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PubMed1993

Dietary aluminum chloride reduces the amount of intestinal calbindin D-28K in chicks fed low calcium or low phosphorus diets.

The mechanism of aluminum (Al) toxicity may involve disturbances in calcium (Ca) metabolism. Aluminum compounds have been reported to reduce vitamin D-dependent Ca absorption in chicks, rats and humans. To investigate the mechanism by which Al reduces Ca absorption, we studied the effect of dietary aluminum chloride (AlCl3) on the relative amounts of intestinal calbindin D-28K in chicks fed diets varying in Ca and phosphorus concentration. AlCl3 was added so that Al constituted 0, 0.15 or 0.3 g/100 g of diets that were either adequate, low in Ca, low in P, or contained supplemental P. Diets were fed for 2 wk. Intestinal calbindin D-28K levels were assayed using SDS-PAGE and 45Ca binding to Western blots. Added dietary Al greatly reduced the amount of intestinal calbindin in chicks fed adequate diets, low Ca diets, or low P diets. When diets with supplemental P were fed, little calbindin was evident with or without added Al. Tibia ash, body weight and food intake were also reduced (P < 0.05) by added Al. These results indicate that dietary AlCl3 inhibits vitamin D-dependent Ca absorption by reducing the amount of intestinal calbindin D-28K. Aluminum, therefore, may interfere with the body's ability to regulate intestinal calbindin D-28K levels. This could have implications for other tissues that contain substantial levels of calbindin D-28K.

Aluminum Chloride↗
PubMed2003

Calbindin and calretinin immunoreactivities identify different types of neurons in the adult lamprey spinal cord.

The central pattern generator for locomotion in vertebrates is composed of different spinal neuronal populations that generate locomotor movement. In the lamprey spinal cord, several classes of interneurons have been identified based on morphologic and physiological criteria and integrated in the spinal cord circuits implicated in the generation of locomotion. However, the lack of histochemical markers for most of the interneurons makes it difficult to study whole populations along the spinal cord. We have investigated the immunoreactivity with antibodies raised against calbindin and calretinin. Several types of neurons could be classified: (1). strongly immunoreactive neurons located dorsomedially, (2). moderately immunoreactive neurons located laterally, (3). small weakly immunoreactive neurons, d). ventromedial neurons, (4). liquor contacting cells, and (5). motoneurons. The ventromedial group of calbindin-immunoreactive neurons also is immunoreactive for serotonin and, therefore, represents the ventromedial group of dopamine/serotonin spinal neurons. Some of the lateral calbindin-immunoreactive neurons may be CC-type cells (cells with caudal-crossed axons), because they are retrogradely labeled by tracer injections into the contralateral spinal cord. Other well-characterized cell types, such as sensory dorsal cells, lateral interneurons, descending propriospinal edge cells, and spinobulbar giant interneurons are negative for both calbindin and calretinin. Therefore, calbindin and calretinin are useful markers for the study of cell populations that may be integrated in locomotor circuits.

Animals↗
PubMedDate not supplied

Opposing effects of ethanol and nicotine on hippocampal calbindin-D28k expression.

Long-term ethanol exposure produces multiple neuroadaptations that likely contribute to dysregulation of Ca(2+) balance and neurotoxicity during ethanol withdrawal. Conversely, nicotine exposure may reduce the neurotoxic consequences of Ca(2+) dysregulation, putatively through up-regulation of the Ca(2+)-buffering protein calbindin-D(28k). The current studies were designed to examine the extent to which 10-day ethanol exposure and withdrawal altered calbindin-D(28k) expression in rat hippocampus. Further, in these studies, we examined the ability of nicotine, through action at alpha(7)(*)-bearing nicotinic acetylcholine receptors (nAChRs), to antagonize the effects of ethanol exposure on calbindin-D(28k) expression. Organotypic cultures of rat hippocampus were exposed to ethanol (50-100 mM) for 10 days. Additional cultures were exposed to 500 nM (-)-nicotine with or without the addition of 50 mM ethanol, 100 nM methyllycaconitine (an alpha(7)*-bearing nAChR antagonist), or both. Prolonged exposure to ethanol (>/=50 mM) produced significant reductions of calbindin-D(28k) immunolabeling in all regions of the hippocampal formation, even at nontoxic concentrations of ethanol. Calbindin-D(28k) expression levels returned to near-control levels after 72 h of withdrawal from 10-day ethanol exposure. Extended (-)-nicotine exposure produced significant elevations in calbindin-D(28k) expression levels that were prevented by methyllycaconitine co-exposure. Co-exposure of cultures to (-)-nicotine with ethanol resulted in an attenuation of ethanol-induced reductions in calbindin-D(28k) expression levels. These findings support the suggestion that long-term ethanol exposure reduces the neuronal capacity to buffer accumulated Ca(2+) in a reversible manner, an effect that likely contributes to withdrawal-induced neurotoxicity. Further, long-term exposure to (-)-nicotine enhances calbindin-D(28k) expression in an alpha(7)* nAChR-dependent manner and antagonizes the effects of ethanol on calbindin-D(28k) expression.

