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PubMed1995

Calmodulin, calbindin-D28k, calretinin and neurocalcin in rat olfactory bulb during postnatal development.

Odorant stimulation of receptor cells results in a calcium influx that activates the transduction pathway. The olfactory neurons extend axons to the olfactory bulb where they synapse onto mitral cells. Ca(2+)-acceptors also may participate in subsequent processing of olfactory information. The present study describes the distribution of calmodulin, calretinin, calbindin-D28k and neurocalcin during rat main olfactory bulb development. From postnatal day 1 (P1) we observed in the olfactory nerve layer a thin external bundle containing calbindin and calretinin whereas calmodulin was present in a large internal bundle. In tufted cells, neurocalcin immunoreactivity was detected at P10 and increased until P20. In mitral cells calmodulin was intensively immunoreactive at P1 but decreased during development to disappear at adulthood whereas calretinin was weakly labelled at P1 but raised in intensity until P20. In granule cells calbindin-D28k and calretinin were detected from P1. Giant neurons were positive for both calretinin and calbindin-D28k from postnatal day 20.

Aging↗
PubMed1993

Calcium buffering properties of calbindin D28k and parvalbumin in rat sensory neurones.

1. We have examined the ability of the Ca(2+)-binding proteins (CABP) calbindin D28k and paravalbumin to modulate increases in the intracellular free Ca2+ concentration ([Ca2+]i), produced by brief depolarizations, in rat dorsal root ganglion (DRG) neurones. 2. In order to obtain good voltage control, we replated DRG neurones prior to performing these experiments. Immunocytochemical staining of these cells revealed that approximately 10% stained for CABPs. 3. Using fluorescently labelled parvalbumin, we demonstrated that in the whole-cell voltage clamp mode the protein freely entered the cell soma with a mean half-life t0.5 of 6 min 22 s +/- 54 s. 4. Analysis of the effects of calbindin D28k (370 microM) and parvalbumin (1 mM) on Ca2+ currents in the whole-cell voltage clamp mode, revealed that neither protein changed the rate of inactivation of the Ca2+ current or its rate of run-down. 5. Introducing either calbindin D28k (370 microM) or parvalbumin (1 mM) into the cell soma did not significantly alter the basal [Ca2+]i when compared to control cells. 6. Compared to control cells, both CABPs significantly reduced the peak [Ca2+]i obtained for a Ca2+ influx of an equivalent charge density, whereas lysozyme (1 mM), a protein with low affinity for Ca2+, failed to do so. 7. Calbindin D28k caused an 8-fold decrease in the rate of rise in [Ca2+]i and altered the kinetics of decay of [Ca2+]i to a single slow component. Parvalbumin also slowed the rate of rise in [Ca2+]i. Parvalbumin selectively increased a fast component in the decay of the Ca2+ signal. 8. These data demonstrate that both calbindin D28k and paravalbumin effectively buffer Ca2+ in a cellular environment and may therefore regulate Ca(2+)-dependent aspects of neuronal function.

Animals↗
PubMed1993

Effect of 1,25,28-trihydroxyvitamin D2 and 1,24,25-trihydroxyvitamin D3 on intestinal calbindin-D9K mRNA and protein: is there a correlation with intestinal calcium transport?

Although analogs and metabolites of vitamin D have been tested for their calciotropic activity, very little information has been available concerning the effects of these compounds on gene expression. In this study one analog of vitamin D, 1,25,28-trihydroxyvitamin D2 [1,25,28-(OH)3D2], and one metabolite, 1,24,25-trihydroxyvitamin D3 [1,24,25-(OH)3D3], were tested for their effect on intestinal calbindin-D9K mRNA and protein as well as for their effect on intestinal calcium absorption and bone calcium mobilization. These compounds were also evaluated for their ability to compete for rat intestinal 1,25-(OH)2D3 receptor sites and to induce differentiation of human leukemia (HL-60) cells as indicated by reduction of nitro blue tetrazolium. In vivo studies involved intrajugular injection of 12.5 ng 1,25-(OH)2D3 or test compound to vitamin D-deficient rats and sacrifice after 18 h. 1,25,28-Trihydroxyvitamin D2 had no effect on intestinal calcium absorption, bone calcium mobilization, or intestinal calbindin-D9K protein and mRNA. Competitive binding to 1,25-(OH)2D3 receptors was 0.8% of that observed using 1,25-(OH)2D3. However, 20- and 40-fold higher doses of 1,25,28-(OH)3D2 (250 and 500 ng) resulted in significant inductions in calbindin-D9K protein and mRNA (3.5 to 7.4-fold), although doses as high as 800 ng were found to have no effect on intestinal calcium absorption or bone calcium mobilization.(ABSTRACT TRUNCATED AT 250 WORDS)

