Incorporation of Cestrum diurnum leaf improves intestinal Ca transport in broilers

The economy of Ca utilization is under the control of vitamin D₃, particularly its active metabolite 1,25-dihydroxy cholecalciferol [1,25(OH)₂D₃]. In sufficient Ca absorption leads to tibial dyschondroplasia resulting in not attaining optimum body weight. Our earlier studies [T.P. Prema, N. Raghuramulu, Phytochemistry 37 (1994) 167] have shown that the Cestrum diurnum (CD) leaves contain vitamin D₃ metabolites. It was felt whether incorporation of CD as a source of 1,25(OH)₂D₃ could improve the Ca absorption in broilers. Four groups of 60 birds each were fed with either normal diet or normal diet + 0.25% CD or normal diet without vitamin D₃ or normal diet without vitamin D₃ + 0.25% CD leaf powder for 45 days. In subsample of six birds it was observed that incorporation of CD leaves in the feed had the maximal effect on all the parameters studied. The results indicate that the intestinal Ca transport as represented by Serosa/Mucosa (S/M) ratio was found to be significantly (p < 0.01) higher in broilers fed diet with CD leaf powder and the 1 hydroxylase activity in kidney is significantly (p < 0.001) higher in negative controls. On the other hand the supplementation of CD leaves enhanced the serum Ca, body weight, tibia weight, density and strength resulting in the disappearance of tibial dyschondroplasia. No lesions of toxicity were observed in any of the soft tissue examined. The results suggest that the incorporation of CD leaf powder in poultry feed could be beneficial to the poultry.     

Calbindin D9k is not required for 1,25-dihydroxyvitamin D3-mediated Ca2+ absorption in small intestine

The exact role of calbindin D9k in vitamin D-mediated calcium absorption has been debated but remains unsettled. In 129/OlaHsd mice, calbindin D9k was found highest in duodenum (36-50%) and kidney (24-34%) followed by stomach, lung and uterus. Age does not affect the relative distribution of calbindin D9k but it does decline with age in duodenum of both male and female 129/Ola mice. Recently, we produced a null calbindin D9k mutant 129/OlaHsd mouse; this mouse proved to be indistinguishable from the wild-type in phenotype and in a serum calcium level regardless of age or gender. We have now examined directly whether the mutant mouse can absorb calcium from the intestine in response to the active form of vitamin D. The calbindin D9k null mutant mouse is fully able to absorb calcium from the intestine in response to 1,25-dihydroxyvitamin D3. It is, therefore, clear that calbindin D9k is not required for vitamin D-induced intestinal calcium absorption.     

Clusterin over-expression modulates proapoptotic and antiproliferative effects of 1,25(OH)2D3 in prostate cancer cells in vitro

Prostate cancer is the most commonly diagnosed cancer in the majority of western countries. Due to their antiproliferative and proapoptotic activity, vitamin D analogues have been introduced recently as an experimental therapy for prostate cancer. Clusterin (CLU) is a glycoprotein that has two known isoforms generated in human cells. A nuclear form of CLU protein (nCLU) is pro-apoptotic, and a secretory form (sCLU) is pro-survival. In this study, we analyzed whether proapoptotic and antiproliferative effects of 1,25(OH)₂D₃ on LNCaP prostate cancer cells are modulated by expression of sCLU. Using colony forming assay, we studied the effect of treatment with different doses of 1,25(OH)₂D₃ (10⁻⁶, 10⁻⁷, 10⁻¹⁰ M) on proliferation of LNCaP cells that were stable transfected and over-express sCLU (LNT-1) as compared to empty vector-transfected cells (LN/C). We also measured apoptosis using TUNEL assay. sCLU over-expression protected against both antiproliferative (30%) and proapoptotic (15%) effects of 1,25(OH)₂D₃, although this effect was statistically not significant. In conclusion, our findings demonstrate that expression of sCLU modulates growth regulatory effects of 1,25(OH)₂D₃ in prostate cancer indicating that CLU interferes with vitamin D signalling pathways.     

