Biomedical subjects

K V Clemons

Publications and source records attributed to K V Clemons.

83 records · Page 5Linked to original sources

PubMed1989

Steroid metabolism as a mechanism of escape from progesterone-mediated growth inhibition in Trichophyton mentagrophytes.

It has been shown by us and others that progesterone inhibits the growth of Trichophyton mentagrophytes and that the organism escapes from this inhibition over time. We report here studies which show that escape from growth inhibition is related to the enzymatic transformation of progesterone to polar metabolites. Isolation and identification of the progesterone metabolites confirm the production of 15 alpha-hydroxyprogesterone. In addition, three other metabolites were isolated. Two of these were determined to be 1-dehydroprogesterone and 11 alpha-hydroxyprogesterone. The third metabolite was a 1-dehydro-hydroxyprogesterone, but the location of the hydroxyl group could not be determined unequivocally. Studies using authentic 15 alpha-hydroxyprogesterone, 1-dehydroprogesterone, and 11 alpha-hydroxyprogesterone reveal that these derivatives are significantly less inhibitory to the growth of T. mentagrophytes than progesterone. Pretreatment of organisms with progesterone augments the rate of metabolism and enhances escape. We have described previously a progesterone-binding protein (PBP) in cytoplasmic extracts of T. mentagrophytes and hypothesized that progesterone mediates growth inhibition by binding to the PBP of this organism. The relative binding affinity that progesterone and its metabolites display for PBP correlates with the relative growth inhibitory potency of these compounds. These results suggest that metabolism of progesterone to more polar and less inhibitory compounds, which exhibit lower affinity for PBP, is the mechanism of escape from progesterone-mediated inhibition of growth in this organism.

Binding, Competitive↗

PubMed1989

Influence of oestradiol on protein expression and methionine utilization during morphogenesis of Paracoccidioides brasiliensis.

The temporal sequence of cytosolic protein expression during phase transition of Paracoccidioides brasiliensis was examined. Electrophoretic analysis of cytosol proteins by one-dimensional SDS-PAGE revealed numerous differences between the mycelial and yeast forms as well as alterations induced by 17 beta-oestradiol. Using either protein staining or fluorography of [35S]methionine-labelled proteins 30 phase-specific bands were detected, 12 mycelial-associated bands (range 30 to 140 kDa) and 18 yeast-associated bands (range 22 to 127 kDa). In cells undergoing mycelial to yeast transition after a shift from 25 degrees C to 37 degrees C, the protein patterns showed a temporal progression toward the yeast profile with the accumulation of yeast bands prior to observable morphogenesis. Five novel protein bands (range 23 to 50 kDa) were detected by silver staining during transition. Treatment of temperature-shifted mycelial cultures with 2.6 x 10(-7) M-oestradiol altered observed profiles; 4 of 12 mycelial-associated bands were maintained whereas the appearance of the 5 novel transition bands and 9 of 18 yeast-associated bands was blocked or delayed. Analysis of [35S]methionine-labelled proteins revealed that oestradiol induced label uptake by mycelial cells, blocked the synthesis of a 92 kDa yeast-specific band 72 h into transition, and diminished label incorporation 120 h into transition. In conjunction with these steroid-induced alterations of protein expression, little or no morphological transformation occurred. These results support our hypothesis that, analogous to mammalian steroid receptor action, the functional responses of P. brasiliensis to oestradiol are related to regulation of protein expression, presumably mediated via a specific binding protein-ligand complex.

Cytosol↗

PubMed1988

Dermatophyte-hormone relationships: characterization of progesterone-binding specificity and growth inhibition in the genera Trichophyton and Microsporum.