25-Hydroxyvitamin D 2↗
PubMed1986

Effects of alpha-amanitin and cycloheximide on 1,25-dihydroxyvitamin D3-dependent calbindin-D28K and its mRNA in vitamin D3-replete chick intestine.

We have examined the effects of the transcriptional inhibitor alpha-amanitin and the translational inhibitor cycloheximide on levels of calbindin-D28K (28-kDa calcium binding protein, CaBP) and CaBP-mRNA in the vitamin D-replete chick intestine. Chicks were raised on one of four diets: "normal" (1% calcium, 0.6% phosphorus); high calcium (3.3% calcium, 0.5% phosphorus); low calcium (0.3% calcium, 0.6% phosphorus); or low phosphorus (1% calcium, 0.09% phosphorus). Chicks were then treated either with alpha-amanitin (20 micrograms/chick) or cycloheximide (600 micrograms/chick) 2 h prior to a dose of 6.5 nmol of 1,25-dihydroxyvitamin D3. Duodenal mucosa was collected from 0 to 120 min afterward and assayed for CaBP-mRNA by dot blot hybridization and for CaBP using an enzyme-linked immunosorbent assay. In the absence of inhibitor, CaBP levels were depressed by high calcium and elevated by low calcium or low phosphorus, as expected. These changes occurred, however, without a change in CaBP-mRNA levels. alpha-Amanitin had no effect on CaBP or on CaBP-mRNA levels in chicks raised on any of the diets. Cycloheximide inhibited CaBP levels, and surprisingly also inhibited CaBP-mRNA levels in all four dietary groups. These results indicate that continual protein synthesis is necessary for the expression of CaBP-mRNA, suggesting the existence of a rapidly turned over protein that may be required for stabilization or for processing of the chick intestinal CaBP messenger RNA.

Amanitins↗
PubMed2004

Biological actions and mechanism of action of calbindin in the process of apoptosis.

Although it was originally proposed that the major role of calbindin is to facilitate the vitamin D dependent movement of calcium through the cytosolic compartment of the intestinal or renal cell, we found that calbindin also has a major role in different cell types in protecting against apoptotic cell death. Calbindin, which buffers calcium, can inhibit apoptosis induced by different proapoptotic stimuli. Expression of calbindin-D(28k) in neural cell suppressed the proapoptotic actions of presenilin-1, which is causally linked to familial Alzheimer's disease, by preventing calcium mediated mitochondrial damage and the subsequent release of cytochrome c. Calbindin, by buffering intracellular calcium can also protect HEK 293 kidney cells from parathyroid hormone induced apoptosis that was found to be mediated by a phospholipase C dependent increase in intracellular calcium. In addition, cytokine mediated destruction of pancreatic beta cells can be prevented by calbindin. Induction by cytokines of nitric oxide, peroxynitrite and lipid hydroperoxide production was significantly decreased in calbindin expressing beta cells. Thus, calbindin-D(28k), by inhibiting free radical formation, can protect islet beta cells from autoimmune destruction in type 1 diabetes. Calbindin-D(28k) can also protect against apoptosis in bone cells. Calbindin was found to block apoptosis in osteocytic and osteoblastic cells. Our findings suggest that calbindin is capable of directly inhibiting the activity of caspase-3, a common downstream effector of multiple apoptotic signaling pathways, and that this inhibition results in an inhibition of tumor necrosis factor (TNFalpha) and glucocorticoid induced apoptosis in bone cells. Thus, while part of calbindin's protective effect may result from buffering rises in intracellular calcium, other mechanisms of action, such as inhibition of caspase activity, also play a significant role in the prevention of apoptosis by calbindin-D(28k). These findings have implications for the prevention of degeneration in different cell types and therefore could prove important for the therapeutic intervention of many diseases, including diabetes and osteoporosis.