Screening of Vitamin D activity (VDA) of Solanum glaucophyllum leaves measured by radioimmunoassay (RIA)

The ingestion of Solanum glaucophyllum (SG) causes a calcinosis of cattle named Enteque Seco (ES). The toxic principle is the 1,25-(OH)₂D₃, mainly conjugated as glycoside. This study aims to validate a simple novel method of evaluation of the VDA of SG leaves. Aqueous extracts of SG were purified using C₁₈ minicolumns and assayed by RIA with an antibody raised in rabbits by injection of the acid—C22, 1α-(OH)Vitamin D₃. Data were expresed as glycoside equivalent to 1,25-(OH)₂D₃ in ng/g of dry leaves. We compared this data with 1,25-(OH)₂D₃ levels measured, in the same samples, by liquid chromatography (HPLC) after enzyme cleavage. This procedure involved the incubation of SG leaves with rumen fluid, followed by C₁₈-OH solid phase extraction. The 1,25-(OH)₂D₃ fraction was run by HPLC and detection was achieved using a photodiode array detector. Data were expressed as micrograms of 1,25-(OH)₂D₃/g dry leaves. A significant regression of 1,25-(OH)₂D₃ levels (Y) as a function of glycoside RIA 1,25-(OH)₂D₃ equivalents (X) was found: Y = 12.02 + 0.35X [R = 0.81; P = 0,0002; N = 15], allowing us to conclude that this novel assay could be used to estimate the amount of this active principle contained in SG leaves.     

Caffeine decreases vitamin D receptor protein expression and 1,25(OH)2D3 stimulated alkaline phosphatase activity in human osteoblast cells

Of the various risk factors contributing to osteoporosis, dietary/lifestyle factors are important. In a clinical study we reported that women with caffeine intakes >300 mg/day had higher bone loss and women with vitamin D receptor (VDR) variant, tt were at a greater risk for this deleterious effect of caffeine. However, the mechanism of how caffeine effects bone metabolism is not clear. 1,25-Dihydroxy vitamin D₃ (1,25(OH)₂D₃) plays a critical role in regulating bone metabolism. The receptor for 1,25(OH)₂D₃, VDR has been demonstrated in osteoblast cells and it belongs to the superfamily of nuclear hormone receptors. To understand the molecular mechanism of the role of caffeine in relation to bone, we tested the effect of caffeine on VDR expression and 1,25(OH)₂D₃ mediated actions in bone. We therefore examined the effect of different doses of caffeine (0.2, 0.5, 1.0 and 10 mM) on 1,25(OH)₂D₃ induced VDR protein expression in human osteoblast cells. We also tested the effect of different doses of caffeine on 1,25(OH)₂D₃ induced alkaline phosphatase (ALP) activity, a widely used marker of osteoblastic activity. Caffeine dose dependently decreased the 1,25(OH)₂D₃ induced VDR expression and at concentrations of 1 and 10 mM, VDR expression was decreased by about 50–70%, respectively. In addition, the 1,25(OH)₂D₃ induced alkaline phosphatase activity was also reduced at similar doses thus affecting the osteoblastic function. The basal ALP activity was not affected with increasing doses of caffeine. Overall, our results suggest that caffeine affects 1,25(OH)₂D₃ stimulated VDR protein expression and 1,25(OH)₂D₃ mediated actions in human osteoblast cells.     

夜香树均一化全长cDNA文库的构建及EST分析

为构建高质量的夜香树(Cestrum nocturnum)均一化全长c DNA文库,以其花朵为材料,采用DSN均一化技术与改进的SMART技术相结合,将连接产物进行脱盐浓缩后电转化进行构建。结果表明,未脱盐连接产物的转化效率为1μL连接产物有7000个菌落,理论重组率为96%;脱盐后,提升为4.1×106个菌落,理论重组率为98%;蓝斑也有插入片段,实际重组率应为100%;插入片段大小平均为1.6 kb。随机挑取500个单克隆(含50个蓝斑)测序,共获得464条EST,单一序列(unigene)为426条,占91.8%,其中片段重叠群26个,单基因400个,冗余率仅为8.1%,表明构建文库的均一化效果较好,可满足后续功能基因的筛选和基因信息的研究。     

Quantifying division of aortal smooth muscle cells in culture stimulated by 1,25(OH)2D3

