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Microbiology and Immunology 1992The arginine deiminase gene of Mycoplasma hominis was amplified by the polymerase chain reaction, and its entire nucleotide sequence was determined. This gene consists... (Comparative Study)
Comparative Study
The arginine deiminase gene of Mycoplasma hominis was amplified by the polymerase chain reaction, and its entire nucleotide sequence was determined. This gene consists of 1227 base pairs encoding 409 amino acids, and has 35.2% guanine plus cytosine content. Nucleotide sequence homologies of the arginine deiminase gene between M. hominis and M. arginini, and between M. hominis and M. orale were 82.1 and 80.8%, respectively, suggesting that this gene is highly conserved among arginine-utilizing Mycoplasma species.
Topics: Amino Acid Sequence; Base Sequence; Cloning, Molecular; DNA, Bacterial; Genes, Bacterial; Hydrolases; Molecular Sequence Data; Mycoplasma; Polymerase Chain Reaction; Sequence Homology, Nucleic Acid
PubMed: 1522817
DOI: 10.1111/j.1348-0421.1992.tb02068.x -
Microbiology and Immunology 1992Various species of mycoplasmas were tested for their ability to induce cytokine production in human peripheral blood mononuclear cells (PBMC). Human PBMC were incubated...
Various species of mycoplasmas were tested for their ability to induce cytokine production in human peripheral blood mononuclear cells (PBMC). Human PBMC were incubated with Mycoplasma pneumoniae, M. hyorhinis, M. arginini, M. salivarium, M. orale, M. gallisepticum or A. laidlawii for 48 hr, and the activities of interleukin-1 beta (IL-1 beta), IL-2, IL-4, IL-6, tumor necrosis factor-alpha (TNF-alpha) and interferon (IFN) in the supernatants were determined by ELISA or bioassay. All mycoplasma species induced IL-1 beta, IL-6 and TNF-alpha, although IL-2 was induced only by M. pneumoniae. IFN was induced by 5 of the 7 species, and the IFN produced was antigenically confirmed to be mainly IFN-alpha. On the other hand, mycoplasma-stimulated cultures did not contain detectable amounts of IFN-beta and IL-4 activities. Furthermore, the cytokines were induced by mycoplasmal contaminating cells in human PBMC as well as by mycoplasma alone. These results suggest that many kinds of cytokines induced by mycoplasma contamination in cell culture affect immunological experiments in vitro.
Topics: Biological Assay; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Humans; Interferons; Interleukins; Leukocytes, Mononuclear; Mycoplasma; Tumor Necrosis Factor-alpha
PubMed: 1381037
DOI: 10.1111/j.1348-0421.1992.tb02048.x -
Radiation Research Sep 1991Mycoplasma infection of mammalian cells in culture is a common occurrence that can affect the results of experimental protocols. Current methods of eliminating...
Mycoplasma infection of mammalian cells in culture is a common occurrence that can affect the results of experimental protocols. Current methods of eliminating mycoplasma from cell cultures are usually tedious, time-consuming, and sometimes unsuccessful. In the present study, four cultured brain tumor cell lines (human U-251 MG, U-87 MG, SF-126, and rat 9L) were heavily contaminated with Mycoplasma orale. Heating the cultures to 41 degrees C for at least 96 h eliminated the contamination for up to 7 months, the maximum period of observation. The time chosen to assay for the presence of mycoplasma in cultures was critical: in some cultures heated for less than 96 h that initially appeared to be free of contamination, mycoplasma began to appear after 2 weeks. Heat-treated cells grew at the same rate as unheated control cells. Infected cells were more sensitive to X rays than uncontaminated cells, but the sensitivity reverted to normal after mycoplasma was eliminated by hyperthermia. The heating method does not require a cell cloning procedure or the use of exogenous materials. Treated cell cultures exhibit normal growth and radiation sensitivity, and the technique seems to be reliable and efficient.
Topics: Animals; Brain Neoplasms; Hot Temperature; Humans; Mycoplasma; Rats; Time Factors; Tumor Cells, Cultured
PubMed: 1886990
DOI: No ID Found -
Revista Argentina de Microbiologia 1991Over a period of 4 years 200 cell cultures were analysed for the presence of mycoplasma. Cultures were established cell lines from different origins, namely monkey,...