Animals↗
PubMed1995

Ageing affects cytosolic Ca2+ binding proteins and synaptic markers in the retina but not in cerebral cortex neurons of the rat.

Two cerebral cortex areas (frontal and occipital) and the retina of rats varying in age from 0.4 to 30 months were investigated for the expression levels and distribution of two cytosolic high affinity Ca2+ binding proteins, calbindin-28 and calretinin, and of two presynaptic protein markers. Of these latter proteins, one is integral (synaptophysin) the other peripheral (synapsin I) to the synaptic vesicle membranes. In the cortex areas, no significant changes of the markers were observed, except for a drop of calretinin from 0.4 to 2 months, probably related to a stage of neuronal development. In the retina, calbindin-28 decreased progressively during ageing (-40% at 30 months) while calretinin remained unchanged. Concomitantly, the two synaptic vesicle proteins dropped, synaptophysin > 50% and synapsin I > 85%. The role of these changes in sustaining the functional alterations previously described in the retina of aged animals remains to be investigated.

Aging↗
PubMed2002

Calbindin-d(28k): a marker of recurrence for medulloblastomas.

BACKGROUND: The expression of the Ca(2+)-binding protein calbindin-D(28k) was analyzed in medulloblastomas in relation to clinical features and other biologic markers related to cell proliferation, differentiation, p53, and cerebellar developmental regulated gene expression. METHODS: Immunohistochemistry was carried out on histologic slides from a first retrospective series of 29 nonmetastatic and 10 metastatic medulloblastoma formalin-fixed, paraffin-embedded tissues, using specific antibodies against calbindin-D(28k), calretinin, alpha-parvalbumin and beta-parvalbumin, and S100 proteins. Informed consent was obtained from the subjects and/or guardians. Other biologic markers for differentiation, cell proliferation, the expression of the p53 tumor suppressor gene protein, and cerebellar developmental regulated genes were similarly investigated. A second series of 16 medulloblastomas from young patients (younger than 15 years) was added in order to validate the results obtained in the first series. RESULTS: Of all the markers investigated, only calbindin-D(28k) was significantly associated with prognosis. Survival and remission (i.e. recurrence free) time analysis performed on all the cases (n = 55) confirmed a high risk of death (P = 0.004) and recurrence (P = 0.003) associated with calbindin-positivity. As calbindin-positivity was predominantly observed in tumors from young patients, the authors confirmed its prognostic value in the subgroup of patients younger than 15 years (n = 37). Cox regression analysis showed a significant and independent prognostic value for calbindin expression and, to a lesser extent, the type of surgery (total or subtotal). Three risk groups were thus identified, distinguishing among the cases characterized by a total resection and calbindin-negativity (good prognosis), by a subtotal resection and calbindin-negativity (intermediary), and by calbindin-positivity (bad prognosis). CONCLUSIONS: The current study suggests that calbindin-positive medulloblastomas represent a subclass of aggressive tumors more frequently seen in younger patients.

Adolescent↗
PubMed2002

The decreases in calcium binding proteins and neurofilament immunoreactivities in the Purkinje cell of the seizure sensitive gerbils.

