Apoptosis- and endoplasmic reticulum stress-related genes were regulated by estrogen and progesterone in the uteri of calbindin-D(9k) and -D(28k) knockout mice

Calcium (Ca(2+)) is an important regulator of apoptotic signaling. Calbindin-D(9k) (CaBP-9k) and -D(28k) (CaBP-28k) have a high affinity for Ca(2+) ions. Uterine calbindins appear to be involved in the regulation of myometrial activity by intracellular Ca(2+). In addition, uterine calbindins are expressed in the mouse endometrium and are regulated by steroid hormones during implantation and development. The aim of the present study was to evaluate the regulation of apoptosis in the uteri of CaBP-9k, CaBP-28k, and CaBP-9k/28k knockout (KO) mice. Our findings indicated that Bax protein was enhanced in the uteri of CaBP-28k and CaBP-9k/28k KO mice compared to wild-type (WT) and CaBP-9k KO mice, but no difference was observed in Bcl-2 protein expression. The expressions of caspase 3, 6, and 7 proteins were higher in both CaBP-28k and CaBP-9k/28k KO mice than in WT and CaBP-9k KO mice. These results suggest that the absence of CaBP-28k increases apoptotic signaling. We also investigated the expression of endoplasmic reticulum (ER) stress genes by Western blot analysis in calbindin KO mice. C/EBP homologous protein and immunoglobulin heavy chain-binding protein protein levels were elevated in CaBP-28k KO mice compared to WT mice. When immature mice were treated with 17β-estradiol (E2) or progesterone (P4) for 3 days, we found that the expressions of Bax and caspase 3 protein were increased by E2 treatment in WT and CaBP-9k KO mice, and by P4 treatment in CaBP-28k KO mice. These results indicate that CaBP-28k blocks the up-regulation of apoptosis-related genes and ER stress genes, implying that CaBP-28k may decrease the expression of genes involved in apoptosis and ER stress in murine uterine tissue.     

Estrogen responsiveness of renal calbindin-D28k gene expression in rat kidney

In women, calcium excretion in the urine rises after menopause and falls with estrogen replacement therapy. The amount of calcium lost in the urine following estrogen therapy is less than should occur based on changes in serum calcium and the amount of calcium filtered by the kidney. This suggests there may be a direct effect of estrogen therapy to increase renal calcium reabsorption. Calbindin D_28k is a putative calcium ferry protein located in the distal renal tubules which has been shown to increase transcellular calcium transport. We proposed that estrogen loss after menopause may diminish gene expression of renal calbindin D_28k and subsequently diminish renal calcium reabsorption. We used the ovariectomized rat model of estrogen deficiency to investigate changes at the messenger RNA level of calbindin D_28k in ovariectomized rats (OVX), sham ovariectomized rats (S-OVX), and estrogen treated ovariectomized rats (E-OVX). We have demonstrated that ovariectomy in rats diminishes the gene expression of renal calbindin D_28k. The mRNA levels were approximately three times lower in OVX rats than S-OVX rats. Administration of 17β estradiol to OVX rats produced a significant increase in mRNA level to greater than the S-OVX rats by 4 h. Measurement of serum 1,25 dihydroxyvitamin D₃ showed lower level in OVX rats than S-OVX rats but no significant change in E-OVX animals. In conclusion, our results indicate that estrogen increases renal calbindin D_28k mRNA levels, by a mechanism independent of changes in 1,25 dihydroxyvitamin D₃. This may result in increased expression of calbindin D_28k protein which may have a role in reducing renal calcium excretion. J. Cell. Biochem. 65:340–348. © 1997 Wiley-Liss, Inc.     

