Shigeyuki , Ooshima Takashi , Kotani Shozo , Kato Keijiro
Published: November 2013

The susceptibility to a cell wall lytic L‐11 enzyme from Flavobacterium sp. and the quantitative and/or qualitative composition of the cell walls of some strains of cariogenic Streptococcus mutans and a non‐cariogenic strain of Streptococcus mitis were determined. The purified cell walls of S. mutans strains HS‐1 (serotype a), BHT (b), NCTC10449 (c), C67–1 (c), C67–25 (c), OMZ 176 (d), MT703 (e), MT557 (f), OMZ65 (g), and AHT (g), and S. mitis CHT contained glutamic acid, alanine, and lysine as well as muramic acid and glucosamine as a peptidoglycan component. Besides these amino acids, significant amounts of threonine were detected in strains HS‐1, OMZ65, and AHT cell walls, and considerable amounts of aspartic acid and/or threonine as well as several other amino acids in OMZ 176, OMZ65, and CHT cell walls. Rhamnose was a common special component of the cell walls of S. mutans strains BHT, NCTC10449, MT703, B2 (e), MT557, and AHT, and S. mitis CHT. An additional sugar component, glucose, was detected in the cell walls of all of these strains except BHT, and galactose was found in BHT, AHT, and CHT cell walls. Galactosamine was present in S. mitis CHT cell walls. Varying amounts of phosphorus were detected in the cell walls of all the strains examined. The cell walls of all these streptococcal strains except MT703, 6715, and AHT were susceptible to the lytic action of the L‐11 enzyme to various extents. No consistent relationship was observed between the amino acid and sugar composition of these cell walls and their susceptibility to the L‐11 enzyme. The chemical composition of these cell walls is discussed in terms of the serological classification of S. mutans.
Seroconversion to Human‐Cytomegalovirus‐Specific Pre‐Early Nuclear Antigens in Renal Transplant Recipients
Authors: Ohtsuka Masahiro , Tanaka Shigeaki , Saitho Hiroshi , Hashimoto Kazuo
Published: November 2013

Seroconversion to human cytomegalovirus (H‐CMV)‐specific antigens was observed in 8 out of 9 renal transplant recipients. In 7 of the 8 recipients antibody to “pre‐early nuclear antigens” (PENA), which are detectable in human embryonic lung cells within 1 hr of H‐CMV infection by anti‐complement immunofluorescence staining, developed concomitantly with the increase in other antibodies including anti‐early antigens (EA), anti‐nuclear inclusions (NI), and complement‐fixing (CF) antibody in 1–2 months after transplantation. About 1 year later, anti‐PENA and anti‐EA titers were concomitantly decreased in 2 recipients, whereas anti‐NI and CF antibody titers were maintained at elevated levels in all the seroconverted recipients. These results support the idea that the development of antibody to PENA, like antibody to EA, may represent a current or recent infection with (or reactivation of) H‐CMV. In one patient, antibody to PENA did not develop through the observation period despite increases in antibody to EA and other antibodies; this lends support to immunological distinctness of PENA from EA.
Effect of Capsular Polysaccharide of Klebsiella pneumoniae on Host Resistance to Bacterial Infections
Authors: Kato Nobuo , Kato Osamu , Nakashima Izumi , Naito Setsuko , Asai Junpei
Published: November 2013

The mechanism for the infection‐promoting effect of the capsular polysaccharide of Klebsiella pneumoniae (CPS‐K) was investigated using the experimental system in which mice were infected intraperitoneally (i.p.) with a virulent strain of Salmonella enteritidis immediately after i.p. injection of CPS‐K. In the peritoneal phagocytes of CPS‐K‐untreated control mice, approximately 70, 3, and 10% of phagocytized bacteria survived 6, 12, and 24 hr after challenge, respectively, when calculated from the ratio of the number of cell‐associated viable bacteria, which was estimated by direct plate count, to the number of phagocytized bacteria, which was estimated by microscopic observation of stained smears. In contrast, almost all of the phagocytized bacteria were viable throughout the experimental period in mice treated with CPS‐K. The electron microscopical findings of the phagocytes obtained 12 hr after challenge showed that in the cells of mice treated with CPS‐K almost all of phagocytized bacteria were morphologically intact, with some of them in the stages of cell division, whereas in those of untreated control mice, almost all of the phagocytized bacteria underwent digestive changes. When the reaction product of acid phosphatase was examined by electron microscopy in the phagocytes obtained 12 hr after challenge, the enzyme activity in the phagosomes was very low in mice treated with CPS‐K in comparison with that in untreated control mice. Enzyme assays of the lysosomal and extralysosomal fractions of peritoneal cells obtained at various times after challenge also showed that release of acid phosphatase from the lysosomal fraction to the extralysosomal fraction after bacterial challenge was inhibited in peritoneal cells of mice treated with CPS‐K.
Isolation and Partial Characterization of Exosporium from Spores of a Highly Sporogenic Mutant of Clostridium botulinum Type A
Authors: Takumi Kenji , Kinouchi Takemi , Kawata Tomio
Published: November 2013

