Molecular Cloning of Two Additional Members of the Neural Visinin-Like Ca2+-Binding Protein Gene Family

Abstract: We have isolated a rat cDNA clone encoding a neural visinin-like Ca²⁺-binding protein (NVP), which we designate NVP-1. To identify additional molecular forms of NVP, a rat brain cDNA library was screened for their presence using an NVP-1 cDNA probe under low-stringency hybridization conditions. Two types of cDNA clones encoding structurally related proteins, designated NVP-2 and NVP-3, have been isolated. The deduced amino acid sequences of NVP-2 and NVP-3 are 89.0% and 68.6% identical to that of NVP-1, respectively, and contain consensus sequences for EF-hand Ca²⁺-binding sites. Northern blot analysis shows that NVP-1, NVP-2, and NVP-3 mRNAs are most highly expressed in brain and are differentially expressed in various regions of rat brain. These results suggest that NVP-2 and NVP-3 are additional members of the NVP gene family.     

Possible mechanism for the influence of weak magnetic fields on biological systems

A physical mechanism is suggested for a resonant interaction of weak magnetic fields with biological systems. An ion inside a Ca(2+)-binding protein is approximated by a charged oscillator. A shift in the probability of ion transition between different vibrational energy levels occurs when a combination of static and alternating magnetic fields is applied. This in turn affects the interaction of the ion with the surrounding ligands. The effect reaches its maximum when the frequency of the alternating field is equal to the cyclotron frequency of this ion or to some of its harmonics or sub-harmonics. A resonant response of the biosystem to the magnetic field results. The proposed theory permits a quantitative explanation for the main characteristics of experimentally observed effects.     

Criticism of Lednev's mechanism for the influence of weak magnetic fields on biological systems.

V. V. Lednev has proposed a mechanism that he suggests would allow very weak magnetic fields, at the cyclotron resonance frequency for Ca2+ ions in the earth's field, to induce biological effects. I show that for four independent reasons no such mechanism can operate.     

New perspectives on S100 proteins: a multi-functional Ca 2+ -, Zn 2+ - and Cu 2+ -binding protein family

S100 proteins (16 members) show a very divergent pattern of cell- and tissue-specific expression, of subcel-lular localizations and relocations, of post-translational modifications, and of affinities for Ca 2+ , Zn 2+ , and Cu 2+ , consistent with their pleiotropic intra- and extracellular functions. Up to 40 target proteins are reported to interact with S100 proteins and for S100A1 alone 15 target proteins are presently known. Therefore it is not surprising that many functional roles have been proposed and that several human disorders such as cancer, neurodegenerative diseases, cardiomyopathies, inflammations, diabetes, and allergies are associated with an altered expression of S100 proteins. It is not unlikely that their biological activity in some cases is regulated by Zn 2+ and Cu 2+ , rather than by Ca 2+ Despite the numerous putative functions of S100 proteins, their three-dimensional structures of, e.g., S100B, S100A6, and S100A7 are surprisingly similar. They contain a compact dimerization domain whose conformation is rather insensitive to Ca 2+ binding and two lateral a-helices III and III, which project outward of each subunit when Ca 2+ is bound. Target docking depends on the two hydrophobic patches in front of the paired EF-hand generated by the binding of Ca 2+. The selec-tivity in target binding is assured by the central linker between the two EF-hands and the C-terminal tail. It appears that the S100-binding domain in some target proteins contains a basic amphiphilic a-helix and that the mode of interaction and activation bears structural similarity to that of calmodulin.© Kluwer Academic Publishers     

Structures of EF-hand Ca 2+-binding proteins: Diversity in the organization, packing and response to Ca 2+ Binding

The growing database of three-dimensional structures of EF-hand calcium-binding proteins is revealing a previously unrecognized variability in the coformations and organizations of EF-hand binding motifs. The structures of twelve different EF-hand proteins for which coordinates are publicly available are discussed and related to their respective biological and biophysical properties. The classical picture of calcium sensors and calcium signal modulators is presented, along with variants on the basic theme and new structural paradigms.© Kluwer Academic Publishers     

Biochemical Characterization of Annexins I and II Isolated from Pig Nervous Tissue

Five proteins having molecular masses of 90, 67, 37, 36, and 32 kDa (p90, p67, p37, p36, and p32, respectively) were identified in the particulate fractions of pig brain cortex and pig spinal cord prepared in the presence of 0.2 mM Ca2+ and further purified using a protocol previously described for the purification of calpactins. Proteins p90, p37, and p36 are related to annexins I and II. Annexin II, represented by p90, is found as an heterotetramer, composed of two heavy chains of 36 kDa and two light chains of 11 kDa, and as a monomer of 36 kDa. Protein p37, which differs immunologically from p36, is a monomer and could be related to annexin I. All three proteins are Ca(2+)-dependent phospholipid- and F-actin-binding proteins; they are phosphorylated on a serine and on a tyrosine residue by protein kinases associated with synaptic plasma membranes. Purified p36 monomer and p36 heterotetramer proteins bind to actin at millimolar Ca2+ concentrations. The stoichiometry of p36 binding to F-actin at saturation is 1:2, corresponding to one tetramer or monomer of calpactin for two actin monomers (KD, 3 x 10(-6) M). Synaptic plasma membranes supplemented with the monomeric or tetrameric forms of p36 phosphorylate the proteins on a serine residue. The monomer is phosphorylated on a serine residue by a Ca(2+)-independent protein kinase, whereas the heterotetramer is phosphorylated on a serine residue and a tyrosine residue by Ca(2+)-dependent protein kinases. Antibodies to brain p37 and p36 together with antibodies to lymphocytes lipocortins 1 and 2 were used to follow the distribution of these proteins in nervous tissues. Polypeptides of 37, 34, and 36 kDa cross-react with these antibodies. Anti-p37 and antilipocortin 1 cross-react on the same 37- and 34-kDa polypeptides; anti-p36 and antilipocortin 2 cross-react only on the 36-kDa polypeptides.     

