Calcium Binding Protein in Squid Brain: Biochemical Similarity to the 28,000-Mr Vitamin D-Dependent Calcium Binding Protein (Calbindin-D28k)

A calcium binding protein that is biochemically similar to vertebrate 28,000-Mr vitamin D-dependent calcium binding protein (calbindin-D28k) has been purified from squid brain. Squid brain calbindin was found to have an isoelectric point of 5.0, was heat stable up to 60 degrees C, and showed increased electrophoretic mobility in the presence of chelator. Amino acid analysis revealed a high content of glutamic and aspartic acids and a low level of methionine, histidine, and tyrosine, a finding similar but not identical to the composition of vertebrate calbindin-D28k. The molecular weight of the squid protein, determined by Ferguson plot analysis of data obtained from sodium dodecyl sulfate-gel electrophoresis, was calculated to be 25,700, as compared with 27,800 for rat renal calbindin. Immunocytochemical analysis demonstrated immunoreactive protein in a selected population of neurons and fibers in several areas of the molluscan nervous system. This study represents the first purification from an invertebrate of a calcium binding protein that is biochemically similar to vitamin D-dependent calcium binding protein. These results demonstrate that calbindin, although not identical in vertebrates and cephalopods, may be phylogenetically conserved in structure. The restricted distribution of immunoreactive calbindin in both the cephalopod and mammalian brain suggests that the function of neuronal calbindin may also be conserved in evolution.     

Vitamin D and disease prevention with special reference to cardiovascular disease

Circulating 25-hydroxyvitamin D [25(OH)D] is the hallmark for determining vitamin D status. Serum parathyroid hormone [PTH] increases progressively when 25(OH)D falls below 75 nmol/l. Concentrations of 25(OH)D below 50 nmol/l or even below 25 nmol/l are frequently observed in various population groups throughout the world. This paper highlights the relationship of vitamin D insufficiency with cardiovascular disease and non-insulin dependent diabetes mellitus, two diseases that account for up to 50% of all deaths in western countries. There is evidence from patients with end-stage renal disease that high PTH concentrations are causally related to cardiovascular morbidity and mortality. Activated vitamin D is able to increase survival in this patient group significantly. Moreover, already slightly enhanced PTH concentrations are associated with ventricular hypertrophy and coronary heart disease in the general population. Experimental studies have demonstrated that a lack of vitamin D action leads to hypertension in mice. Some intervention trials have also shown that vitamin D can reduce blood pressure in hypertensive patients. In young and elderly adults, serum 25(OH)D is inversely correlated with blood glucose concentrations and insulin resistance. Sun-deprived lifestyle, resulting in low cutaneous vitamin D synthesis, is the major factor for an insufficient vitamin D status. Unfortunately, vitamin D content of most foods is negligible. Moreover, fortified foods and over-the-counter supplements usually contain inadequate amounts of vitamin D to increase serum 25(OH)D to 75 nmol/l. As a consequence, legislation has to be changed to allow higher amounts of vitamin D in fortified foods and supplements.     

Regulation of Brain and Cerebrospinal Fluid Calcium by Brain Barrier Membranes Following Vitamin D-Related Chronic Hypo- and Hypercalcemia in Rats

Male Fischer-344 rats, 21 days old, were fed diets containing 0 (LOD), 2,200 (CONT), or 440,000 (HID) international units of vitamin D3 per kilogram for 12 weeks. [Ca] was measured in plasma, CSF, brain, and choroid plexus. In addition, 45Ca and 36Cl transfer coefficients (KCa and KCl) for uptake from blood into CSF and brain were determined. Although plasma ionized [Ca]s in LOD and HID rats were 50% and 136%, respectively, of values in CONT animals, CSF and brain [Ca]s ranged from only 85% to 110% of respective CONT values. Choroid plexus [Ca] was increased by 37% after HID diet, but was decreased only 10% after LOD. KCa values at CSF, parietal cortex, and pons-medulla were negatively correlated with plasma ionized [Ca], whereas KCl values at CSF and brain were not different between the diet groups. The findings demonstrate that central nervous system [Ca] is maintained during chronic hypo- or hypercalcemia by saturable transport of Ca at brain barrier membranes. This transport does not seem to involve modulation by 1,25-dihydroxyvitamin D3.     