Homogeneous fragments of exosporium were isolated and purified from mature spores of a highly sporogenic mutant derived from Clostridium botulinum type A strain 190L. The exosporium was composed of three lamellae and showed a hexagonal array when negatively stained. The hexagonal array of isolated exosporium was resistant to sodium dodecyl sulfate, urea, dithiothreitol, and proteolytic enzymes such as trypsin, pronase, and nagarse, except for pepsin. The hexagonal array was partially disintegrated with 5 M guanidine‐HCl and almost completely disrupted with 8 M urea in combination with 1 % mercaptoethanol under alkaline conditions. The purified exosporium fraction was composed mainly of protein (69.1 %) and lipids (13.8%). A small amount of amino sugars (2.5%) was present, but neutral sugars could not be detected. The exosporium protein had a predominantly acidic amino acid composition accompanied by low levels of cystine, methionine, and histidine.
Thymus‐Independent Anti‐DNP Antibody Responses to DNP‐Casein and DNP‐Gelatin
Authors: Ishizaka Shigeaki , Otani Shuzo , Morisawa Seiji
Published: November 2013

Although studies on the molecular nature of thymus‐independent antigens suggested that the polymeric structure with repeated antigenic determinants and slow metabolism are responsible for thymus‐independence, we found that anti‐DNP antibody responses to DNP‐casein and DNP‐gelatin were thymus‐independent as well as macrophage‐independent. These antibody responses were not affected by in vivo treatment with carrageenan or anti‐thymocyte serum. In addition, responses of athymic nude mice to both antigens did not show any significant differences when compared with heterologous nu/+ mice. These findings were confirmed by in vitro experiments; non‐adherent spleen cells or T cell‐depleted spleen cells responded well to both antigens to the same extent as normal spleen cells. Since both casein and gelatin are polyclonal B cell activators and are not presumed to be high polymer or slow‐metabolizing substances, we suggest that thymus‐independence in many kinds of antibody response should be reconsidered.
Accumulation of Eosinophils and Monocytes in Lymphoid Organs of Chick‐Embryos
Authors: Moriya Osamu , Ighikawa Yoichi
Published: November 2013

The mitogen effect on migration of eosinophils and monocytes was studied in embryonic chickens. On the 13th embryonic day, chickens were injected with mitogens, such as concanavalin A (Con A), phytohemagglutinin‐P (PHA‐P), and lipopolysaccharide (LPS), into the allantoic cavity, and the mitogenic effect was estimated from the relative frequencies of eosinophils and monocytes by enumerating the number of oxidase positive cells (OPC) in the spleen, thymus, and bursa of Fabricius. Splenic frequencies of OPC increased in the embryos treated with mitogens. Similar influences were also detected in the thymic OPC. Higher responses were seen on the 18th embryonic day in the number of splenic OPC when embryos were treated with Con A or PHA‐P than with LPS. These findings suggest that Con A and PHA‐P are preferential OPC accumulation promoters. However, bursal frequencies of OPC in the cortex were low after mitogenic stimulations when compared with controls, although appreciable responses were detected in the bursal medulla after LPS stimulation. These results suggest that the migration pattern in the population of eosinophils and monocytes is affected not only by T cell mitogens but is also derived from LPS stimulation.
Integration of Temperature‐Sensitive Nonconjugative Plasmid Carrying Kanamycin Gene into Conjugative R Plasmids
Authors: Katsumata Ryoichi , Hashimoto Hajime , Mitsuhashi Susumu
Published: November 2013