Presence of an acidic glycoprotein in the serum of arthritic rats: Modulation by capsaicin and curcumin

Voltage-dependent L-type Ca²⁺ channels form highly selective pores for Ca²⁺ ions in the membranes of excitable cells. We investigated the functional role of negatively charged residues, within or near the selectivity region, in ion permeation of a human cardiac L-type Ca²⁺ channel. Glutamates in each of the four repeats, and an aspartate in repeat IV, were substituted with positively charged lysine. Wild-type and mutant Ca²⁺ channels were expressed in Xenopus oocytes. Block by Ca²⁺ and Mg² of inward Li⁺ currents through the channels was used to assess the effects of amino acid substitutions on high-affinity divalent cation binding. The rank order of IC₅₀'s for Ca²⁺ block of I_Li was: E677K > E1086K > E334K > E1387K > D1391K > wild-type. The order of IC₅₀'s for Mg²⁺ block of I_Li indicated differential involvement of the same residues in Mg²⁺ binding: E1387K > E334K > E1086K > E677K > D1391K wild-type. Mutants E1387K and D1391K effectively permeated Ba²⁺, but exhibited a decreased single-channel conductance. The unitary current amplitude carried by Na+, in the absence of external divalent cations, was slightly decreased in the E1387K mutant but not in the D1391K mutant. The results confirm that each of the four glutamates participate unequally in high-affinity Ca²⁺ binding. Additionally, our results indicate that these glutamate residues participate in Mg²⁺ binding. The glutamate at position 1387 may be only peripherally involved in the formation of a high-affinity Ca²⁺ -binding site but is central to a Mg²⁺ binding site accessible from the external side of the pore. The aspartate at position 1391 is most likely located just external to the selectivity region. (Mol Cell Biochem 166: 125-134, 1997)     

S100A1 codistributes with synapsin I in discrete brain areas and inhibits the F-actin-bundling activity of synapsin I

The Ca2+-sensor protein S100A1 was recently shown to bind in vitro to synapsins, a family of synaptic vesicle phosphoproteins involved in the regulation of neurotransmitter release. In this paper, we analyzed the distribution of S100A1 and synapsin I in the CNS and investigated the effects of the S100A1/synapsin binding on the synapsin functional properties. Subcellular fractionation of rat brain homogenate revealed that S100A1 is present in the soluble fraction of isolated nerve endings. Confocal laser scanning microscopy and immunogold immunocytochemistry demonstrated that S100A1 and synapsin codistribute in a subpopulation (5-20%) of nerve terminals in the mouse cerebral and cerebellar cortices. By forming heterocomplexes with either dephosphorylated or phosphorylated synapsin I, S100A1 caused a dose- and Ca2+-dependent inhibition of synapsin-induced F-actin bundling and abolished synapsin dimerization, without affecting the binding of synapsin to F-actin, G-actin or synaptic vesicles. These data indicate that: (i) synapsins and S100A1 can interact in the nerve terminals where they are coexpresssed; (ii) S100A1 is unable to bind to SV-associated synapsin I and may function as a cytoplasmic store of monomeric synapsin I; and (iii) synapsin dimerization and interaction with S100A1 are mutually exclusive, suggesting an involvement of S100A1 in the Ca2+-dependent regulation of synaptic vesicle trafficking.     

Parvalbumin in Human Brain

Parvalbumin was isolated from human cerebral cortex and biceps and triceps muscles by HPLC. The immunological properties of the human protein and the mobility in two-dimensional polyacrylamide gels were similar to that of parvalbumin isolated from the muscles of rat, mouse, rabbit, and chicken. The tryptic peptide maps of the human parvalbumin, however, differed considerably from all other parvalbumins, indicating a distinct primary structure. The immunolabeled cells in the hippocampus of the human brain were of different sizes and forms; they occurred in all subfields and probably represent interneurons.     