Neuronal primary cilia: a review

Primary cilia in neurons have often been regarded as rare, vestigial curiosities. However, neuronal cilia are now gaining recognition as ubiquitous organelles in the mammalian brain, raising speculation about what their functions may be. They might have some features tailored for the nervous system and others that serve needs shared by a spectrum of other cell types. Here we review clues from the literature and present new data supporting several possibilities for the significance of neuronal cilia. Our immunocytochemical results show regional heterogeneity in neuronal cilia. Brain regions nearer to the cerebral ventricles had longer cilia, suggesting that they might sense chemicals such as peptides, originating from cerebrospinal fluid. In mutant Tg737(orpk)mice, most brain regions appeared to be missing cilia. The importance of intraflagellar transport proteins establishes a functional link between neuronal cilia and other primary cilia.     

Antibody Recognition of Calcium-Binding Proteins Depends on Their Calcium-Binding Status

Abstract: Previous studies have revealed changes in immunohistochemical stains for calcium-binding proteins after manipulations that influence intracellular calcium. Cases have been revealed in which these changes in immunoreactivity were not correlated with changes in protein amounts. The present experiments examined whether these effects might be explained by changes in antiserum recognition due to calcium-induced changes in protein conformation. Calretinin, calbindin D28k, and parvalbumin incubated in high calcium were recognized by antisera better than when they were incubated in low calcium. Using a calbindin D28k antibody, it was shown that this effect occurs within physiological calcium concentrations. Formalin fixation of the proteins in the presence of calcium resulted in greater antibody recognition than did fixation of proteins in calcium-free states. The calretinin antiserum appeared to recognize a portion of the molecule previously shown to undergo calcium-dependent conformational changes. A calcium-insensitive antiserum was made to a different fragment of calretinin. These results indicate that some antibodies to calcium-binding proteins preferentially recognize particular calcium-induced protein conformations. Given the potential for wide fluctuations in neuronal calcium, the present results indicate that quantitative estimates of intracellular calcium-binding proteins obtained from immunohistochemical studies of neurons must be interpreted with caution.     

Analysis of the orientation of primary cilia in growth plate cartilage: a mathematical method based on multiphoton microscopical images

The chondrocytic primary cilium has been hypothesized to act as a mechano-sensor, analogously to primary cilium of cells in epithelial tissues. We hypothesize that mechanical inputs during growth, sensed through the primary cilium, result in directed secretion of the extracellular matrix, thereby establishing tissue anisotropy in growth plate cartilage. The cilium, through its orientation in three-dimensional space, is hypothesized to transmit to the chondrocyte the preferential direction for matrix secretion. This paper reports on the application of classical mathematical methods to develop an algorithm that addresses the particular challenges relative to the assessment of the orientation of the primary cilium in growth plate cartilage, based on image analysis of optical sections visualized by multiphoton microscopy. Specimens are prepared by rapid cold precipitation-based fixation to minimize possible artifactual post-mortem alterations of ciliary orientation. The ciliary axoneme is localized by immunocytochemistry with antibody acetylated-alpha-tubulin. The method is applicable to investigation of ciliary orientation in different zones of the growth plate, under either normal or altered biomechanical environments. The methodology is highly flexible and adaptable to other connective tissues where tissue anisotropy and directed secretion of extracellular matrix components are hypothesized to depend on the tissue's biomechanical environment during development and growth.     

Histone variants and melanoma: facts and hypotheses

Melanoma is the most aggressive form of skin cancer with rising incidence and morbidity. Despite advances in treatment, the 10‐yr survival for patients with metastatic disease is less than 10%. During the past few years, ongoing research on different epigenomic aberrations in melanoma has catalyzed better understanding of its pathogenesis and identification of new therapeutics. In our review, we will focus on the role of histone variants, key epigenetic players in melanoma initiation and progression. Specifically, incorporation of histone variants enables additional layers of chromatin structure, and here, we will describe how alterations in this epigenetic behavior impact melanoma.     