A nonconjugative R plasmid, rMS3, whose molecular weight was 2.4 × 107 daltons, possessed a kanamycin resistance gene and was thermosensitive in its maintenance in Escherichia coli strains. We mobilized rMS3 with a conjugative R plasmid, R100 or T‐tet, and obtained cointegrates carrying all the parental resistance markers. Various markers of the cointegrates were frequently deleted by P1 transduction and the deletion patterns among the different cointegrates were differed from each other. The cointegrates were thermoresistant, but the thermosensitive replicon could be segregated from the thermoresistant cointegrate by deletion. Some cointegrates between rMS3 and T‐tet showed a derepressed state of transferability because of the integration of rMS3 into the regulator gene of the transfer loci. The genome size of the cointegrate so far tested was the sum of the sizes of the parental plasmids, indicating that the whole genome of rMS3 could integrate into various sites of the conjugative plasmids R100 and T‐tet.
Changes in the Activities of Enzymes, Especially Lactate Dehydrogenase, in Endotoxin‐Poisoned Mice
Authors: Sakaguchi Osamu , Sakaguchi Shuhei , Tsunoda Nobuko
Published: November 2013

Carbohydrate metabolic disorders were investigated by means of enzyme activities in mice (ddYS) injected intraperitoneally with endotoxin from Salmonella typhimurium. The mice exhibited hyperglycemia 2 hr after administration of endotoxin and hypoglycemia at 18 hr. Activity of hepatic phosphorylase in the endotoxin‐poisoned mice at 2 hr was slightly higher than that in the control mice, whereas the level of this activity was not significantly different from that in the controls after 18 hr. Glucose‐6‐phosphatase activity in the poisoned mice increased by 2 hr after injection, but decreased by 18 hr.

The blood lactate level in the poisoned mice transiently decreased until 3 hr after injection, but the mice exhibited a marked lactacidemia by 8–24 hr. The time course of lactate dehydrogenase (LDH) activity in various tissues was examined in mice injected with endotoxin. The activity of hepatic LDH declined to about two‐thirds of that of the control mice after 16 hr, and was restored to the normal level by 48 hr. LDH in the cardiac muscle was markedly activated (by about 37%) in the early period (3–6 hr) after administration of endotoxin, and this activity gradually declined. However, the activity of LDH in the skeletal muscle showed a tendency similar to the rise and fall of the levels of blood lactate, and was restored to the normal value at 72 hr after injection. On the other hand, the serum LDH activity in the poisoned mice increased about 1.75‐fold by 16 hr after injection. Mice injected with endotoxin exhibited a leakage of the isozymes LDH 3 and 5, but the origin of the leakage is uncertain. Similar elevation in the activities of transaminases (GPT and GOT) and malate dehydrogenase was found in the mouse serum at 16 hr after injection of endotoxin.
Studies on the Neutralization of Herpes Simplex Virus
Authors: Yoshino Kamesaburo , Isono Noriko , Tada Aiko , Urayama Mari
Published: November 2013

A sample of late IgG from a rabbit hyperimmunized with herpes simplex virus was analyzed for neutralizing (N) and complement‐requiring neutralizing (CRN) antibodies. In a usual endpoint test, N and CRN titers were 1 : 40 and 1 : 160, respectively, but when virus‐IgG mixtures were incubated at 0 C overnight before addition of complement (C), an endpoint of 1 : 1280 was obtained. Virus sensitized at 0 C overnight required more C for inactivation than did sensitized virus formed earlier. Sensitization kinetic curve experiments employing a proper initial virus concentration, which permitted differentiation of sensitized viruses requiring different amounts of C, indicated that formation of sensitized virus detectable only with a relatively large amount of C proceeded slowly at IgG dilutions where the ordinary CRN antibody requiring a smaller amount of C was negligible. The results strongly suggested that the IgG sample contained slow‐reacting CRN (s‐CRN) antibody in excess of the hitherto known CRN antibody. As to the mechanism of formation of s‐CRN complexes, experiments failed to prove the occurrence of complexes initially insensitive to C, and it appears more likely that s‐CRN antibody has a comparatively low avidity for virus.
A B Lymphocyte Mitogen Extracted from a Fungus Peziza vesiculosa1
Authors: Yadomae Toshiro , Suzuki Iwao , Kumazawa Yoshio , Miyazaki Toshio
Published: November 2013

A water‐soluble mitogen was extracted with hot‐water from the fruiting bodies of a fungus, Peziza vesiculosa, collected in the wild. The active substance, named vesiculogen, was able to stimulate selectively murine B cells because mitogenic activity was observed in the spleen cell cultures of congenitally athymic nude mice, but not in the thymus cell cultures. The possibility that the mitogenicity of vesiculogen was due to lipopolysaccharide was denied completely by the following evidence: 1) lipopolysaccharide in vesiculogen was undetectable (less than 0.001% in the Limulus test), 2) vesiculogen was able to stimulate strongly DNA synthesis of spleen cells from C3H/HeJ mice, and 3) the mitogenic activity of vesiculogen was not inhibited by polymyxin B.

