Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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The aim of this work was to study the role of different parameters involved in the motility of human spermatozoa. Human spermatozoa were totally demembranated with 0.05% Triton X-100, and the demembranation was checked using electron microscopy. We have shown that, with a concentration of ATP-Mg lower than 2 mM, a pH effect was observed with a dose-dependent motility reactivation at pH 7.1, with 14% +/- 2.0% motile cells at 1 mM ATP-Mg and a straight line velocity (VSL) of 12.0 +/- 1.4 microns/sec. However, at pH 7.8, more than 65% of the spermatozoa were reactivated with as low as 0.02 mM ATP-Mg and 77.8% +/- 2.5% of them were motile at 1 mM ATP-Mg and had a VSL of 23.4 +/- 3.9 microns/sec. The depletion of free calcium by the addition of 0.5 mM EGTA in the reactivation medium (RM) improved the percentage of motile cells and the VSL most markedly at low ATP-Mg and low pH. If no MgSO4 was added in RM, cells were not motile at pH 7.8, but 30-40% reactivated at pH 7.1. If 5 mM Ca2+ was added to the RM, up to 88% of the cells became reactivated at both pHs, but the beat frequencies were very low, suggesting different mechanisms of reactivation when Mg2+ or when Ca2+ is present in the RM.
Mol Reprod Dev 1991 Jun
PMID:Effects of pH on the reactivation of human spermatozoa demembranated with Triton X-100. 187 24

The crystal structure of the Ca-loaded form of pike 4.10 parvalbumin (minor component from pike muscle belonging to the beta phylogenetic series), with both its primary sites CD and EF occupied by Ca2+ ions and its third site occupied by an ammonium ion, as previously determined at 1.93 A resolution, has now been refined to a resolution of 1.65 A. The crystallization of this parvalbumin in different ionic environments has allowed three novel non-isomorphous crystalline forms to be obtained: (1) a first form, crystallized in the presence of a mixture of ammonium sulphate and manganese sulphate, for which all the cation binding sites in the protein are occupied by Mn2+; (2) a second form crystallized in the presence of MgSO4 as the precipitating agent, only differs from the Ca/NH4 form by the occupation of the third site by Mg2+, whereas the primary sites remain occupied by Ca2+; (3) a third form, also crystallized in the presence of MgSO4, corresponds to a well-defined molecular species with both the primary EF site and the third site occupied by Mg2+, whereas the primary CD site remains occupied by CA2+. The corresponding molecular structures reported here have been determined at resolutions between 1.8 and 2.4 A. The comparison of the different crystal structures allows the structural modifications accompanying the substitution of the primary sites by cations differing significantly in their ionic radii (Ca2+, Mn2+, Mg2+) to be investigated in detail, and it also leads to a precise description of the third site in a typical beta parvalbumin. The substitution Ca2+ by Mg2+ within the primary site EF is characterized by a "contraction" of the co-ordination sphere, with a decrease of the mean oxygen-metal distance by a value of 0.25 A and a decrease of the co-ordination number from 7 to 6, as a consequence of the loss of a bidentate ligand (Glu101), which becomes a monodentate one. Such an adaptation of the co-ordination sphere around a cation of smaller size involves, among others, the transformation of the Glu101 side-chain from the stable gauche(+) form to the less stable gauche(-) form. The third site is clearly described as a satellite of the CD primary site, since both sites possess common protein ligands, such as Asp53 and Glu59. Furthermore, Asp61 appears as a specific ligand of the third site in the different environments investigated in this work. We finally discuss the relevance of the third site to parvalbumin phylogeny.
J Mol Biol 1991 Aug 20
PMID:Ionic interactions with parvalbumins. Crystal structure determination of pike 4.10 parvalbumin in four different ionic environments. 188 Jul 97