Inhibitory effect of 1α,25dihydroxycholecalciferol (1,25D₃ = calcitriol) in different cell type is well recognized but its promoting effect on vascular smooth muscle cells (SMCs) is poor established. Therefore, the aim of this study was to determine stimulatory effect of calcitriol on aortal SMCs proliferation in culture. We used the cell division analysis procedure based on the quantitative sequential halving of the stably incorporating fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE). This technique allowed the visualization of cycles of SMCs division by flow cytometry. Rat aortal SMCs were labeled with CFSE and cultured for up to 10 days with defined concentration of calcitriol in medium. Proliferative activity as the percentage of SMCs in different phases of the cell cycle using propidium iodide was determined. Apoptosis was assessed using Annexin-V/CFDA method. The results suggest that low concentrations of an active form of vitamin D—1α,25dihydroxycholecalciferol applied in supraphysiological concentration of 10 nmol/l is a mitogenic factor for aortal SMCs. None of the applied concentrations of calcitriol caused apoptosis. The findings well support our morphological (LM) and ultrastructural (TEM and SEM) observations.     

Phosphate uptake in chick kidney cells: effects of 1,25(OH)2D3 and 24,25(OH)2D3

Phosphate homeostasis is controlled in part by absorption from the intestine, and reabsorption in the kidney. While the effect of Vitamin D metabolites on enterocytes is well documented, in the current study we assess selected responses in primary cultures of kidney cells. Time course studies revealed a rapid stimulation of phosphate uptake in cells treated with 1,25(OH)(2)D(3), relative to controls. Dose-response studies indicated a biphasic curve with optimal stimulation at 300 pM 1,25(OH)(2)D(3) and inhibition at 600 pM seco-steroid. Antibody 099--against the 1,25D(3)-MARRS receptor - abolished stimulation by the steroid hormone. Moreover, phosphate uptake was mediated by the protein kinase C pathway. The metabolite 24,25(OH)(2)D(3), which was found to inhibit the rapid stimulation of phosphate uptake in intestinal cells, had a parallel effect in cultured kidney cells. Finally, the 24,25(OH)(2)D(3) binding protein, catalase, was assessed for longer term down regulation. In both intestinal epithelial cells and kidney cells incubated with 24,25(OH)(2)D(3) for 5-24h, both the specific activity of the enzyme and protein levels were decreased relative to controls, while 1,25(OH)(2)D(3) increased both parameters over the same time periods. We conclude that the Vitamin D metabolites have similar effects in both kidney and intestine, and that 24,25(OH)(2)D(3) may have effects at the level of gene expression.     

1α,25-Dihydroxyvitamin D3-3β-bromoacetate, a potential cancer therapeutic agent: synthesis and molecular mechanism of action

Synthesis of 1α,25-dihydroxyvitamin D₃-3β-bromoacetate (1,25(OH)₂D₃-3-BE), a potential anti-cancer agent is presented. We also report that mechanism of action of 1,25(OH)₂D₃-3-BE may involve reduction of its catabolism, as evidenced by the reduced and delayed expression of 1α,25-dihydroxyvitamin D₃-24-hydroxylase (CYP24) gene in cellular assays.     

25(OH)D3 and 1,25(OH)2D3 serum concentration and breast tissue expression of 1alpha-hydroxylase, 24-hydroxylase and Vitamin D receptor in women with and without breast cancer

1,25(OH)2D3 is an antiproliferative agent that may inhibit proliferation of breast cancer (BC) cells in vitro and BC development in animals. Epidemiological studies have shown a high incidence of BC in people less exposed to solar rays. To unravel the role of Vitamin D3 in BC patients, we have investigated serum levels of 25(OH)D3 and its active form 1,25(OH)2D3 as well as tissue expression of 1alpha-hydroxylase, 24-hydroxylase, and Vitamin D-receptor (VDR), determined by semiquantitative RT-PCR, in 88 Brazilian BC patients and 35 women without cancer (submitted to mammoplasties or resection of benign lesions). Median age of women with and without cancer was 51 and 46 years, respectively, and the majority of BC patients were classified as clinical stage II (67%). Although no differences in 25(OH)D3 serum concentration were found, 1,25(OH)2D3 (40+/-21 pg/ml) levels in BC patients were lower than in women without cancer (53+/-23). Our results indicate that 24-hydroxylase, VDR and 1alpha-hydroxylase mRNA tissue expression is similar in both groups and no correlation between 24-hydroxylase, 1alpha-hydroxylase, and VDR expression in breast tumors was found. A low 1,25(OH)2D3 serum concentration seems to be associated to breast cancer, however, the mechanism involved in this regulation is still unclear.     