Vesiculogen increased antigen‐nonspecifically the number of direct plaque forming cells to sheep erythrocytes, horse erythrocytes, and trinitrophenylated‐horse erythrocytes. This result shows that vesiculogen acts as a polyclonal B cell activator on murine spleen cells.
Purification and Some Physicochemical Properties of Slow‐Reacting Substance of Anaphylaxis (SRS‐A) from Sensitized Guinea Pig Lung
Authors: Watanabe Shigekatsu , Koda Akihide
Published: November 2013

The slow‐reacting substance of anaphylaxis (SRS‐A) generated by antigen challenge of sensitized guinea pig lung fragments was partially purified and the physicochemical properties of this activity were studied. The SRS‐A recovered from antigen challenged lung preparations of 600 animals was used for the purification procedure. Treatment with organic solvents, extraction with 80% ethanol, Sephadex LH‐20 chromatography with 80% ethanol, and DEAE‐Sephadex A‐25 chromatography in 60% methanol eluted with 0.0 to 0.1 M NaCl in 60% methanol was the purification sequence finally adopted. Overall recovery of SRS‐A bioactivity was 60% with a specific activity of 2.52 units/ng of dry weight. This represented a 1.67 million‐fold purification over the starting material. The DEAE‐Sephadex A‐25 step alone provided a 7600‐fold purification. This highly purified SRS‐A had an apparent molecular weight of 380 to 400 daltons. The bioactivity was acid labile and alkaline stable and was blocked by low concentrations of the SRS‐A antagonist FPL 55712. The SRS‐A was thermostable in aqueous media and displayed enhanced bioactivity after heating at 60 C for 60 min. These results indicate that we have developed a highly efficient new approach to the isolation of guinea pig SRS‐A, which also may be useful in the study of SRS‐A from other tissues or species. The physicochemical properties of guinea pig SRS‐A appear to be very similar to those of SRS‐A from other species.
Disassembly of Tobacco Mosaic Virus by Membrane Lipid Isolated from Tobacco Leaves and Polyornithine
Authors: Kiho Yukio , Abe Tetsuya , Ohashi Yuko
Published: November 2013

In vitro disassembly of tobacco mosaic virus (TMV) virions occurred in the presence of both polyornithine and a lipid fraction isolated from tobacco leaf membrane. The latter could be replaced by lecithine. Disassembly of 10 μg of TMV virions was attained in the presence of a 500‐mg leaf equivalent of membrane lipid and 20 μg of polyornithine in 1 ml of 0.01 M Tris‐HCl buffer, pH 7.4 at 30 C. Similarity and dissimilarity between the in vitro disassembly and the in vivo uncoating mechanisms are discussed.
Lipopolysaccharide‐Induced Mediators Assisting the Proliferative Response of C3H/HeJ Thymocytes to Concanavalin A
Authors: Tanabe Masao J. , Nakano Masayasu
Published: November 2013

3H‐Thymidine uptake of thymocytes from LPS‐responder Balb/c mice in the presence of a submitogenic dose (0.5 μg/ml) of con A in vitro was significantly enhanced by adding LPS (0.1 to 2.5 μg/ml), while the uptake of thymocytes from LPS‐nonresponder C3H/HeJ was not enhanced by LPS. However, “endotoxin soups,” which were prepared from the supernatants of LPS‐responder murine spleen cell cultures in the presence of LPS, clearly increased the incorporation of 3H‐thymidine into C3H/HeJ thymocytes in the presence of this small amount of con A. The soup prepared from C3H/HeJ spleen cell cultures did not show any synergistic effect with con A. Even if the major histocompatibility between soup‐producer cells and responder cells to con A was different, the soups were still effective. The active substance in the “endotoxin soups” was eluted through a Sepharose CL‐4B column, and its molecular size was estimated to be about 20,000 daltons. The activity of the soups was destroyed by heating at 70 C for 30 min or at 80 C for 10 min. Digestion with trypsin destroyed the activity of the soups, but digestion with DNase or RNase did not. The role of the active substance in the soups in synergy with con A and its relation to the synergistic effect of con A and LPS are discussed.
Antagonistic Effects of Bacillus natto and Streptococcus faecalis on Growth of Candida albicans
Authors: Ozawa Kyosuke , Yabu‐Uchi Kanji , Yamanaka Kumiko , Yamashita Yutaka , Ueba Keizo , Miwatani Toshio
Published: November 2013

