|
| ||
|
|
||
Calcium Binding Protein (calcium + binding_protein)
Selected AbstractsA novel protein kinase from Brassica juncea stimulated by a protozoan calcium binding proteinFEBS JOURNAL, Issue 11 2000Purification, partial characterization A novel protein kinase (BjCCaBPk) from etiolated Brassica juncea seedlings has been purified and partially characterized. The purified enzyme migrated on SDS/PAGE as a single band with an apparent molecular mass of 43 kDa. The optimum pH for the kinase activity was 8.0. It was stimulated more than sixfold by the protozoa Entamoeba histolytica calcium binding protein EhCaBP (10.5 nm) but not by calmodulin (CaM) when used at equimolar concentration. Moreover the kinase also did not bind CaM,Sepharose. There was neither inhibition of the kinase activity in the presence of W-7 (a CaM antagonist), KN-62 (a specific calcium/CaM kinase inhibitor) and anti-CaM Ig, nor any effect on BjCCaBPk activity of staurosporine (a protein kinase C inhibitor). Furthermore a CaM-kinase specific substrate, syntide-2, proved to be a poor substrate for the BjCCaBPk compared with histone III-S. The phosphorylation of histone III-S involved serine residues. Southern and Northern blot analysis showed the presence of EhCaBP homologues in Brassica. The data suggest that BjCCaBPk may be a novel protein kinase with an affinity towards a calcium binding protein like EhCaBP. [source] S100A6 (calcyclin) deficiency induces senescence-like changes in cell cycle, morphology and functional characteristics of mouse NIH 3T3 fibroblastsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2010omnicki, ukasz P. S Abstract S100A6 (calcyclin) is a calcium binding protein with two EF-hand structures expressed mostly in fibroblasts and epithelial cells. We have established a NIH 3T3 fibroblast cell line stably transfected with siRNA against S100A6 to examine the effect of S100A6 deficiency on non-transformed cell physiology. We found that NIH 3T3 fibroblasts with decreased level of S100A6 manifested altered cell morphology and proliferated at a much slower pace than the control cells. Cell cycle analysis showed that a large population of these cells lost the ability to respond to serum and persisted in the G0/G1 phase. Furthermore, fibroblasts with diminished S100A6 level exhibited morphological changes and biochemical features of cellular senescence as revealed by ,-galactosidase and gelatinase assays. Also, S100A6 deficiency induced changes in the actin cytoskeleton and had a profound impact on cell adhesion and migration. Thus, we have shown that the S100A6 protein is involved in multiple aspects of fibroblast physiology and that its presence ensures normal fibroblast proliferation and function. J. Cell. Biochem. 109: 576,584, 2010. © 2009 Wiley-Liss, Inc. [source] Mouse spermatozoa contain a nuclease that is activated by pretreatment with EGTA and subsequent calcium incubationJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2008Segal M. Boaz Abstract We demonstrated that mouse spermatozoa cleave their DNA into ,50 kb loop-sized fragments with topoisomerase IIB when treated with MnCl2 and CaCl2 in a process we term sperm chromatin fragmentation (SCF). SCF can be reversed by EDTA. A nuclease then further degrades the DNA in a process we term sperm DNA degradation (SDD). MnCl2 alone could elicit this activity, but CaCl2 had no effect. Here, we demonstrate the existence of a nuclease in the vas deferens that can be activated by ethylene glycol tetraacetic acid (EGTA) to digest the sperm DNA by SDD. Spermatozoa were extracted with salt and dithiothreitol to remove protamines and then incubated with EGTA. Next, the EGTA was removed and divalent cations were added. We found that Mn2+, Ca2+, or Zn2+ could each activate SDD in spermatozoa but Mg2+ could not. When the reaction was slowed by incubation on ice, EGTA pretreatment followed by incubation in Ca2+ elicited the reversible fragmentation of sperm DNA evident in SCF. When the reactions were then incubated at 37°C they progressed to the more complete degradation of DNA by SDD. EDTA could also be used to activate the nuclease, but required a higher concentration than EGTA. This EGTA-activatable nuclease activity was found in each fraction of the vas deferens plasma: in the spermatozoa, in the surrounding fluid, and in the insoluble components in the fluid. These results suggest that this sperm nuclease is regulated by a mechanism that is sensitive to EGTA, possibly by removing inhibition of a calcium binding protein. J. Cell. Biochem. 