Apoptosis↗
PubMed1992

Estrogen inhibits calbindin-D28k expression in mouse uterus.

The cellular localization and hormonal controls of calbindin-D9k expression in the rodent reproductive tract have suggested new functions for this protein. The present studies were undertaken to extend the earlier studies of calbindin-D9k to the related protein, calbindin-D28k. Immunohistochemical studies revealed that calbindin-D28k was absent from female rat reproductive tissues, but was abundantly expressed in immature mouse uterus and oviduct. Immunoreactivity was restricted to the endometrial and glandular epithelium of the uterus and the oviductal epithelium. Neither 1,25-dihydroxyvitamin D- nor strontium-containing diets (to blunt 1,25-dihydroxyvitamin D production) affected expression of calbindin-D28k. Uterine, but not oviductal, calbindin-D28k decreased markedly at sexual maturity; this pattern persisted in pregnant mice and was reproduced in immature mice by the administration of estradiol (3 micrograms/day for 3 days). RNA extraction and Northern analyses demonstrated that estrogen markedly decreased calbindin-D28k mRNA abundance in the uterus, but not in the oviduct. These findings suggest that estrogen affects mammalian calbindin-D28k expression and represent a rare example of estrogen-induced down-regulation of gene expression.

Animals↗
PubMed1994

Immunolocalization of calcitriol receptor, 24-hydroxylase cytochrome P-450, and calbindin D28k in human kidney.

The precise localization of the calcitriol (1 alpha,25-dihydroxyvitamin D3) receptor (VDR) and the 25-hydroxyvitamin D3 [25(OH)D3] 24-hydroxylase cytochrome P-450 in the human kidney is unknown. Using newly developed polyclonal antibodies against the human VDR, we demonstrate that the receptor is present in cells of the distal tubule, the collecting duct, the proximal tubule, and in the parietal epithelial cells of the glomerulus. In the distal tubule and collecting duct not all cells contain epitopes for the receptor. The protein is not detected in glomerular capillaries, in the glomerular mesangium, in the interstitium, or in blood vessels. Specific polyclonal antibodies directed against the 25(OH)D3 24-hydroxylase cytochrome P-450 demonstrate epitopes for the cytochrome in cells of the proximal tubule, the distal tubule, glomerular parietal epithelial cells, and mesangial cells. The protein is absent from interstitial cells. Calbindin D28k is present exclusively in principal cells of the distal tubule and collecting duct. In the human kidney, the VDR is present in cells where vitamin D-inducible proteins are found; conversely it is absent from cells where vitamin D-dependent proteins are not present.

Animals↗
PubMed1996

Effect of vitamin D metabolites and analogs on renal and intestinal calbindin-D in the rat.