The growth‐inhibitory effects of Bacillus natto and Streptococcus faecalis on Candida albicans were investigated. When inoculated into the filtrate of a long‐term culture of B. natto strain BN (BN), a stock culture of C. albicans RIMD 0301020 lost its viability completely, whereas C. albicans RIMD 0301011, a fresh isolate from a clinical source, did not. In continuous flow (CF) culture the growth of both strains of C. albicans was suppressed by mixed cultivation with BN. On the other hand, in classical batch culture BN did not suppress the growth of C. albicans. S. faecalis BIO‐4R, a multi‐drug resistant strain, was also antagonistic to C. albicans RIMD 0301011 but symbiotic with BN in CF culture. These findings suggest that BN in concert with S. faecalis BIO‐4R may inhibit the growth of C. albicans in the intestinal tract.
Immune Interferon Produced In Vitro as a Quantitative Indicator of Cell‐Mediated Immunity
Authors: Ito Yasuhiko , Nishiyama Yukihiro , Shimokata Kaoru , Takeyama Hideo , Kunii Akira
Published: November 2013

The present study was constructed to provide some information on the possibility of utilizing immune interferon as a quantitative indicator of cell‐mediated immunity and to clarify some of the nature of immune interferon‐producing cells (IIPC).

When spleen cells derived from L cell‐sensitized mice were co‐cultivated with L cells, interferon appeared in the culture fluid. It was shown by additional experiments that the cells responsible for immune interferon production in this system were T‐lymphocytes assisted by macrophages. The pattern of kinetics of immune interferon production in vitro was similar to that of migration inhibitory factor or T cell‐mediated cytotoxicity.
Isolation and Characterization of Heat‐Resistant (HR) Mutants of Newcastle Disease Virus1
Authors: Iinuma Masao , Nishiyama Yukihiro , Hamaguchi Michinari , Yoshida Tetsuya , Nagai Yoshiyuki , Maeno Koichiro , Matsumoto Toshisada
Published: November 2013

Heat‐resistant (HR) mutants (HR 70 and HR 74) of Newcastle disease virus (NDV) which exhibited significantly higher thermostability in their infectivity than wild‐type virus were isolated and characterized. They differ from each other in their plaque morphology; HR 70 produces small turbid plaques, whereas those of HR 74 are large and clear. Cytopathogenicity of these mutants is much lower than that of wild‐type virus in cultured cells such as CEF, LLCMK2, and HeLa cells. Moreover, these HR mutants exhibited extended mean embryo survival times. Synthesis of cellular RNAs and proteins in cells infected with HR mutants was not significantly reduced under conditions in which synthesis of these macromolecules was strongly reduced in cells infected with wild‐type virus. No significant differences were observed between HR mutants and wild‐type virus in their other phenotypic characteristics such as the capacity for interferon production, growth characteristics at a low multiplicity of infection, and cleavage of viral glycoproteins in infected cells.

From these findings, it was suggested that the inhibitory effect of virus infection on cellular macromolecular synthesis is a possible determinant of cytopathogenicity of NDV.
Intestinal Microflora and Bile Acids
Authors: Morotomi Masami , Kawai Yasuo , Mutai Masahiko
Published: November 2013

In vitro cholic acid (CA) transformation by mixed fecal culture was investigated. Concentrations of glucose, peptone, and yeast extract in the medium and the initial pH of the medium markedly affected the CA transformation. Yeast extract enhanced the transformation, whereas high concentrations of glucose and peptone inhibited it. When the initial pH of the medium was below 6.5, CA was converted to 7‐keto‐deoxycholic acid (7KD),and formation of deoxycholic acid (DC) was not observed. In contrast, with an initial pH of 7.0, about 60% of the CA was converted to 7KD after 3 days of incubation, and then DC gradually formed after 4 days of incubation, following the disappearance of 7KD. The formation of DC in the cultured samples was paralleled in each case by disappearance of 7KD. In pure culture systems, Escherichia coli and some strains of Bacteroides formed 7KD from CA. No DC formation was observed in pure cultures of any of the strains examined.
Resistance System of Mycobacterium bovis B.C.G. to Aminoglycoside‐ and Peptide‐Antibiotics
Authors: Tsukamura Michio
Published: November 2013

