Bovine cumulus-granulosa cells contain biologically active retinoid receptors that can respond to retinoic acid

Retinoids, a class of compounds that include retinol and its metabolite, retinoic acid, are absolutely essential for ovarian steroid production, oocyte maturation, and early embryogenesis. Previous studies have detected high concentrations of retinol in bovine large follicles. Further, administration of retinol in vivo and supplementation of retinoic acid during in vitro maturation results in enhanced embryonic development. In the present study, we hypothesized that retinoids administered either in vivo previously or in vitro can exert receptor-mediated effects in cumulus-granulosa cells. Total RNA extracted from in vitro cultured cumulus-granulosa cells was subjected to reverse transcription polymerase chain reaction (RT-PCR) and mRNA expression for retinol binding protein (RBP), retinoic acid receptor alpha (RARalpha), retinoic acid receptor beta (RARbeta), retinoic acid receptor gamma (RARgamma), retinoid X receptor alpha (RXRalpha), retinoid X receptor beta (RXRbeta), retinaldehyde dehydrogenase-2 (RALDH-2), and peroxisome proliferator activated receptor gamma (PPARgamma). Transcripts were detected for RBP, RARalpha, RARgamma, RXRalpha, RXRbeta, RALDH-2, and PPARgamma. Expression of RARbeta was not detected in cumulus-granulosa cells. Using western blotting, immunoreactive RARalpha, and RXRbeta protein was also detected in bovine cumulus-granulosa cells. The biological activity of these endogenous retinoid receptors was tested using a transient reporter assay using the pAAV-MCS-betaRARE-Luc vector. Addition of 0.5 and 1 micro molar all-trans retinoic acid significantly (P < 0.05) increased the activity of the pAAV-MCS-betaRARE-Luc reporter compared to cells transfected with the control reporter lacking a retinoic acid response element. Addition of 5 or 10 micro molar all-trans retinol stimulated a mild increase in reporter activity, however, the increase was not statistically significant. Based on these results we conclude that cumulus cells contain endogenously active retinoid receptors and may also be competent to synthesize retinoic acid using the precursor, retinol. These results also indirectly provide evidence that retinoids administered either in vivo previously or in vitro may have exerted a receptor-mediated effect on cumulus-granulosa cells

Application of Molecular Methods in Diagnostics of Bacterial Pathogens

The present study investigated the applications of recent advances in molecular techniques for developing novel diagnostics of bacterial pathogens. The study adapted an in vitro/ selection method known as s?ystematic e?volution of l?igands by &#60;u&#62;ex&#60;/u&#62;ponential enrichment (SELEX) for generation of ssDNA aptamers against Francisella tularensis/ subspecies tularensis/ (Strain OSU 10) lipopolysaccharide (LPS). The study also reports the sup#1/sup#H- and sup#13/sup#C-NMR spectroscopy structural analysis of O-antigen of F. tularensis/ subspecies tularensis/ (strain OSU 10). Further, microarray based immunoassays were developed for the detection of antibodies to bacterial antigens. Single stranded DNA aptamers were selected against the purified carbohydrate part of LPS (containing core oligosaccharide and O-polysaccharide) from F. tularensis/ subspecies tularensis/ (Strain OSU 10). A selected aptamer Ft1 showed specific binding to LPS from F. tularensis/ subspecies tularensis/ and subspecies holaVeterinary Pathobiolog

Structure-Based Vaccines Provide Protection in a Mouse Model of Ehrlichiosis

infection. were protected against the pathogen.Our results demonstrate the power of structural vaccines and could be a new strategy in the development of vaccines to provide protection against pathogenic microorganisms

Persistent Infection Contributes to Heterologous Protective Immunity against Fatal Ehrlichiosis▿

Human monocytotropic ehrlichiosis (HME), an emerging and often life-threatening tick-transmitted disease, is caused by the obligately intracellular bacterium Ehrlichia chaffeensis. HME is modeled in C57BL/6 mice using Ehrlichia muris, which causes persistent infection, and Ixodes ovatus Ehrlichia (IOE), which is either acutely lethal or sublethal depending on the dose and route of inoculation. A persistent primary E. muris infection, but not a sublethal IOE infection, protects mice against an ordinarily lethal secondary IOE challenge. In the present study, we determined the role of persistent infection in maintenance of protective memory immune responses. E. muris-infected mice were treated with doxycycline or left untreated and then challenged with an ordinarily lethal dose of IOE. Compared to E. muris-primed mice treated with doxycycline, untreated mice persistently infected with E. muris had significantly greater numbers of antigen-specific gamma interferon-producing splenic memory T cells, significant expansion of CD4+ CD25+ T regulatory cells, and production of transforming growth factor β1 in the spleen. Importantly, E. muris-primed mice treated with doxycycline showed significantly greater susceptibility to challenge infection with IOE compared to untreated mice persistently infected with E. muris. The study indicated that persistent ehrlichial infection contributes to heterologous protection by stimulating the maintenance of memory T-cell responses

Reactivity of <i>Ehrlichia</i> Hsp60 antibody.

<p>The <i>Ehrlichia</i> Hsp60 _43–63 peptide was sensitive in detecting different dilutions of the <i>Ehrlichia</i> Hsp60 antibody. The <i>Ehrlichia</i> Hsp60 (250ng) was probed with different dilutions of <i>Ehrlichia</i> Hsp60 specific sera or sera from naïve mice. Finally they were probed with goat anti-mouse-AP (1∶2000) and the OD measured at 650 nm after the addition of substrate. Each value is the mean of 3 replicates (***<i>p</i><0.001 as determined by two way ANOVA).</p