In previous studies, it has been reported that Purkinje cell degeneration during seizure is evoked by excitotoxicity due to an increase in the intracellular Ca(2+) level, though calbindin D-28k (CB) and parvalbumin (PV), intracellular free calcium buffers, are abundantly colocalized in these cells. In the present study, we investigated the expressions of CB, PV, neurofilament (NF) 68, 150, 200, and polyphosphorylated epitope in NF (RT 97), in the cerebellum of gerbils to identify the mechanism of Purkinje cell damages induced by seizure. In seizure resistant gerbils, nearly all the Purkinje cells showed CB, PA, NF 150, NF 200 and RT 97 immunoreactivity. In SS gerbils, however, a clear decrease in the number of CB(+) and PV(+) Purkinje cells was observed. The NF and RT 97 immunoreactivities, in the Purkinje cells was also lower (except NF 68), but not absent. These results suggest several points. First, the decrease in the concentrations of CB and PV may render the Purkinje cells more susceptible to intermittent Ca(2+) fluctuations and more prone to accumulating intolerable quantities of Ca(2+). Second, during the Ca(2+)-PV interaction PV plays an important role in facilitating donations of Mg(2+), which is a potent enzyme activator in phosphorylation. Thus the decline in PV concentration also implicated the defects of phosphorylation in the NF. Third, increases in both the intracellular Ca(2+) level and dephosphorylation trigger the degradation of the NF, particularly NF 200. Finally, these degradations in the NF induce the functional defects in Purkinje cell, which then cause Purkinje cell degeneration.

Animals↗
PubMed2005

Coordinated control of renal Ca(2+) transport proteins by parathyroid hormone.

BACKGROUND: The kidney is one of the affected organs involved in the clinical symptoms of parathyroid hormone (PTH)-related disorders, like primary hyperparathyroidism and familial hypocalciuric hypercalcemia. The molecular mechanism(s) underlying alterations in renal Ca(2+) handling in these disorders is poorly understood. METHODS: Parathyroidectomized and PTH-supplemented rats and mice infused with the calcimimetic compound NPS R-467 were used to study the in vivo effect of PTH on the expression of renal transcellular Ca(2+) transport proteins, including the epithelial Ca(2+) channel transient receptor potential, vanilloid, member 5 (TRPV5), calbindins, and the Na(+)/Ca(2+)-exchanger (NCX1). In addition, the effect of PTH on transepithelial Ca(2+) transport in rabbit connecting tubule/cortical collecting duct (CNT/CCD) primary cultures was determined. RESULTS: Decreased PTH levels in parathyroidectomized rats or NPS R-467-infused mice, resulted in reduced expression of these proteins, which is consistent with diminished Ca(2+) reabsorption, causing the development of the observed hypocalcemia. PTH supplementation of parathyroidectomized rats restored the expression of the renal Ca(2+) transport machinery and serum Ca(2+) levels, independent of serum 1,25-dihydroxyvitamin D(3) levels and renal vitamin D or Ca(2+)-sensing receptor mRNA abundance. Inhibition of the PTH-stimulated transepithelial Ca(2+) transport by the TRPV5-specific inhibitor ruthenium red reduced the PTH-stimulated expression of calbindin-D(28K) and NCX1 in rabbit CNT/CCD primary cultures. CONCLUSION: PTH stimulates renal Ca(2+) reabsorption through the coordinated expression of renal transcellular Ca(2+) transport proteins. Moreover, the PTH-induced stimulation is enhanced by the magnitude of the Ca(2+) influx through the gatekeeper TRPV5, which in turn facilitates the expression of the downstream Ca(2+) transport proteins. Therefore, the renal transcellular Ca(2+) transport proteins, including TRPV5, could contribute to the pathogenesis of PTH-related disorders.

Aniline Compounds↗
PubMed1994

The role of calcium, pH, and cell proliferation in the programmed (apoptotic) death of androgen-independent prostatic cancer cells induced by thapsigargin.