The resistance system of Mycobacterium bovis (B.C.G.) to aminoglycoside‐and peptide‐antibiotics has been studied. The phenotype of mutants isolated from the parent B.C.G. strain by a single‐step selection with an antibiotic were classified into the following three types: (1) resistant only to a low concentration (200 μg/ml) of kanamycin in Ogawa egg medium (k1R); (2) resistant to a low concentration (200 μg/ml) of viomycin and of capreomycin (2R); and (3) resistant to a high concentration (1,000 μg/ml or more) of kanamycin and low concentrations (100 to 200 μg/ml) of lividomycin and of paromomycin (KR). The mutants showing these phenotypes, k1R, 2R, and KR, were isolated from the parent strain by inoculating the strain into media containing 100 μg/ml of kanamycin, and 100 μ/g/ml of viomycin or capreomycin, and 1,000 μg/ml of kanamycin, respectively, at rates of 10−5‐10−6, 10−5‐10−6, and 10−6‐10−7, respectively, in a total viable population of the parent strain. Unlike in the case of M. tuberculosis, no mutant could be isolated from the parent strain by use of enviomycin, lividomycin, and/or paromomycin. In contrast to the fact that quadruply resistant mutants were isolated directly from the parent H37Rv strain of M. tuberculosis, such mutants could be isolated only by two‐step selections. Furthermore, the phenotypes of the quadruply resistant mutants were those showing a higher resistance or a broader spectrum than expected by the addition of phenotypes of individual mutations. In addition, it was shown that, in contrast to the fact that hextuply resistant mutants could be isolated directly from the parent strain of M. tuberculosis, such mutants were not isolated directly from the parent B.C.G. strain, but could be isolated only after pre‐incubation of the strain on a medium containing Tween 80.
Growth of Poliovirus in HeLa Cells Persistently Infected with HVJ (Sendai Virus)
Authors: Kimura Yoshinobu , Yoshii Saiji , Maeno Koichiro , Matsumoto Toshisada
Published: November 2013

The growth of poliovirus in a HeLa cell culture persistently infected with the hemagglutinating virus of Japan (HVJ, the Sendai strain of parainfluenza 1 virus) (HeLaHVJ) was studied. Plaques produced by poliovirus on HeLaHVJ cell monolayers were hazier, smaller and fewer than those on HeLa cells. HeLaHVJ cells were indistinguishable from normal HeLa cells with respect to adsorption rate and penetration efficiency of poliovirus. Extracellular yields of poliovirus in HeLaHVJ cells were lower, and the cytopathic changes were less than those in normal HeLa cells, while cell‐associated virus growth in HeLaHVJ cells was nearly equal to that in HeLa cells. HeLaHVJ cells responded more effectively to the action of magnesium chloride, which facilitates virus release from infected cells, resulting in an increase of virus yield to the level of normal HeLa cells, with an early appearance of cytopathic effects. No reduction in poliovirus yield could be detected in HeLa cells acutely infected with HVJ. The relationship between the inhibition of the release of poliovirus from HeLaHVJ cells and the persistent infection of the cells with HVJ is discussed.
Analysis of Nuclear Accumulation of Influenza Nucleoprotein Antigen in the Presence of p‐Fluorophenylalanine
Authors: Hamaguchi Michinari , Maeno Koichiro , Nagai Yoshiyuki , Iinuma Masao , Yoshida Tetsuya , Matsumoto Toshisada
Published: November 2013

When p‐fluorophenylalanine (FPA) was added to influenza virus RI/5+‐infected cells 4 hr after infection, virus‐specific proteins were synthesized but infectious progeny virus was not produced. In these cells, synthesis of viral RNA was strongly inhibited and nucleoprotein (NP) antigen was found predominantly in the nucleus in contrast to untreated cells in which NP antigen was distributed throughout the whole cell. The intracellular location and migration of NP were examined by isotope labeling followed by fractionation of infected cells. In untreated cells, a large portion of the NP was present in the cytoplasm and most of it was detected in the form of ribonucleoprotein (RNP). In contrast, in FPA‐treated cells little viral RNP was detectable and NP was present predominantly in the nucleus in a nonassembled, soluble form. When FPA was removed from the culture, synthesis of viral RNA was soon restored and a large amount of viral RNP appeared in the cytoplasm; this was followed by the production of infectious virus. The results of the experiments suggest that the NP synthesized in the presence of FPA is not assembled into viral RNP because of the lack of available RNA, and such NP migrates readily into the nucleus and accumulates there.
Effect of Molar Ratio of Fluorescent Dye to IgG on the Detection of Lymphocyte Surface Immunoglobulins by Immunofluorescence
Authors: Murata Michisato , Kawamura Akiyoshi
Published: November 2013

Labeled antibodies with different F/P molar ratios of FITC to protein (F/P molar ratio) were used for the detection of surface immunoglobulin (S‐Ig) of human and mouse lymphocytes by membrane immunofluorescence, and the following results were obtained.

