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Biomedical subjects

D Gerard

Publications and source records attributed to D Gerard.

At least 37 records · Page 2Linked to original sources

Brain alterations induced by vitamin E deficiency and intoxication with methyl ethyl ketone peroxide.

Rats fed a vitamin E-deficient diet from age 3-10 weeks were either maintained on a vitamin E-deficient diet or fed a vitamin E-enriched diet for 8 subsequent weeks. The content of vitamin E, endoperoxide-derived malonaldehyde, lipofluorescent material and polyunsaturated fatty acids, and the activities of catalase, glutathione reductase, and glutathione peroxidase were then measured in cerebral tissues, with or without intoxication with methyl ethyl ketone peroxide (MEKP). For this purpose, one half of the animals in each vitamin E group received an ip injection of 5 mg MEKP per kg of body weight, which was followed 44 hours later, i.e., 4 hours before sample collection, by a second ip injection of 15 mg MEKP per kg of body weight. Despite the fact that the vitamin E concentration was 12-times lower in the brain of vitamin E-deficient rats, no significant change in other cerebral parameters was found between the two groups of animals. In contrast, the activity of selenium-glutathione peroxidase was markedly decreased in the liver of 10-week old vitamin E-deficient rats. Unexpectedly, acute systemic intoxication with MEKP caused only a small, albeit significant, decrease in glutathione reductase activity in the brain of vitamin E-sufficient rats, while no significant change in other cerebral parameters was observed in either group of animals. These results suggest that the central nervous system (CNS) is still substantially protected when its vitamin E content has been decreased to 3 micrograms/g fresh weight, and that systemic intoxication with MEKP may not cause lipid peroxidation in the CNS.

Animals↗

Comparative effects of calmodulin inhibitors on calmodulin's hydrophobic sites and on the activation of cyclic nucleotide phosphodiesterase by calmodulin.

Experiments were designed to investigate the effect of inhibitors on calmodulin's hydrophobic sites and their consequences on the activation of a target enzyme, cyclic nucleotide phosphodiesterase. Two fluorescent probes, 2-(p-toluidinyl)-naphthalene-6-sulfonate (TNS) and 9-anthroylcholine (9AC) were used to study the interactions with calmodulin of inhibitors devoid of direct effect on the probes. Contrary to W-7, nicergoline, nicardipine and quercetin, which decreased the fluorescence of the two probes bound to calmodulin, bepridil only decreased 9AC fluorescence but increased the fluorescence intensity at the wavelength of the emission maximum of TNS. In spite of this difference, bepridil as well as W-7 and nicergoline competitively inhibited calmodulin activation of phosphodiesterase. In addition, nicergoline also inhibited phosphodiesterase activity competitively to cyclic GMP. These results show differences in the interactions of inhibitors with calmodulin; these differences are not detected in functional studies of the effect of inhibitors on phosphodiesterase activation.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

Natural antibodies to human lymphocytes and erythrocytes in the serum of Orcinus orca killer whale.

The existence of naturally occurring heterophile antibodies to antigenic determinants on human blood cell membranes has long been known. It has been shown that the serum of Orcinus orca (Killer whale) does contain similar antibody. Absorption techniques in concert with either microagglutination or complement-dependent microcytotoxicity assays revealed at least three antibody specificities erythrocyte (RBC), B-lymphocyte and T-lymphocyte. Human erythrocyte specificity has been separated from other mammalian RBC specificity, and higher microagglutination titers and/or scores were observed with human group A RBC's than with group B,O, or AB. Tests run at 4 degrees, 20 degrees and 37 degrees C). Higher microcytotoxicity and microagglutination activity was demonstrated with B versus T lymphocytes. It is hoped that the characterization of the antigenic specificity of these heterophile agglutinins will prove to be useful as a biological reagent-tool which may be applied to the identification of a new receptor on human lymphocytes and/or erythrocytes. Also, if isolated, these agglutinins could be useful in the study of the occurrence and presence of specific receptors on cell membranes and give insight as to how these receptors change in health, disease and malignancy.

Agglutinins↗

Spectroscopic evidence of two melittin molecules bound to Ca2+-calmodulin.