The effects on renal and intestinal calbindin-D of vitamin D3 metabolites and synthetic 20-epi-vitamin D3 analogs with different calcemic actions were examined in Wistar rats. The compounds were administered intraperitoneally once daily for 5 days. The dosages of the metabolites were 1,25-(OH)2D3 0.01, 0.05, 0.1, and 0.4 microg/kg x d, 24,25-(OH)2D3 0.1, 1 and 10 microg/kg x d, and 25-(OH)D3 10 and 400 microg/kg x d. The dosage of the synthetic analogs were MC903 0. 1, 10, and 100 microg/kg x d, EB1213 0.1 and 10 microg/kg x d, KH1060 0.1 and 0.4 microg/kg x d, and GS1725 0.01 and 0.1 microg/kg x d. Two control groups had either vehicle alone or no treatment. N = 8 in each group. 1,25-(OH)2D3 increased renal and intestinal calbindin-D levels, induced hypercalcemia, and suppressed plasma PTH and magnesium concentrations. 24,25-(OH)2D3 increased intestinal calbindin-D9k and plasma calcium, but had no effect on renal calbindin-D28k, plasma PTH, and magnesium. The dosage of 24, 25-(OH)2D3 that was required to increase plasma calcium was larger than the dosage required to increase intestinal calbindin-D9k. 25-(OH)D3 did not change the calcium metabolic parameters. MC903, a low calcemic analog with a relative high affinity for the vitamin D receptor and a short half-life, increased renal calbindin-D28k without increasing ionized calcium or intestinal calbindin-D9k. EB1213, an analog with a reduced calcemic action and short half-life, increased renal calbindin-D28k and ionized calcium without increasing intestinal calbindin-D9k. The effect of the high calcemic vitamin D analogs KH1060 and GS1725 on calbindin-D was directly related to their calcemic activity. In conclusion, these results demonstrate that 24,25-(OH)2D3 increases intestinal calbindin-D9k, but has no effect on renal calbindin-D28k, that low calcemic analogs may increase renal calbindin-D28k without increasing intestinal calbindin-D9k, and that the effect of high calcemic analogs on calbindin-D is directly related to their calcemic activity.

Administration, Cutaneous↗
PubMed2004

Altered hippocampal expression of calbindin-D-28k and calretinin in GABA(B(1))-deficient mice.

Balb/c GABA(B(1))(-/-) mice develop complex epileptiform activity, including spontaneous and audiogenic generalized seizures, 6-8 weeks after birth. The neuronal systems involved in these epilepsies have not been identified yet. Because the hippocampus is critically involved in epileptiform activity, we now investigated whether this brain region exhibits seizure-related alterations. Using semi-quantitative immunohistochemistry, we studied the temporal and cellular hippocampal expression pattern of two seizure-sensitive calcium-binding proteins, calbindin-D-28k and calretinin, in GABA(B(1))(-/-) mice. One month after birth, before the onset of overt epileptiform activity, wild-type (WT) and GABA(B(1))(-/-) mice exhibit comparable expression profiles for the two calcium-binding proteins. Three months after birth, once the epileptic phenotype is established, we observe clear alterations in the expression of calcium-binding proteins in the dentate gyrus area. GABA(B(1))(-/-) mice exhibit a 50% decline in the staining intensity of calbindin-D-28k expressing neurons and a 70% increase in the number of calretinin-positive neurons when compared to WT littermates. Six months after birth, the down-regulation of calbindin-D-28k protein is even more pronounced, while the calretinin expression in GABA(B(1))(-/-) mice reverts to the pattern seen in WT littermates. Our data demonstrate that the absence of functional GABA(B) receptors causes epileptiform activity through a mechanism that crucially involves dentate gyrus granule cells, and that this pathological activity is accompanied by adaptive changes.

Animals↗
PubMed2001

Aluminum toxicity alters the regulation of calbindin-D28k protein and mRNA expression in chick intestine.

Previous studies have shown that aluminum inhibits vitamin D-dependent calcium absorption. The mechanism involves reduced sensitivity to 1,25-dihydroxycholecalciferol and reduced expression of the calcium transport protein, calbindin-D28k. Reduced expression of calbindin protein may be due to decreased levels of calbindin mRNA. To test this hypothesis, we measured calbindin mRNA levels in chicks fed diets with and without added aluminum. Groups of chicks were fed one of four diets: control, control plus aluminum, low calcium, or low calcium plus aluminum. A fifth group was fed a vitamin D-free diet as a negative control. Calbindin protein was measured by immunoblotting. Serum calcium and inorganic phosphorus were determined. Intestinal mRNA was isolated and assayed by slot-blot hybridization to a fluorescein-conjugated oligonucleotide probe complementary to calbindin-D28k mRNA. Antifluorescein antibodies conjugated to alkaline phosphatase were used to detect hybrids and mRNA levels were quantified by densitometry. Specificity of the probe was verified by Northern analysis. Intestinal calbindin protein was greater in the control plus aluminum group than in controls, but no difference in calbindin mRNA was observed. These changes were associated with small decreases in serum phosphorus and calcium, suggesting a postranscriptional effect of aluminum. Chicks fed the low calcium diet had greater intestinal calbindin protein and mRNA levels relative to the control group in association with a 45% decrease in serum calcium. In contrast, no difference in calbindin protein, and significantly less mRNA were found in the low calcium plus aluminum group compared with controls, despite a decrease in serum calcium similar to that of chicks fed the low calcium diet without aluminum. These results show that in chicks fed a low calcium diet, aluminum intake decreases transcription and/or stability of intestinal calbindin mRNA, and that aluminum may inhibit the expression of vitamin D-dependent genes.