Estimation of the cutoff value of vitamin D: the Dong-gu study

Background

Vitamin D plays an essential role in bone health and growth, but the optimal serum 25-hydroxyvitamin D (25(OH)D) concentration is not known. This study was performed to investigate the optimal 25(OH)D concentration in regard to parathyroid hormone (PTH) concentration in the Korean general population aged 50 years or older.

Findings

The study population consisted of 8,857 subjects (3,545 men and 5,312 women) who participated in the baseline survey of the Dong-gu study, conducted in Korea between 2007 and 2010. Serum 25(OH)D and PTH concentrations were measured by chemiluminescent microparticle immunoassay. The optimal 25(OH)D concentration was estimated by using nonlinear regression model. Our data show that PTH concentration reached a theoretical plateau at 38.2 pg/ml and corresponding 25(OH)D concentration was 21.1 ng/ml in men and PTH concentration at 42.9 pg/ml and 25(OH)D concentration at 13.8 ng/ml in women.

Conclusions

These results indicate that, for Korean general population aged 50 years or older, the optimal 25(OH)D concentration is 21.1 ng/ml in men and 13.8 ng/ml in women.     

Distribution of Ankyrin Isoforms and Their Proteolysis After Ischemia and Reperfusion in Rat Brain

Abstract: The distribution of brain-type ankyrin (ankyrin_B, 212 kDa) and erythrocyte-type ankyrin (ankyrin_R, 239 kDa) was investigated in the subcellular fractions of rat forebrain (P1, 1,000 g pellet; P2, 15,000 g pellet; P3, 100,000 g pellet; S, 100,000 g supernatant) by immunoblotting using specific antibodies. The P2 fraction contained ∼40% of the 212- and 163-kDa isoforms of ankyrin_B and the 239-kDa isoform of ankyrin_R. Further subfractionation of the P2 by Percoll gradient centrifugation followed by separation of myelin showed association of the three ankyrin isoforms with the synaptosome-rich fraction but not with the myelin-rich fraction. The plasma membrane-rich P3 fraction contained a concentration of ankyrin isoforms similar to that in the P2 fraction. In vitro proteolysis of ankyrin in the P2 fraction with calpain showed that the 212-kDa ankyrin_B was more susceptible to calpain than was ankyrin_R. In the two-vessel occlusion model, ischemia for 30 min generated the 160-kDa fragment of ankyrin_R, and reperfusion for 60 min after 30 min of ischemia remarkably increased the 160-kDa fragment. The reperfusion also significantly decreased the 212-kDa isoform of ankyrin_B. Both ischemia-reperfusion and in vitro proteolysis with calpain generated the 160-kDa fragment of ankyrin_R, suggesting the involvement of calpain.     

Calcium uptake and membrane trafficking in response to PTH or 25(OH)D3 in polarized intestinal epithelial cells

Cell culture techniques providing retention of the polarized enterocyte morphology has allowed, for the first time, comparison of parathyroid hormone (PTH)- and 25-hydroxyvitamin D(3) [25(OH)D(3)]-induced (45)Ca uptake with membrane trafficking events discerned using confocal microscopy. Treatment of cells with 65 pM bPTH(1-34) promoted enhanced (45)Ca uptake between 1 and 10 min after peptide. The protein kinase A (PKA) antagonist, RpcAMP inhibited hormone-mediated uptake. At the microscopic level, cells labeled with the endocytic tracking dye FM1-43 revealed increased punctate staining 50-550s after hormone. Pretreatment of cells with RpcAMP abolished this pattern of staining. The calcium indicator dye fluo-3 AM revealed faint punctate labeling in controls, with increased bands of punctate labeling in the apical region of the cells after peptide hormone, and ultimately the basal region. Parallel studies conducted with the metabolite 25(OH)D(3) resulted in a slower stimulation of (45)Ca uptake 5-10 min after steroid, which was also inhibited by preincubation with RpcAMP. Cells labeled with FM1-43 and then treated with steroid showed no change in distribution of fluorescence during the 10 min incubation period. Confocal microscopy with fluo-3 revealed intense apical fluorescence--that after steroid --streamed to a perinuclear position, and ultimately the basal area. Uniformly diffuse staining, which would indicate cytoplasmic calcium transport, was observed only in controls. Membrane trafficking and compartmentalized calcium appear to be integral to agonist mediated cation transport.     