Calcium (Ca2+) accumulates within the endoplasmic reticulum of cells through function of the sarcoplasmic reticulum and endoplasmic reticulum Ca(2+)-dependent ATPase family of intracellular Ca(2+)-pumping ATPases. The resulting pools have important signaling functions. Thapsigargin (TG) is a sesquiterpene gamma-lactone which selectively inhibits the sarcoplasmic reticulum and endoplasmic reticulum Ca(2+)-dependent ATPase pumps with a 50% inhibitory concentration of approximately 30 nM. Treatment of androgen-independent prostate cancer cells of both rat and human origin with TG inhibits their endoplasmic reticulum Ca(2+)-dependent ATPase activity, resulting in a 3-4-fold elevation in the level of intracellular free Ca2+ (Cai) within minutes of exposure. Due to a secondary influx of extracellular Ca2+, this increase in Cai is sustained, resulting in morphological (cell rounding) and biochemical changes within 6-12 h (enhanced calmodulin, glucose regulated protein, and tissue transglutaminase expression, and decreased expression of the G1 cyclins). Within 24 h of exposure, androgen-independent prostatic cancer cells stop progression through the cell cycle, arrest out of cycle in G0, and irreversibly lose their ability to proliferate with a median effective concentration value of 31 nM TG. During the next 24-48 h, the genomic DNA of the G0-arrested cells undergoes double-strand fragmentation. This is followed by the loss of plasma membrane integrity and fragmentation of the cell into apoptotic bodies. During this process, there is no acidification in the intracellular pH. Using cells transfected with the avian M(r) 28,000 calbindin D Ca(2+)-buffering protein, it was demonstrated that the programmed death initiated by TG is critically dependent upon an adequate (i.e., 3-4-fold) sustained (> 1 h) elevation in Cai and not depletion of the endoplasmic reticulum pools of Ca2+. These results demonstrate that TG induces programmed cell death in androgen-independent prostatic cancer cells in a dose-dependent manner and that this death does not require proliferation or intracellular acidification but is critically dependent upon an adequate, sustained (i.e., > 1 h) elevation in Cai.

Androgens↗
PubMed1999

Sequence analysis of an 800-kb genomic DNA region on chromosome 8q21 that contains the Nijmegen breakage syndrome gene, NBS1.

An 800-kb region on chromosome 8q21, which complements the phenotype of cells from Nijmegen breakage syndrome patients, is a candidate for the locus of the underlying gene, termed NBS1. The sequence of this 800-kb region of DNA indicated that the size of this segment is 755,832 bp with an additional 36-kb gap. From this region, we identified four genes including NBS1, a gene coding for a 27-kDa vitamin D-dependent calcium-binding protein (27-kDa calbindin), the mitochondrial 2,4-dienoyl-CoA reductase gene, and a novel gene, C8orf1/hT41. All four genes were aligned in a 250-kb centromeric portion of the region, and no gene was found in the remaining telomeric portion containing 500 kb. The genomic organization of the C8orf1/hT41 and NBS1 genes has been analyzed using the computer programs GRAIL 2 and GENSCAN. They predicted and successfully found more than 93% of the exons, even a small 54-bp exon, indicating that one or more exons in any gene can be identified by these programs. GENSCAN was more efficient at locating the four genes than GRAIL 2 and identified 15 of the 16 exons of the NBS1 gene. This 800-kb region contained repetitive sequences, including 179 copies of the Alu sequence (1 copy/4.2 kb), 123 copies of the L1 sequence (1 copy/6.1 kb), 107 copies of the LTR sequence (1 copy/7.1 kb), and 63 copies of the MER sequence (1 copy/12 kb). There was a slight but not significant difference in the repetitive content of the gene-rich region and the remaining noncoding region. Our results indicate that computer-assisted methods are useful and powerful for identifying exons of both known and novel genes.

Abnormalities, Multiple↗
PubMed2002

A supramicromolar elevation of intracellular free calcium ([Ca(2+)](i)) is consistently required to induce the execution phase of apoptosis.

Many agents, such as the endoplasmic reticulum Ca(2+) ATPase inhibitor, thapsigargin, or the ionophore, ionomycin, induce apoptosis by transiently elevating [Ca(2+)](i). The role of [Ca(2+)](i) in apoptosis induced by agents that do not immediately increase [Ca(2+)](i), such as 5-FdUr, TGF beta-1, doxorubicin, or radiation, is far more controversial. In the present paper, [Ca(2+)](i) was measured continuously for 120 h. in prostate and bladder cancer cell lines exposed to these four agents: 5-FdUR, TGF beta-1, doxorubicin, or radiation. Each of them consistently induced a delayed [Ca(2+)](i) rise associated with the morphological changes that characterize the execution phase of apoptosis (i.e. rounding, blebbing). This [Ca(2+)](i) rise occurred in two consecutive steps (< or = 10 microM and >10 microM) and resulted from a Ca(2+) influx from the extracellular medium. This delayed supramicromolar [Ca(2+)](i) rise was also observed previously in breast, prostate and bladder cancer cell lines exposed to thapsigargin. This influx regulated transcriptional reprogramming of Gadd153 and is required to activate cytochrome c release, caspase-3 activation, loss of clonal survival and DNA fragmentation. When cells were maintained in low extracellular Ca(2+) media, these phenomena were temporarily delayed but occurred on return to normal Ca(2+) medium. Similarly, apoptosis could be delayed by overexpressing the Ca(2+)-binding proteins, Calbindin-D(28K) and parvalbumin. As this delayed >or = 10 microM [Ca(2+)](i) elevation was observed in a number of cell lines exposed to a variety of different agents, we conclude that such elevation constitutes a key and general event of apoptosis in these malignant cells.