Calbindin D-28k Immunoreactivity and Its Protein Level in Hippocampal Subregions During Normal Aging in Gerbils

The hippocampus is associated with learning and memory function and shows neurochemical changes in aging processes. Calbindin D-28k (CB) binds calcium ion with a fast association rate. We examined age-related changes in CB immunoreactivity and its protein level in the gerbil hippocampus during normal aging. In the hippocampal CA1 region (CA1) and CA2, CB immunoreaction was found in some neurons in the stratum pyramidale (SP) at postnatal month 1 (PM 1). CB immunoreactivity in neurons was markedly increased at PM 3. Thereafter, CB immunoreactivity was decreased with time: CB-immunoreactive (⁺) neurons were fewest at PM 24. In the CA3, a few CB⁺ neurons were found only in the SP at PM 1 and in the stratum radiatum at PM 18 and 24. In addition, mossy fibers were stained with CB at PM 1. CB immunoreactivity in mossy fibers was markedly increased at PM 3, thereafter it was decreased with time. In the dentate gyrus, many granule cells (GC) in the granule cell layer were stained with CB at PM 1. CB immunoreactivity in GC was markedly increased at PM 3, thereafter CB immunoreactivity was decreased with time. In Western blot analysis, CB protein level in the gerbil hippocampus was highest at PM 3, thereafter CB protein levels were decreased with time. This result indicates that CB in the gerbil hippocampus is abundant at PM 3 and is decreased with age.     

Coral reef bleaching: facts, hypotheses and implications

Coral reef bleaching, the temporary or permanent loss of photosynthetic microalgae (zooxanthellae) and/or their pigments by a variety of reef taxa, is a stress response usually associated with anthropogenic and natural disturbances. Degrees of bleaching, within and among coral colonies and across reef communities, are highly variable and difficult to quantify, thus complicating comparisons of different bleaching events. Small-scale bleaching events can often be correlated with specific disturbances (e.g. extreme low/high temperatures, low/high solar irradiance, subaerial exposure, sedimentation, freshwater dilution, contaminants, and diseases), whereas large scale (mass) bleaching occurs over 100s to 1000s of km², which is more difficult to explain. Debilitating effects of bleaching include reduced/no skeletal growth and reproductive activity, and a lowered capacity to shed sediments, resist invasion of competing species and diseases. Severe and prolonged bleaching can cause partial to total colony death, resulting in diminished reef growth, the transformation of reef-building communities to alternate, non-reef building community types, bioerosion and ultimately the disappearance of reef structures. Present evidence suggests that the leading factors responsible for large-scale coral reef bleaching are elevated sea temperatures and high solar irradiance (especially ultraviolet wavelengths), which may frequently act jointly.     

High expression of the taurine transporter TauT in primary cilia of NIH3T3 fibroblasts

Taurine, present in high concentrations in various mammalian cells, is essential for regulation of cell volume, cellular oxidative status as well as the cellular Ca2+ homeostasis. Cellular taurine content is a balance between active uptake through the saturable, Na(+)-dependent taurine transporter TauT, and passive release via a volume-sensitive leak pathway. Here we demonstrate that: (i) TauT localizes to the primary cilium of growth-arrested NIH3T3 fibroblasts, (ii) long-term exposure to TNF(alpha) or hypertonic sucrose medium, i.e., growth medium supplemented with 100 mM sucrose, increases ciliary TauT expression and (iii) long-term exposure to hypertonic taurine medium, i.e., growth medium supplemented with 100 mM taurine, reduces ciliary TauT expression. These results point to an important role of taurine in the regulation of physiological processes located to the primary cilium.     