Aluminum↗
PubMed1995

Calbindin-D28k, calretinin, and S-100 immunoreactivities in rat pineal gland during postnatal development.

Profound morphological modifications occur during postnatal development of the rat pineal gland. We have immunohistochemically followed those events from postnatal day 1 to 20 by using three cytoarchitectonic markers (S-100, calbindin-D28k, and calretinin) that belong to the calmodulin/troponin C calcium-binding protein family. In the developing rat pineal, anticalbindin-D28k antibody labels three cell types: immature and mature astrocytes and perivascular type II pinealocytes. During development, calbindin-D28k positive cells migrate from the base of the pineal stalk into the superficial part of the pineal. Calbindin-D28k, usually used as a neuronal marker in the central nervous system, recognizes in rat pineal precursor astrocytes 5 days before S-100 and labels a subpopulation somewhat different from S-100 positive astrocytes. Calretinin immunoreactivity appeared in the postero-superior part of the pineal and was abundant until postnatal day 5, then its density dramatically felt to leave, after postnatal day 20, an occasional population of cells whose morphology is compatible with neuron-like cells.

Animals↗
PubMed1996

Calbindin-D9k and calbindin-D28k expression in rat mineralized tissues in vivo.

Following their terminal differentiation, highly specialized cells, ameloblasts, odontoblasts, and osteoblasts sequentially elaborate mineralized tissues. While the developmental expression pattern of matrix proteins has been studied extensively, less attention has been paid to the molecules involved in calcium handling, such as calcium-binding proteins. This shortcoming, as well as previous conflicting data, led us to conduct studies on calbindin-D9k and calbindin-D28k in rat mandibular bone and incisor based on several methods established on rat ameloblasts in vivo. Radioimmunoassays showed that calbindin-D28k accounts for approximately 0.1% of cytosolic proteins in the ectomesenchymal fraction and 1% in the epithelial fraction of the rat incisor and is 100-fold more concentrated than calbindin-D9k in both tissue types. Western blot analysis confirmed that the anticalbindin-D28k reactive species corresponded to the well characterized renal calbindin-D28k in the ectomesenchyme. In this tissue, calbindin-D28k was ultrastructurally immunolocalized in the odontoblasts. Quantitative immunocytochemistry showed that labeling was distributed throughout their nucleus and cytoplasm. The similar cytoplasmic distribution of both calbindin-D proteins and mRNAs suggests that their expression is regulated at the subcellular level. In particular, immunoreactive calbindin-D28k appeared to be associated with rough endoplasmic reticulum. Calbindin-D9k antisense probe showed negligible labeling in odontoblasts, in parallel with the protein quantities measured (approximately 10 ng/mg of total protein). Finally, in situ hybridization showed transcripts for both calbindins-D in ameloblasts and also in osteoblasts. In summary, the present results support the concept that an elevated expression of these vitamin D-dependent calcium-binding proteins may characterize the phenotype of cells directly involved in the elaboration of mineralized tissues, enamel, dentine, and bone.

Age Factors↗
PubMed1994

Human striatum: chemoarchitecture of the caudate nucleus, putamen and ventral striatum in health and Alzheimer's disease.