Apoptosis↗
PubMed1995

Calbindin-D28K and -D9K and 1,25(OH)2 vitamin D3 receptor immunolocalization and mineralization induction in long-term primary cultures of rat epiphyseal chondrocytes.

Rat epiphyseal plat chondrocytes were grown on glass slides, as nonadhering monolayer cultures for up to 6 weeks. Chondrocyte growth, differentiation and maturation, matrix formation and mineralization, and the temporospatial distribution of the vitamin D-dependent calcium-binding proteins, calbindin-D9K and -D28K, and the 1,25(OH)2D3 receptor (VDR), were all monitored. Chondrocytes became confluent in 2.5 weeks, differentiated to acquire a chondrocyte (polygonal) morphology, produced extracellular matrix, and finally formed a true monolayer mineralizing cartilaginous tissue, with all the stages of chondrocyte development within a single culture. beta-Glycerophosphate promoted initial matrix mineralization in 4 weeks and accelerated cell differentiation. High nominal calcium and ascorbic acid were needed for abundant matrix formation. VDR occurred at all differentiation stages, in the nuclei and nucleoli and in the cytoplasm. Calbindin-D28K and -D9K were not coexpressed. Calbindin-D28K was found in prechondroblasts, chondroblasts, and in newly differentiated chondrocytes. It was cytoplasmic in prechondroblasts and subsequently also in the perinuclear region and in nuclei, suggesting migration to the nuclear chromatin. Calbindin-D28K was nuclear only in newly differentiated chondrocytes in vitro and was not found in mature chondrocytes. In contrast, calbindin-D9K was present in the cytoplasm of mature and hypertrophic chondrocytes only. It was first in the cell body and eventually migrated within and to the far end of long cell processes with a decreasing cytoplasmic concentration showed by decreased immunostaining intensity, and ultimately hypertrophy of chondrocytes in culture. These in vitro patterns of calbindins-D and VDR accurately reflect their in vivo distributions. The genomic action of vitamin D, in vitro, resulted in the synthesis of nuclear VDR and calbindins-D.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

Immunohistochemistry of human cutaneous Meissner and pacinian corpuscles.

This paper reviews the immunohistochemical characteristics of two kinds of human cutaneous sensory nerve formations (SNFs), the Meissner and Pacinian corpuscles. In both kinds of SNF the central axon might be easily identifiable because it displays immunoreactivity (IR) for the neuroendocrine markers neuron-specific enolase and protein gene product 9.5, as well as for neuron-specific intermediate filament proteins, i.e., neurofilaments. Other intermediate filament proteins such as vimentin are localized in the lamellar cells of Meissner corpuscles, and in the inner core, outer core and capsule of Pacinian corpuscles. However, they lack cytokeratins or glial fibrillary acidic protein IR. On the other hand, and in agreement with ultrastructural data, IR for basement membrane constituents laminin and type IV collagen is found underlying all SNF constituents, with the exception of the axon. One of the mechanisms involved in the maintenance of intracellular calcium ions (Ca2+) homeostasis is the calcium binding proteins. Ca2+ play a key role in the mechanoelectric transduction and have been localized in SNFs. In this way IR for the Ca(2+)-binding proteins calbindin D28K, parvalbumin and calretinin, is present and colocalized in both Meissner and Pacinian corpuscles; furthermore, S-100 protein is exclusively localized in the lamellar cells and the inner core. On the other hand, the skin is a main source of neurotrophins for a subset of neural crest sensory neurons, some of which end forming SNF. These factors are conveyed via retrograde axonal transport from the skin to the cell body of the responsive neurons. Interestingly, Meissner and Pacinian corpuscles also display IR for the pan-neurotrophin low-affinity receptor (p75), and for the trkA receptor protein, a basic constituent of the high-affinity receptor for some neurotrophins. Moreover, they express IR for the epidermal growth factor receptor. Finally, other antigens not proper to the cells forming human cutaneous SNF, such as the epithelial membrane antigen and the leucocytary antigen-7, have also been detected.

