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J E Wilson

Publications and source records attributed to J E Wilson.

At least 19 recordsLinked to original sources
PubMed1995

Application of a double isotopic labeling method to a study of the interaction of mitochondrially bound rat brain hexokinase with intramitochondrial compartments of ATP generated by oxidative phosphorylation.

gamma-Labeled ATP was produced by rat brain mitochondria utilizing [32P]Pi as substrate for oxidative phosphorylation. The 32P/14C ratio of Glc-6-P produced by the endogenous mitochondrially bound hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) using [U-14C]Glc as substrate was determined as a function of time after initiation of oxidative phosphorylation. This same ratio was determined for Glc-6-P formed by added yeast hexokinase using extramitochondrial ATP as substrate. The specific activity of ATP formed by oxidative phosphorylation was manipulated either by initiating the reaction with labeled Pi and subsequently adding excess unlabeled Pi or by initiating the reaction with unlabeled Pi and introducing the labeled substrate at a later time. The 32P/14C ratio of Glc-6-P formed by yeast hexokinase, reflecting the specific activity of ATP in the extramitochondrial space, was rapidly responsive to such manipulations, but the corresponding changes in the 32P/14C ratio of Glc-6-P produced by the endogenous hexokinase were markedly different. The results are consistent with the view that mitochondrially bound hexokinase does not utilize extramitochondrial ATP as substrate but rather is functionally coupled to a discrete intramitochondrial compartment of ATP produced by oxidative phosphorylation.

Adenosine Triphosphate↗
PubMed1995

Residues putatively involved in binding of ATP and glucose 6-phosphate to a mammalian hexokinase: site-directed mutation at analogous positions in the N- and C-terminal halves of the type I isozyme.

Despite extensive sequence similarity between the N- and C-terminal halves of the Type I isozyme of mammalian hexokinase (ATP:D-hexose 6-phosphotransferase; EC 2.7.1.1), they are functionally distinct, the C-terminal half being responsible for catalysis and the N-terminal half thought to play a regulatory role. We have examined the effects of several site-directed mutations on kinetic and regulatory properties of the rat Type I isozyme. Mutation of the C-terminal residues, Asp 532 to Asn, Arg 539 to Met, and Gly 896 or Gly 898 to Val, resulted in drastic loss of catalytic activity (< 10% of wild-type enzyme), consistent with previous suggestions that these residues are involved in binding of ATP. Mutation of the corresponding residues in the N-terminal half of the enzyme caused much less marked (> 50% of wild type), but significant, effects on activity which are presumed to result from subtle effects on conformation of the enzyme. Mutation of Lys 899 to Met resulted in an approximately 50% decrease in specific activity and an approximately fivefold increase in the Km for ATP, consistent with the view that Lys 899 participates in binding of ATP through electrostatic interactions with the phosphate sidechain. Cys residues corresponding to Cys 158 and Cys 606 of Type I hexokinase are found in other hexokinases that exhibit marked sensitivity to inhibition by the product, glucose 6-phosphate (Glc-6-P), but analogous residues are not found in hexokinases insensitive to Glc-6-P. However, this correlation appears to be coincidental since neither the mutation of Cys 158 or Cys 606 to Ala nor any of the other mutations examined abolished sensitivity of Type I hexokinase to inhibition by the Glc-6-P analog 1,5-anhydroglucitol-6-P or to antagonism of this inhibition by P(i).

Adenosine Triphosphate↗
PubMed1995

Functional organization of mammalian hexokinases: characterization of chimeric hexokinases constructed from the N- and C-terminal domains of the rat type I and type II isozymes.