The morphology and distribution of perikarya positive for choline acetyltransferase, somatostatin, calcium binding protein (calbindin D28K) and nicotinamide adenine dinucleotide phosphate diaphorase were surveyed in the human striatum. Choline acetyltransferase and somatostatin antibodies labeled separate populations of large striatal interneurons. Somatostatin immunoreactivity and nicotinamide adenine dinucleotide phosphate diaphorase (nitric oxide synthase) activity were completely co-localized. Calbindin antibody identified two distinct groups of striatal neurons: (1) numerous medium-sized, lightly stained neurons, probably analogous to striatopallidal projection neurons in the rat, and (2) much less numerous, large, darkly stained neurons. Half of the latter group, but none of the former, were also nicotinamide adenine dinucleotide phosphate diaphorase-positive. Somatostatin-positive and medium-sized, calbindin-positive neurons were more numerous in the caudate nucleus than in the putamen or ventral striatum. By contrast, large calbindin-immunoreactive neurons were more frequently encountered in the putamen. Choline acetyltransferase-positive neurons were evenly distributed across striatal components. In aged control subjects, the size of large, darkly stained calbindin-positive neurons was reduced relative to young subjects. Aging had no effect on somatostatin-, medium-sized calbindin-, or choline acetyltransferase-positive neurons. However, in histologically confirmed cases of Alzheimer's disease, there was a selective, 75% loss of choline acetyltransferase-immunoreactive perikarya from the ventral striatum, but not from the dorsal striatum, compared to aged controls. Furthermore, the remaining cholinergic neurons in the ventral striatum of Alzheimer's disease cases were significantly smaller than similar neurons in controls. These results indicate that various striatal components which have been shown to differ in their anatomical connectivity and functional specialization, also differ in their neurochemical signatures. The specific and marked loss of choline acetyltransferase-positive neurons from the ventral striatum in Alzheimer's disease is consistent with the characteristic cholinergic and 'limbic' pathology in this disease.

Adult↗
PubMed1996

Prolonged subordination stress increases Calbindin-D28k immunoreactivity in the rat hippocampal CA1 area.

Previously we observed that corticosteroids alter Calbindin-D28k immunoreactivity in the rat hippocampus. In the present study we investigated whether prolonged subordination stress, presumably producing elevated plasma corticosterone levels (1) altered the immunocytochemical distribution of the Ca(2+)-binding proteins Calbindin-D28k (CBir) and Parvalbumin (PVir) in the rat hippocampus, and (2) induced ongoing neurodegenerative changes using a silver impregnation method. Eight days of subordination stress reduced body weight, increased adrenal weight corrected for body weight and reduced thymus weight, indicating its effectiveness to produce a stressful situation. Stress increased CBir selectively in the CA1 pyramidal cell layer whereas PVir was not altered. Silver-impregnation revealed no ongoing neurodegenerative changes in any of the hippocampal subfields.

Animals↗
PubMed1995

Calbindin-D28k: role in determining intrinsically generated firing patterns in rat supraoptic neurones.

1. Physiological activation of rat supraoptic nucleus (SON) neurones leads to phasic firing in vasopressin neurones and fast, continuous firing in oxytocin neurones. Using whole-cell patch clamp methods in brain slices, we investigated the role of endogenous calbindin-D28k (calbindin) in determining these intrinsically generated patterns of firing. 2. Direct introduction of calbindin (0.1-0.2 mM) into twelve of twelve phasically firing neurones suppressed Ca(2+)-dependent depolarizing after-potentials (DAPs) and changed activity from phasic to continuous firing. Bovine calcium binding protein (0.3 mM), an analogue of calbindin, had similar effects on both DAPs and firing patterns in five of five cells tested. 3. Introduction of anti-calbindin antiserum (1:2000-5000) into thirteen of thirteen continuously firing neurones unmasked DAPs and converted continuous into phasic firing. Such effects could not be mimicked either by diffusion of normal rabbit serum or antibodies directed against glial fibrillary acidic protein or against neurophysin. 4. Immunocytochemical staining with antisera directed against calbindin revealed more intense staining in the dorsal, oxytocin-rich and less intense staining in the ventral, vasopressin-rich areas of the SON. 5. Elevated intracellular Ca2+ concentration ([Ca2+]i; 0.1 mM) induced DAPs and phasic firing in all twenty-nine SON cells recorded. During chelation of intracellular Ca2+ with (1.1-11 mM) BAPTA, fifty-eight of fifty-eight neurones recorded displayed regular continuous activity and had no DAPs. 6. These data suggest that firing activities in SON cells are dependent on [Ca2+]i and that calbindin, acting as an endogenous Ca2+ buffer, is involved in regulation of intrinsic firing patterns. It is likely that calcium binding proteins have a similar influence on the firing patterns of many neuronal types throughout the nervous system.