We reported previously that Trichophyton mentagrophytes contains a cytoplasmic macromolecule which specifically binds progesterone. Progesterone is also an effective inhibitor of growth of the fungus. We report here studies which characterize more fully the specific binding properties and the functional responses of T. mentagrophytes and taxonomically related fungi to a series of mammalian steroid hormones. Scatchard analysis of [3H]progesterone binding in both the + and - mating types of Arthroderma benhamiae and in Microsporum canis revealed a single class of binding sites with approximately the same affinity as that in T. mentagrophytes (Kd, 1 X 10(-7) to 2 X 10(-7) M). Trichophyton rubrum had a protein with a higher binding affinity (Kd, 1.6 X 10(-8) M). Characterization of the [3H]progesterone-binding sites in T. mentagrophytes showed the binder to be a protein which was destroyed by trypsin and heating to 56 degrees C. Previous examination of the steroid-binding specificity in T. mentagrophytes had demonstrated that deoxycorticosterone (DOC) and dihydrotestosterone (DHT) were effective competitors for [3H]progesterone binding. Expansion of this study to include other competitors revealed that R5020 (a synthetic progestin), androstenedione, and dehydroepiandosterone possessed relative binding affinities which were 20, 11, and 9% of that of progesterone, respectively. Other ligands tested were less effective. Competition studies for the binder in M. canis resulted in similar findings: DOC and DHT were effective competitors for [3H]progesterone binding. The growth of A. benhamiae + and -, M. canis, and T. rubrum were all inhibited by progesterone in a dose-responsive manner, with 50% inhibition achieved at concentrations of 9.8 x 10(-6), 1.2 x 10(-5), 1.5 x 10(-5), and 2.7 x 10(-6) M. respectively,.

Binding, Competitive↗

PubMed1986

Estradiol-binding proteins from mycelial and yeast-form cultures of Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis, causes disease much more frequently in men than it does in women, suggesting that the hormonal milieu of the host might influence P. brasiliensis pathogenicity. We recently demonstrated that cytosol from yeast cultures of P. brasiliensis contains a high-affinity, low-capacity, tritiated 17 beta-estradiol [( 3H]estradiol)-binding protein. Estradiol and, to a lesser degree, diethylstilbestrol (DES), inhibited the transformation of P. brasiliensis cultures from the mycelial to the yeast form, an event critical to the establishment of infection. Our current studies demonstrated a somewhat higher affinity (apparent dissociation constant [Kd], approximately equal to 6 to 12 nM) of the estrogen-binding protein for [3H]estradiol than was previously described for yeast cytosol. The presence of both high- and low-affinity estrogen-binding sites in yeast-form P. brasiliensis cytosol was detected after warming the cytosol to 37 degrees C. The high-affinity protein was labile to further heating (56 degrees C), although the low-affinity protein was stable. Additional experiments demonstrated the presence of an estrogen-binding protein in cytosol prepared from mycelial-form P. brasiliensis. This estrogen-binding protein had a slightly lower affinity for [3H]estradiol (Kd approximately equal to 13 nM), and its cytosol contained somewhat fewer binding sites (approximately equal to 78 fmol/mg of protein) than did yeast-form P. brasiliensis cytosol. Of particular interest was the finding that DES, a weak competitor for [3H]estradiol binding in yeast cytosol, displaced [3H]estradiol from the mycelial-form binding moiety. DES had a 50- to 100-fold-lower affinity for the [3H]estradiol-binding protein than did estradiol, consistent with its lower bioactivity in the mycelial-to-yeast-form transformation studies. The current results lend further support to our hypothesis that endogenous estrogens in the host, acting through the cytosol binding protein in the fungus, inhibit mycelial-to-yeast-form transformation, thus explaining the resistance of women to paracoccidioidomycosis.

Binding, Competitive↗

PubMed1986

Progesterone binding and inhibition of growth in Trichophyton mentagrophytes.

