Prevalence of Mobiluncus spp among women with and without bacterial vaginosis as detected by polymerase chain reaction.

BACKGROUND: Mobiluncus spp are highly associated with bacterial vaginosis, but their role in its pathogenesis is unknown. The authors used polymerase chain reaction (PCR) to compare the prevalence of Mobiluncus in women with and without bacterial vaginosis. GOAL: To compare the prevalence of Mobiluncus spp among women with and without bacterial vaginosis and to compare the sensitivities of PCR and Gram stain for detection. STUDY DESIGN: Vaginal specimens from 74 women were analyzed by PCR and Gram stain for the presence of Mobiluncus spp. Comparisons were made between the prevalence of this organism between the two cohorts and between the Gram stain and PCR detection methods. RESULTS: Mobiluncus was detected by PCR in 84.5% of women with bacterial vaginosis and in 38% of women without infection. M curtisii was rarely detected in the latter group, though it was found in 65.3% of women with bacterial vaginosis. The sensitivity and specificity of Gram stain compared with PCR were 46.9% and 100%, respectively. CONCLUSIONS: Mobiluncus is more common in healthy women than previously suspected, with M mulieris as the predominant species. The significant difference in the prevalence of M curtisii between women with bacterial vaginosis and uninfected women suggests that this species could be involved in the pathogenesis of bacterial vaginosis.

Detection of trichomonosis in vaginal and urine specimens from women by culture and PCR.

Vaginal trichomonosis is a highly prevalent infection which has been associated with human immunodeficiency virus acquisition and preterm birth. Culture is the current "gold standard" for diagnosis. As urine-based testing using DNA amplification techniques becomes more widely used for other sexually transmitted diseases (STDs) such as gonorrhea and chlamydia, a similar technique for trichomonosis would be highly desirable. Women attending an STD clinic for a new complaint were screened for Trichomonas vaginalis by wet-preparation (wet-prep) microscopy and culture and for the presence of T. vaginalis DNA by specific PCR of vaginal and urine specimens. The presence of trichomonosis was defined as the detection of T. vaginalis by direct microscopy and/or culture from either vaginal samples or urine. The overall prevalence of trichomonosis in the population was 28% (53 of 190). The sensitivity and specificity of PCR using vaginal samples were 89 and 97%, respectively. Seventy-four percent (38 of 51) of women who had a vaginal wet prep or vaginal culture positive for trichomonads had microscopic and/or culture evidence of the organisms in the urine. Two women were positive for trichomonads by wet prep or culture only in the urine. The sensitivity and specificity of PCR using urine specimens were 64 and 100%, respectively. These results indicate that the exclusive use of urine-based detection of T. vaginalis is not appropriate in women. PCR-based detection of T. vaginalis using vaginal specimens may provide an alternative to culture.

Tumor cell invasion of basement membrane in vitro is regulated by amino acids.

Because most cancer deaths result from disseminated disease, understanding the regulation of tumor invasion and metastasis is a central theme in tumor cell biology. Interactions between extracellular matrices (ECM) and cellular microenvironment play a crucial role in this process. We have tested selected amino acids and polyamines for their ability to regulate RL95-2 cell invasion through both intact human amniotic basement membrane and a novel human ECM (Amgel). Three major systems for neutral amino acid transport, systems L, A, and ASC, are operational in these neoplastic cells. Amino acids entering the cell via transport system A or N, i.e., (methyl amino)-isobutyrate (MeAIB) or Asn, markedly enhanced invasiveness of these human adenocarcinoma cells as measured by a standard 72-hr amnion or Amgel invasion assay. Addition of 2-amino-2-norborane carboxylic acid (BCH; 1 mM), a model substrate of the L transport system, caused a significant decrease in invasive activity when tested in the Amgel assay. Interestingly, Val lowers steady-state levels of MeAIB uptake and blocks the increase in cell invasion elicited by MeAIB. At the same time, these amino acids do not influence cell proliferation activity. Neither the charged amino acid Lys or Asp (not transported by A/N/L systems) nor the polyamines putrescine, spermidine, or spermine modulate invasiveness under similar experimental conditions. Moreover, the observed time-dependent stimulation of system A activity (cellular influx of MeAIB) by substrate depletion is prevented by the addition of actinomycin D (5 microM) or cycloheximide (100 microM), suggesting the involvement of de novo RNA and protein synthesis events in these processes. MeAIB treatment of tumor cells selectively increased the activities of key invasion-associated type IV collagenases/gelatinases. These results indicate that in the absence of defined regulators (growth factors or hormones), certain amino acids may contribute to the epigenetic control of human tumor cell invasion and, by extension, metastasis. We propose that amino acids, acting via specific signaling pathways, modulate phenotypic cell behavior by modulating the levels of key regulatory enzymatic proteins.

Identification and characterization of cytosolic sulfotransferase activities in MCF-7 human breast carcinoma cells.

MCF-7 human mammary carcinoma cells have been reported to possess beta-estradiol and dehydroepiandrosterone sulfotransferase activities. These steroid sulfotransferase activities may be important in the metabolism and activity of different steroids in these cells. This report describes and characterizes both the enzymatic activity of three cytosolic sulfotransferases found in MCF-7 cells and the corresponding immunoblot analysis of these enzymes with specific anti-sulfotransferase antibodies. Two cytosolic sulfotransferases have been purified and characterized from human tissues which are capable of sulfating estrogens. These are the phenol-sulfating form of phenol sulfotransferase (P-PST) and the hydroxysteroid sulfotransferase, dehydroepiandrosterone sulfotransferase (DHEA-ST). The results of this study show that P-PST is the major cytosolic sulfotransferase found in MCF-7 cytosol and is responsible for most of the beta-estradiol sulfation in these cells. Although DHEA-ST activity was found in MCF-7 cytosol, this activity was only about 3% of the P-PST activity. Immunoblot analysis of MCF-7 cytosol detected both P-PST and lower levels of the monoamine-sulfating form of PST; however DHEA-ST could not be detected apparently because of low levels of expression. Human liver P-PST was expressed in Cos-7 Green monkey kidney fibroblasts and the ability of the cloned enzyme to sulfate beta-estradiol was investigated. This study indicates that P-PST is the prevalent cytosolic sulfotransferase in MCF-7 cytosol and is responsible for the majority of beta-estradiol sulfation in these cells.

Antioxidant activity of uric acid and 3-N-ribosyluric acid with unsaturated fatty acids and erythrocyte membranes.

Uric acid and 3-N-ribosyluric acid have been proposed to be important antioxidants in human plasma and in the bovine erythrocyte, respectively. In this study both of these compounds at 0.2 mM protected linolenate and arachidonate from air oxidation in the presence of 5 microM copper sulfate. The unsaturated fatty acids were protected when the urates were added at the beginning of the incubation or at 12 and 24 h of incubation for linolenate and at 3, 6, and 12 h for arachidonate. Both urates protected human and bovine erythrocyte membranes from peroxidation by t-butylhydroperoxide. These data support the proposal that urates are important antioxidants and free radical scavengers.