Novel N-terminal variant of human VDR.

The importance of N-terminal regions of nuclear hormone receptors in transcriptional regulation is increasingly recognized. As variant VDR gene transcripts indicated possible N-terminally extended receptors, we investigated their natural occurrence, transactivation capacity, and subcellular localization. A novel 54-kDa VDRB1 protein, in addition to the previously recognized 48-kDa VDRA form, was detected in human kidney tissue as well as in osteoblastic (MG63), intestinal (Int-407, DLD-1, and COLO 206F), and kidney epithelial (786) human cell lines by Western blots using isoform-specific and nonselective anti-VDR antibodies. VDRB1 was present at approximately one-third the level of VDRA. Isoform-specific VDRB1 expression constructs produced lower ligand-dependent transactivation than VDRA when transiently transfected with a vitamin D-responsive promoter into cell lines with low endogenous VDR. Intracellular localization patterns of the green fluorescent protein-tagged VDR isoforms differed. VDRB1 appeared as discrete intranuclear foci in the absence of 1,25-dihydroxyvitamin D3, whereas VDRA produced diffuse nuclear fluorescence. After 1,25-dihydroxyvitamin D3 treatment, both VDR isoforms exhibited similar diffuse nuclear signal. In the absence of 1,25-dihydroxyvitamin D3, the VDRB1 foci partially colocalized with SC-35 speckles and a subset of promyelocytic leukemia nuclear bodies. These data provide the first evidence of VDRB1, a novel N-terminally variant human VDR that is expressed at a level comparable to VDRA in human tissue and cell lines. It is characterized by reduced transactivation activity and a ligand-responsive speckled intranuclear localization. The intranuclear compartmentalization and altered functional activity of VDRB1 may mediate a specialized physiological role for this receptor isoform.

Human and murine osteocalcin gene expression: conserved tissue restricted expression and divergent responses to 1,25-dihydroxyvitamin D3 in vivo.

Human and murine osteocalcin genes demonstrate similar cell-specific expression patterns despite significant differences in gene locus organization and sequence variations in cis-acting regulatory elements. To investigate whether differences in these regulatory regions result in an altered response to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in vivo, we compared the response of the endogenous mouse osteocalcin gene to a bacterial reporter gene directed by flanking regions of the human osteocalcin gene in transgenic mice. Transgene expression colocalized with endogenous osteocalcin expression in serial sections, being detected in osteoblasts, osteocytes and hypertrophic chondrocytes. In calvarial cell culture lysates from transgenic and nontransgenic mice, the endogenous mouse osteocalcin gene did not respond to 1,25-(OH)2D3 treatment. Despite this, transgene activity was significantly increased in the same cells. Similarly, Northern blots of total cellular RNA and in situ hybridization studies of transgenic animals demonstrated a maximal increase in transgene expression at 6 h after 1,25-(OH)2D3 injection (23.6+/-3.6-fold) with a return to levels equivalent to uninjected animals by 24 h (1.2+/-0.1-fold). This increase in transgene expression was also observed at 6 h after 1,25-(OH)2D3 treatment in animals on a low calcium diet (25.2+/-7.7-fold) as well as in transgenic mice fed a vitamin D-deficient diet containing strontium chloride to block endogenous 1,25-(OH)2D3 production (7.5+/-0.9-fold). In contrast to the increased transgene expression levels, neither endogenous mouse osteocalcin mRNA levels nor serum osteocalcin levels were significantly altered after 1,25-(OH)2D3 injection in transgenic or nontransgenic mice, regardless of dietary manipulations, supporting evidence for different mechanisms regulating the response of human and mouse osteocalcin genes to 1,25-(OH)2D3. Although the cis- and trans-acting mechanisms directing cell-specific gene expression appear to be conserved in the mouse and human osteocalcin genes, responsiveness to 1,25-(OH)2D3 is not. The mouse osteocalcin genes do not respond to 1,25-(OH)2D3 treatment, but the human osteocalcin-directed transgene is markedly upregulated under the same conditions and in the same cells. The divergent responses of these homologous genes to 1,25-(OH)2D3 are therefore likely to be due to differences in mouse and human osteocalcin-regulatory sequences rather than to variation in the complement of trans-acting factors present in mouse osteoblastic cells. Increased understanding of these murine-human differences in osteocalcin regulation may shed light on the function of osteocalcin and its regulation by vitamin D in bone physiology.

Expression of the parathyroid Ca(2+)-sensing receptor in cytotrophoblasts from human term placenta.