Parvalbumin in Cat Brain: Isolation, Characterization, and Localization

Because of the increasing evidence that Ca2+-binding proteins have important regulating functions in nerve cells and because of the indications that there are species differences in the structures of these proteins, parvalbumin was purified from cat brain and muscle. Brain and muscle parvalbumins were found to be indistinguishable from each other in their biochemical and immunological properties. However, cat parvalbumin differs from all other mammalian parvalbumins by its apparently lower Mr on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 10-11K (compared to rat parvalbumin, 12K), and a lower pI of 4.6 (rat parvalbumin, 4.9), in the tryptic peptide maps, and in the immunological properties, indicating a distinct primary structure. With the purified parvalbumin as antigen, polyclonal antibodies were raised in rabbits and these were subsequently used for immunohistochemical localizations of parvalbumin in the cat brain. In the visual cortices of adult cats immunoreactive neurons were present throughout layers II and IV. In cerebellar cortex, Purkinje, basket, and stellate cells were immunoreactive. Comparison with staining patterns obtained with antiserum against rat parvalbumin revealed some cross-reactivity but confirmed the existence of species differences in the antigenic structure of rat and cat parvalbumin.     

Calcium in fungi

Abstract. Recently much experimental evidence has accumulated concerning intracellular calcium and its fundamental role as a regulator in eukaryotic cells. The literature relating to Ca²⁺ in fungi is large and diverse and this paper draws together the available information and discusses the particular functions of the ion in this group of organisms.
Uptake mechanisms in fungi are considered with special reference to the effect of Ca²⁺ on permeability and the systems responsible for transport of ions, sugars and amino acids. Discussion of the subcellular locations and distribution of Ca²⁺ is accompanied by a critique of methodology used in determination of subcellular sites of Ca²⁺ in fungi. The role of Ca²⁺ in morphogenesis in fungi is considered with particular reference to selected groups.     

Parvalbumin, a Neuronal Protein in Brain Cell Cultures

Dissociated brain cell cultures were derived from 14-day-old embryonic as well as from newborn mice. The cells were grown in a medium containing 10% fetal calf serum. Indirect immunofluorescence was performed using antisera directed against the Ca2+-binding protein parvalbumin (Mr 12,000). In embryonic cultures a large proportion of cells was intensely stained by antiparvalbumin . In double-labelling experiments involving the simultaneous application of antisera against parvalbumin and the neuron-specific enolase, the enolase-containing cells were also parvalbumin-positive and both antisera revealed identical intracellular staining patterns. Conversely, almost no parvalbumin- and enolase-positive cells were present in cultures derived from newborn mice. However, in these cultures many cells were immunoreactive toward the myelin basic protein, an accepted marker for oligodendrocytes. The presence of parvalbumin within the embryonic brain cell cultures was confirmed by analyses of the culture extracts (4 mM EDTA, pH 7.5) by HPLC on reverse-phase supports, two-dimensional polyacrylamide gel electrophoresis, and immunoblotting. The present study suggests that in mouse brain cell cultures, parvalbumin is localized in neurons.     

Regulation of the sarcoplasmic reticulum Ca(2+)-release channel requires intact annexin VI.

Annexin VI has eight highly conserved repeated domains; all other annexins have four. Díaz-Mu?oz et al. (J Biol Chem 265:15894, 1990) reported that annexin VI alters the gating properties of the ryanodine-sensitive Ca(2+)-release channel isolated from sarcoplasmic reticulum. The investigate the domain structure of rat annexin VI (67 kDa calcimedin) required for this channel regulation, various proteolytic digestions were performed. In each case, protease-resistant core polypeptides were produced. Annexin VI was digested with V8 protease and two core polypeptides were purified by Ca(2+)-dependent phospholipid binding followed by HPLC. The purified fragments were shown to be derived from the N- and C-terminal halves of annexin VI, and demonstrated differential immunoreactivity with monoclonal antibodies to rat annexin VI. While both core polypeptides retained their ability to bind phospholipids in a Ca(2+)-dependent manner, they did not regulate the sarcoplasmic reticulum Ca(2+)-dependent manner, they did not regulate the sarcoplasmic reticulum Ca(2+)-release channel as did intact annexin VI.     

NaHCO3胁迫下星星草根中Ca2＋与Ca2＋-ATPase的超微细胞化学定位

利用焦锑酸盐和磷酸铅沉淀技术分别对NaHCO3胁迫条件下星星草（Puccinellia tenuiflora）根中Ca2＋和Ca2＋-ATPase进行超微细胞化学定位研究,旨在进一步探讨Ca2＋在NaHCO3胁迫诱导胞内信号转导过程中的作用,以及Ca2＋-ATPase活性定位变化与NaHCO3胁迫下星星草抗盐碱能力的关系。结果表明：在正常状态下,根毛区细胞质内Ca2＋较少,主要位于质膜附近和液泡中,Ca2＋-ATPase主要定位于质膜和液泡膜,有一定活性。在0.448%NaHCO3胁迫下,根毛区细胞质中Ca2＋增多,液泡中Ca2＋减少,且主要集中于液泡膜附近,质膜和液泡膜Ca2＋-ATPase活性明显升高。在1.054%NaHCO3胁迫下,细胞质中分布的Ca2＋增多,而液泡中Ca2＋极少,Ca2＋-ATPase活性也降低。以上结果表明,Ca2＋亚细胞定位和Ca2＋-ATPase活性变化在星星草响应NaHCO3胁迫的信号传递过程中具有重要作用。