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Biomedical subjects

D Jay

Publications and source records attributed to D Jay.

At least 19 recordsLinked to original sources
PubMed2000

Determination of a cAMP-dependent protein kinase phosphorylation site in the C-terminal region of human endothelial actin-binding protein.

Three different C-terminal regions of human endothelial actin-binding protein-280 (ABP-280 or ABP; nonmuscle filamin) were subcloned and efficiently expressed in the Escherichia coli BL21 (DE3) system as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As predicted by the aminoacid sequence one of the fragments, a 109-kDa peptide (residues 1671-2647), contained a calpain cleavage site and two potential cAMP-dependent protein kinase (PKA) phosphorylation sites (serine 2152 and threonine 2336). A second fragment, a 74-kDa peptide (residues 1671-2331), contained a calpain cleavage site and one of the three presumptive PKA phosphorylation sites (serine 2152). The third fragment, a 48-kDa peptide (residues 2223-2647), contained only one of the PKA sites (threonine 2336). Phosphorylation of these truncated peptides indicated that only the fragments containing serine 2152 incorporated phosphate after PKA treatment. Site-directed mutagenesis analysis confirmed that serine 2152 is the unique substrate for PKA in the C-terminal region of ABP. The functional significance of phosphorylation of this residue, which belongs to a serine-proline motif, is discussed.

Amino Acid Motifs↗
PubMed1997

Expression in Escherichia coli, phosphorylation with cAMP-dependent protein kinase and proteolysis by calpain of a 71-kDa domain of human endothelial actin binding protein.

A middle region of human endothelial actin-binding protein (ABP) was subcloned and expressed in the pT7-7/E. coli BL21 (DE3) system. As predicted by the amino acid sequence this 71 kD truncated protein (residues 1717-2360) contained a calpain cleavage site and two of the three presumptive cAMP-dependent protein kinase phosphorylation sites. This peptide fragment comprised all the elements needed to confer stability against calpain proteolysis to ABP after PKA phosphorylation.

Binding Sites↗
PubMed1997

Expression of Meis2, a Knotted-related murine homeobox gene, indicates a role in the differentiation of the forebrain and the somitic mesoderm.

Knotted (Kn) genes are expressed within restricted domains of the plant meristems and play a key role in the control of plant morphogenesis. We have isolated the Kn-related gene Meis2 in mouse, which labels the lateral somitic compartment and its derivatives during early mouse embryogenesis and later becomes a marker for the dorso-ectodermal region overlying cells of the paraxial mesoderm. Meis2 is also highly expressed in specific areas of the developing central nervous system from embryonic day 9 (e9) onward. In later developmental stages, a strong expression is detectable in differentiating nuclei and regions of the forebrain, midbrain, hindbrain, and spinal cord. This temporal and spatial expression pattern suggests that Meis2 may play an important role in the cascade of induction leading to somitic differentiation as well as in brain regionalization and histogenesis.

Amino Acid Sequence↗
PubMed1996

Effect of cadmium on calcium transport in a human fetal hepatic cell line (WRL-68 cells).

Toxic metals appear to use the transport pathways that exist for biologically essential metals. Calcium uptake in cells occurs through specific membrane channels. Since cadmium inhibits calcium uptake, this study was carried on to elucidate the mechanism of Cd interference with calcium transport using the fetal hepatic cell line WRL-68 as an in vitro model. Ca accumulation by WRL-68 cells presented an initial rapid phase, followed by a sustained phase of slower accumulation over a 60 min period. A concentration of 50 microM CdCl2 produced 39% inhibition of the uptake of CaCl2 (100 microM), while 100 microM nifedipine or verapamil decreased Ca accumulation by 35 and 63%, respectively. All Cd concentrations tested produced significant decrease in Ca uptake in a concentration-dependent manner at 1 min and thereafter, although with 10 microM CdCl2 no significant difference was found after 30 min of incubation. From the Lineweaver-Burk plot, we found that Cd exerted a competitive inhibition on Ca uptake, since there was no significant effect on the Vmax but an increased K(m). A second order rate constant of Cd inactivation of 0.061 mM-1.s-1 was determined from the course of Ca uptake during Cd inhibition. SH groups seemed to play an essential role in Ca inhibition uptake by Cd because the inhibition of Ca accumulation by 50 microM Cd was practically reversed after the addition of dithiothreitol.