Increased Concentrations of 3-Hydroxykynurenine in Vitamin B6 Deficient Neonatal Rat Brain

Increased concentrations of the endogenous tryptophan metabolite 3-hydroxykynurenine (3-HK) were measured in the brains of vitamin B6 deficient neonatal rats. Mean concentrations of 3-HK in B6 deficient cerebellum, corpus striatum, frontal cortex, and pons/medulla ranged from 9.7 to 18.6 and 102 to 142 nmol/g of wet tissue at 14 and 18 days of age, respectively. 3-HK was not significantly increased in control neonatal or adult rat brain, vitamin B6 deficient rat brain at 7 days of age, or in brains from adult rats deprived of vitamin B6 for 58 days. The administration of daily intraperitoneal injections of vitamin B6 from the 14th to the 18th day of age decreased the concentration of 3-HK to control levels. 3-HK has been shown by other investigators to produce seizures when injected into the cerebral ventricles of adult rodents. Thus, our studies show the accumulation in brain of a putative endogenous convulsant as the result of a nutritional deficiency.     

Renal parathyroid hormone-dependent adenylate cyclase activity after repletion of vitamin D-deficient rats with vitamin D-2

Rats fed a diet deficient in both vitamin D and Ca²⁺ exhibited a greater depression of the renal parathyroid hormone (PTH)-dependent adenylate cyclase than was observed in rats fed diets deficient in either vitamin D or calcium. Total serum Ca²⁺ was decreased from a control level of 11.2 mg/dl to 8.5 mg/dl in rats fed the diet deficient in calcium alone, and to 5.4 mg/dl in rats fed the diet deficient in vitamin D. Serum calcium was decreased further to 4.3 mg/dl in rats fed the diet deficient in both vitamin D and Ca²⁺. Serum immunoreactive PTH was significantly elevated over control levels when rats were fed the test diets; however, there were no significant differences between the elevated levels in the three experimental groups. Repletion of rats deficient in vitamin D only with a single oral dose of 3200 I.U. vitamin D-2 resulted in restoration of serum calcium to normal levels, a return of serum PTH to the control state, and an associated increase in PTH-dependent adenylate cyclase activity to the control level by 72 h. Repletion of rats deficient in both vitamin D and Ca²⁺ with the same dose of vitamin D-2 raised serum Ca²⁺ to 7.2 mg/dl by 72 h, but did not cause a reduction in circulating PTH, nor did it result in any significant improvement in the responsiveness of the membrane adenylate cyclase to PTH. These results suggest that elevated PTH is a factor in the down regulation of the PTH-dependent adenylate cyclase, but do not rule out a role for calcium as a regulatory factor.     

Homeostasis of Brain and Cerebrospinal Fluid Calcium Concentrations During Chronic Hypo-and Hypercalcemia

Three-week-old rats were made hypocalcemic or hypercalcemic by being fed diets low or high in Ca. Both total and ionized [Ca]s in the plasma decreased about 40% and remained depressed for 4 weeks in rats fed a low-Ca diet. Plasma [Ca]s in rats fed a high-Ca diet increased by 30% and remained elevated for 7 weeks. After 8 weeks on the diets, cerebrospinal fluid (CSF) [Ca] changed by less than 30% whereas brain [Ca] changed by less than 20% of the chronic changes in plasma ionized [Ca]. Assuming a brain extracellular volume of 20% and noting that brain extracellular volume equilibrates freely with CSF, the findings demonstrate only small perturbations in the Ca content of the brain cellular compartment during sustained hypo or hypercalcemia. Partial regulation of CSF and brain extracellular Ca suggests a role for the blood-brain barrier in regulating CNS [Ca] during chronic changes in plasma [Ca].     