Metabolism of 1α,25-dihydroxyvitamin D2 by human CYP24A1

The metabolism of 1α,25-dihydroxyvitamin D₂ (1α,25(OH)₂D₂) by human CYP24A1 was examined using the recombinant enzyme expressed in Escherichia coli cells. HPLC analysis revealed that human CYP24A1 produces at least 10 metabolites, while rat CYP24A1 produces only three metabolites, indicating a remarkable species-based difference in the CYP24A1-dependent metabolism of 1α,25(OH)₂D₂ between humans and rats. LC-MS analysis and periodate treatment of the metabolites strongly suggest that human CYP24A1 converts 1α,25(OH)₂D₂ to 1α,24,25,26(OH)₄D₂, 1α,24,25,28(OH)₄D₂, and 24-oxo-25,26,27-trinor-1α(OH)D₂ via 1α,24,25(OH)₃D₂. These results indicate that human CYP24A1 catalyzes the C₂₄-C₂₅ bond cleavage of 1α,24,25(OH)₂D₂, which is quite effective in the inactivation of the active form of vitamin D₂. The combination of hydroxylation at multiple sites and C-C bond cleavage could form a large number of metabolites. Our findings appear to be useful to predict the metabolism of vitamin D₂ and its analogs in the human body.     

Mechanisms of 1,25(OH)2D3-induced rapid changes of membrane potential in proximal tubule: Role of Ca2+-dependent K+ channels

Summary Eleven different secosteroids or steroids (10^–10 to 10^–8 m) were acutely and reversibly introduced in solutions delivered to the lumen of single proximal tubules of the amphibianNecturus kidney while recording basolateral cell membrane potentialV _m. Seven of these molecules (1,25(OH)₂D₃, 25(OH)D₃, 24,25(OH)₂D₃, 5,6-trans-25(OH)D₃, 19-diol-cholesterol, estradiol and testosterone) resulted in changes ofV _m (V _m) occurring in a few seconds, the largest V _m being observed with 1,25(OH)₂D₃, +6.5±0.75 mV (n=19); these seven (seco)steroids but not the four inactive sterols (vitamin D₃, cholesterol, 1D₃ and aldosterone) possess a hydroxyl group on at least one carbon of the C₁₇ to C₂₅ lateral chain of the sterol ring. The V _m effect was present in Na⁺-free or Cl^–-free media, but it was abolished in HCO₃-free media. Depolarization of cell membrane potential by addition of glucose, 11mm, in luminal perfusion fluid abolished the 1,25(OH)₂D₃-evoked V _m effect, suggesting dependence of the latter on the absolute value of membrane potential. Barium, a blocking agent of K⁺ conductances, suppressed the 1,25(OH)₂D₃-evoked V _m effect, even when the proper effects of barium of cell membrane potential were canceled by current clamp. Pretreatment with quinine, a putative blocker of Ca²⁺-dependent K⁺ channels also abolished the 1,25(OH)₂D₃-evoked depolarization. Such observations are consistent with the presence of Ca²⁺-dependent K⁺ channels at the apical cell membrane of the proximal tubule, these channels being inactivated by 1,25(OH)₂D₃ and probably by other (seco)steroids.     

Glucose 1-phosphate increases active transport of calcium in intestine

Active calcium transport in intestine is essential for serum calcium homeostasis as well as for bone formation. It is well recognized that vitamin D is a major, if not sole, stimulator of intestinal calcium transport activity in mammals. Besides vitamin D, endogenous glucose 1-phosphate (G1P) affects calcium transport activity in some microorganisms. In this study, we investigated whether G1P affects intestinal calcium transport activity in mammals as well. Of several glycolytic intermediates, G1P was the sole sugar compound in stimulating intestinal calcium uptake in Caco-2 cells. G1P stimulated net calcium influx and expression of calbindin D9K protein in rat intestine, through an active transport mechanism. Calcium uptake in G1P-supplemented rats was greater than that in the control rats fed a diet containing adequate vitamin D3. Bone mineral density (BMD) of aged rat femoral metaphysis and diaphysis was also increased by feeding the G1P diet. G1P did not affect serum levels of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] at all. These results suggest that exogenously applied G1P stimulates active transport of calcium in intestine, independent of vitamin D, leading to an increase of BMD.     

