Direct effects of vitamin D3 analogues on G-protein mediated signalling systems in rat osteosarcoma cells and rat pituitary adenoma cells

In normal rats treated with 1,25(OH)₂D₃ or 24,25(OH)₂D₃, serum Ca²⁺, ALP, PRL and GH are significantly altered. In order to study the primary effect of vitamin D₃ analogues on target organ function, rat UMR 106 osteosarcoma and GH₃ pituitary adenoma cells in monolayer culture were exposed accordingly.Surprisingly, prolonged exposure of these cell lines to physiological levels of either 1,25(OH)₂D₃ or 24,25(OH)₂D₃ did not significantly affect the secretory parameters (ALP, PRL or GH) tested. However, 1,25(OH)₂D₃ exposure significantly reduced PTH- and Gpp(NH)p-elicited AC as well as Gpp(NH)p-stimulated PLC activities in the UMR 106 cells. These changes were accompanied by an increase and decrease in the membrane contents of the G-protein subunits G₃₆ and G_q/11, respectively. In contrast, 24,25(OH)₂D₃ remained without significant biological effect on these signalling systems despite concomitantly augmented levels of G₃₆. TRH- and Gpp(NH)p-elicited PLC activities in the GH₃ cells were significantly reduced by 1,25(OH)₂D₃ with a concurrent reduction in cellular amounts of G_q/11, however, 24,25(OH)₂D₃ did not significantly alter any signalling systems nor G-proteins analyzed.It is concluded that the osteoblastic and pituitary cell secretion of ALP, PRL and GH remain unaffected by the presence of 1,25(OH)₂D₃ and 24,25(OH)₂D₃, despite distinct alterations in components of G-protein mediated signalling pathways. Hence, other factors like ambient Ca²⁺ may be responsible for the perturbed secretory patterns of ALP and PRL seen in vitamin D₃ treated rats.Abbreviations AC adenylate cyclase - ALP alkaline phosphatase - BGP osteocalcin - BSA bovine serum albumin - DA dopamine - DAG diacylglycerol - GH growth hormone - GHRH growth hormone releasing hormone - Gpp(NH)p guanosine 5-[-imido]triphosphate - G-protein guanine nucleotide-binding regulatory protein - G_s etc. G_s protein -subunit - IP₃ inositol 1,4,5 trisphosphate - OAF osteoclast activating factor - PGE₂ prostaglandin E₂ - PKA & PKC protein kinase A & C - PLC phospholipase C - PRL prolactin - PTH parathyroid hormone - SRIF somatostatin - TRH thyrotropin releasing hormone - VIP vasoactive intestinal peptide - 25(OH)D₃ 25 hydroxy vitamin D₃ - 1,25(OH)₂D₃ 1·25 dihydroxy vitamin D₃ - 24,25(OH)₂D₃ 24,25 dihydroxy vitamin D₃     

IPS-1 Is Essential for the Control of West Nile Virus Infection and Immunity

The innate immune response is essential for controlling West Nile virus (WNV) infection but how this response is propagated and regulates adaptive immunity in vivo are not defined. Herein, we show that IPS-1, the central adaptor protein to RIG-I-like receptor (RLR) signaling, is essential for triggering of innate immunity and for effective development and regulation of adaptive immunity against pathogenic WNV. IPS-1^−/− mice exhibited increased susceptibility to WNV infection marked by enhanced viral replication and dissemination with early viral entry into the CNS. Infection of cultured bone-marrow (BM) derived dendritic cells (DCs), macrophages (Macs), and primary cortical neurons showed that the IPS-1-dependent RLR signaling was essential for triggering IFN defenses and controlling virus replication in these key target cells of infection. Intriguingly, infected IPS-1^−/− mice displayed uncontrolled inflammation that included elevated systemic type I IFN, proinflammatory cytokine and chemokine responses, increased numbers of inflammatory DCs, enhanced humoral responses marked by complete loss of virus neutralization activity, and increased numbers of virus-specific CD8+ T cells and non-specific immune cell proliferation in the periphery and in the CNS. This uncontrolled inflammatory response was associated with a lack of regulatory T cell expansion that normally occurs during acute WNV infection. Thus, the enhanced inflammatory response in the absence of IPS-1 was coupled with a failure to protect against WNV infection. Our data define an innate/adaptive immune interface mediated through IPS-1-dependent RLR signaling that regulates the quantity, quality, and balance of the immune response to WNV infection.     

Cutting Edge: Plasmacytoid dendritic cells provide innate immune protection against mucosal viral infection in situ

Dendritic cells (DCs) are powerful APCs capable of activating naive lymphocytes. Of the DC subfamilies, plasmacytoid DCs (pDCs) are unique in that they secrete high levels of type I IFNs in response to viruses but their role in inducing adaptive immunity remains divisive. In this study, we examined the importance of pDCs and their ability to recognize a virus through TLR9 in immunity against genital HSV-2 infection. We show that a low number of pDCs survey the vaginal mucosa at steady state. Upon infection, pDCs are recruited to the vagina and produce large amounts of type I IFNs in a TLR9-dependent manner and suppress local viral replication. Although pDCs are critical in innate defense against genital herpes challenge, adaptive Th1 immunity developed normally in the absence of pDCs. Thus, by way of migrating directly into the peripheral mucosa, pDCs act strictly as innate antiviral effector cells against mucosal viral infection in situ.     

