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Cell-free Fractions (cell-free + fraction)
Selected AbstractsA NEW LARVAL FISH BIOASSAY FOR TESTING THE PATHOGENICITY OF PFIESTERIA SPP. (DINOPHYCEAE),JOURNAL OF PHYCOLOGY, Issue 3 2003Vincent J. Lovko Water quality, microbial contamination, prior fish health, and variable results have been major impediments to identifying the cause and mechanism of fish mortality in standard aquarium-format Pfiesteria bioassays. Therefore, we developed a sensitive 96-h larval fish bioassay for assessing Pfiesteria spp. pathogenicity using six-well tissue culture plates and 7-day-old larval cyprinodontid fish. We used the assay to test pathogenicity of several clonal lines of Pfiesteria piscicida Steidinger and Burkholder and P. shumwayae Glasgow and Burkholder that had been cultured with algal prey for 2 to 36 months. The P. shumwayae cultures exhibited 80%,100% cumulative mortality in less than 96 h at initial zoospore densities of approximately 1000 cells·mL,1. No fish mortalities occurred with P. piscicida at identical densities or in controls. In a dose-response assay, we demonstrated a strong positive correlation between dinospore density and fish mortality in a highly pathogenic culture of P. shumwayae, generating a 96-h LD50 of 108 zoospores·mL,1. Additionally, we applied the assay to evaluate a 38-L P. shumwayae bioassay that was actively killing fish and compared results with those from exposures of juvenile tilapia (Oreochromis niloticus) in a 500-mL assay system. Water from the fish-killing 38-L assay was filtered and centrifuged to produce fractions dominated by dinoflagellates, bacteria, or presumed ichthyotoxin (cell-free fraction). After 96 h, the larval fish assay exhibited 50%,100% cumulative mortality only in fractions containing dinoflagellates, with no mortalities occurring in the other fractions. The 500-mL bioassay with tilapia produced inconsistent results and demonstrated no clear correlation between mortality and treatment. The new larval fish bioassay was demonstrated as a highly effective method to verify and evaluate dinoflagellate pathogenicity. [source] Human laminin-332 degradation by Candida proteinasesJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 6 2008P. Pärnänen Background:, Human laminin-332 (Lm-332) degradation by 12 Candida strains and effects of synthetic proteinase inhibitors [Ilomastat (ILM), EDTA, chemically modified tetracycline-3(CMT-3), CMT-308, synthetic peptide CTT-2, and Pefabloc] were studied. Materials and methods:, Laminin-332 was incubated with sonicated cell fractions and 10 times concentrated cell-free fractions of reference and clinical strains of C. albicans, C. dubliniensis, C. guilliermondii, C. glabrata, C. krusei, and C. tropicalis. Proteolysis, pH effects, and inhibitors were analyzed by fluorography and zymography. Results:, Cell fractions of all species except C. guilliermondii and cell-free fractions of C. albicans, and C. dubliniensis showed 20,70 kDa gelatinases at pH 5.0 and 6.0. At pH 7.6, C. glabrata, C. krusei, and C. tropicalis cell fractions and C. tropicalis cell-free fractions showed 55,70 kDa gelatinases. CMT-3, CMT-308, and CTT-2 inhibited Candida gelatinases slightly better than Pefabloc, ILM, and EDTA. No Candida fractions degraded Lm-332 at pH 7.6, but at pH 5.0, 100 kDa bands were generated by cell fractions of C. dubliniensis and C. tropicalis; C. albicans and C. glabrata clinical strains; and C. guilliermondii reference strain. C. krusei reference strain yielded three 100,130 kDa bands. C. albicans, C. dubliniensis, and C. tropicalis reference and clinical strain's cell-free fractions generated 100 kDa band. Conclusions:, Laminin-332 degradation is pH-dependent and differences exist between studied Candida strains. Lm-332 degradation can exert functional disturbances on basement membrane integrity, possibly aiding Candida cell invasion into tissues. Certain synthetic matrix metalloproteinase inhibitors (CMTs, CTT) can inhibit Candida proteinases and may be therapeutically useful in future. [source] Two distinct hemolysins in Trichomonas tenax ATCC 30207MOLECULAR ORAL MICROBIOLOGY, Issue 6 2000E. Nagao An oral protist Trichomonas tenax ATCC 30207 was investigated for the ability to lyse erythrocytes of sheep, rabbits, horses and humans. Five fractions, including intact cells, culture supernatant, culture filtrate, cell debris and lipid-enriched fractions, were prepared from the protozoan cells, and their hemolytic activities were assayed under various conditions. All the samples except culture supernatant had hemolytic activities, which were due to two different kinds of hemolysins. One hemolysin was protein-like and mainly found in cell-free fractions: culture supernatant and culture filtrate. It was heat-labile and inhibited by various cysteine-proteinase inhibitors. The other hemolysin was lipid-like and found in cell-associated fractions: intact cells, cell-debris and lipid-enriched fractions. It was heat-stable, organic solvent,tolerant and unaffected by various proteinase inhibitors and stimulators. These results suggested that T. tenax ATCC 30207 possessed two distinct hemolysins, protein and lipid. [source] | ||||||||||