Specific binding of [3H]progesterone to cytosol of Trichophyton mentagrophytes was demonstrated. Scatchard analysis of [3H]progesterone binding showed a single class of binding sites with a dissociation constant of 9.5 X 10(-8) [corrected] +/- 2.4 X 10(-8) M (standard deviation) and a maximal binding capacity of 4,979 +/- 3,489 fmol/mg of cytosol protein. Deoxycorticosterone and dihydrotestosterone competitively inhibited binding by 50% at molar ratios of 10:1 and 20:1, respectively. Other steroid hormones that were tested had minimal activity, indicating binding specificity. Steroid hormone actions in T. mentagrophytes were examined in growth studies. Growth was assessed by determination of cellular ATP content. Progesterone inhibited growth in a dose-responsive manner, with a 50% inhibition concentration of 5.5 X 10(-6) M. Partial recovery from inhibition occurred after 24 to 48 h; inhibition could be enhanced by dividing the amount of added progesterone every 24 h. In the same rank order as was their relationship to each other and progesterone in binding studies, deoxycorticosterone and dihydrotestosterone were less effective inhibitors; other steroid hormones that were tested showed no consistent effect. We hypothesize that the binder described, acting as a hormone receptor, is the molecular site of action for the functional effect of the hormone. The functional effect may be related to the observed resistance of females to dermatophytosis.

Binding, Competitive↗

PubMed1985

Systemic Coccidioides immitis infection in nude and beige mice.

The course of experimental systemic Coccidioides immitis infection was assessed quantitatively and histologically in beige mice, congenitally athymic nude mice, and their respective normal counterparts. After intravenous inoculation with 50 arthroconidia, the number of viable C. immitis cultured from the spleens, livers, and lungs progressively increased throughout the assay in the organs of all mice. During the first 2 weeks of infection, significantly greater numbers of CFU were recovered from the spleens and livers, but not the lungs, of nude mice than from the respective organs of their phenotypically normal littermates. Significantly greater numbers of CFU were cultured from the lungs and spleens of beige mice compared with the number recovered from their functionally normal littermates. After intranasal inoculation, extrapulmonary dissemination of C. immitis occurred at an equal rate and resulted in similar organ burdens in nude mice and their normal littermates. Histological examination of infected tissues revealed a characteristic mixed inflammatory cell infiltrate in euthymic mice; the response in nude mice was less severe, consisting predominantly, if not solely, of granulocytes. In addition, in tissue sections from nude mice, but not in those from their euthymic counterparts, mature spherules were frequently observed to be devoid of an associated inflammatory response. The inflammatory lesion in beige mice contained a predominance of mononuclear cells, whereas their littermates responded with a typical mixed granulomatous infiltrate. Collectively, these results provide evidence supporting the hypothesis that resistance to C. immitis infection involves two primary cell populations, one under the direct influence of T-cells and the other independent of T-lymphocytes.

Animals↗

PubMed1982

Increased indole detection for Pasteurella multocida.

A supplemented 2% peptone broth is described for the detection of indole production by Pasteurella multocida. The 96 isolates of P. multocida that were utilized in this evaluation were indole positive within 18 to 24 h.

Bacteriological Techniques↗

PubMedDate not supplied

Efficacy of oral saperconazole in systemic murine aspergillosis.

Saperconazole is a fluorinated bis-triazole. Groups of ten 5-week-old female CD-1 mice were infected intravenously with 5.5 x 10(7) Aspergillus conidia. Saperconazole, dissolved in hydroxypropyl-beta-cyclodextrin (HPBC), was given orally twice a day for 11 days, beginning 1 day post-infection, at 50, 100 or 200 mg kg-1 day-1. At day 18 post-infection, survivors were killed and residual infection quantified in the kidneys. With Aspergillus fumigatus isolate 10AF, 70% given no therapy, 100% given daily oral HPBC and 40% given intraperitoneal amphotericin B at 3.3 mg kg-1 three times a week for 2 weeks died, whereas all mice given saperconazole survived. Each saperconazole regimen prolonged survival compared to untreated or HPBC treated mice (P < 0.01). Saperconazole at 200 mg kg-1 day-1 reduced colony forming units of aspergillus in kidneys more than 1000-fold compared to untreated or HPBC treated mice (P < 0.001) and saperconazole regimens were superior to amphotericin B therapy (P < 0.01). In another study of the same design with A. fumigatus isolate 15AF, 90% of untreated and 20% of mice treated with saperconazole at 50 mg kg-1 day-1 died; all others survived. Any saperconazole regimen prolonged survival (P < 0.001). Residual infection was also significantly reduced by all saperconazole regimens (P < 0.01). With Aspergillus terreus isolate 4AT, 80% of untreated mice, 50% of mice treated with saperconazole at 50 mg kg-1 day-1 and 10% of mice treated at 200 mg kg-1 day-1 died. Any saperconazole regimen prolonged survival (P < 0.05). Saperconazole at 100 and 200 mg kg-1 day-1 also reduced residual infection (P < 0.001). No adverse effects were noted in any study. Thus, saperconazole was efficacious in vivo against different Aspergillus isolates.