The percentage of S‐Ig bearing cells increased markedly when labeled anti‐human H‐ or L‐chains antibodies were used with higher F/P molar ratios. The investigation of frozen kidney sections of mice injected with human immunoglobulin revealed that such an increase of the positive ratio in S‐Ig was caused by increased non‐specific adsorption of the fraction of labeled antibody with a high F/P molar ratio.

This non‐specific adsorption phenomenon was observed at various intensities in materials from different species; materials from mice showed less non‐specific adsorption than those from humans.

It was possible to exclude reactivity with an Fc receptor using the top one third of the supernatant of labeled antibody centrifuged at 150,000 for 30 min.
Adenylate Cyclase Activity of Bordetella Organisms
Authors: Endoh Masahiko , Takezawa Toshiyuki , Nakase Yasukiyo
Published: November 2013

Abundant adenylate cyclase activity was found in the phase I cultures not only of Bordetella pertussis but also of B. parapertussis and B. bronchiseptica. The enzyme activity in the culture fluid increased rapidly and reached a peak during the logarithmic growth phase. B. parapertussis and B. bronchiseptica especially produced a high activity of the enzyme in the culture fluid during the logarithmic phase, but little or no activity was detected in the cells throughout the growth period. In the culture of B. pertussis, the intracellular activity was higher than that in the culture fluid. Phase III cultures of these species lacked both the extracellular and intracellular enzyme activities throughout their growth.

In the culture of B. parapertussis, accumulation of cyclic AMP was parallel to that of adenylate cyclase activity through the growth periods, but in B. pertussis there was no parallelism from the stationary through the declining phases. The difference in production patterns of the enzyme activity among the species is discussed.
The Molar Combining Ratio of Anti‐Albumin Fab' Fragments to Homologous and Heterologous Serum Albumins
Authors: Kamiyama Tsuneo , Katsube Yasuji , Imaizumi Kiyoshi
Published: November 2013

The number of antibody combining sites on bovine (BSA), goat (GSA) and sheep (SSA) serum albumins was studied using rabbit and chicken antibodies. In homologous reactions, the profiles of quantitative precipitations with chicken antibody were similar to those with rabbit antibody reported previously (12), and the antigenic valence in the extreme antibody excess zone was found to be 6–7 for each albumin.

The univalent Fab' fragments of rabbit and chicken antibodies were prepared. The stoichiometry of the soluble complex formed with the Fab' fragment and fluorescence labeled albumin was analyzed by gel filtration, and the number of Fab' fragment molecules capable of binding to an albumin molecule was estimated. In a homologous reaction with both rabbit and chicken Fab' fragments, the Fab' to albumin combining ratio revealed from the molecular weight of the soluble complex was 14:1. In the heterologous reactions, the combining ratio was 5:1 for rabbit Fab' fragment to BSA, and 9:1 for chicken Fab' fragment to BSA. From the heterologous reactions between GSA and SSA, it was demonstrated that the combining ratios were 10–11:1 with rabbit Fab' fragment and 11–13:1 with chicken Fab' fragment.
Electron Microscopy of the Spherical Body of Oral Spirochetes In Vitro: Further Studies
Authors: Umemoto Toshihiko , Namikawa Isamu
Published: November 2013

The surface and inner structure of the spherical bodies (SB) produced by the human oral treponeme strain G7201, similar to Treponema macrodentium, were studied by electron microscopy. Ultrathin sectioning and scanning techniques demonstrated that in the presence of a high concentration of sucrose, the outer envelope of one or both terminal ends of this oral spirochete changed into a swollen structure, the SB. Spirochetal cells adhered firmly to the surface of the resultant body. The membrane of the SB, i.e. the outer envelope, enclosed the coiled protoplasmic cylinder and five axial fibrils which were located between the envelope and the cylinder. Large expanded protoplasmic cylinders were observed, surrounded by a partially disrupted double membrane in some SBs. A number of frizzly fibrous structures, which differed from axial fibrils in number and shape, were also observed within these SBs. Except for abnormal or partially broken cylinders, the protoplasmic cylinders tended to be located close to the inner surface of the SB membrane, resulting in a central vacant space with occasional axial fibrils.

