Studies on Thin‐Layer Chromatography after Incubation with [35S]Methionine as an Aid to Identification of MycobacteriaTsukamura, Michio; Mizuno, Shoji
doi: 10.1111/j.1348-0421.1981.tb00025.xpmid: 7253963
Thin‐layer chromatography (TLC) of ethyl ether‐ethanol extracts of mycobacteria obtained after incubation with [35S] methionine is useful for differentiation among mycobacterial species, as the distribution of radioactive spots in TLC shows a characteristic pattern except for a few species, including M. intracellulare and M. gordonae. Some supplementary studies have been carried out in the present investigation and the following results have been obtained.
Pretreatment of bacterial cells with trichloroacetic acid (TCA) is required to remove radioactive sulfur‐containing amino acids.
The radioactive spots that appear in TLC of the ethyl ether‐ethanol extract are divided into the following groups: (1) Petroleum ether‐soluble fraction (the spot at Rf value 0.95); (2) acetone‐soluble (petroleum ether‐insoluble) fraction (the spots at Rf values 0.50, 0.30, and 0.13–0.20); (3) ethanol‐soluble (petroleum ether‐ and acetone‐insoluble) fraction (the spot at Rf value 0.10); (4) petroleum ether‐, acetone‐, and ethanol‐insoluble fraction (the spot at Rf value 0.00). The spot which appears in the region of Rf values 0.13–0.20 (it may appear as two spots) in the TLC of ethyl ether‐ethanol extracts is composed of two compounds, one soluble in acetone and the other insoluble in acetone but soluble in ethanol.
Strains of the M. avium‐M. intracellulare complex have been divided into three subgroups by the pattern of radioactive spots B, C, and D, which show Rf values of 0.13–0.20, 0.30, and 0.50, respectively, in the TLC of ethyl ether‐ethanol extracts. The first group shows none of these spots, and strains of the serotypes 1, 2, and 8 belong in this group. The second group shows spot B, and strains of the serotypes 4, 18, and 20 belong in this group. The third group shows spots B, C, and D, and strains of the serotype 16 belong in it.
Lipid Peroxide Formation and Membrane Damage in Endotoxin‐Poisoned MiceSakaguchi, Shuhei; Kanda, Norio; Hsu, Cheng‐Chin; Sakaguchi, Osamu
doi: 10.1111/j.1348-0421.1981.tb00026.xpmid: 6973056
Lipid peroxide formation and plasma membrane damage in mouse liver following the administration of Salmonella endotoxin were examined. The liver lipoperoxide level was markedly elevated in animals given endotoxin compared with that in the controls, and returned to its normal range after 2 days. On the other hand, superoxide dismutase activity was decreased by 18–48 hr after endotoxin injection, thereafter tending to increase. Glutathione reductase and glutathione peroxidase activities declined in the liver 18 hr after the injection. The endotoxin resulted in much lower lipoperoxide formation in the livers of tolerant mice than in those of the poisoned mice. The lipoperoxide level in endotoxin‐poisoned mice after the administration of α‐tocopherol was lower than that in the controls, and α‐tocopherol administration prevented completely the membrane protein damage that arose from endotoxin challenge. After glutathione administration the membranes of the poisoned mice also returned to almost the normal disk electrophoretic profile. These results suggest that lipid peroxide formation in the liver plasma membrane caused by free radicals might occur in a tissue ischemic state in endotoxicosis.
Studies on Streptococci: I. Distribution of Fecal Streptococci in ManWatanabe, Tsugio; Shimohashi, Hirotaka; Kawai, Yasuo; Mutai, Masahiko
doi: 10.1111/j.1348-0421.1981.tb00028.xpmid: 7253964
To understand the significance to the host of streptococci as part of the intestinal microflora, we first tried to investigate the distribution of human fecal streptococci on the species level. Of the selective media compared, KMN agar was more effective than the other media for the isolation of streptococci from human feces. We made an effort to improve streptococcal classification. Especially we used utilization of 1% pyruvate, 1% arginine, and 1% citrate for differentiation between Streptococcus faecalis and S. faecium. In a tellurite tolerance test, S. faecalis was distinguished more clearly from S. faecium in the medium containing 0.16 or 0.32% tellurite. We devised methods of presumptive identification of fecal streptococci from the results of the characteristics of 1,442 isolates. These methods enabled us to identify many strains rapidly. Different results in the distribution of species of streptococci between children and adults were observed. S. faecalis and S. faecium were isolated constantly from both groups. S. bovis and S. avium were isolated frequently from the feces of children. On the other hand, “viridans” streptococci, e.g. S. salivarius, S. mitis and S. MG‐intermedius were present at a high frequency in, and no S. avium could be isolated from, the feces of adults.
Transformation of Bile Acids by Mixed Microbial Cultures from Human Feces and Bile Acid Transforming Activities of Isolated Bacterial StrainsHirano, Seiju; Masuda, Noriyuki; Oda, Hiroshi; Imamura, Teisuke
doi: 10.1111/j.1348-0421.1981.tb00029.xpmid: 7253965
Microbial transformation of cholic acid and chenodeoxycholic acid by anaerobic mixed cultures of human fecal microorganisms was investigated, and the results were examined in relation to the bile acid transforming activities of 75 bacterial strains isolated from the same fecal cultures. The reactions involved in the mixed cultures were dehydrogenation and dehydroxylation of the 7α‐hydroxy group in both primary bile acids and epimerization of the 3α‐hydroxy group in all metabolic bile acids. Extensive epimerization of the 7α‐hydroxy group of chenodeoxycholic acid yielding ursodeoxycholic acid was also demonstrated by certain fecal samples. 7α‐Dehydrogenase activity was widespread among the fecal isolates (88% of 16 facultative anaerobes and 51% of 59 obligate anaerobes), and 7α‐dehydroxylase activity was revealed in one of the isolates, an unidentified gram‐positive nonsporeforming anaerobic bacterium. 3α‐Epimerization was effected by seven strains assigned to Eubacterium lentum, which were also active for 3α‐ and 7α‐dehydrogenations. No microorganism accounting for 7α‐epimerization was recovered among the isolates. Splitting of conjugated bile acid was demonstrated by the majority of obligate anaerobes but the activity was rare among facultative anaerobes.