According to Comte et al. (Comte, M., Maulet, Y. and Cox, J.A., (1983), Biochem.J., 209, 269-272), melittin (Mel) gives rise to a one:one complex. We evidence here, by fluorescence anisotropy and gel filtration binding assay (in the presence of 5 mM CaCl2 and 100 mM NaCl) the existence of two complexes: the well-known CaM.Ca4.Mel and a second CaM.Ca4.Mel2 which had not yet been reported. The affinity of Mel for the CaM.Ca4.Mel species is about three orders of magnitude lower than the affinity of Mel for the CaM-Ca4 species.

Animals↗

Ions binding to S100 proteins. I. Calcium- and zinc-binding properties of bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) protein: Zn2+ regulates Ca2+ binding on S100b protein.

Flow dialysis measurements of calcium binding to bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) proteins in 20 mM Tris-HCl buffer at pH 7.5 and 8.3 revealed that S100 proteins bind specifically 4 Ca2+ eq/mol of protein dimer. The specific calcium-binding sites had, therefore, been assigned to typical amino acid sequences on the alpha and beta subunit. The protein affinity for calcium is much lower in the presence of magnesium and potassium. Potassium strongly antagonizes calcium binding on two calcium-binding sites responsible for most of the Ca2+-induced conformational changes on S100 proteins (probably site II alpha and site II beta). Zinc-binding studies in the absence of divalent cations revealed eight zinc-binding sites/mol of S100b protein dimer that we assumed to correspond to 4 zinc-binding sites/beta subunit. Zinc binding to S100b studied with UV spectroscopy methods showed that the occupation of the four higher affinity sites and the four lower affinity sites on the protein dimer were responsible for different conformational changes in S100b structure. Zinc binding on the higher affinity sites regulates calcium binding to S100b by increasing the protein affinity for calcium and decreasing the antagonistic effect of potassium on calcium binding. Zinc-binding studies on S100a and S100 alpha alpha protein showed that the Trp-containing S100 proteins bind zinc more weakly than S100b protein. Calcium-binding studies on zinc-bound S100a proved that calcium- and zinc-binding sites were distinct although there was no increase in zinc-bound S100a affinity for calcium, as in S100b protein. Finally we provide evidence that discrepancies between previously published results on the optical properties of S100b protein probably result from oxidation of the sulfhydryl groups in the protein.

Animals↗

Ions binding to S100 proteins. II. Conformational studies and calcium-induced conformational changes in S100 alpha alpha protein: the effect of acidic pH and calcium incubation on subunit exchange in S100a (alpha beta) protein.

A rapid separation method for bovine brain S100 alpha alpha, S100a, and S100b protein using fast protein liquid chromatography on a Mono Q column and its application in preparation of a large amount of S100 alpha alpha protein are described. The conformation of S100 alpha alpha in the metal-free forms as well as in the presence of calcium were studied by UV absorption, circular dichroism, intrinsic fluorescence, sulfhydryl reactivity, and interaction with a hydrophobic fluorescent probe. The alpha-subunit appears to have nearly identical conformation in S100 alpha alpha and S100a protein dimers. We also confirmed that only the alpha-subunit exposes hydrophobic domains to solvent in the presence of calcium and that cysteine residues exposed upon Ca2+ binding to S100 proteins correspond to Cys 85 alpha and Cys 84 beta. Incubation of S100a with calcium and KCl proved that calcium binding to the putative calcium-binding sites (site I alpha, I beta) triggers a time- and temperature-dependent conformational change in the protein structure which decreases the antagonistic effect of KCl on calcium binding to sites II alpha and II beta and provokes subunit exchanges between protein dimers and the emergence of S100 alpha alpha and S100b (beta beta) proteins. Dynamic fluorescence measurements showed that incubating calcium at high S100a protein concentrations (greater than 10(-5) M) induces an apparent slow dimer-monomer equilibrium which might result in total subunit dissociation at lower protein concentrations. The effect of acidic pH on subunit dissociation in S100a protein (Morero, R. D., and Weber, G. (1982) Biochim. Biophys. Acta 703, 231-240) arises from conformational changes in the protein structure that are similar to those induced by Ca2+ incubation.

Animals↗

Purification, calcium-binding properties, and conformational studies on a 28-kDa cholecalcin-like protein from bovine brain.