Biological Transport↗
PubMed1995

Superoxide dismutase activity of the captopril-iron complex.

With an assay that generates superoxide anion radicals without the intervention of metal ions we investigated the antioxidant properties of captopril, an angiotensin-converting enzyme inhibitor with a sulfhydryl group. Under these conditions, increasing concentrations of the drug were seen not to scavenge O.-2 directly. However, a combination of captopril and iron could bring about the breakdown of the superoxide anion; a result that may help to understand the free radical-scavenging properties of captopril.

Antioxidants↗
PubMed1994

Expression in Escherichia coli and phosphorylation with cAMP-dependent protein kinase of the N-terminal region of human endothelial actin-binding protein.

The N-terminal region of human endothelial actin-binding protein was subcloned and expressed in the pT 7-7/E. coli BL 21 (DE 3) system. This peptide was efficiently expressed in E. coli as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by western analysis using a monoclonal antibody raised against this part of the molecule. As predicted by the amino acid sequence this truncated protein (residues 21-1569), corresponding to 164 KD and containing the actin-binding domain near the amino-terminus, could be phosphorylated by cAMP-dependent protein kinase unmasking a phosphorylation site which is not apparent in the native ABP protein.

Actins↗
PubMed1994

Existence of multiple phosphorylated forms of human platelet actin binding protein.

Platelet actin binding protein (ABP) as isolated from human platelets exists in at least four phosphorylated forms which we have designated ABP-0, ABP-1, ABP-2, and ABP-3 whose phosphate content ranges from 18 (ABP-0) to 40 (ABP-3) moles Pi/mole ABP. These forms differ in their resistance to calpain cleavage and ability to cross-link F-actin with ABP-3 being the best in each of these properties. Attempts to phosphorylate ABP-1, two or three with protein kinase C (PKC) were unsuccessful except if the proteins were pretreated with Escherichia coli alkaline phosphatase. All of the forms could be phosphorylated with cAMP-dependent kinase (PKA) and subsequent resistance to calpain cleavage conferred. Phosphorylation/dephosphorylation of ABP may be an important regulatory mechanism by which the cytoskeletal architecture is stabilized or transformed.

Actins↗
PubMed1993

Inhibition of membrane-bound succinate dehydrogenase by fluorescamine.

Fluorescamine rapidly inactivated membrane-bound succinate dehydrogenase. The inhibition of the enzyme by this reagent was prevented by succinate and malonate, suggesting that the group modified by fluorescamine was located at the active site. The modification of the active site sulfhydryl group by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) did not alter the inhibitory action of fluorescamine. However, the protective effect of malonate against fluorescamine inhibition was abolished in the enzyme modified at the thiol.

Animals↗
PubMed1992

Study of a Fenton type reaction: effect of captopril and chelating reagents.

The purpose of this study was to determine if captopril, an angiotensin-converting enzyme inhibitor, could interact with iron ions and so modify a Fenton type reaction. Results indicate that different degrees of thiobarbituric acid-reactive substance from deoxyribose are obtained in an ascorbate-driven Fenton system depending on the order of addition of captopril and iron to the incubation medium. Similar results were obtained with the chelating reagents ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid, indicating that the buffer solution plays a relevant role when a particular iron complex is formed with a chelating agent. These metal complexes produce oxidizing species in a Fenton type system whose nature is discussed.

Captopril↗
PubMed1991

Captopril does not scavenge superoxide: captopril prevents O2-. production by chelating copper.

The purpose of this study was to use a direct method, that of electron spin resonance spectroscopy, to evaluate the ability of captopril, an angiotensin-converting enzyme inhibitor, to prevent the superoxide-mediated formation of phenyl radicals. Results indicate that, under certain conditions, captopril is a potent inhibitor of the generation of phenyl radicals, produced by the autoxidation of phenylhydrazine. The inhibitory effect of captopril, however, was better understood as a direct interaction of the drug with the metals that catalyze the autoxidation process rather than as a reaction of captopril with the free radicals generated. This last conclusion was supported by the finding that captopril was not able to inhibit the superoxide anion-mediated reduction of nitroblue tetrazolium.

Captopril↗
PubMed1991

Inhibition of membrane-bound succinate dehydrogenase by disulfiram.