Action Potentials↗
PubMed1995

Regulation by estrogen through the 5'-flanking region of the mouse calbindin-D28k gene.

Mouse calbindin-D28k expression is regulated in vivo by estradiol in ovaries, uterus, and oviduct. To determine whether estrogen can have an effect on the transcription of the calbindin-D28k gene, the human breast cancer cells T47D were transiently transfected with a plasmid containing a 1.1 kilobase (kb) PstI/SacII fragment (-1075/+34) of the mouse calbindin gene ligated to the chloramphenicol acetyltransferase (CAT) gene and cotransfected with human estrogen receptor expression vector. T47D cells, transfected and treated with estradiol (10(-11) - 10(-7) M for 64-65 h), exhibited a dose-dependent increase in CAT activity (up to 6.2-fold). Transfection of MCF-7 breast cancer cells with the chimeric gene construct alone also resulted in an estradiol-dependent induction in CAT activity. Deletion mutant analysis demonstrated that there are two regions of the mouse calbindin-D28k promoter (between -1075/-702 and between -175/-78) that contribute to the induction by estradiol. These fragments, when linked to the thymidine kinase promoter to construct a heterologous promoter chimera, were able to convert the thymidine kinase promoter to estrogen responsiveness. In these regions there are multiple imperfect half-palindromic estrogen-responsive elements. Gel retardation assays demonstrated weak protein-DNA interactions that were competed with cold oligonucleotide containing the vitellogenin estrogen-response element. These findings indicate that the mouse calbindin-D28k promoter is capable of conferring estrogen responsiveness, which may be mediated by several imperfect half-palindromic estrogen-responsive elements, and suggest, in light of previous studies concerning 1,25-dihydroxyvitamin D regulation, multiple steroid regulation of the calbindin-D28k gene.

Animals↗
PubMed1994

Cellular distribution of calbindin D28K mRNAs in the adult mouse brain.

We have determined the cellular distribution of calbindin D28K mRNAs throughout the mouse brain by in situ hybridization. While these studies identified neuronal populations similar to those previously identified in rat brain by immunohistochemistry, some discrepancies exist. These may derive from species differences or from the immunological cross-reactivity of calbindin D28K antiserum with other proteins. We note an intriguing association between the distribution of neurons containing calbindin D28K mRNA and those reported by others to contain the inositol 1,4,5-triphosphate (InsP3) receptor.

Animals↗
PubMed1993

A functional and degenerate pair of EF hands contains the very high affinity calcium-binding site of calbindin-D28K.

Calbindin-D28K, a member of the troponin C superfamily of calcium-binding proteins, had six putative EF hand domains containing one very high affinity and two to three lower affinity calcium-binding sites. The location and binding activity of the calcium-binding sites were examined with a recombinant calbindin-D28K protein. This protein (Calb I-II) only contained EF hand domains 1 and 2 of calbindin-D28K. Binding of calcium and calcium analogs, the lanthanides, by the recombinant protein was determined in fluorescence emission experiments. Calb I-II bound 1 mol of terbium/mol of protein. Terbium was displaced from Calb I-II by other lanthanides and calcium. Fluorescence from terbium was not quenched by holmium. These results and Hill plots of calcium binding activity, determined from intrinsic protein fluorescence measurements, indicated the presence of a single high affinity calcium-binding site on Calb I-II. The properties of the binding site indicated that the very high affinity site of calbindin-D28K was located in EF hand domains 1 and 2 of the protein. In addition, these findings indicated the NH2-terminal pair of EF hands in calbindin-D28K did not depend on interactions with other domains in the protein for high affinity calcium binding activity. The results suggested at least one calcium-binding domain of calbindin-D28K can exist as an independent EF hand pair.

Amino Acid Sequence↗