These findings suggest that the oral spirochete produces an SB by terminal expansion of the outer envelope in the presence of high concentrations of sucrose. The outer envelope of the SB, which consists of two electron‐dense layers, has the property of binding spirochetal cells to its outer layer and the protoplasmic cylinder and axial fibrils to the inner layer. Some protoplasmic cylinders were also observed to be swollen in the presence of high sucrose concentrations.
Mycoplasma pulmonis Arthritis in Congenitally Athymic (Nude) Mice
Authors: Kono Masako , Tanaka Hideo , Yayoshi Masumi , Araake Minako , Yoshioka Morimasa , Imai Miki
Published: November 2013

Histopathological examinations were performed on arthritic joints and other organs of strain BALB/cA nu/nu and nu/+ mice intravenously injected with Mycoplasma pulmonis strain m53. In both groups of mice suffering from polyarthritis, acute inflammatory lesions with infiltration of polymorphonuclear leukocytes in the synovia and periarticular tissues were observed one to two weeks after injection. In nu/nu mice, the acute inflammation appeared repeatedly up to 20 weeks after inoculation, when the experiment was terminated, and furthermore, extensive synovial and periarticular necrosis were characteristically present after the 4th week. Only a small number of lymphocytes and plasma cells were in the lesions. In nu/+ mice, after the early acute inflammation of arthritis, relapses of the infiltration of polymorphonuclear leukocytes were also observed in some mice in and after the 10th week. In addition, infiltration of lymphocytes and plasma cells was substantial after the 15th week. Focal necrosis was sometimes found in the liver of nu/nu mice. Perivascular infiltration of small lymphocytes and plasma cells was found in the lungs, liver and kidney of nu/+ mice in and after the 15th week. Repair mechanisms of injured articular tissues in nu/nu mice were histopathologically poor, while those in nu/+ mice seemed to be progressive and quite similar to those reported by many investigators for mice with the thymus intact. The histopathological differences are discussed in respect to the thymus‐dependent immune responses.
Morphological Changes during Conversion of Clostridium saccharoperbutylacetonicum to Protoplasts by Sucrose‐Induced Autolysis
Authors: Ogata Seiya , Choi Kyoung Ho , Hongo Motoyoshi
Published: November 2013

When exponentially growing cells of Clostridium saccharoperbutylacetonicum (ATCC 13564) were exposed to hypertonic concentrations of sucrose (0.3–0.5 M), rapid degradation of the cell wall occurred (sucrose‐induced autolysis). The morphological changes from the original rod‐shaped cells to protoplasts during the sucrose‐induced autolysis were investigated by phase contrast and electron microscopy. When the cells were autolysed in the sucrose solution (0.35 M), each cell began to swell at the middle or at one pole and then formed a small bulb at the swollen part. The bulb consisted of the cytoplasm which was enveloped by the plasma membrane and extruded from the small gap produced by the degradation of the cell wall. The bulb gradually enlarged as lysis progressed, and finally became a protoplast which had no cell wall. The large pre‐division cell frequently formed the bulb at the middle (septal site), while the small post‐division cell formed the bulb at the pole.
Heat‐Stable Enterotoxin Produced by Yersinia enterocolitica Isolated from Patients
Authors: Okamoto Keinosuke , Ichikawa Hidetaka , Kawamoto Yasuko , Miyama Akio , Yoshii Saiji
Published: November 2013

Twenty‐three strains of Yersinia enterocolitica were isolated from children with gastrointestinal illness and examined for the production of enterotoxins by using both suckling mouse and Chinese hamster ovary (CHO) cell assay systems. Six strains were found to be enterotoxigenic in the suckling mouse assay, but all strains were negative in the CHO cell assay. Enterotoxin was detected in the culture supernatant when organisms were grown at 25 C but not at 37 C. Enterotoxin in a 15‐fold concentrated culture supernatant was precipitated by adding absolute ethanol to a concentration of 90%. However, after being dialyzed against distilled water in Spectra/por 6 membrane tubing, it was soluble in 80% acetone. One unit dose of partially purified enterotoxin was 5.0 μg of protein/mouse in the suckling mouse assay. The molecular weight of enterotoxin was between 10,000 and 50,000 daltons as determined by ultrafiltration. It was stable to heat (121 C × 20 min or 100 C × 60 min). These observations indicate that Y. enterocolitica isolated in Japan also produce an enterotoxin similar to the heat‐stable enterotoxin of Escherichia coli. However its physicochemical properties seem to be different from those of E. coli.