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101.
Abstract In an in vitro direct assay with tissue-type plasminogen activator (tPA), plasminogen and the chromogenic substrate S-2251, the ability of Mycoplasma fermentans KL4 to stimulate tPA-mediated activation of plasminogen to plasmin was studied. Mycoplasma cells markedly enhanced the activation of plasminogen by tPA in a concentration-, temperature- and pH-dependent manner. Nonidet P-40 (0.01%), sonication, and freezing and thawing of the cells substantially increased the stimulatory effect of mycoplasma on tPA activity. In contrast, the activation of plasminogen by urokinase was refractory to mycoplasma cells. The mycoplasma-mediated stimulation of tPA activity was prevented by ϵ-aminocaproic acid (EACA), a lysine analogue known to block lysine-binding sites (LBS) in plasminogen and tPA. Among several Mycoplasma fermentans strains tested, incognitus strain demonstrated the highest stimulation activity. These results suggest that mycoplasma cells interact with LBS in tPA and plasminogen to enhance plasminogen activation. 相似文献
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Molecular survey of haemoplasmas in shelter dogs and associations with Rhipicephalus sanguineus sensu lato 下载免费PDF全文
This study aimed to assess the occurrence of canine haemoplasma infection in domestic dogs and its possible trans‐stadial transmission by Rhipicephalus sanguineus sensu lato (Ixodida: Ixodidae) in shelter dogs in Diyarbak?r Province in southeast Turkey. Blood samples (n = 282) collected from domestic dogs were analysed by polymerase chain reaction (PCR) for the presence of canine haemoplasma. Fully engorged nymphs (n = 204) were removed from dogs that were positive for canine haemoplasma by PCR and maintained in an incubator at 28 °C for moulting. Unfed ticks (n = 2185) comprising 2100 nymphs and 85 adults collected from the grounds of the same shelter were also screened. Of 282 dogs, 108 [38.3%, 95% confidence interval (CI) 32.6–44.2] were PCR‐positive for canine haemoplasmas. Mycoplasma haemocanis (Mhc) infection (26.2%, 95% CI 21.2–31.8) was observed in a significantly higher number of dogs than was Candidatus Mycoplasma haematoparvum (CMhp) infection (6.7%, 95% CI 4.1–10.3). Co‐infections were seen in 15 (5.3%, 95% CI 3.0–8.6) dogs. None of the tick specimens examined were found to be positive for haemoplasma. Partial sequences of the 16S ribosomal RNA (rRNA) gene shared 99–100% identity with the corresponding published sequences for Mhc and CMhp. The present results revealed no trans‐stadial transmission of canine haemoplasma species by R. sanguineus s.l. in field conditions. 相似文献
104.
Erica J. Fratz-Berilla Phillip Angart Ryan J. Graham David N. Powers Adil Mohammad Casey Kohnhorst Talia Faison Sai Rashmika Velugula-Yellela Nicholas Trunfio Cyrus Agarabi 《Biotechnology and bioengineering》2020,117(9):2802-2815
A mycoplasma contamination event in a biomanufacturing facility can result in costly cleanups and potential drug shortages. Mycoplasma may survive in mammalian cell cultures with only subtle changes to the culture and penetrate the standard 0.2-µm filters used in the clarification of harvested cell culture fluid. Previously, we reported a study regarding the ability of Mycoplasma arginini to persist in a single-use, perfusion rocking bioreactor system containing a Chinese hamster ovary (CHO) DG44 cell line expressing a model monoclonal immunoglobulin G 1 (IgG1) antibody. Our previous work showed that M. arginini affects CHO cell growth profile, viability, nutrient consumption, oxygen use, and waste production at varying timepoints after M. arginini introduction to the culture. Careful evaluation of certain identified process parameters over time may be used to indicate mycoplasma contamination in CHO cell cultures in a bioreactor before detection from a traditional method. In this report, we studied the changes in the IgG1 product quality produced by CHO cells considered to be induced by the M. arginini contamination events. We observed changes in critical quality attributes correlated with the duration of contamination, including increased acidic charge variants and high mannose species, which were further modeled using principal component analysis to explore the relationships among M. arginini contamination, CHO cell growth and metabolites, and IgG1 product quality attributes. Finally, partial least square models using NIR spectral data were used to establish predictions of high levels (≥104 colony-forming unit [CFU/ml]) of M. arginini contamination, but prediction of levels below 104 CFU/ml were not reliable. Contamination of CHO cells with M. arginini resulted in significant reduction of antibody product quality, highlighting the importance of rapid microbiological testing and mycoplasma testing during particularly long upstream bioprocesses to ensure product safety and quality. 相似文献
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Choline deficiency induced by Mycoplasma fermentans enhances apoptosis of rat astrocytes 总被引:1,自引:0,他引:1
Ben-Menachem G Mousa A Brenner T Pinto F Zähringer U Rottem S 《FEMS microbiology letters》2001,201(2):157-162
A choline uptake system accumulating free choline in an energy-dependent process is described in Mycoplasma fermentans. The uptake system has a K(m) of 2.2x10(-5) M and a V(max) of 0.15 nmol 10 min(-1) mg(-1) cell protein and the choline incorporated could be recovered in the soluble fraction as free choline, phosphorylcholine and CDP-choline. Choline accumulation by M. fermentans resulted in a marked choline depletion of the growth medium. The choline depletion of an astrocyte cell culture induced by M. fermentans was associated with the apoptotic death of the cells. Apoptosis was not obtained with heat-inactivated mycoplasmas and could be reversed by the addition of free choline to the growth medium. 相似文献
109.
Yet another improved silver staining method for the detection of proteins in polyacrylamide gels 总被引:11,自引:0,他引:11
Silver staining is very sensitive for detection of proteins in polyacrylamide gels and different procedures have been published. By combining and modifying some of the recipes, a very reproducible method, which is based upon staining with diamine complexes of silver, has been developed. The background staining is negligible and reduced silver does not precipitate on the gel surface. The technique works very well for sodium dodecyl sulfate-polyacrylamide gel electrophoresis in both homogeneous and in gradient gels as well as for two-dimensional (2-D) PAGE. It was possible to detect 1-10 ng of protein corresponding to approximately 50 pg/mm2, provided that a discontinuous buffer system was used, which gives sharp bands. 相似文献
110.
Monoclonal antibodies specific for cell culture mycoplasmas 总被引:3,自引:0,他引:3
David W. Buck Roger H. Kennett Gerard McGarrity 《In vitro cellular & developmental biology. Plant》1982,18(4):377-381
Summary Mycoplasma infection of cell cultures is still a major problem in some laboratories. Although several methods can be used
for their detection, identification is normally by serological procedures. As no commercial source for the necessary antibodies
is available we have prepared monoclonal antibodies to the five mycoplasma species that account for the majority of cell culture
infections. These antibodies have been characterized by the growth inhibition test (GIT), immunofluorescence, and enzyme linked
immunosorbent assay (ELISA) and have shown perfect correlation in all tests when compared to conventional antisera raised
in rabbits or donkeys. In addition, a monoclonal antibody toMycoplasma pneumoniae was produced.M. pneumoniae is an infrequent cell culture contaminant but is a human pathogen, and the monoclonal antibody described here could be useful
in the clinical diagnosis ofM. pneumoniae infection in man.
These studies were supported by Grants Al-15748 from the National Institute of Allergy and Infectious Diseases, and GM20138-07
from the National Institutes of Health, Bethesda, MD. 相似文献