A transformation system for Ophiostoma ulmi (Buism.) Nannf. was developed and analyzed. Protoplasts were generated from actively budding yeastlike cells by digestion with NovoZym 234 in MgSO4 after pretreatment with 2-mercaptoethanol. Protoplast regeneration was most efficient when 0.6 M sucrose was used as the osmoticum. Several plasmids containing fusions between fungal promoters and a bacterial gene for hygromycin phosphotransferase successfully transformed O. ulmi to hygromycin resistance. One of these vectors, pPS57, which contains a promoter for isopenicillin N synthetase from Penicillium chrysogenum, consistently conferred the greatest resistance to hygromycin. Linearization of the vector and inclusion of 2-mercaptoethanol in the transformation reaction resulted in enhanced transformation efficiency. Approximately 4 x 10(3) transformants/micrograms DNA per 10(7) protoplasts were obtained using the optimized procedure. Southern hybridization after alternating field and standard electrophoresis suggested random insertion of tandem repeats (some greater than 250 kb) into the fungal chromosomes. Antibiotic resistance was stable through mitosis. However, expression of the transforming DNA after meiosis was highly variable.
Mol Gen Genet 1991 Jan
PMID:Analysis of a high frequency transformation system for Ophiostoma ulmi, the causal agent of Dutch elm disease. 200 87

The effect of hypoxia on myocardial lipolysis (glycerol release) was investigated in freshly isolated, calcium-tolerant rat ventricular myocytes. Hypoxia was produced by gassing the incubation medium (Joklik-minimum essential medium, supplemented with 1.2 mM MgSO4, 1 mM DL-carnitine, 1.5 mM CaCl2 and 0.6 mM palmitate bound to 0.15 mM fatty acid free bovine serum albumin) with 95% N2-5% CO2. Control (normoxic) incubations were carried out under air-5% CO2 atmosphere. Basal glycerol release increased from 46.6 +/- 3.0 nmol/10(6) cells.30 min in normoxia to 64.5 +/- 4.3 nmol/10(6) cells.30 min in hypoxia (p less than 0.05). Addition of isoprenaline (10 microM) resulted in a significant (p less than 0.05) stimulation of the glycerol release both in normoxia and in hypoxia, but the enhancement above basal rates was apparently lower in hypoxia (8.7 +/- 2.5 nmol/10(6) cells.30 min) than in normoxia (12.2 +/- 2.7 nmol/10(6) cells.30 min). Furthermore, whereas the isoprenaline-induced rise in lipolysis both in normoxia and hypoxia was prevented by inclusion of propranolol (10 microM), propranolol did not affect the hypoxia-induced increase in lipolysis. Thus, the above findings suggest that myocardial lipolysis may be stimulated by local non-adrenergic mechanisms during hypoxia.
Mol Cell Biochem
PMID:Effects of hypoxia on lipolysis in isolated rat myocardial cells. 277 33

In the presence of a 30 nM prazosin mask, [3H]-2-(2,6-dimethoxyphenoxyethyl) aminomethyl-1,4-benzodioxane ([3H]WB4101) can selectively label 5-HT1 serotonin receptors. Serotonin exhibits high affinity (Ki = 2.5 nM) and monophasic competition for [3H] WB4101 binding in cerebral cortex. Furthermore, we have found a significant correlation (r = 0.96) between the affinities of a number of serotonergic and nonserotonergic compounds at [3H]WB4101-binding sites in the presence of 30 nM prazosin and [3H] lysergic acid diethylamide ([3H]LSD)-labeled 5-HT1 serotonin receptors in homogenates of rat cerebral cortex. Despite similar pharmacological profiles, distribution studies indicate that, in the presence of 5 mM MgSO4, the Bmax of [3H]WB4101 is significantly lower than the Bmax of [3H]LSD in various brain regions. WB4101 competition for [3H] LSD-labeled 5-HT1 receptors fits best to a computer-derived model assuming two binding sites, with the KH for WB4101 being similar to the KD of [3H]WB4101 binding derived from saturation experiments. This suggests that [3H]WB4101 labels only one of the subtypes of the 5-HT1 serotonin receptors labeled by [3H]LSD. Interestingly, the selective 5-HT1A serotonin receptor antagonist, spiperone, and the selective 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino) tetraline, exhibit high affinity and monophasic competition for [3H]WB4101 but compete for multiple [3H]LSD 5-HT1 binding sites. These data indicate that [3H]WB4101 selectively labels the 5-HT1A serotonin receptor, whereas [3H] LSD appears to label both the 5-HT1A and the 5-HT1B serotonin receptor subtypes. The divalent cations, Mn2+, Mg2+, and Ca2+ were found to markedly increase the affinity and Bmax of [3H]WB4101 binding in cerebral cortex. Conversely, the guanine nucleotides guanylylimidodiphosphate and GTP, but not the adenosine nucleotide ATP, markedly reduce the Bmax of [3H]WB4101 binding. These characteristics are typical of agonists interacting with receptors which modulate cellular function via a guanine nucleotide-regulatory subunit.
Mol Pharmacol 1985 Dec
PMID:[3H]WB4101 labels the 5-HT1A serotonin receptor subtype in rat brain. Guanine nucleotide and divalent cation sensitivity. 286 62