Chimeric hexokinases consisting of either the N-terminal half of Type I hexokinase fused with the C-terminal half of the Type II isozyme (NICII) or the inverse pair (NIICI), along with the parental isozymes, were expressed in COS-1 cells. The thermal stability of the chimeras was intermediate between that of the highly labile Type II isozyme and the relatively stable Type I hexokinase. In their Kms for substrates, Glc and ATP, the chimeric enzymes were similar to the parental isozyme from which the C-terminal half was derived. Although the Type I and Type II isozymes were similar in their sensitivity to inhibition (competitive vs ATP) by the Glc-6-P analogs, 1,5-anhydroglucitol 6-phosphate (AnGlc-6-P), and Glc-1,6-bisphosphate, the chimeric enzymes differed markedly, with the NIICI chimera being much more sensitive and the NICII chimera much less sensitive than either parental form to these inhibitors. In contrast, the response of the chimeras to Pi, either as an antagonist of inhibition by AnGlc-6-P or, at higher concentrations, as an inhibitor, was correlated with the origin of the N-terminal domain. The results are consistent with the view that catalytic function is associated with the C-terminal domain of the Type I isozyme, with regulatory function--inhibition by Glc-6-P and its analogs and antagonism of this inhibition by Pi--being mediated by the N-terminal domain.

Adenosine Triphosphate↗
PubMed1995

Hexokinases.

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Animals↗
PubMed1995

Synergistic effects of tacrolimus and human interferon-alpha A/D in murine viral myocarditis.

The effects of interferon-alpha A/D (IFN) therapy in combination with various immunosuppressants were investigated in a murine model of viral myocarditis. Viral infection is an important cause of morbidity in immunocompromised hosts and transplant recipients. Human IFN therapy reduces viral replication, reducing the virus-induced myocardial destruction. Groups consisting of 25 C3H/He mice received i.p. injections of prednisolone, azathioprine, 15-deoxyspergualin, cyclosporine or tacrolimus (FK506), for 16 days beginning 2 days before inoculation with 500 plaque-forming units of encephalomyocarditis virus (EMCV). IFN, 10(4) U/g daily, was administered i.p. alone or in combination with immunosuppressants to separate groups of mice beginning on the day of viral inoculation. Animals were sacrificed at random at 4 or 10 days after inoculation with EMCV. The survival rate was significantly higher in mice treated with azathioprine, 15-deoxyspergualin, cyclosporine or FK506 in combination with IFN than in infected controls (P < .01) and was similar to the rate in the IFN monotherapy group. Survival in mice treated with prednisolone resembled that in infected controls and was significantly lower than in mice treated with IFN (P < .01). Heart weight was lower and cellular infiltration in the myocardium was reduced in mice treated with both FK506 and IFN compared with mice given IFN monotherapy. The results suggest that the effect of IFN therapy in viral myocarditis differs depending on which immunosuppressants is used. The findings suggest that the combination of FK506 and IFN may have beneficial effects in hosts with viral myocarditis by reducing cellular infiltration of heart.

Animals↗
PubMed1995

Prominence of coronary arterial wall lipids in human heart allografts. Implications for pathogenesis of allograft arteriopathy.

Transplant arteriopathy is a major late complication in human heart allograft recipients and the pathogenesis of such arteriopathy remains uncertain. The degree to which lipids and atheromata are involved in the arteriopathic lesions remains unsettled, and there is uncertainty regarding the significance of insudation or retention of lipids within the coronary artery walls of transplanted hearts. On current immunosuppressive regimens, most patients experience an increased serum total cholesterol and low-density lipoprotein cholesterol after transplant. Elevation of these blood lipids has an undetermined relationship to arteriopathy. We carried out morphological, morphometric, immunohistochemical, ultrastructural, and biochemical studies of particular coronary artery segments from 23 unselected explant or autopsy allografts and donor age-matched native coronary controls. Patients died of cardiac and non-cardiac reasons over a period of 4 to 1610 days after transplant. Atheromata were frequent, and diffuse intra- and extra-cellular accumulation of lipids in both intimal and medial walls was documented by oil red O positivity, immunohistochemical staining (muscle-specific alpha-actin), transmission and scanning electron microscopy, and biochemical analysis. Mean total cholesterol, esterified cholesterol, free cholesterol, and phospholipid content (microgram/cm2 intimal surface area) and concentration (microgram/mg dry defatted weight) in arteriopathic coronaries were > 10-fold higher than in comparable native coronary segments. Extent of lipids in the arterial walls was highly correlated with digitized percent luminal narrowing, mean daily and cumulative cyclosporin dose, and mean cumulative prednisone dose. Our data suggests strongly that lipid accumulation is an important early and persistent phenomenon in the development of transplant arteriopathy.