Over a period of 4 years 200 cell cultures were analysed for the presence of mycoplasma. Cultures were established cell lines from different origins, namely monkey, mouse and human, hybrid cell cultures and primary cultures. The cultures belonged to various research and industrial laboratories located in different areas of the country. Seventy per cent of investigated cultures were found to be contaminated with mycoplasma using a DNA fluorescent stain. Fifty cultures, selected at random out of the contaminated cultures, were further investigated to identify the prevalent serotype. For that purpose immunofluorescent reactions were performed using immune sera raised against several mycoplasma strains routinely found among contaminated cultures. Forty one cultures were contaminated with a single type of mycoplasma, whereas in the remaining nine, two or even three serotypes were detected. Mycoplasma orale II contaminated 40% of single infected cultures, followed by M. hyorhinis and A. laidlawii-A (12% each), M. arginini (5%), M. orale III (8%), A. laidlawii-B (2%). We were unable to serotype the remaining positive cultures, because of the lack of a full battery of immune sera against all known serotypes. The prevalence of M. orale in mycoplasma contaminated cultures thus far tested, indicates that human handling would be the main source of infection. This situation could be modified by avoiding mouth pipetting and adopting good microbiological techniques.
Topics: Acholeplasma; Animals; Argentina; Cells, Cultured; Culture Techniques; Humans; Mycoplasma
PubMed: 1815279
DOI: No ID Found -
Infection and Immunity Jun 1991Antisera were produced by inoculation of mycoplasma cells grown in PPLO broth supplemented with rabbit serum alone or rabbit serum plus Freund complete or incomplete...
Antisera were produced by inoculation of mycoplasma cells grown in PPLO broth supplemented with rabbit serum alone or rabbit serum plus Freund complete or incomplete adjuvant. By using an enzyme-linked immunosorbent assay, immunoglobulin G antibodies to cholesterol were detected in antisera to mycoplasmas (Mycoplasma salivarium ATCC 23064, M. orale ATCC 15539, M. buccale IID 802, M. faucium IID 996, and M. hominis IID 801) and rabbit serum.
Topics: Animals; Antibodies, Bacterial; Cholesterol; Enzyme-Linked Immunosorbent Assay; Immunoglobulin G; Mycoplasma; Rabbits
PubMed: 2037380
DOI: 10.1128/iai.59.6.2200-2202.1991 -
FEMS Microbiology Letters Jun 1991Alignment of published 16S rRNA sequences allowed the definition of a pair of oligonucleotides suitable for polymerase chain reaction (PCR). Using this pair of PCR...
Alignment of published 16S rRNA sequences allowed the definition of a pair of oligonucleotides suitable for polymerase chain reaction (PCR). Using this pair of PCR primers, several mycoplasmas including the four human parasites Mycoplasma genitalium, M. hominis, M. salivarium and M. orale were detected. This DNA amplification was restricted to species of the genus Mycoplasma while no cross-reaction was observed with DNA from other bacteria and eukaryotic cells. Subsequent analysis of amplified products by either specific oligonucleotide hybridization or dideoxy sequencing specified the identity of the detected mycoplasmas. This method offers a highly discriminating and sensitive assay for the direct detection and identification of these microorganisms without the need for prior cultivation.
Topics: Base Sequence; DNA, Ribosomal; Molecular Sequence Data; Mycoplasma; Polymerase Chain Reaction; RNA, Ribosomal, 16S
PubMed: 1874400
DOI: 10.1016/0378-1097(91)90467-o -
Infection and Immunity Mar 1991Proteins resistant to proteinase K are rare because of the potency, wide pH optimum, and low peptide bond specificity of this enzyme. Previously, only the prion proteins...
Proteins resistant to proteinase K are rare because of the potency, wide pH optimum, and low peptide bond specificity of this enzyme. Previously, only the prion proteins associated with transmissible spongiform encephalopathies, possibly related proteins in the mollicute Spiroplasma mirum, and proteinase K itself have been reported. We identified a new proteinase K-resistant protein, p40-pr, in two strains of Mycoplasma hyorhinis and in extracts of these organisms. p40-pr's are similar to prion proteins in their resistance to high doses of proteinase K and in the reversal of this resistance by strong denaturing conditions. However, p40-pr's were distinct immunologically, in relative molecular mass, and in their method of extraction. Two immunologically related forms of p40-pr were identified on sodium dodecyl sulfate (SDS) gels and Western immunoblots, a 40-kDa species in boiled samples and a 120-kDa species dissociable by boiling in SDS. Reduction with 2-mercaptoethanol did not affect the mass of p40-pr's or the 120-kDa forms. The development of proteinase K resistance of p40-pr correlated to age-dependent increases in organism protein-lipid ratios. p40-pr-like proteinase K-resistant proteins of 46 to 50 kDa were identified in four of eight additional species of the class Mollicutes but not in S. mirum. However, these mycoplasmal proteins did not react with antibody to the denatured 40-kDa form of M. hyorhinis p40-pr purified by electroelution. The chromatographically purified 46-kDa proteinase K-resistant protein of Mycoplasma orale was an arginine deiminase.