Rat thymocytes were exposed in vitro to the corticosteroid dexamethasone, 10 nM, for 10 min, or to oleic acid, 500 nM for 2 min. This results in cytolysis after 6 hr, if incubation is continued. Instead, the cells were centrifuged, the supernatant fluid decanted, and the cells subjected to osmotic shock in 1.5 mM MgCl2. The naked nuclei were incubated at 37 degrees C and examined by light and electron microscopy. Nuclear edema was evident early, and most nuclei showed damage with variation in shape and size and distinct folds, which was maximal by 1-2 hr as a result of these treatments. This was true also if nuclei were incubated in MgF2 or Mg(NO3)2 but not in MgBr2, MgI2, MgSO4 or Mg-citrate. Spleen lymphocyte nuclei showed similar damage but only after incubation with 20 microM oleic acid, and not at all with corticosteroids. The effects of both steroid and fatty acid, even at greatly increased concentrations, were inhibited by tri-n-butyl tin chloride, 10 microM, and by 4-4'-diisothiocyanostilbene-2,2'-disulfonic acid, sodium salt, 10 microM, both of which block chloride ion transport. It is concluded that the cytolytic effects of both corticosteroids and free fatty acids involve influx of chloride ion resulting in nuclear edema, which subsequently leads to fragmentation of chromatin, karyorrhexis and, ultimately, cytolysis.
Mol Cell Biochem 1984 Sep
PMID:Role of anions in the lymphocytolytic action of corticosteroids and fatty acids. 649 16

The binding of [3H]nicotine to mouse brain has been measured and subsequently compared with the binding of [125I]alpha-bungarotoxin (alpha-BTX) and L-[3H]quinuclidinyl benzilate (QNB). The binding of nicotine was saturable, reversible, and stereospecific. The average KD and Bmax were 59 nM and 88 fmoles/mg of protein, respectively. Although the rates of association and dissociation of nicotine were temperature-dependent, the incubation temperature had no effect on either KD or Bmax. When measured at 20 degrees or 37 degrees, nicotine appeared to bind to a single class of binding sites, but a second, very low-affinity, binding site was observed at 4 degrees. Nicotine binding was unaffected by the addition of NaCl, KCl, CaCl2, or MgSO4 to the incubation medium. Nicotinic cholinergic agonists were potent inhibitors of nicotine binding; however, nicotinic antagonists were poor inhibitors. The regional distribution of binding was not uniform: midbrain and striatum contained the highest number of receptors, whereas cerebellum had the fewest. Differences in site densities, regional distribution, inhibitor potencies, and thermal denaturation indicated that nicotine binding was not the same as either QNB or alpha-BTX binding, and therefore that receptors for nicotine may represent a unique population of cholinergic receptors.
Mol Pharmacol 1982 Nov
PMID:Characterization of nicotine binding in mouse brain and comparison with the binding of alpha-bungarotoxin and quinuclidinyl benzilate. 715 23