Extracellular calcium is a direct effecter of VDR levels in proximal tubule epithelial cells that counter-balances effects of PTH on renal Vitamin D metabolism

In renal proximal tubules, VDR is transiently decreased by parathyroid hormone (PTH) during times of hypocalcemia and returns to normal levels with the rise in serum calcium (Ca). In this study we tested the hypothesis that elevated extracellular Ca induces VDR in a human renal proximal cell line (HK-2G) stably expressing PTH receptor type I. Exposure of HK-2G cells to increasing Ca concentration, up to 3 mM, induced the expression of VDR. The increase in VDR occurred within 1 h and was sustained over 24 h. The increase in VDR was also dose-dependently increased using 20–100 nM gadolinium, suggesting the induction of VDR is regulated via the extracellular Ca sensing receptor (CaSR) with is naturally expressed in HK-2G cells. In conclusion, an extracellular Ca concentration in the physiological range is capable of direct increase of renal proximal VDR expression, and the induction mechanism represents a strategy the body may use to counterbalance effects of PTH on renal Vitamin D metabolism.     

Selective analogs of 1α,25-dihydroxyvitamin D3 for the study of specific functions of Vitamin D

New analogs of 1α,25-dihydroxyvitamin D₃ synthesized in our research group that show selective activity in vivo are presented along with supporting biological results. Compounds that act preferentially on intestine are 2-(3′-propylidene-19-nor-(20S or 20R))-1α,25-dihydroxyvitamin D₃ and 2-methylene-19-21-dinor-1α,25-dihydroxyvitamin D₃. Compounds that act anabolically to induce bone formation are 2-methylene-19-nor-(20S)-1α,25-dihydroxyvitamin D₃ (2MD), the 2α-methyl derivative, the 26,27-dimethyl derivative, and the 26-dimethylene derivative. Compounds that act preferentially on parathyroid glands are 2-methylene-19-nor-1α-hydroxy-homopregnacalciferol, the 20S-bishomo derivative and the 2-methylene-19,26,27-trinor-1α,25-dihydroxyvitamin D₃. These latter compounds do not elevate serum calcium until doses of the order of >300 μg/kg body weight are used, while parathyroid hormone levels are suppressed at much lower doses. Some of these novel analogs may ultimately be useful as new and safer therapeutic agents. Regardless of their clinical utility, they represent valuable research tools that can be used to study the specific functions of the Vitamin D hormone in vivo.     

Regional Localization and Subcellular Compartmentalization of Thyrotropin-Releasing Hormone in Adult Human Brain

In the current study, we sought to define the subcellular compartmentalization of thyrotropin-releasing hormone (TRH) in adult human brain tissues. Upon evaluating tissues (3-24 h post mortem) from 62 humans, ranging in age from 5 to 75 years, we found that TRH was widely distributed throughout the brain. The highest TRH concentration (ng/mg protein) was in the stalk-median eminence region of the hypothalamus (19.3 +/- 3.3, mean +/- SE); the TRH concentration in the hypothalamus, exclusive of the stalk-median eminence, was much lower (1.7 +/- 0.2). Substantial quantities of TRH also were detected in the medulla oblongata (0.26 +/- 0.08), mammillary bodies (0.33 +/- 0.25), and optic chiasm (0.14 +/- 0.07). Lower levels of TRH were found in the amygdala (0.060 +/- 0.015) and the corpus striatum (0.033 +/- 0.010). TRH was near or below the limits of detection in tissues of the cerebral and cerebellar cortices, the olfactory bulbs, the pons, and the hippocampus. When homogenates of medial basal hypothalamic tissue (prepared in 0.32 M sucrose-10 microM CaCl2) were fractionated by means of differential centrifugation, most of the TRH was recovered in subcellular particles which were pelleted at 10,000 X g and which contained the highest amounts of occluded LDH activity. When the nuclei-free supernatant fluid (900 X g S) was fractionated on discontinuous sucrose density gradients or continuous sucrose density gradients, most of the TRH was recovered in subcellular fractions containing synaptosomes. The subcellular distribution of TRH appeared to be stable for up to 24 h post mortem in rat and human brain tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