Topics: Animals; Bacterial Proteins; Blotting, Western; Chromatography, High Pressure Liquid; Cross Reactions; Electrophoresis, Polyacrylamide Gel; Endopeptidase K; Hydrolases; Immunoenzyme Techniques; Lipid Metabolism; Male; Mycoplasma; Prions; Rabbits; Serine Endopeptidases
PubMed: 1997407
DOI: 10.1128/iai.59.3.1037-1042.1991 -
Journal of Immunological Methods Aug 1990The detection of mycoplasmas in cell cultures is still a problem, especially in those laboratories in which the detection and identification of microorganisms is not...
The detection of mycoplasmas in cell cultures is still a problem, especially in those laboratories in which the detection and identification of microorganisms is not established as a routine procedure. In our laboratory, monoclonal antibodies (mAbs) have been prepared to Acholeplasma laidlawii, Mycoplasma hyorhinis, Mycoplasma orale, Mycoplasma arginini and Mycoplasma salivarium. 30 mAbs were obtained and one of these, designated CCM-2, was shown to bind to all five mycoplasma species. It also bound to Mycoplasma fermentans, Mycoplasma hominis and to all wild types (n = 54), isolated from cell cultures submitted to our laboratory. The mAb was used in a immunofluorescent assay (IF) and the method correlated with the microbiological assay. Using this mAb immunofluorescent staining of cells is a fast and simple procedure for mycoplasma detection in cell cultures.
Topics: Animals; Antibodies, Monoclonal; Antigens, Bacterial; Cells, Cultured; Epitopes; Female; Fluorescent Antibody Technique; Mice; Mice, Inbred BALB C; Mycoplasma
PubMed: 1697315
DOI: 10.1016/0022-1759(90)90191-w -
Journal of Cellular Biochemistry Jun 1990Deoxycoformycin-treated P388 and L1210 mouse leukemia cells salvage 2'-deoxyadenosine from the medium only inefficiently, because deoxyadenosine deamination is blocked... (Comparative Study)
Comparative Study
Deoxycoformycin-treated P388 and L1210 mouse leukemia cells salvage 2'-deoxyadenosine from the medium only inefficiently, because deoxyadenosine deamination is blocked and its phosphorylation is limited by feedback controls. Mycoplasma contamination at a level that had no significant effect on the growth of the cells increased the salvage of deoxyadenosine greater than 10 fold over a 90 min period of incubation at 37 degrees C, but in this case deoxyadenosine was mainly incorporated into ribonucleotides and RNA via adenine formed from deoxyadenosine by mycoplasma adenosine phosphorylase. Deoxyadenosine was an efficient substrate for this enzyme, in contrast to 2',3'-dideoxyadenosine which was not phosphorolyzed. Mycoplasma infection was confirmed by the presence of uracil phosphoribosyltransferase activity and by culture isolation. The contaminant has been identified as Mycoplasma orale. Mycoplasma infection had no effect on the deamination and phosphorylation of deoxyadenosine and adenosine, on the salvage of hypoxanthine and adenine, or on the degradation of dAMP and dATP by the cells or on their acid and alkaline phosphatase activities.
Topics: Animals; Deoxyadenosines; Leukemia L1210; Mice; Mycoplasma Infections; Pentostatin; Pentosyltransferases; Phosphorylation; Tumor Cells, Cultured
PubMed: 2380261
DOI: 10.1002/jcb.240430207 -
Journal of Medical Microbiology Mar 1990All of five lyophilised cultures of Mycoplasma orale kept for 23 years at room temperature were still viable, as were all but one of 12 lyophilised cultures of six...
All of five lyophilised cultures of Mycoplasma orale kept for 23 years at room temperature were still viable, as were all but one of 12 lyophilised cultures of six Mycoplasma spp. that had been stored for 18-22 years at 4 degrees C. Similarly, 11 of 13 lyophilised ureaplasma cultures were viable after 8-22 years at 4 degrees C; the titre of organisms in the viable cultures had diminished no more than 100-fold. Seven broth cultures of five different Mycoplasma spp. all proved viable 5-13 years after being frozen and stored at -70 degrees C, although there was up to 10(4)-fold reduction in the titre of organisms in some cultures. Furthermore, 18 (82%) of 22 different Mycoplasma spp., originally lyophilised and then reconstituted and stored at -70 degrees C, were viable after 16 years. Viable organisms were found, with little or no reduction in titre, in all of seven broth cultures of Ureaplasma urealyticum, comprising six serotypes, after storage for 6-10 years at -70 degrees C, but five of 18 broth cultures of other human and animal ureaplasmas stored likewise were not viable after 13-14 years and in a further seven of them the titre of viable organisms had diminished greater than or equal to 10(4)-fold.
Topics: Animals; Bacteriological Techniques; Cell Division; Cryopreservation; Culture Media; Freeze Drying; Humans; Mycoplasma; Time Factors; Ureaplasma
PubMed: 2179556
DOI: 10.1099/00222615-31-3-203