Antigens, Differentiation↗
PubMed1991

Co-cultures of inferior olive and cerebellum: electrophysiological evidence for multiple innervation of Purkinje cells by olivary axons.

Slices of inferior olive (IO) and cerebellum were co-cultured for several weeks by means of the roller tube technique. Recordings were carried out intracellularly from Purkinje cells (PCs) which were identified morphologically by intracellular injection of the fluorescent dye Lucifer yellow, or by immunohistochemical stainings with antibodies raised against the 28 kD Ca(2+)-binding protein calbindin. Following stimulation of olivary tissue, an all-or-none full complex spike response was recorded in some PCs consisting of a fast rising spike followed by a depolarizing potential. In other PCs, graded stimulation of the olivary explant induced synaptic potentials which were characterized by step-wise variation in their amplitude and resembled the ones occurring spontaneously. In contrast, only smoothly graded synaptic potentials were observed in cerebellar mono-cultures. These results indicate that some of the PCs in olivo-cerebellar co-cultures are innervated by several olivary neurons.

Action Potentials↗
PubMed2005

Cell type analysis of functional fetal dopamine cell suspension transplants in the striatum and substantia nigra of patients with Parkinson's disease.

We report the first post-mortem analysis of two patients with Parkinson's disease who received fetal midbrain transplants as a cell suspension in the striatum, and in one case also in the substantia nigra. These patients had a favourable clinical evolution and positive 18F-fluorodopa PET scans and did not develop motor complications. The surviving transplanted dopamine neurons were positively identified with phenotypic markers of normal control human substantia nigra (n = 3), such as tyrosine hydroxylase, G-protein-coupled inward rectifying current potassium channel type 2 (Girk2) and calbindin. The grafts restored the cell type that provides specific dopaminergic innervation to the most affected striatal regions in the parkinsonian brain. Such transplants were able to densely reinnervate the host putamen with new dopamine fibres. The patients received only 6 months of standard immune suppression, yet by post-mortem analysis 3-4 years after surgery the transplants appeared only mildly immunogenic to the host brain, by analysis of microglial CD45 and CD68 markers. This study demonstrates that, using these methods, dopamine neuronal replacement cell therapy can be beneficial for patients with advanced disease, and that changing technical approaches could have a favourable impact on efficacy and adverse events following neural transplantation.

Aged↗
PubMed2005

Quantitative neuropathologic correlates of changes in ratio of N-acetylaspartate to creatine in macaque brain.

PURPOSE: To elucidate the neuropathologic basis of transient changes in the ratio of N-acetylaspartate (NAA) to creatine (Cr) in the primate brain by using a simian immunodeficiency virus (SIV)-infected macaque model of the neurologic manifestation of acquired immune deficiency syndrome. MATERIALS AND METHODS: This study was approved by the Massachusetts General Hospital Subcommittee on Research and Animal Care and the Institutional Animal Care and Use Committee of Harvard University. Rhesus macaques infected with SIV were evaluated during the 1st month of infection. A total of 11 animals were studied, including four control animals, three animals sacrificed 12 days after infection, three animals sacrificed 14 days after infection, and one animal sacrificed 28 days after infection. All animals underwent in vivo proton ((1)H) magnetic resonance (MR) spectroscopy, and postmortem frontal lobe tissue was investigated by using high-spectral-resolution (1)H MR spectroscopy of brain extracts. In addition, quantitative neuropathologic analyses were performed. Stereologic analysis was performed to determine neuronal counts, and immunohistochemical analysis was performed to analyze three neuronal markers: synaptophysin, microtubule-associated protein 2 (MAP2), and calbindin. Analysis of variance (ANOVA) was used to determine substantial changes in neuropathologic and MR spectroscopic markers. Spearman rank correlations were calculated between plasma viral load and neuropathologic and spectroscopic markers. RESULTS: During acute infection with SIV, the macaque brain exhibited significant changes in NAA/Cr (P < .02, ANOVA) and synaptophysin (P < .013, ANOVA). There was no significant change in the concentration of Cr. No significant changes were found in neuronal counts or other immunohistochemical neuronal markers. With the Spearman rank test, a significant direct correlation was detected between synaptophysin and ex vivo NAA/Cr (r(s) = 0.72, P < .013). No correlation between NAA/Cr and neuronal counts, calbindin, or MAP2 was found. CONCLUSION: NAA/Cr is a sensitive marker of neuronal injury, not necessarily neuronal loss, and best correlates with synaptophysin, a marker of synaptodendritic dysfunction.