Adolescent↗
PubMed1995

Infrequency of cytomegalovirus genome in coronary arteriopathy of human heart allografts.

In heart transplantation, long-term engraftment success is severely limited by the rapid development of obliterative disease of the coronary arteries. Data from various groups have been suggestive of a pathogenetic role of herpesviruses, particularly human cytomegalovirus, in accelerated allograft coronary artery disease; however, results are not yet conclusive. This study examines the hypothesis that human cytomegalovirus infection of allograft tissues is related pathogenetically and directly to accelerated coronary artery disease. Using in situ DNA hybridization and polymerase chain reaction, we examined particular coronary artery segments from 41 human heart allografts (ranging from 4 days to greater than 4 years after transplantation; mean, 457 days) and 22 donor age- and gender-comparable, coronary site-matched trauma victims for presence of human cytomegalovirus DNA. Human cytomegalovirus genome was detected in 8 of 41 (19.5%) allografts and in 1 of 22 (4.5%) control hearts. This difference in positivity was not statistically significant (P = 0.10). In the human cytomegalovirus-positive hearts, viral genome was localized to perivascular myocardium or coronary artery media or adventitia. Human cytomegalovirus genome was not detected in arterial intima of any allograft or control heart, although human cytomegalovirus genome was readily identified within intima of small pulmonary arteries from lung tissue with human cytomegalovirus pneumonitis. By statistical analyses, the presence of human cytomegalovirus genome was not associated with the nature or digitized extent of transplant arteriopathy, evidence of rejection, allograft recipient or donor serological data suggestive of human cytomegalovirus infection, duration of allograft implantation, or causes of death or retransplantation. Thus, our data indicate a low frequency of detectable human cytomegalovirus genome in accelerated coronary artery disease and do not support a direct role for human cytomegalovirus in vascular wall infection or in the development of accelerated coronary artery disease.

Adolescent↗
PubMed1994

The 50-kDa glucose 6-phosphate-sensitive hexokinase of Schistosoma mansoni.

Hexokinase has been purified from adult Schistosoma mansoni worms and the activity shown to be associated with a single protein species having an M(r) about 50,000. This protein is recognized on Western blots probed with antisera against rat Type I hexokinase or against a recombinant S. mansoni hexokinase that had been expressed in Escherichia coli using a previously cloned cDNA. An 18-residue N-terminal sequence determined for the purified S. mansoni hexokinase is identical to that deduced from the nucleotide sequence of the cDNA, consistent with the view that the cloned cDNA encodes the hexokinase characterized in the present study. The S. mansoni enzyme has a relatively low Km (approximately 60 microM) for glucose and is sensitive to inhibition (competitive versus ATP, Ki approximately 50 microM) by its product, glucose 6-phosphate (Glc-6-P). With these kinetic properties and 50 kDa molecular mass, S. mansoni hexokinase resembles the ancestral hexokinase predicted to have given rise, by gene duplication and fusion, to the present day 100-kDa Glc-6-P-sensitive mammalian hexokinases. The schistosomal hexokinase represents the first 50-kDa Glc-6-P-sensitive hexokinase whose sequence has been obtained. The schistosomal hexokinase does not bind to mitochondria, consistent with its lack of a hydrophobic segment at the N terminus which is required for binding of the mammalian Type I and II isoenzymes to mitochondria. The marked Crabtree effect exhibited by S. mansoni cercariae may be at least partly attributed to the expression of rather high levels of a hexokinase having a high affinity for glucose but only a moderate sensitivity to product inhibition by Glc-6-P.

Amino Acid Sequence↗
PubMed1994

Crystals of HIV-1 reverse transcriptase diffracting to 2.2 A resolution.