Analysis of Nuclear Accumulation of Influenza NP Antigen in von Magnus Virus‐Infected CellsMaeno, Koichiro; Aoki, Hiizu; Hamaguchi, Michinari; Iinuma, Masao; Nagai, Yoshiyuki; Matsumoto, Toshisada; Takeura, Shigeki; Shibata, Motohiro
doi: 10.1111/j.1348-0421.1981.tb00030.xpmid: 7253966
When 1–5C‐4 cells were infected with von Magnus virus derived from influenza A/RI/5+ virus by four successive undiluted passages in chick embryos, virus‐specific proteins were synthesized but production of infectious virus was inhibited. In these cells the synthesis of viral RNA was suppressed and the nucleoprotein (NP) antigen was found predominantly in the nucleus in contrast to standard virus‐infected cells in which the antigen was distributed throughout the whole cell. The intracellular location and migration of NP were determined by isotope labeling and sucrose gradient centrifugation of subcellular fractions. In standard virus‐infected cells NP polypeptide was present predominantly in the cytoplasm in the form of viral ribonucleoprotein (RNP) and intranuclear RNP was detected in reduced amounts. In contrast, in von Magnus virus‐infected cells NP polypeptide was present predominantly in the nucleus in a nonassembled, soluble form and the amount of cytoplasmic RNP was considerably reduced. After short‐pulse labeling NP was detected exclusively in the cytoplasm in a soluble form and after a chase a large proportion of such soluble NP was seen in the nucleus. It is suggested that a large proportion of the NP synthesized in von Magnus virus‐infected cells is not assembled into cytoplasmic RNP because of the lack of available RNA and the NP migrated into the nucleus and remained there.
Anticomplement Immunofluorescence for the Titration of Antibody to Varicella‐Zoster VirusShigeta, Shiro; Baba, Masanori; Ogata, Masahiro; Iijima, Susumu; Murai, Chiaki
doi: 10.1111/j.1348-0421.1981.tb00031.xpmid: 6166838
Anticomplement immunofluorescence (ACIF) was tested for its use for the titration of antibody against varicella‐zoster virus (VZV). ACIF antibody responses of patients with VZV infection were specific for VZV antigen and heterotypic responses to herpes simplex virus type‐1 and cytomegalovirus antigens were not observed. Comparative studies of ACIF, membrane immunofluorescence (MIF) and indirect immunofluorescence (IF), using acetone‐fixed antigen, were carried out with nonimmune sera and convalescent sera of patients who had recovered from varicella, herpes zoster and Rumsey Hunt disease. Nonspecific staining occurred with some nonimmune sera at a 1:4 dilution in the MIF and IF tests, after freezing and thawing of the serum, but not in the ACIF test. The antibody titers in convalescent sera agreed well in these three methods and the highest titer was obtained by MIF. The titers in ACIF and IF were similar but the ACIF antibody decreased earlier than the IF antibody during convalescence. On the other hand there was a discrepancy between the titers of ACIF and those of MIF and IF antibody in the sera of healthy adults, all sera with titers higher than 10 in the MIF and IF tests had titers below 10 in the ACIF test. The average titer of ACIF antibody declined to less than 10 with increasing age (13 to more than 20 years), whereas the MIF antibody increased during the same period of life.
Interferon‐Inducing Activity of an Immuno‐therapeutic Anticancer Agent, SSM, Prepared from Mycobacterium tuberculosis Strain Aoyama BHayashi, Yoshiro; Ebina, Takusaburo; Suzuki, Fujio; Ishida, Nakao
doi: 10.1111/j.1348-0421.1981.tb00032.xpmid: 6789037
The interferon‐inducing capacity of arabinomannan‐lipid preparation (SSM) extracted from Mycobacterium tuberculosis Aoyama B in both BCG‐sensitized and unsensitized mice was studied in comparison with that of purified protein derivative (PPD) prepared from the same tubercle bacillus. Although it is known that PPD cannot stimulate interferon production in BCG‐unsensitized mice, interferon activity was found in sera of both groups of mice after intravenous injection of SSM at a dose of 5 mg/kg. The maximum titer was detected 5 hr after injection. The interferon induced by SSM in both groups of mice shared certain physicochemical properties with the immune interferon induced by PPD in BCG‐sensitized mice. In BCG‐unsensitized mice, interferon induction by SSM was markedly inhibited by pretreatment with trypan blue and carrageenan, whereas it was not depressed in BCG‐sensitized mice given the same treatment or when interferon was induced by PPD. In addition, induction of interferon in BCG‐sensitized mice by SSM and PPD and in unsensitized mice by SSM was completely abrogated by pretreatment with hydrocortisone acetate and whole‐body x‐irradiation (700 R). These results suggest that in BCG‐unsensitized mice macrophages, in addition to X‐ray or hydrocortisone‐sensitive cells, may be required for interferon induction by SSM.