不明原因复发性流产再次妊娠患者血清1,25(OH)2D3、sTim-3与Th17/Treg免疫失衡和妊娠结局的关系

摘要 目的：探讨不明原因复发性流产（URSA）再次妊娠患者血清1,25-二羟维生素D3[1,25（OH)）₂D₃]、可溶性T细胞免疫球蛋白黏蛋白分子3（sTim-3）与辅助性T细胞17（Th17）/调节性T细胞（Treg）免疫失衡和妊娠结局的关系。方法：选择于湖南省妇幼保健院2020年1月~2022年1月就诊的62例URSA再次妊娠患者作为研究组,另选择同期进行孕检的正常早孕妇女30例作为对照组。比较两组孕早期血清1,25(OH) ₂D₃、sTim-3及外周血Th17细胞、Treg细胞水平、Th17/Treg比值。Pearson法分析URSA再次妊娠患者血清1,25(OH) ₂D₃、sTim-3与外周血Th17细胞、Treg细胞水平、Th17/Treg比值平的相关性。根据URSA再次妊娠患者妊娠结局的不同分为妊娠成功分娩组和妊娠再次流产组,比较两组孕早期血清1,25(OH) ₂D₃、sTim-3与外周血Th17细胞、Treg细胞水平、Th17/Treg比值。受试者工作特征（ROC）曲线分析血清1,25(OH) ₂D₃、sTim-3与外周血Th17细胞、Treg细胞水平、Th17/Treg比值对妊娠结局的预测价值。结果：研究组血清sTim-3、外周血Th17细胞水平、Th17/Treg比值高于对照组,血清1,25(OH) ₂D₃、外周血Treg细胞水平低于对照组（P<0.05）。Pearson相关分析显示,URSA再次妊娠患者血清1,25(OH) ₂D₃与血清sTim-3、外周血Th17细胞水平、Th17/Treg比值呈负相关,与Treg细胞水平呈正相关（P<0.05）;血清sTim-3与外周血Treg细胞水平呈负相关,与Th17细胞水平、Th17/Treg比值呈正相关（P<0.05）。妊娠再次流产组血清sTim-3、外周血Th17细胞水平、Th17/Treg比值高于妊娠成功分娩组,血清1,25(OH) ₂D₃、外周血Treg细胞水平低于妊娠成功分娩组（P<0.05）。ROC曲线分析显示,血清1,25(OH) ₂D₃、sTim-3及外周血Th17细胞、Treg细胞水平及Th17/Treg比值均可预测URSA再次妊娠患者妊娠再次流产的发生风险,且上述指标联合检测的预测效能更高。结论：血清1,25(OH) ₂D₃水平异常降低、sTim-3水平异常升高可导致Th17/Treg免疫失衡,导致URSA再次妊娠患者再次发生流产。上述指标联合检测对URSA再次妊娠患者妊娠再次流产的预测效能更高。     

Involvement of 1,25D3-MARRS (membrane associated, rapid response steroid-binding), a novel vitamin D receptor, in growth inhibition of breast cancer cells