103: 1636,1645, 2008. © 2007 Wiley-Liss, Inc. [source] Calgizarrin like gene (Cal) deficient mice undergo normal spermatogenesisMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2003Ashraf U. Mannan Abstract The murine calgizzarin like gene (Cal) encodes for a calcium binding protein, which belongs to the S100 family of EF-hand proteins. It is specifically expressed in Sertoli cells in the testis and its expression is down-regulated by unknown factor(s) from spermatocytes/spermatids. In this paper, we show by transfection of a fusion protein of green fluorescent protein and Cal protein into NIH3T3 cells, that the expression of Cal is restricted only in the cytoplasm of the cell. A differentially regulated cytoplasmic expression of the Cal in Sertoli cells during mouse development suggests that Cal might play an important role during spermatogenesis. In order to elucidate the function of the Cal protein in the spermatogenesis, we disrupted the Cal locus in mouse by homologous recombination. In our knockout mouse, we deleted exon 2 and exon 3 of the Cal gene and replaced them with a neomycin cassette, which resulted in a complete loss of the Cal transcript. Male and female Cal4+/, and Cal4,/, mice from genetic backgrounds C57BL/6J,× 129X1/SvJ hybrid and 129X1/SvJ inbred exhibited normal phenotype and were fertile. An intensive phenotypic analysis showed no gross abnormalities in testis morphology. The lack of the Cal protein also does not affect the parameters of sperm, as they are able to fertilize the oocytes in a competent manner, which is comparable to wild-type sperm. Collectively our results demonstrate that Cal is a nonessential protein and it does not play an important role in mouse spermatogenesis or in process of fertilization. Mol. Reprod. Dev. 66: 431,438, 2003. © 2003 Wiley-Liss, Inc. [source] Differential detection of S100A8 in transitional cell carcinoma of the bladder by pair wise tissue proteomic and immunohistochemical analysisPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 2 2006Jonathan Abstract The search for novel molecular markers of tumor invasion is vital if strategies are to become more effective in the diagnostic and prognostic management of transitional cell carcinoma of the bladder. Up to 50% of tumors detected at stage,1 (pT1) progress to a higher grade even after endoscopic surgical resection, and there are currently no protein markers of this aggressive, invasive phenotype. We have combined SELDI-TOF-MS, ClinProt magnetic bead enrichment, Nano-LC-ESI-ion trap tandem mass spectrometry and immunohistochemical analysis to the study of 12,invasive bladder cancer tissue biopsies paired with normal bladder tissue samples obtained from the same patients for the definition and identification of proteins up-regulated in the tumors. We report the inflammation-associated calcium binding protein,S100A8 (MRP-8, calgranulin,A) to be highly expressed in tumor cells in contrast to normal urothelium in 50% of the samples, as well as two unidentified protein markers at 5.75 and 6.89,kDa that were differentially detected in 9/12 and 10/12,tumor samples, respectively. These new markers, when fully characterized, may contribute to new target proteins for the prediction of aggressive, invasive bladder tumors. [source] Increased glycated calmodulin in the submandibular salivary glands of streptozotocin-induced diabetic ratsCELL BIOCHEMISTRY AND FUNCTION, Issue 4 2009José Nicolau Abstract Non-enzymatic glycosylation, a post translational protein modification may be implicated in the diabetes complications. Calmodulin is an important calcium binding protein that complexed with Ca2+ may be implicated in salivary gland secretory process. Glycated calmodulin has shown to be less effective in binding calcium. The aim of this study was to determine whether the concentration of glycated-calmodulin may be elevated in the submandibular salivary glands of streptozotocin-induced diabetic rats. Diabetes was induced by an intraperitoneal injection of spreptozotocin, and hyperglycemia was confirmed 72,h after injection using a glucosimeter. Thirty days after the induction of diabetes, submandibular salivary glands were used for the analysis of glycated and non-glycated calmodulin, using a glycogel B columns for separation. Glycated and non-glycated calmodulin were assayed by an enzymatic method and by ELISA. The overall concentration of CaM (non-glycated + glycated) in induced diabetic rats was significantly lower than in controls (p,<,0.05). The concentration of non-glycated CaM in controls was significantly higher than in experimental group (p,<,0.05), while the concentration of glycated calmodulin between these groups was statistically similar (p,>,0.05). Copyright © 2009 John Wiley & Sons, Ltd. [source] Backbone-Only Protein Solution Structures with a Combination of Classical and Paramagnetism-Based Constraints: A Method that Can Be Scaled to Large MoleculesCHEMPHYSCHEM, Issue 6 2004Renato Barbieri Dr. Abstract Herein, it is shown that a medium-resolution solution structure of a protein can be obtained with the sole assignment of the protein backbone and backbone-related constraints if a derivative with a firmly bound paramagnetic metal is available. The proof-of-concept is provided on calbindin D9k, a calcium binding protein in which one of the two calcium ions can be selectively substituted by a paramagnetic lanthanide ion. The constraints used are HN (and H,) nuclear Overhauser effects (NOEs), hydrogen bonds, dihedral angle constraints from chemical shifts, and the following paramagnetism-based constraints: 1) pseudocontact shifts, acquired by substituting one (or more) lanthanide(s) in the C-terminal calcium binding site; 2) NHN residual dipolar couplings due to self-orientation induced by the paramagnetic lanthanide(s); 3) cross-correlations between the Curie and internuclear