Administration, Oral↗

PubMedDate not supplied

Therapy of systemic histoplasmosis in immunosuppressed mice with the triazole D0870.

Because histoplasmosis is a life-threatening disease in AIDS and other compromised patients, we examined the efficacy of D0870 (Zeneca) in immunosuppressed mice against systemic histoplasmosis. Oral therapy with fluconazole given once daily (QD) was ineffective in prolonging survival, whereas itraconazole given once or twice daily (BID), fluconazole given BID or D0870 given QD or given every other day (QOD) were efficacious (P < 0.001). Burdens of Histoplasma capsulatum in the liver and spleen of survivors showed that D0870 given QD or QOD and itraconazole given BID caused dose-responsive reduction of infectious burden. Infection was cleared more readily from the liver than from the spleen. Overall, D0870 was > or = 20-fold more efficacious than fluconazole or itraconazole and itraconazole was > ten-fold better than fluconazole for the treatment of systemic histoplasmosis in the immunosuppressed model.

Animals↗

PubMedDate not supplied

Colony phenotype switching in clinical and non-clinical isolates of Saccharomyces cerevisiae.

During studies on the virulence and genetics of isolates of Saccharomyces cerevisiae we noted colony phenotype switching. Virulent clinical (YJM128, YJM436) and avirulent non-clinical (Y55, YJM237) isolates of S. cerevisiae were scored for colony phenotypes and reversion rates of the switched second-generation colonies on YEPD with phloxine B. YJM128 produced six colony phenotypes; 99.7% were large (8-9 mm diameter), smooth, pink with a white edge (type A) and the other five types arose at frequencies ranging from 2 x 10(-3) to 5 x 10(-5). On replacing of type A, 98.8% of colonies scored arose as type A, 1.1% type J and 0.1% larger, but similar to type A indicating both stability and reversibility. YJM436 also varied, with 91.3% arising as a single phenotype; five other phenotypes were observed at frequencies ranging from 7.6 x 10(-2) to 1.5 x 10(-4). For Y55, 0.2% of the colonies were 5-7 mm in size with a ridged pink edge (type D); 36.4% of YJM237 were a 5-7 mm, smooth pink colony (type H). The remaining 99.8% of Y55 and 63.4% of YJM237 colonies arose as sectored or non-sectored non-reverting petites. These results indicate that virulent isolates of S. cerevisiae tend to produce multiple colony phenotypes, which are reversible. In contrast, avirulent isolates of S. cerevisiae showed single colony phenotypes and a high frequency of non-reverting petites. Whether selected colony phenotypes and switching are associated with the virulence of S. cerevisiae remains to be determined.

Humans↗

PubMedDate not supplied

Experimental systemic cryptococcosis in SCID mice.

The susceptibility of severe combined immune deficiency (SCID) mice to experimental systemic cryptococcosis was studied. SCID mice were at least 16-fold more susceptible to lethal infection than were immunocompetent control mice (P < 0.001). Histological assessment showed that control mice responded with a granulomatous inflammatory response, whereas SCID mice showed no evidence of a cellular host response. In a therapeutic study, 5 mg kg-1 day-1 of fluconazole caused a significant reduction in the infectious burden in the spleen, liver, kidney and lungs of treated mice compared with no treatment. These results indicate that a model of systemic cryptococcosis with meningitis in SCID mice may have utility in the study of immunology and therapy of this disease in the severely immunocompromised host.

Animals↗