中国北方地区血清钙、维生素D水平与乳腺癌的相关性研究

目的:探讨中国北方地区血清钙、维生素D水平与乳腺癌及相关临床因素的关系.方法:选取2007年12月至2012年7月哈尔滨医科大学附属肿瘤医院794例女性乳腺癌患者及976例乳腺良性肿瘤患者,并以128例健康妇女为对照,取空腹血清采用原子吸收分光光度法检测三组血清钙含量,采用放免法检测三组中162例血清25(OH)D含量,结合相关临床资料进行分析.结果:乳腺癌组血清钙含量为2.26± 0.12 mmol/L,乳腺良性肿瘤组血清钙含量为2.26±0.09 mmol/L,正常对照组血清钙含量为2.25±0.24 mmol/L,经方差分析,三组总体均数差别无统计学意义(P＞0.05);乳腺癌患者的血清钙水平与年龄、TNM分期、BMI、绝经情况、乳腺癌家族遗传史无关(P＞0.05).乳腺癌组血清25(OH)D含量为41.91±7.55 ng/mL,乳腺良性肿瘤血清25(OH)D含量为54.62±7.48 ng/mL,正常对照组血清25(OH)D含量为56.15±8.87 ng/mL,经方差分析,乳腺癌患者血清25(OH)D含量低于乳腺良性肿瘤组,差别有统计学意义(P＜0.05),乳腺良性肿瘤组与正常对照组差别无统计学意义(P＞0.05);乳腺癌患者的维生素D水平与年龄、TNM分期、BMI、绝经情况有关(P＜0.05),而与乳腺癌家族遗传史无关(P＞0.05).结论:中国北方地区的乳腺癌患者血清钙水平与乳腺良性肿瘤患者无明显差异.乳腺癌患者的维生素D水平低于乳腺良性肿瘤患者,并且与年龄、TNM分期、BMI、绝经情况有关.维生素D水平降低可能与乳腺癌的发生有关,高水平的维生素D可能会降低女性患乳癌的风险.     

Vitamin D and calcium signaling in epidermal stem cells and their regeneration

Epidermal stem cells (SCs) residing in the skin play an essential role for epidermal regeneration during cutaneous wound healing. Upon injury, distinct epidermal SCs residing in the interfollicular epidermis and/or hair follicles are activated to proliferate. Subsequently, SCs and progeny migrate, differentiate and restore the epidermis. We review a role of the vitamin D signaling through its receptor of vitamin D receptor (Vdr) in these processes. Vdr conditional knockout (cKO) mouse skin experiences a delay in wound re-epithelialization under low dietary calcium conditions, stimulating our efforts to examine a cooperative role of Vdr with calcium signaling through the calcium sensing receptor in the epidermis. We review the role of vitamin D and calcium signaling in different processes essential for injury induced epidermal regeneration during cutaneous wound repair. First, we discuss their roles in self-renewal of epidermal SCs through β-catenin signaling. Then, we describe epidermal remodeling, in which SCs and progeny migrate and differentiate to restore the epidermis, events controlled by the E-cadherin mediated adherens junction signaling. Finally, we discuss the potential mechanisms for vitamin D and calcium signaling to regulate injury induced epidermal regeneration mutually and interdependently.     