Intracellular regions of potassium channels: Kv2.1 and heag

Intracellular regions of voltage-gated potassium channels often comprise the largest part of the channel protein, and yet the functional role of these regions is not fully understood. For the Kv2.1 channel, although there are differences in activation kinetics between rat and human channels, there are, for instance, no differences in movement of the S4 region between the two channels, and indeed our mutagenesis studies have identified interacting residues in both the N- and C -terminal intracellular regions that are responsible for these functional effects. Furthermore, using FRET with fluorescent-tagged Kv2.1 channels, we have shown movement of the C-termini relative to the N-termini during activation. Such interactions and movements of the intracellular regions of the channel appear to form part of the channel gating machinery. Heag1 and heag2 channels also display differing activation properties, despite their considerable homology. By a chimeric approach, we have shown that these differences in activation kinetics are determined by multiple interacting regions in the N-terminus and membrane-spanning regions. Furthermore, alanine mutations of many residues in the C-terminal cyclic nucleotide binding domain affect activation kinetics. The data again suggest interacting regions between N- and C- termini that participate in the conformational changes during channel activation. Using a mass-spectrometry approach, we have identified α-tubulin and a heat shock protein as binding to the C-terminus of the heag2 channel, and α-tubulin itself has functional effects on channel activation kinetics. Clearly, the intracellular regions of these ion channels (and most likely many other ion channels too) are important regions in determining channel function. EBSA Satellite Meeting: Ion channels, Leeds, July 2007.     

Ca2+ and Reactive Oxygen Species in Staurosporine-Induced Neuronal Apoptosis

Abstract: Staurosporine (0.03–0.5 µ M ) induced a dose-dependent, apoptotic degeneration in cultured rat hippocampal neurons that was sensitive to 24-h pretreatments with the protein synthesis inhibitor cycloheximide (1 µ M ) or the cell cycle inhibitor mimosine (100 µ M ). To investigate the role of Ca²⁺ and reactive oxygen species in staurosporine-induced neuronal apoptosis, we overexpressed calbindin D_28K, a Ca²⁺ binding protein, and Cu/Zn superoxide dismutase, an antioxidative enzyme, in the hippocampal neurons using adenovirus-mediated gene transfer. Infection of the cultures with the recombinant adenoviruses (100 multiplicity of infection) resulted in a stable expression of the respective proteins assessed 48 h later. Overexpression of both calbindin D_28K and Cu/Zn superoxide dismutase significantly reduced staurosporine neurotoxicity compared with control cultures infected with a β-galactosidase overexpressing adenovirus. Staurosporine-induced neuronal apoptosis was also significantly reduced when the culture medium was supplemented with 10 or 30 m M K⁺, suggesting that Ca²⁺ influx via voltage-sensitive Ca²⁺ channels reduces this apoptotic cell death. In contrast, neither the glutamate receptor agonist NMDA (1–10 µ M ) nor the NMDA receptor antagonist dizocilpine (MK-801; 1 µ M ) was able to reduce staurosporine neurotoxicity. Cultures treated with the antioxidants U-74500A (1–10 µ M ) and N -acetylcysteine (100 µ M ) also demonstrated reduced staurosporine neurotoxicity. These results suggest a fundamental role for both Ca²⁺ and reactive oxygen species in staurosporine-induced neuronal apoptosis.     

KcsA closed and open: modelling and simulation studies

Bacterial homologues of mammalian potassium channels provide structures of two states of a gated K channel. Thus, the crystal structure of KcsA represents a closed state whilst that of MthK represents an open state. Using homology modelling and molecular dynamics simulations we have built a model of the transmembrane domain of KcsA in an open state and have compared its conformational stability with that of the same domain of KcsA in a closed state. Approximate Born energy calculations of monovalent cations within the two KcsA channel states suggest that the intracellular hydrophobic gate in the closed state provides a barrier of height ~5 kT to ion permeation, whilst in the open state the barrier is absent. Simulations (10 ns duration) in an octane slab (a simple membrane mimetic) suggest that closed- and open-state models are of comparable conformational stability, both exhibiting conformational drifts of ~3.3 Å C RMSD relative to the respective starting models. Substantial conformational fluctuations are observed in the intracellular gate region during both simulations (closed state and open state). In the simulation of open-state KcsA, rapid (<5 ns) exit of all three K⁺ ions occurs through the intracellular mouth of the channel. Helix kink and swivel motion is observed at the molecular hinge formed by residue G99 of the M2 helix. This motion is more substantial for the open- than for the closed-state model of the channel.     

