Conformational coupling of Mg2+ and Ca2+ on the three-state folding of calexcitin B

Calexcitin (CE) is a calcium sensor protein that has been implicated in associative learning through the Ca(2+)-dependent inhibition of K(+) channels and activation of ryanodine receptors. CE(B), the major CE variant, was identified as a member of the sarcoplasmic Ca(2+) binding protein family: proteins that can bind both Ca(2+) and Mg(2+). We have now determined the intrinsic Ca(2+) and Mg(2+) binding affinities of CE(B) and investigated their interplay on the folding and structure of CE(B). We find that urea denaturation of CE(B) displays a three-state unfolding transition consistent with the presence of two structural domains. Through a combination of spectroscopic and denaturation studies we find that one domain likely possesses molten globule structure and contains a mixed Ca(2+)/Mg(2+) binding site and a Ca(2+) binding site with weak Mg(2+) antagonism. Furthermore, ion binding to the putative molten globule domain induces native structure formation. The other domain contains a single Ca(2+)-specific binding site and has native structure, even in the absence of ion binding. Ca(2+) binding to CE(B) induces the formation of a recessed hydrophobic pocket. On the basis of measured ion binding affinities and intracellular ion concentrations, it appears that Mg(2+)-CE(B) represents the resting state and Ca(2+)-CE(B) corresponds to the active state, under physiological conditions.     

Nine novel germline mutations of STK11 in ten families with Peutz-Jeghers syndrome

Peutz-Jeghers Syndrome (PJS) is an autosomal dominant hereditary disease characterized by hamartomatous polyposis involving the entire bowel. Recently STK11, a gene bearing a mutation responsible for PJS, was isolated. We investigated the entire coding region of STK11 in 15 unrelated PJS families by the PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism) method and PCR-direct sequence analysis, and found nine different, novel mutations among ten of those families. One nonsense mutation and five different frameshift mutations (two families carried the same mutation), all of which would cause truncation of the gene product, were found in seven families; mutations found in five families were clustered within exon 6. Among these five mutations, three occurred at the mononucleotide-repeat region (CCCCCC) of codons 279–281, suggesting that this region is likely to be a mutational hotspot of this gene. One of the remaining three families carried a 3-bp in-frame deletion that would eliminate an asparagine residue within a kinase domain of the product; the other two carried intronic mutations at or adjacent to the consensus dinucleotide sequences of splice-acceptor or -donor sites, which were likely to lead to aberrant splicing. Received: 9 March 1998 / Accepted: 1 May 1998     

Hanganutziu-Deicher antibodies in infectious mononucleosis and other diseases

An enzyme immunoassay (EIA) was developed for the detection of heterophile, Hanganutziu-Deicher (H-D) antibodies in sera of patients with infectious mononucleosis (IM) and various other diseases. The EIA with a high m.w. glycoprotein (HMWGP) isolated from bovine erythrocyte stromata was shown to detect H-D antibodies directly, in spite of higher titers of Paul-Bunnell (P-B) antibodies in the IM sera. Absorption and inhibition studies of IM sera demonstrated H-D specificity of the antibodies combining with HMWGP in the EIA. The H-D antibodies were found in sera of 56% Caucasians and 27% of Japanese suffering from IM. The vast majority of the H-D antibodies in IM sera was of IgM class. Sera of patients with various other diseases also gave positive results: rheumatoid arthritis, 22%; syphilis, 19%; cancer of the gastrointestinal tract, 13%; and lepromatous leprosy 9.7%. The incidence of positive results in control sera from apparently healthy subjects was less than 4%. Results of this study confirmed our previous observation that whereas P-B antigens appear in immunogenic form in only IM, the H-D antigen is expressed as an immunogen in various diseases including IM.     

Calpain activation in shear-induced platelet aggregation

Fluid shear stress has been known to activate platelet reaction such as aggregation, but the exact mechanism of shear-induced platelet aggregation (SIPA) has not been fully understood. Calpain, an intracellular calcium-activated cysteine protease, is abundant in platelets and is considered to be activated and involved in the proteolytic processes during platelet activation. A possible activation of calpain in SIPA was investigated, employing a newly developed aggregometer and specific monoclonal antibodies to detect activation of calpain. When a shear stress gradient varying between 6 and 108 dyn/cm² was applied to platelets, activation of μ-calpain was observed only in high-shear-stressed platelets, resulting in the proteolysis of talin. At 1 min after the onset of constant high shear stress of 108 dyn/cm², μ-calpain activation and proteolysis of talin were detected and increased in a time-dependent manner. Constant shear stress more than 50 dyn/cm², applied for 5 min, caused μ-calpain activation and proteolysis of talin, which were increased in a shear-force-dependent manner. Calpeptin, a calpain-specific peptide antagonist, caused the complete inhibition of both μ-calpain activation and proteolysis of talin, while SIPA profiles with calpeptin showed almost no change compared to those without calpeptin. These results suggest the possibility of calpain involvement in late phases of shear-induced platelet activation such as cytoskeletal reorganization. J. Cell. Biochem. 66:54–64, 1997. © 1997 Wiley-Liss, Inc.     