In addition to classical roles in calcium homeostasis and bone development, 1,25 dihydroxyvitamin D₃ [1,25(OH)₂D₃] inhibits the growth of several cancer types, including breast cancer. Although cellular effects of 1,25(OH)₂D₃ traditionally have been attributed to activation of a nuclear vitamin D receptor (VDR), a novel receptor for 1,25(OH)₂D₃ called 1,25D₃-MARRS (membrane-associated, rapid response steroid-binding) protein was identified recently. The purpose of this study was to determine if the level of 1,25D₃-MARRS expression modulates 1,25(OH)₂D₃ activity in breast cancer cells.Relative levels of 1,25D₃-MARRS protein in MCF-7, MDA MB 231, and MCF-10A cells were estimated by real-time RT-PCR and Western blotting. To determine if 1,25D₃-MARRS receptor was involved in the growth inhibitory effects of 1,25(OH)₂D₃ in MCF-7 cells, a ribozyme construct designed to knock down 1,25D₃-MARRS mRNA was stably transfected into MCF-7 cells. MCF-7 clones in which 1,25D₃-MARRS receptor expression was reduced showed increased sensitivity to 1,25(OH)₂D₃ ( IC₅₀ 56 ± 24 nM) compared to controls (319 ± 181 nM; P < 0.05). Reduction in 1,25D₃-MARRS receptor lengthened the doubling time in transfectants treated with 1,25(OH)₂D₃. Knockdown of 1,25D₃-MARRS receptor also increased the sensitivity of MCF-7 cells to the vitamin D analogs KH1060 and MC903, but not to unrelated agents (all-trans retinoic acid, paclitaxel, serum/glucose starvation, or the isoflavone, pomiferin). These results suggest that 1,25D₃-MARRS receptor expression interferes with the growth inhibitory activity of 1,25(OH)₂D₃ in breast cancer cells, possibly through the nuclear VDR. Further research should examine the potential for pharmacological or natural agents that modify 1,25D₃-MARRS expression or activity as anticancer agents.     

Thyrotropes in the pituitary are target cells for 1,25 dihydroxy vitamin D3

Summary In the anterior pituitary of the rat, target cells of 1,25 (OH)₂ vitamin D₃ are identified as those that secrete thyroid stimulating hormone by means of a combined technique of thaw-mount autoradiography and immunohistochemistry. The results for the first time provide evidence that suggests a central effect of 1,25 (OH)₂ vitamin D₃ on the modulation of thyrotropin secretion in a manner similar to that of other steroid hormones at the level of the pituitary.Supported by PHS grant NS09914     

Effect of 1,25(OH)(2)D(3) on rat peritoneal mesothelial cells treated with high glucose plus lipopolysaccharide

1,25(OH)₂D₃, the active metabolite of vitamin D₃, its activity is not limited to mineral and skeletal homeostasis. In recent years, there has been increasing evidence pointing to the role of its activity in the regulation of cell proliferation, cell differentiation and immunomodulation. Here we report lipopolysaccharide (LPS), a glycolipid that is produced and secreted by gram-negative bacteria during peritonitis, plus high glucose (HG) can significantly inhibit mesothelial cell viability while induce more apoptosis in rat peritoneal mesothelial cells (RPMC). Pretreatment with 1,25(OH)₂D₃ can reverse the above effect in a concentration dependent manner. HG plus LPS can down-regulate the levels of both mRNA and protein of VDR, and up-regulate the expression of TGF-β1 and TNF-α in RPMC, which can also be effectively reversed by pretreatment with 1,25(OH)₂D₃. The above results suggest that HG plus LPS may induce changes in RPMC’s viability and apoptosis, leading to peritoneal injury. 1,25(OH)₂D₃ can reverse the inhibition of cell viability, the increase of apoptotic rate and induction of fibrosis related cytokine TGF-β1 and TNF-α by HG plus LPS in RPMC, thus protect peritoneal membrane.     

PTH(7-84) inhibits PTH(1-34)-induced 1,25-(OH)2D3 production in murine renal tubules

To elucidate whether PTH(7-84), a degradation product of PTH(1-84), which inhibits PTH(1-84)-induced bone resorption, also exerts an antagonistic effect on the kidney, we studied the effect of PTH(7-84) on PTH(1-34)-induced production of 1,25-(OH)₂D₃ in primary cultured murine renal tubules.Neonatal mouse renal tubules cultured in serum-free MEM for 7 days were treated with PTH(1-34) and/or PTH(7-84). Three hours after addition of 25-OHD₃ (10⁻⁶ M), 1,25-(OH)₂D₃ was determined. PTH(1-34) stimulated the conversion of 25-OHD₃ to 1,25-(OH)₂D₃, and PTH(7-84) dose-dependently inhibited this process. Real-time PCR revealed that PTH(1-34) increased the expression level of 1α-hydroxylase mRNA, whereas PTH(7-84) did not affect the expression level 1α or 24-hydroxylase mRNA.These in vitro data suggest that PTH(7-84) elicits an antagonistic effect in renal tubules through receptors different from the type I PTH/PTHrP receptor. This may at least partly account for the decreased serum level of 1,25-(OH)₂D in patients with severe primary hyperparathyroidism with renal failure.