A large-scale preparation method for bovine brain 28-kDa cholecalcin-like protein is described. Flow dialysis binding studies revealed that the protein binds at least 3 mol of Ca2+/mol of protein. The protein undergoes conformational changes on binding calcium as shown by UV differential absorption spectroscopy, near and far UV circular dichroism, and intrinsic fluorescence. Circular dichroism (CD) studies in the far UV indicate an apparent increase in helical content in the presence of Ca2+. The effect of calcium on the protein structure is nearly maximum for 1 Ca2+ bound/protein molecule. UV differential absorption studies on the binding of the Ca2+ agonist Tb3+ and Tb3+ luminescence induced by energy Trp----Tb3+ transfer indicate that Tb3+ binds to two higher affinity Ca2+-binding sites. These sites are probably very close to the single Trp residue. Analysis of the fluorescence parameters of the single tryptophan residue in the apoprotein and its accessibility to ionic and neutral quenchers suggests that this residue is located in a highly hydrophobic domain on the protein surface.

Animals↗

Rat brain S100b protein: purification, characterization, and ion binding properties. A comparison with bovine S100b protein.

We purified to homogeneity rat brain S100b protein, which constitutes about 90% of the soluble S100 protein fraction. Purified rat S100b protein comigrates with bovine S100b protein in nondenaturant system electrophoresis but differs in its amino acid composition and in its electrophoretic mobility in urea-sodium dodecyl sulfate-polyacrylamide gel with bovine S100b protein. The properties of the Ca2+ and Zn2+ binding sites on rat S100b protein were investigated by flow dialysis and by fluorometric titration, and the conformation of rat S100b in its metal-free form as well as in the presence of Ca2+ or Zn2+ was studied. The results were compared with those obtained for the bovine S100b protein. In the absence of KCl, rat brain S100b protein is characterized by two high-affinity Ca2+ binding sites with a KD of 2 X 10(-5) M and four lower affinity sites with KD about 10(-4) M. The calcium binding properties of rat S100b protein differ from bovine S100b only by the number of low-affinity calcium binding sites whereas similar Ca2+-induced conformational changes were observed for both proteins. In the presence of 120 mM KCl rat brain S100b protein bound two Zn2+-ions/mol of protein with a KD of 10(-7) M and four other with lower affinity (KD approximately equal to 10(-6) M). The occupancy of the two high-affinity Zn2+ binding sites was responsible for most of the Zn2+-induced conformational changes in the rat S100b protein. No increase in the tyrosine fluorescence quantum yield after Zn2+ binding to rat S100b was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

Purification, characterization and ion binding properties of human brain S100b protein.

Human brain S100b (beta beta) protein was purified using zinc-dependent affinity chromatography on phenyl-Sepharose. The calcium- and zinc-binding properties of the protein were studied by flow dialysis technique and the protein conformation both in the metal-free form and in the presence of Ca2+ or Zn2+ was investigated with ultraviolet spectroscopy, sulfhydryl reactivity and interaction with a hydrophobic fluorescence probe 6-(p-toluidino)naphthalene-2-sulfonic acid (TNS). Flow dialysis measurements of Ca2+ binding to human brain S100b (beta beta) protein revealed six Ca2+-binding sites which we assumed to represent three for each beta monomer, characterized by the macroscopic association constants K1 = 0.44 X 10(5) M-1; K2 = 0.1 X 10(5) M-1 and K3 = 0.08 X 10(5) M-1. In the presence of 120 mM KCl, the affinity of the protein for calcium is drastically reduced. Zinc-binding studies on human S100b protein showed that the protein bound two zinc ions per beta monomer, with macroscopic constants K1 = 4.47 X 10(7) M-1 and K2 = 0.1 X 10(7) M-1. Most of the Zn2+-induced conformational changes occurred after the binding of two zinc ions per mole of S100b protein. These results differ significantly from those for bovine protein and cast doubt on the conservation of the S100 structure during evolution. When calcium binding was studied in the presence of zinc, we noted an increase in the affinity of the protein for calcium, K1 = 4.4 X 10(5) M-1; K2 = 0.57 X 10(5) M-1; K3 = 0.023 X 10(5) M-1. These results indicated that the Ca2+- and Zn2+-binding sites on S100b protein are different and suggest that Zn2+ may regulate Ca2+ binding by increasing the affinity of the protein for calcium.