Animals↗
PubMed1991

The properties of intracellular calcium stores in cultured rat cerebellar neurons.

Cerebellar Purkinje neurons contain a remarkable array of cellular components potentially concerned with regulation of the free cytoplasmic Ca2+ concentration, [Ca2+]i. These include high concentrations of Ca(2+)-binding proteins, inositol 1,4,5-triphosphate receptors (IP3R), and ryanodine receptors (RyR). The latter two molecules are thought to be associated with intracellular Ca2+ stores. We have examined the properties of such stores in cultured rat cerebellar neurons taken from 16 d rat embryos. In this system, about half of the neurons could be identified as Purkinje-like cells, as indicated by staining for the Ca(2+)-binding protein calbindin D-28k, as well as for IP3R and RyR. In double immunofluorescent staining, the IP3R and RyR immunoreactivity primarily colocalized with the staining for calbindin. The cells responded to glutamate, kainate, and quisqualate with large increases in the somatic [Ca2+]i but failed to respond directly to NMDA (10-50 microM). Furthermore, the neurons expressed active membrane conductances, repetitive action potential firing, and spontaneous firing patterns similar to those reported for cerebellar Purkinje neurons in vivo. Action potential firing produced changes in somatic [Ca2+]i that were quite small or absent in most cells. However, blocking spike repolarization with tetraethylammonium (5 mM) produced substantial transient elevations in somatic [Ca2+]i, suggesting the expression of some Ca2+ channels in the somatic membrane. Caffeine (10 mM) released Ca2+ from intracellular stores in about one-half of the cultured neurons. This effect could be repeated if the stores were first reloaded by a depolarization-induced elevation in [Ca2+]i. The effects of caffeine were reduced by prolonged application of ryanodine (10 microM). We were also able to demonstrate that the caffeine-sensitive Ca2+ stores could regulate electrophysiological events in some cells, altering patterns of spontaneous activity. Furthermore, in the presence of caffeine, [Ca2+]i signals induced by an evoked spike train were larger and accompanied by long-lasting after hyperpolarizations. We conclude that in addition to providing a releasable pool of Ca2+, the caffeine-sensitive stores also influence cellular events by their contribution to Ca2+ buffering.

Animals↗
PubMed1995

Expression of human copper/zinc-superoxide dismutase inhibits the death of rat sympathetic neurons caused by withdrawal of nerve growth factor.

Rat superior cervical ganglion neurons require the presence of nerve growth factor (NGF) to develop and survive in culture. If NGF is removed from the culture medium, then the neurons die of programmed cell death. We investigated the potential role of Ca2+ and reactive oxygen species in this process. We found that overexpression of human wild-type copper/zinc-superoxide dismutase in cultured superior cervical ganglion neurons, using an adenovirus-based vector, substantially protected the cells from the effects of NGF withdrawal, although overexpression of the Ca(2+)-binding protein calbindin D28k or the enzyme beta-galactosidase did not. We also observed that treatment of the cells with the cytokine transforming growth factor-beta 1, which has been shown to protect neurons against oxidative injury, delayed cell death produced by NGF withdrawal. These data suggest a role for reactive oxygen species in triggering programmed cell death of rat sympathetic neurons upon growth factor withdrawal.

Animals↗
PubMed1996

Mechanisms of intestinal calcium absorption and availability of dietary calcium in pigs.

Calcium absorption from the intestine may be active via the transcellular route or passive via the paracellular route, but the active component is necessary for the maintenance of calcium homeostasis. The active transport depends on vitamin D and is more restricted to the proximal part of the small intestine. Calcitriol, the active metabolite of vitamin D, induces the synthesis of calbindin, a Ca(2+)-binding protein which seems to be involved in transcellular Ca(2+)-transport, increases the number of Ca(2+)-transport components in the apical membrane, but is without direct influence on Ca(2+)-pumps of the basolateral membrane. A phytate-phosphorus containing diet not only reduces the availability of phosphorus but also of calcium. However, with sufficient supply of vitamin D, this may be compensated by the hormonal regulation of calcium homeostasis. Addition of lactose to the diet increases the apparent digestibility of calcium in the postileal segment of the gastrointestinal tract by an so far unknown mechanism.

Animals↗