Gene Transfer of Calbindin D28k cDNA via Herpes Simplex Virus Amplicon Vector Decreases Cytoplasmic Calcium Ion Response and Enhances Neuronal Survival Following Glutamatergic Challenge but Not Following Cyanide

Abstract: Excitatory amino acid overstimulation of neurons can lead to a marked rise in cytoplasmic Ca²⁺ concentration ([Ca²⁺])_i) and be followed by neuron death from hours to days later. If the rise in [Ca²⁺]_i is prevented, either by removing Ca²⁺ from the extracellular environment or by placing Ca²⁺ chelators in the cytosol of the stimulated cells, the neurotoxicity associated with excitotoxins can be ameliorated. We have recently shown that neurons infected with a herpes simplex virus amplicon vector expressing cDNA for calbindin D_28k responded to hypoglycemia with decreased [Ca²⁺]_i and increased survival relative to controls. We now report that vector-infected neurons respond to glutamatergic insults with lower [Ca²⁺]_i than controls and with increased survival. Infected neurons exposed to sodium cyanide did not respond with lower [Ca²⁺]_i than controls, nor did they demonstrate increased survival postinsult. We examine these results in light of our earlier report and in the context of the potential of vectors like this for neuronal gene therapy.     

Ion permeation of pores in model membranes: selectivity,fluctuations and the role of surface charge

Fluctuation of surface charge on pore walls provides a realistic, additional mechanism for generating fluctuation of ionic current and ionic selectivity in narrow pores.     

Calretinin and calbindin D28k have different domain organizations

The domain organization of calretinin (CR) was predicted to involve all six EF-hand motifs (labeled I to VI) condensed into a single domain, as characterized for calbindin D28k (Calb), the closest homolog of calretinin. Unperturbed (1)H,(15)N HSQC NMR spectra of a (15)N-labeled calretinin fragment (CR III-VI, residues 100-271) in the presence of the unlabeled complimentary fragment (CR I-II, residues 1-100) show that these fragments do not interact. Size exclusion chromatography and affinity chromatography data support this conclusion. The HSQC spectrum of (15)N-labeled CR is similar to the overlaid spectra of individual (15)N-labeled CR fragments (CR I-II and CR III-VI), also suggesting that these regions do not interact within intact CR. In contrast to these observations, but in accordance with the Calb studies, we observed interactions between other CR fragments: CR I (1-60) with CR II-VI (61-271), and CR I-III (1-142) with CR IV-VI (145-271). We conclude that CR is formed from at least two independent domains consisting of CR I-II and CR III-VI. The differences in domain organization of Calb and CR may explain the specific target interaction of Calb with caspase-3. Most importantly, the comparison of CR and Calb domain organizations questions the value of homologous modeling of EF-hand proteins, and perhaps of other protein families.     

Developmental and Functional Studies of Parvalbumin and Calbindin D28K in Hypothalamic Neurons Grown in Serum-Free Medium

The Ca2+-binding proteins parvalbumin (Mr = 12K) and calbindin D28K [previously designated vitamin D-dependent Ca2+-binding protein (Mr = 28K)] are neuronal markers, but their functional roles in mammalian brain are unknown. The expression of these two proteins was studied by immunocytochemical methods in serum-free cultures of hypothalamic cells from 16-day-old fetal mice. Parvalbumin is first detected in all immature neurons, but during differentiation, the number of parvalbumin-immunoreactive neurons greatly declines to a level reminiscent of that observed in vivo, where only a subpopulation of neurons stains for parvalbumin. In contrast, calbindin D28K was expressed throughout the period investigated only in a distinct subpopulation of neurons. Depolarization of fully differentiated hypothalamic neurons in culture resulted in a dramatic decrease of parvalbumin immunoreactivity but not of calbindin D28K immunoreactivity. The parvalbumin staining was restored on repolarization. Because the anti-parvalbumin serum seems to recognize only the metal-bound form of parvalbumin, the loss of immunoreactivity may signal a release of Ca2+ from intracellular parvalbumin during depolarization of the cells. We suggest that parvalbumin might be involved in Ca2+-dependent processes associated with neurotransmitter release.