Binding Sites↗

Fluorescence investigations of calmodulin hydrophobic sites.

Calmodulin activation of target enzymes depends on the interaction between calmodulin hydrophobic regions and some enzyme areas. The Ca2+ induced exposure of calmodulin hydrophobic sites was studied by means of 2-p-toluidinylnaphthalene-6-sulfonate, a fluorescent probe. Scatchard and Job plots showed that the calmodulin-Ca42+ complex bound two molecules of this hydrophobic probe, with KD congruent to 1.4 X 10(-4) M. These sites are not totally exposed until calmodulin has bound four Ca2+ per molecule, so the conformational change is not over before the four specific Ca2+ - binding sites are saturated with Ca2+.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

Intrinsic fluorescence of a non-myelin apoproteolipid and evidence for the existence of conformational flexibility.

An extremely hydrophobic protein (Mr = 16000), which in its native form is only soluble in organic solvents and which differs from the myelin proteolipid (Mr = 24000), was purified to homogeneity. Intrinsic fluorescence studies on this apoproteolipid have revealed a large conformational flexibility. In the water-soluble form the emitting residues appear to be buried in a hydrophobic core while in organic solvents they are exposed to the external medium. Structural changes depending on the organic solvent are also observed. The emission characteristics of reconstituted proteoliposomes may be due to the formation of a membrane-linked complex between several proteolipid monomers.

Journal Article↗

The effect of S-100a and S-100b proteins and Zn2+ on the assembly of brain microtubule proteins in vitro.

The homologous proteins S-100a and S-100b affect the microtubule system in a distinctly different way in the presence of low molar ratios of Zn2+. Assembly of brain microtubule proteins can be almost completely inhibited and rapid disassembly can be induced by low molar amounts of S-100b in the presence of low molar ratios [2-4] of Zn2+. Higher molar ratios per S-100b (greater than 4) potentiate the general Zn2+ effect, promoting the formation of sheets of microtubules. However, the effect of S-100a is quite different, no inhibition of assembly can be observed and the presence of S-100a seems to protect the microtubule proteins against the effect of Zn2+ by chelating the Zn2+ and decreasing the free metal-ion concentration. S-100a or S-100b cannot bind to the microtubule polymer-form, either in the absence or in the presence of Zn2+.

Animals↗

Kainate-enhanced release of D-[3H]aspartate from cerebral cortex and striatum: reversal by baclofen and pentobarbital.

A study was made of the actions of the excitant neurotoxin, kainic acid, on the uptake and the release of D-[2,3-3H]aspartate (D-ASP) in slices of guinea pig cerebral neocortex and striatum. The slices took up D-ASP, reaching concentrations of the amino acid in the tissue which were 14-23 times that in the medium. Subsequently, electrical stimulation of the slices evoked a Ca2+-dependent release of a portion of the D-ASP. Kainic acid (10(-5)-10(-3) M) produced a dose-dependent inhibition of D-ASP uptake. The electrically evoked release of D-ASP was increased 1.6-2.0 fold by 10(-5) and 10(-4)M kainic acid. The kainate-enlarged release was Ca2+-dependent. Dihydrokainic acid, an analogue of kainic acid with little excitatory or toxic action, did not increase D-ASP release but depressed D-ASP uptake. Attempts were made to block the action of kainic acid with baclofen and pentobarbital, compounds which depress the electrically evoked release of L-glutamate (L-GLU) and L-aspartate (L-ASP). Baclofen (4 X 10(-6)M), an antispastic drug, and pentobarbital (10(-4)M), an anesthetic agent, each inhibited the electrically evoked release of D-ASP and prevented the enhancement of the release above control levels usually produced by 10(-4)M kainic acid. It is proposed that 10(-5) and 10(-4)M kainic acid may enhance the synaptic release of L-GLU and L-ASP from neurons which use these amino acids as transmitters. This action is prevented by baclofen and pentobarbital. In view of the possibility that cell death in Huntington's disease could involve excessive depolarization of striatal and other cells by glutamate, baclofen might be effective in delaying the loss of neurons associated with this condition.

Animals↗