Reverse transcriptase (RT) from the human immunodeficiency virus type 1 has been crystallized in four closely related forms, the best of which diffract X-rays to 2.2 A resolution. The RT was crystallized as a complex with a non-nucleoside inhibitor, either nevirapine or a nevirapine analogue. Crystals grew from 6% PEG 3400 buffered at pH 5. These were of space group P2(1)2(1)2(1) with unit cell parameters a = 147 A, b = 112 A, c = 79 A (form A), with one RT heterodimer in the asymmetric unit. Changes in unit cell parameters and degree of crystalline order were observed on soaking pregrown crystals in various solutions, giving three further sets of unit cells. These were a = 143 A, b = 112, A, c = 79 A (form B), a = 141 A, b = 111 A, c = 73 A (form C), a = 143 A, b = 117 A, c = 66.5 A (form D). The last two forms diffract X-rays to 2.2 A resolution. Structure determinations of these latter crystal forms of RT should give a detailed atomic model for this therapeutically important drug target.

Crystallography, X-Ray↗
PubMed1994

Mitochondrial hexokinase in brain: coexistence of forms differing in sensitivity to solubilization by glucose-6-phosphate on the same mitochondria.

Hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) is associated with mitochondria from brain of various species. The fraction of the bound activity that can be released in soluble form after incubation of the mitochondria with glucose-6-phosphate (Glc-6-P) has been shown to vary markedly among species (F. Kabir and J.E. Wilson, Arch. Biochem. Biophys. 300, 641-650, 1993). A method has been developed by which mitochondria bearing significant amounts of bound hexokinase can be selectively immunoprecipitated by Staphylococcus aureus cells coated with anti-Type I hexokinase antibodies. Treatment of mitochondria from guinea pig, bovine, and human brain with Glc-6-P solubilized approximately 60, 40, and 20% of the bound hexokinase activity, respectively, but had no effect on the extent to which the mitochondria could be immunoprecipitated. This is consistent with the view that the residual hexokinase, resistant to solubilization with Glc-6-P, coexists on the same mitochondria bearing the Glc-6-P-sensitive form, i.e., removal of the latter does not prevent immunoprecipitation mediated by the Glc-6-P-resistant form. Mitochondrial porin has previously been shown to be involved in binding of hexokinase to mitochondria. Four porin species, having a common molecular weight but differing in isoelectric point, were detected, in the same relative amounts, in mitochondria from bovine and rat brain. The extent to which Glc-6-P solubilizes hexokinase from rat and bovine brain mitochondria is approximately 90 and approximately 40%, respectively. Thus the marked difference in sensitivity to solubilization with Glc-6-P cannot be attributed to differences in the relative amounts of different porin species present in rat and bovine mitochondria.

Animals↗
PubMed1994

Morphology and microtubule organization in Arabidopsis roots exposed to oryzalin or taxol.

In roots of Arabidopsis thaliana, we examined the effects of low concentrations of microtubule inhibitors on the polarity of growth and on the organization of microtubule arrays. Intact 6 d old seedlings were transplanted onto plates containing inhibitors, and sampled 12 h, 24 h and 48 h later. Oryzalin, a compound that causes microtubule depolymerization, stimulates the radial expansion of roots. The amount of radial swelling is linearly proportional to the logarithm of the oryzalin concentration, from the response threshold, 170 nM, to 1 microM. Cells in the zone of division were slightly more sensitive to oryzalin than were cells in the zone of pure elongation. Radial swelling is also stimulated by taxol, a compound that causes microtubule polymerization. Taxol at 1 microM causes little swelling, but at 10 microM causes extensive radial swelling of cells in the elongation zone, and does not affect cells in the division zone. To examine the microtubules in these roots, we used methacrylate sections with immunofluorescence microscopy. At all concentrations of oryzalin, cortical arrays are disorganized and depleted of microtubules, and the microtubules themselves often appear fragmented. These effects increase in severity with concentration, but are unmistakable at 170 nM. In taxol, cortical arrays appear to be more intensely stained than those of controls. At 10 microM, many cells in growing regions of the stele have longitudinal microtubules, whereas many cells in the cortex appear to have transversely aligned microtubules. Taxol affects microtubules in cells of division and elongation zones to the same extent, despite the observed difference in growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Arabidopsis↗
PubMed1994

Lipid overload and proteoglycan expression in chronic rejection of the human transplanted heart.