甲状旁腺高血压因子和甲状旁腺素对细胞钙通道的不同作用

1989年Lewanczuk等[1]报道在自发性高血压大鼠（spontaneouslyhvnertensiverat,SHR）的血浆中发现一种具有独特升压效应的高血压因子．通过激活平滑肌细胞膜钙通道,提高细胞内游离钙（[Ca2 ]）水平起作用．随之证明这种循环血中的高血压因子来源于甲状旁腺,故称“甲状旁腺高血压因子（Parathyroidhypertensivefactor,PHF）”[2]。我们经实验研究证明,当给SD（Sprague-Dawley）大鼠注射经透析的SHR血浆后30min其血压开始升高,45min达高峰,60min恢复到注射前水平．同时经透析的血浆可使SD大鼠尾动脉条的细胞45Ca2 摄取增加,其…     

Effects of Vitamin A and Its Analogs on Nonenzymatic Lipid Peroxidation in Rat Brain Mitochondria

Vitamin A (retinol) and some of its analogs exhibited varying degrees of inhibition on induced iron and ascorbic acid lipid peroxidation of rat brain mitochondria. Malonyldialdehyde production was used as an index of the extent of in vitro lipid peroxidation. The fat-soluble vitamins retinol, retinol acetate, retinoic acid, retinol palmitate, and retinal at concentrations between 0.1 and 10.0 mmol/L inhibited brain lipid peroxidation. Retinol and retinol acetate were the most effective inhibitors. It is concluded from this study that retinol and its analogs can be considered as potential antioxidant factors, more potent than some of the well-known antioxidants such as alpha-tocopherol and butylated hydroxytoluene.     

Insulin-stimulated translocation of glucose transporters in the isolated rat adipose cells: Characterization of subcellular fractions

Insulin stimulates glucose transport in rat adipose cells through the translocation of glucose transporters from an intracellular pool to the plasma membrane. A detailed characterization of the morphology, protein composition and marker enzyme content of subcellular fractions of these cells, prepared by differential ultracentrifugation, and of the distribution of glucose transporters among these fractions is now described. Glucose transporters were measured using specific d-glucose-inhibitable [³H]cytochalasin B binding. In the basal state, roughly 90% of the cells' glucose transporters are associated with a low-density microsomal, Golgi marker enzyme-enriched membrane fraction. However, the distributions of glucose transporters and Golgi marker enzyme activities over all fractions are clearly distinct. Incubation of intact cells with insulin increases the number of glucose transporters in the plasma membrane fraction 4–5-fold and correspondingly decreases the intracellular pool, without influencing any other characteristics of the subcellular fractions examined or the estimated total number of glucose transporters (3.7·10⁶/cell). Insulin does not influence the K_d of the glucose transporters in the plasma membrane fraction for cytochalasin B binding (98 nM), but lowers that in the intracellular pool (from 141 to 93 nM). The calculated turnover numbers of the glucose transporters in the plasma membrane vesicles from basal and insulin-stimulated cells are similar (15·10³ mol of glucose/min per mol of transporters at 37°C), whereas insulin appears to increase the turnover number in the plasma membrane of intact cells roughly 4-fold. These results suggest that (1) the intracellular pool of glucose transporters may comprise a specialized membrane species, (2) intracellular glucose transporters may undergo conformational changes during their cycling to the plasma membrane in response to insulin, and (3) the translocation of glucose transporters may represent only one component in the mechanism through which insulin regulates glucose transport in the intact cell.     

VD缺乏相关疾病及其合理补充的临床意义

众所周知,人体内维生素D水平与少年佝偻病和老年骨质疏松直接相关。最近大量流行病学证据还表明,维生素D(VD)缺乏是多种自身免疫性疾病,癌症,心血管疾病,抑郁症,老年痴呆症,感染性疾病,肌肉骨骼功能下降等的危险因素之一。另外,胰岛素抵抗,高血压和高胆固醇血症也与维生素D缺乏有关。因此,合理补充维生素D可以降低多种疾病风险,并对心血管疾病的风险有益。本文系根据已有的研究结论,阐述维生素D水平与多种临床疾病之间的关联,并对人体血清VD浓度合理监测及合理补充的临床意义做一综述。