Separate analysis of nuclear and cytosolic Ca2+ concentrations in human umbilical vein endothelial cells

Ca²⁺ concentration inside human umbilical vein endothelial cells was studied separately in cytosol and nucleus by a confocal laser scanning microscopy using fluo-3. The in vivo calibration curve for cytosol and nucleus showed good linearity between fluorescence intensity and Ca²⁺ concentration in cytosol ([Ca²⁺]_i) and nuclei ([Ca²⁺]_n). After calibration, [Ca²⁺]_n was constantly higher than [Ca²⁺]_i before and after the chelation of extracellular Ca²⁺ suggesting an active Ca²⁺ accumulation system on nuclear membrane. [Ca²⁺]_n was also constantly higher than [Ca²⁺]_i after the stimulation of thrombin (0.05 U/ml), FCS (10%), and thapsigargin (Tsg, 1μM). The temporal change of [Ca²⁺]_n and [Ca²⁺]_i was identical, and [Ca²⁺]_i gradient towards the nucleus and peripheral or central [Ca²⁺]_n rise was observed after these stimulations. From these results, [Ca²⁺]_n is not only regulated by the active Ca²⁺ accumulation system on nuclear membrane at rest but also the generation of Inositol-triphosphate. FCS caused heterogeneous [Ca²⁺]_n or [Ca²⁺]_i rise from cell to cell; single spike or oscillatory change of [Ca²⁺]_n and [Ca²⁺]_i was observed in about 56% of cells, which were completely abolished by the chelation of extracellular Ca²⁺, suggesting that FCS stimulated [Ca²⁺]_n and [Ca²⁺]_i rise solely depending on Ca²⁺ influx from extracellular medium. The higher concentration of [Ca²⁺]_n and heterogeneous [Ca²⁺]_n rise may have important roles in nuclear-specific cellular responses. © 1996 Wiley-Liss, Inc.     

Blood—Brain Barrier Glucose Transporter

Abstract : The transport of glucose across the blood-brain barrier (BBB) is mediated by the high molecular mass (55-kDa) isoform of the GLUT1 glucose transporter protein. In this study we have utilized the tritiated, impermeant photolabel 2-N-[4-(1-azi-2,2,2-trifluoroethyl)[2-³H]propyl]-1,3-bis(d -mannose-4-yloxy)-2-propylamine to develop a technique to specifically measure the concentration of GLUT1 glucose transporters on the luminal surface of the endothelial cells of the BBB. We have combined this methodology with measurements of BBB glucose transport and immunoblot analysis of isolated brain microvessels for labeled luminal GLUT1 and total GLUT1 to reevaluate the effects of chronic hypoglycemia and diabetic hyperglycemia on transendothelial glucose transport in the rat. Hypoglycemia was induced with continuous-release insulin pellets (6 U/day) for a 12- to 14-day duration ; diabetes was induced by streptozotocin (65 mg/kg i.p.) for a 14- to 21-day duration. Hypoglycemia resulted in 25-45% increases in regional BBB permeability-surface area (PA) values for d -[¹⁴C]glucose uptake, when measured at identical glucose concentration using the in situ brain perfusion technique. Similarily, there was a 23 ± 4% increase in total GLUT1/mg of microvessel protein and a 52 ± 13% increase in luminal GLUT1 in hypoglycemic animals, suggesting that both increased GLUT1 synthesis and a redistribution to favor luminal transporters account for the enhanced uptake. A corresponding (twofold) increase in cortical GLUT1 mRNA was observed by in situ hybridization. In contrast, no significant changes were observed in regional brain glucose uptake PA, total microvessel 55-kDa GLUT1, or luminal GLUT1 concentrations in hyperglycemic rats. There was, however, a 30-40% increase in total cortical GLUT1 mRNA expression, with a 96% increase in the microvessels. Neither condition altered the levels of GLUT3 mRNA or protein expression. These results show that hypoglycemia, but not hyperglycemia, alters glucose transport activity at the BBB and that these changes in transport activity result from both an overall increase in total BBB GLUT1 and an increased transporter concentration at the luminal surface.     

Calmodulin Target Database

The intracellular calcium sensor protein calmodulin (CaM) interacts with a large number of proteins to regulate their biological functions in response to calcium stimulus. This molecular recognition process is diverse in its mechanism, but can be grouped into several classes based on structural and sequence information. We have developed a web-based database (http://calcium.uhnres.utoronto.ca/ctdb) for this family of proteins containing CaM binding sites or, as we propose to call it herein, CaM recruitment signaling (CRS) motifs. At present the CRS motif found in approximately 180 protein sequences in the databases can be divided into four subclasses, each subclass representing a distinct structural mode of molecular recognition involving CaM. The database can predict a putative CRS location within a given protein sequence, identify the subclass to which it may belong, and structural and biophysical parameters such as hydrophobicity, hydrophobic moment, and propensity for a -helix formation. This revised version was published online in July 2006 with corrections to the Cover Date.