dipole,dipole interactions; and 4) paramagnetism-induced relaxation rate enhancements. An upper distance limit for internuclear distances between any two backbone atoms was also given according to the molecular weight of the protein. For this purpose, the paramagnetism-based constraints were collectively implemented in the program CYANA for solution structure determinations, similarly to what was previously done for the program DYANA. The method is intrinsically suitable for large molecular weight proteins. [source] Gene Expression Profiling in Cluster Headache: A Pilot Microarray StudyHEADACHE, Issue 10 2006Christina Sjöstrand MD Background.,Cluster headache (CH) is a primary neurovascular headache disorder characterized by attacks of excruciating pain accompanied by ipsilateral autonomic symptoms. CH pathophysiology is presumed to involve an activation of hypothalamic and trigeminovascular systems, but inflammation and immunological mechanisms have also been hypothesized to be of importance. Objective.,To identify differentially expressed genes during different clinical phases of CH, assuming that changes of pathophysiological importance would also be seen in peripheral venous blood. Methods.,Blood samples were drawn at 3 consecutive occasions from 3 episodic CH patients: during attacks, between attacks and in remission, and at 1 occasion from 3 matched controls. Global gene expression was analyzed with microarray tehnology using the Affymetrix Human Genome U133 2.0 Plus GeneChip® Set, covering more than 54,000 gene transcripts, corresponding to almost 22,000 genes. Quantitative RT-PCR on S100P gene expression was analyzed in 6 patients and 14 controls. Results.,Overall, quite small differences were seen intraindividually and large differences interindividually. However, pairwise comparisons of signal values showed upregulation of several S100 calcium binding proteins; S100A8 (calgranulin A), S100A12 (calgranulin C), and S100P during active phase of the disease compared to remission. Also, annexin A3 (calcium-binding) and ICAM3 showed upregulation. BIRC1 (neuronal apoptosis inhibitory protein), CREB5, HLA-DQA1, and HLA-DQB1 were upregulated in patients compared to controls. The upregulation of S100P during attack versus remission was confirmed by quantitative RT-PCR analysis. Conclusions.,The S100A8 and S100A12 proteins are considered markers of non-infectious inflammatory disease, while the function of S100P is still largely unknown. Furthermore, upregulation of HLA-DQ genes in CH patients may also indicate an inflammatory response. Upregulation of these pro-inflammatory genes during the active phase of CH has not formerly been reported. Data from this pilot microarray study provide a basis for further studies in CH. [source] The Differential Susceptibility of Specific Neuronal Populations: Insights from Huntington's DiseaseIUBMB LIFE, Issue 6 2003Ian Mitchell Abstract Recent successes in identifying the genes and associated proteins underlying several familial neurodegenerative conditions have not always resulted in accounts as to why the associated patterns of neuronal damage are so specific and limited. Here, with reference to Huntington's disease, we present a general scheme to show how the mutant protein could interact with associated proteins to form an aggregation product. This could lead to neuronal death by direct actions on caspases, or by raising the levels of intracellular calcium ions and reactive oxygen species above a threshold that cannot be resisted by the protection normally conferred by endogenous factors such as calcium binding proteins, free radical scavengers and trophic factors. The local distributions of vulnerability and protective factors could ultimately dictate the pattern of damage induced by the mutant gene. IUBMB Life, 55: 293-298, 2003 [source] Proteins of calcified endoskeleton: II Partial amino acid sequences of endoskeletal proteins and the characterization of proteinaceous organic matrix of spicules from the alcyonarian, Synularia polydactylaPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 4 2005M. Azizur Rahman Abstract Calcified organic substances in the skeleton contain a protein-polysaccharide complex taking a key role in the regulation of bio-calcification. However, information concerning the matrix proteins in alcyonarian and their effect on calcification process is still unknown. For this reason, we have studied the organic matrix of endoskeletal spicules from the alcyonarian coral, Synularia polydactyla, to analyze the proteins with their sequences and investigate the functional properties by a molecular approach. The separated spicules from the colony were identified by scanning electron microscope (SEM). The soluble organic matrix comprised 0.04% of spicule weight. By recording decline of pH in the experimental design, the inhibitory effect of the matrix on CaCO3 precipitation was revealed. Prior to electrophoresis, our analysis of proteins extracted from the soluble organic matrix of the spicules revealed an abundance of proteins in molecular weight. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the preparations showed seven bands of proteins with an apparent molecular mass of 109, 83, 70, 63, 41, 30 and 22 kDa. The proteins were electrophoresed on Tricine-SDS-PAGE after electro-elution treatment, and then transferred to polyvinylidene difluoride (PVDF) membranes and their N -termini were sequenced. Two bands of proteins of about 70 and 63 kDa successfully underwent N -terminal amino acid sequencing. For the detection of calcium binding proteins, a Ca2+ overlay analysis was conducted on the extract by 45Ca autoradiography. The 109 and 63 kDa calcium binding proteins were found to be radioactive. Periodic acid schiff staining indicated that 83 and 63 kDa proteins were glycosylated. An assay for carbonic anhydrase, which is thought to play an important role in the process of calcification revealed low level of the activity. These findings suggest that the endoskeletal spicules of alcyonarian corals have protein-rich organic matrices, which might be related to the calcification process. [source] Light and electron microscopic analysis of KChIP and Kv4 localization in rat cerebellar granule cellsTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 2 2005Brian W. Strassle Abstract Potassium channels are key determinants of neuronal excitability. We recently identified KChIPs as a family of calcium binding proteins that coassociate and colocalize with Kv4 family potassium channels in mammalian brain (An et al. [2000] Nature 403:553). Here, we used light microscopic immunohistochemistry and multilabel immunofluorescence labeling, together with transmission electron microscopic immunohistochemistry, to examine the subcellular distribution of KChIPs and Kv4 channels in adult rat cerebellum. Light microscopic immunohistochemistry was performed on 40-,m free-floating sections using a diaminobenzidine labeling procedure. Multilabel immunofluorescence staining was performed on free-floating sections and on 1-,m ultrathin cryosections. Electron microscopic immunohistochemistry was performed using an immunoperoxidase pre-embedding labeling procedure. By light microscopy, immunoperoxidase labeling showed that Kv4.2, Kv4.3, and KChIPs 1, 3, and 4 (but not KChIP2) were expressed at high levels in cerebellar granule cells (GCs). Kv4.2 and KChIP1 were highly expressed in GCs in rostral cerebellum, whereas Kv4.3 was more highly expressed in GCs in caudal cerebellum. Immunofluorescence labeling revealed that KChIP1 and Kv4.2 are concentrated in somata of cerebellar granule cells and in synaptic glomeruli that surround synaptophysin-positive mossy fiber axon terminals. Electron microscopic analysis revealed that KChIP1 and Kv4.2 immunoreactivity is concentrated along the plasma membrane of cerebellar granule cell somata and dendrites. In synaptic glomeruli, KChIP1 and Kv4.2 immunoreactivity is concentrated along the granule cell dendritic membrane, but is not concentrated at postsynaptic densities. Taken together, these data suggest that A-type potassium channels containing Kv4.2 and KChIP1, and perhaps also KChIP3 and 4, play a critical role in regulating postsynaptic excitability at the cerebellar mossy-fiber/granule cell synapse. J. Comp. Neurol. 484:144,155, 2005. © 2005 Wiley-Liss, Inc. [source] Parvalbumin-, calbindin-, and calretinin-immunoreactive hippocampal interneuron density in autismACTA NEUROLOGICA SCANDINAVICA, Issue 2 2010Y. A. Lawrence Lawrence YA, Kemper TL, Bauman ML, Blatt GJ. Parvalbumin-, calbindin-, and calretinin-immunoreactive hippocampal interneuron density in autism. Acta Neurol Scand: 2010: 121: 99,108. © 2009 The Authors Journal compilation © 2009 Blackwell Munksgaard. Background ,, There has been a long-standing interest in the possible role of the hippocampus in autism and both postmortem brain and neuroimaging studies have documented varying abnormalities in this limbic system structure. Aims ,, This study investigates the density of subsets of hippocampal interneurons, immunostained with the calcium binding proteins, calbindin (CB), calretinin (CR) and parvalbumin (PV) to determine whether specific subpopulations of interneurons are impacted in autism. Materials and methods ,, Unbiased stereological techniques were used to quantify the neuronal density of these immunoreactive subpopulations of gamma-aminobutyric acid-ergic (GABAergic) interneurons analyzed in the CA and subicular fields in postmortem brain material obtained from five autistic and five age-, gender- and postmortem interval-matched control cases. Results ,, Results indicate a selective increase in the density of CB-immunoreactive interneurons in the dentate gyrus, an increase in CR-immunoreactive interneurons in area CA1, and an increase in PV-immunoreactive interneurons in areas CA1 and CA3 in the hippocampus of individuals with autism when compared with controls. Discussion/conclusions ,, Although our sample size is small, these findings suggest that GABAergic interneurons may represent a vulnerable target in the brains of individuals with autism, potentially impacting upon their key role in learning and information processing. These preliminary findings further suggest the need for future more expanded studies in a larger number of postmortem brain samples from cases of autism and controls. [source] | ||||||||||||||||