Fura-2-loaded human cytotrophoblasts responded to elevated extracellular Ca2+ concentration ([Ca2+]o) with monophasic or, in the case of large (> 20 microns) extravillous cells, biphasic elevations in intracellular free Ca2+ ion concentration ([Ca2+]i) that returned to baseline levels after restoration of control [Ca2+]o. Large extravillous cytotrophoblasts also responded to elevated [Mg2+]o with transient elevations in [Ca2+]i, consistent with the behaviour of the parathyroid Ca2(+)-sensing receptor. Expression of the parathyroid Ca2(+)-sensing receptor in placental cells was confirmed using Northern blot and reverse transcription (RT)-PCR analysis. However, the major transcript in human placental cells (6.2 kb) differed from that expressed by human parathyroid cells (5.6 kb). RT-PCR analysis and DNA sequencing of key PCR products also revealed the presence of a splice variant in placental and parathyroid cells that lacks exon 3.

Retracted: Risks of avian influenza (H5) in duck farms in the Ayeyarwaddy Delta region, Myanmar.

The Ayeyarwaddy delta region in the south-west of Myanmar is the main agricultural and rice-growing area. The region has a high density of duck and backyard chicken populations with low biosecurity. The objective of this study was to analyse risk factors for avian influenza (H5) in the Ayeyarwaddy delta region, Myanmar. A case­control risk factor study was conducted from April to June 2010 by individual interviews including risk factor questionnaires given to duck farmers (n = 50) in five townships in the Ayeyarwaddy delta region, Myanmar. Risk factor analyses were conducted using univariate analysis and multivariate logistic regression model with backward stepwise (wald) method. The results showed significant risk factors for AI (H5) sero-positivity in ducks were wooden egg box containers (OR = 52.7, 95% CI = 2.34-1188, P = 0.013) and water sourced from wetlands (OR = 30.7, 95% CI = 1.96-481.6, P = 0.015). Conversely, the cleaning of reusable egg containers was determined as a protective factor (OR = 0.03, 95% CI = 0.00-0.42, P = 0.01). In conclusion, this study identified risk factors for AI (H5) in duck farms and the importance of avian influenza prevention and control.

Economic analysis of interventions to improve village chicken production in Myanmar.

A cost-benefit analysis using deterministic and stochastic modelling was conducted to identify the net benefits for households that adopt (1) vaccination of individual birds against Newcastle disease (ND) or (2) improved management of chick rearing by providing coops for the protection of chicks from predation and chick starter feed inside a creep feeder to support chicks' nutrition in village chicken flocks in Myanmar. Partial budgeting was used to assess the additional costs and benefits associated with each of the two interventions tested relative to neither strategy. In the deterministic model, over the first 3 years after the introduction of the interventions, the cumulative sum of the net differences from neither strategy was 13,189Kyat for ND vaccination and 77,645Kyat for improved chick management (effective exchange rate in 2005: 1000Kyat=1$US). Both interventions were also profitable after discounting over a 10-year period; Net Present Values for ND vaccination and improved chick management were 30,791 and 167,825Kyat, respectively. The Benefit-Cost Ratio for ND vaccination was very high (28.8). This was lower for improved chick management, due to greater costs of the intervention, but still favourable at 4.7. Using both interventions concurrently yielded a Net Present Value of 470,543Kyat and a Benefit-Cost Ratio of 11.2 over the 10-year period in the deterministic model. Using the stochastic model, for the first 3 years following the introduction of the interventions, the mean cumulative sums of the net difference were similar to those values obtained from the deterministic model. Sensitivity analysis indicated that the cumulative net differences were strongly influenced by grower bird sale income, particularly under improved chick management. The effects of the strategies on odds of households selling and consuming birds after 7 months, and numbers of birds being sold or consumed after this period also influenced profitability. Cost variations for equipment used under improved chick management were not markedly associated with profitability. Net Present Values and Benefit-Cost Ratios discounted over a 10-year period were also similar to the deterministic model when mean values obtained through stochastic modelling were used. In summary, the study showed that ND vaccination and improved chick management can improve the viability and profitability of village chicken production in Myanmar.

Increased formation and decreased resorption of bone in mice with elevated vitamin D receptor in mature cells of the osteoblastic lineage.

The microarchitecture of bone is regulated by complex interactions between the bone-forming and resorbing cells, and several compounds regulate both actions. For example, vitamin D, which is required for bone mineralization, also stimulates bone resorption. Transgenic mice overexpressing the vitamin D receptor solely in mature cells of the osteoblastic bone-forming lineage were generated to test the potential therapeutic value of shifting the balance of vitamin D activity in favor of bone formation. Cortical bone was 5% wider and 15% stronger in these mice due to a doubling of periosteal mineral apposition rate without altered body weight or calcium homeostatic hormone levels. A 20% increase in trabecular bone volume in transgenic vertebrae was also observed, unexpectedly associated with a 30% reduction in resorption surface rather than greater bone formation. These findings indicate anabolic vitamin D activity in bone and identify a previously unknown pathway from mature osteoblastic cells to inhibit osteoclastic bone resorption, counterbalancing the known stimulatory action through immature osteoblastic cells. A therapeutic approach that both stimulates cortical anabolic and inhibits trabecular resorptive pathways would be ideal for treatment of osteoporosis and other osteopenic disorders.