ATPases of cardiac cells are known to be among the most important enzymes to maintain the fluxes of vital cations by hydrolysis of the terminal high-energy phosphate of ATP. Biochemically the activities of Ca(2+)-pump ATPase, Ca2+/Mg(2+)-ecto ATPase, Na+,K(+)-ATPase and Mg(2+)-ATPase are determined in homogenates and isolated membranes as well as in myofibrillar and mitochondrial fractions of various purities. Such techniques permit estimation of enzyme activities in vitro under optimal conditions without precise enzyme topography. On the other hand, cytochemical methods demonstrate enzyme activity in situ, but not under optimal conditions. Until recently several cytochemical methods have been employed for each enzyme in order to protect its specific activity and precise localization but the results are difficult to interpret. To obtain more consistent data from biochemical and cytochemical point of view, we modified cytochemical methods in which unified conditions for each ATPase were used. The fixative solution (1% paraformaldehyde-0.2% glutaraldehyde in 0.1 M Tris Base buffer, pH 7.4), the same cationic concentrations of basic components in the incubation medium (0.1 M Tris Base, 2 mM Pb(NO2)3, 5 mM MgSO4, 5 mM ATP) and selective stimulators or inhibitors were employed. The results reveal improved localization of Ca(2+)-pump ATPase, Na(+)-K+ ATPase and Ca2+/Mg(2+)-ecto ATPase in the cardiac membrane.
Mol Cell Biochem
PMID:Renaissance of cytochemical localization of membrane ATPases in the myocardium. 749 46

To study the mechanism of action of diflubenzuron (DFB) and other benzoylphenylureas, we have initially hypothesized that their action may be related to exocytosis: to test the hypothesis, we obtained an intracellular vesicle preparation from the homogenate of integument of newly molted American cockroachs (Periplaneta americana L.) in 10 mM MES buffer containing 250 mM sucrose (isotonic) and 2.5 mM MgSO4, at pH 6.6. By studying DFB's effect on various ion transporting activities, we demonstrated that calcium uptake in this intracellular particulate preparation was significantly inhibited by DFB at low concentrations (e.g., 10(-8) M). Such an inhibitory effect on DFB on Ca2+ uptake was eliminated by the addition of ionophores or membrane disruptors, as well as the sonication of vesicle preparation. On the other hand, oligomycin, protein phosphorylation modulators, Na+, and Li+ did not affect the calcium uptake. Among ionophores, agents disrupting H+ gradients (e.g. FCCP and NEM) totally eliminated 45Ca uptaking activity by vesicles as well as the inhibitory effect of DFB. Among calcium ion modulators, calmodulin inhibitors such as calmidazolium and trifluoperazine decreased the Ca2+-uptake, whereas membrane calcium channel blocker, verapamil, did not. ATP and gamma-S-GTP stimulated Ca2+ uptake. However, the former increased only the DFB insensitive portion and the latter largely the DFB sensitive part of Ca2+. Together these data support the hypothesis that the action site of DFB in this preparation is the GTP-dependent Ca2+ transport process which is coupled to vacuolar type intracellular vesicles in the integument cells.
Insect Biochem Mol Biol 1994 Dec
PMID:Diflubenzuron affects gamma-thioGTP stimulated Ca2+ transport in vitro in intracellular vesicles from the integument of the newly molted American cockroach, Periplaneta americana L. 770 84

The influence was investigated of DNA gyrase-inhibiting drugs on the expression of various genes of Bordetella pertussis. We show that the promoters of the virulence regulatory bvg locus and of several bvg-regulated virulence factors, such as the fha, ptx, cya, fim2 and vrg6 loci are very sensitive to the action of novobiocin and coumermycin A, as reflected by transcriptional differences in gene expression. Inhibition of DNA gyrase by the drugs led to a strong decrease in transcription of these genes. Interestingly, one gene belonging to the bvg virulence regulon behaved differently: the promoter of the prn locus, coding for the outer membrane protein pertactin, involved in bacterial adhesion to eukaryotic cells, was induced after inhibition of DNA gyrase. The expression of other genes not belonging to the bvg regulon, such as those encoding porin (POR) and superoxide dismutase (SodB), were not, or only weakly, affected by the drugs. This demonstrates that with respect to drug-induced changes in DNA supercoiling there exist different types of promoters in B. pertussis. In an attempt to identify additional regulatory mechanisms that may modulate virulence gene expression, we investigated the effect of various environmental stimuli on the stability of the bvg-regulated vrg6 and the bvg-independent sodB transcripts. We found that some signals transduced via by the BvgS sensor protein, such as variations in the growth temperature or the presence of nicotinic acid, exerted a strong effect on the half life of these transcripts, whereas another modulating agent, MgSO4, did not have any influence.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Gen Genet 1995 Apr 10
PMID:Global regulatory mechanisms affect virulence gene expression in Bordetella pertussis. 771 7


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