The perception of painful and nonpainful stimuli during voluntary motor activity in man

Previous studies have shown that voluntary movement diminishes the transmission of cutaneous afferent input through the dorsal column-medial lemniscal system, and also raises the threshold for detecting nonpainful, cutaneous stimuli (electrical shocks). Although there is some evidence that pain elicited by electrical stimulation is diminished during movement, no studies have tested the effect of movement on the perception of pain produced by natural stimulation. For this reason, we tested the effects of voluntary motor activity on the perception of noxious thermal stimuli in human volunteers. We first developed a motor paradigm in which the thermal stimulation could be applied to the immobile limb (isometric elbow flexion-extension). Both isometric and isotonic muscle contractions about the elbow increased the threshold for detecting weak cutaneous stimuli (electrical shocks) applied to the forearm, and to a lesser extent the detection of stimuli applied to the dorsum of the hand. Afterwards, noxious and innocuous heat stimuli were applied to the forearm during isometric contractions and at rest. Magnitude estimates for the intensity of the pain, as well as latency measures of the onset of pain, were recorded. We found no evidence that isometric motor activity diminished either the threshold for pain or the subjective intensity of the noxious and innocuous thermal stimuli. Thus, motor activity decreases the ability to detect weak low-threshold cutaneous inputs, but has no effect on the perception of warmth and heat pain.     

Strain-specific ureolytic microbial calcium carbonate precipitation

During a study of ureolytic microbial calcium carbonate (CaCO(3)) precipitation by bacterial isolates collected from different environmental samples, morphological differences were observed in the large CaCO(3) crystal aggregates precipitated within bacterial colonies grown on agar. Based on these differences, 12 isolates were selected for further study. We hypothesized that the striking differences in crystal morphology were the result of different microbial species or, alternatively, differences in the functional attributes of the isolates selected. Sequencing of 16S rRNA genes showed that all of the isolates were phylogenetically closely related to the Bacillus sphaericus group. Urease gene diversity among the isolates was examined by using a novel application of PCR-denaturing gradient gel electrophoresis (DGGE). This approach revealed significant differences between the isolates. Moreover, for several isolates, multiple bands appeared on the DGGE gels, suggesting the apparent presence of different urease genes in these isolates. The substrate affinities (K(m)) and maximum hydrolysis rates (V(max)) of crude enzyme extracts differed considerably for the different strains. For certain isolates, the urease activity increased up to 10-fold in the presence of 30 mM calcium, and apparently this contributed to the characteristic crystal formation by these isolates. We show that strain-specific calcification occurred during ureolytic microbial carbonate precipitation. The specificity was mainly due to differences in urease expression and the response to calcium.     

A study of the reproducibility of a fluorescence in situ hybridization bladder cancer detection assay

OBJECTIVE: To assess the reproducibility of the UroVysion fluorescence in situ hybridization (FISH) bladder cancer detection assay. STUDY DESIGN: Thirteen specimens (2 negative, 3 low-level positive [1-10% abnormal cells], 5 mid-level positive [11-75%], and 3 high-level positive [>75%]) were analyzed by 7 cytotechnologists. Each cytotechnologist rendered an overall diagnosis of positive or negative and determined the percentage of abnormal urothelial cells for all positive specimens. RESULTS: The interobserver reproducibility of the assay was 100% for mid-level and high-level positive specimens, 93% for negative specimens, and 78% for low-level positive specimens. The range of percent abnormal determinations was highest for mid-level positive specimens, with mean SDs of 1.8%, 16.4% and 10.1% for the low-, mid-, and high-level positives, respectively. CONCLUSION: There was a high level of reproducibility among the mid- and high-level positive specimens. The reproducibility for low-level positive specimens was lowest, suggesting that such specimens should be reviewed by a second technologist to ensure an accurate diagnosis. The findings of this study are important for further elucidating the clinical value of quantitative FISH analysis in the management of patients undergoing FISH testing for bladder cancer.     

Development and evolution of adaptive polyphenisms

Phenotypic plasticity is the primitive character state for most if not all traits. Insofar as developmental and physiological processes obey the laws of chemistry and physics, they will be sensitive to such environmental variables as temperature, nutrient supply, ionic environment, and the availability of various macro- and micronutrients. Depending on the effect this phenotypic plasticity has on fitness, evolution may proceed to select either for mechanisms that buffer or canalize the phenotype against relevant environmental variation or for a modified plastic response in which some ranges of the phenotypic variation are adaptive to particular environments. Phenotypic plasticity can be continuous, in which case it is called a reaction norm, or discontinuous, in which case it is called a polyphenism. Although the morphological discontinuity of some polyphenisms is produced by discrete developmental switches, most polyphenisms are due to discontinuities in the environment that induce only portions of what is in reality a continuous reaction norm. In insect polyphenisms, the environmental variable that induces the alternative phenotype is a token stimulus that serves as a predictor of, but is not itself, the environment to which the polyphenism is an adaptation. In all cases studied so far, the environmental stimulus alters the endocrine mechanism of metamorphosis by altering either the pattern of hormone secretion or the pattern of hormone sensitivity in different tissues. Such changes in the patterns of endocrine interactions result in the execution of alternative developmental pathways. The spatial and temporal compartmentalization of endocrine interactions has produced a developmental mechanism that enables substantial localized changes in morphology that remain well integrated into the structure and function of the organism.