The effect of disulfiram on succinate oxidase and succinate dehydrogenase activities of beef heart submitochondrial particles was studied. Results show that disulfiram inhibits both functions. Succinate and malonate suppress the inhibitory action of disulfiram when succinate dehydrogenase is stabilized in an active conformation. Disulfiram is not able to inhibit the enzyme when succinate dehydrogenase is inactivated by oxaloacetate. The inhibitory effect of disulfiram is reverted by the addition of dithiothreitol. From these results, it is proposed that disulfiram inhibits the utilization of succinate by a direct modification of an -SH group located in the catalytically active site of succinate dehydrogenase.

Animals↗
PubMed1991

Study of the interaction of cadmium with membrane-bound succinate dehydrogenase.

Cadmium ions inhibit membrane-bound succinate dehydrogenase with a second-order rate constant of 10.42 mM-1 s-1 at pH 7.35 and 25 degrees C. Succinate and malonate protect the enzyme against cadmium ion inhibition. The protection pattern exerted by succinate and malonate suggests that the group modified by cadmium is located at the active site. The pH curve of inactivation by Cd2+ indicates the involvement of an amino acid residue with pKa of 7.23.

Animals↗
PubMed1987

The mechanism of lead-induced mitochondrial Ca2+ efflux.

Addition of Pb2+ to rat kidney mitochondria is followed by induction of several reactions: inhibition of Ca2+ uptake, collapse of the transmembrane potential, oxidation of pyridine nucleotides, and a fast release of accumulated Ca2+. When the incubation media are supplemented with ruthenium red, the effect of Pb2+ on NAD(P)H oxidation, membrane delta psi, and Ca2+ release are not prevented if malate-glutamate are the oxidizing substrates; however, the latter two lead-induced reactions are prevented by ruthenium red if succinate is the electron donor. It is proposed that in mitochondria oxidizing NAD-dependent substrates, Pb2+ induces Ca2+ release by promoting NAD(P)H oxidation and a parallel drop in delta psi due to its binding to thiol groups, located in the cytosol side of the inner membrane. In addition, it is proposed that with succinate as substrate, the Ca2+ -releasing effect of lead is due to the collapse of the transmembrane potential as a consequence of the uptake of Pb2+ through the calcium uniporter, since such effect is ruthenium red sensitive.

Animals↗
PubMed1987

Control of mitochondrial Ca2+ retention by ADP-stimulated glutamic dehydrogenase.

The protective effect of ADP on unspecific Ca2+ release and collapse of the transmembrane potential was analyzed in mitochondria from kidneys of rats. The presence of ADP in the incubation mixture prevents Ca2+ leakage and collapse of delta psi in sucrose-containing medium, but fails to do so in KCl medium. The effect of the adenine nucleotide in sucrose media correlates with an increase in the level of reduced pyridine nucleotides; the increase was due to a stimulatory effect on the activity of glutamic dehydrogenase. It also was observed that in KCl media, in the presence and in the absence of ADP the rate of NADH oxidation through the respiratory chain was higher than in sucrose; in this latter medium a high level of reduced pyridine nucleotides was found, in comparison to KCl media. It is proposed that the role of ADP is to increase glutamic dehydrogenase activity and in consequence to provoke a higher rate of formation of NADH which in turn controls Ca2+ release.

Adenosine Diphosphate↗
PubMed1986

On the role of K+ on succinic dehydrogenase activity.

The effect of potassium ions on succinic dehydrogenase activity of mitochondria was studied. The results showed that in these organelles K+ induces inhibition of the respiratory control; moreover, in submitochondrial particles potassium inhibits the rate of oxidation of succinate. The results showed also that K+ does not changes the Km for succinate but diminishes the Vmax. In addition, the data provide evidence that mitochondria oxidizing glutamate-malate in a sucrose medium show a higher activity of succinate dehydrogenase than mitochondria incubated in KCl.

Adenosine Triphosphate↗
PubMed1985

Evidence for the involvement of dithiol groups in mitochondrial calcium transport: studies with cadmium.

The effect of cadmium on some functions of mitochondria isolated from kidneys of rat was studied. Addition of cadmium chloride to mitochondria induced stimulation of both State 4 respiratory rate and ATPase activity, which are prevented by the addition of ruthenium red. We also show that cadmium inhibits competitively calcium translocation; this inhibitory effect of cadmium is reverted by the addition of dithiothreitol. From these results, it is proposed that, similarly to Ca2+, cadmium penetrates mitochondria and binds to a membrane dithiol group, which is essential for the translocation of the cation.

Animals↗