The degree to which transplant arteriopathy in solid organ allografts is an atheromatous process remains somewhat controversial. If atheromata develop as common and integral components of the arteriopathic lesions, then the process may be approached therapeutically in a manner analogous to native atheromatous diseases. Approaches to understanding the arteriopathic process may include not only the modulation of alloimmunity, but also the interruption of "storage" phenomena. We have examined the epicardial coronary arteries of nearly 50 explanted human heart allografts using biochemical, morphological, morphometrical, immunohistochemical, and molecular techniques in an effort to establish the degree, nature, and distribution of lipid accumulation in the vessel walls. Concomitantly, we studied the ascertainment of proteoglycan gene expression, represented by biglycan and decorin messenger RNA, and the localization of proteoglycan proteins in the vessels. The degree of lipid and proteoglycan buildup in both the intima and media of transplanted vessels is striking, and correlated strongly with intimal thickening, cross-sectional area reduction of the lumen, cumulative cyclosporine dose, corticosteroid dose, and serum cholesterol levels. Notably, lipid accumulation is not related to implant duration, this being true in an unselected series of "failed" allografts ranging from 4 to 1610 days post-transplant. The profound lipid accumulation in coronary walls of many grafts begins very early post-transplant and appears to contribute substantially to intimal thickening. Whether dysregulation of proteoglycan production leads to entrapment of lipids and lipoproteins remains an important and testable hypothesis.

Chronic Disease↗
PubMed1993

Mechanism of resistance of human immunodeficiency virus type 1 to 2',3'-dideoxyinosine.

A molecular clone containing the wild-type reverse transcriptase (RT) coding region of human immunodeficiency virus type 1 (HIV-1) was constructed, and site-directed mutagenesis was used to introduce mutations--Leu74-->Val (L74V), T215Y, and the combination L74V/T215Y--into the RT coding region. The proteins were purified by immunoaffinity chromatography. Assays were performed with mutant and wild-type RT to determine substrate and inhibitor specificity. All three mutant enzymes catalyzed the incorporation of substrate 2'-deoxynucleoside 5'-triphosphates (dNTPs) as efficiently as wild-type HIV-1 RT. Small changes were observed in the Km values for dNTPs with all three mutant enzymes, while more significant changes were noted in sensitivity to nucleoside 5'-triphosphate analogues that inhibit the enzyme activity. Results suggest that altered substrate recognition by the HIV-1 RT is involved in the mechanism of resistance.

Base Sequence↗
PubMed1993

Biochemical analysis of human immunodeficiency virus-1 reverse transcriptase containing a mutation at position lysine 263.

Site-directed mutagenesis has been used to assess the importance of lysine 263 in substrate binding of human immunodeficiency virus-1 (HIV-1) reverse transcriptase. Previous studies have indicated that lysine 263 functions in the binding of 2'-deoxynucleoside 5'-triphosphate (dNTP) substrates (Basu, A., Tirumalai, R. S., and Modak, M. J. (1989) J. Biol. Chem. 264, 8746-8752). We studied this interaction directly by using site-specific mutagenesis to change lysine 263 to a serine. Highly purified mutant enzyme K263S bound natural dNTP substrates and primed polynucleic acid substrates with equal affinity when compared to the wild type reverse transcriptase. No difference was observed in the binding of 3'-azido-2',3'-dideoxythymidine 5'-triphosphate to the mutant reverse transcriptase on the basis of Km and Ki determinations. The serine substitution had no effect on RNase H activity. These results indicate that lysine 263 is not essential in the binding of substrates to HIV-1 reverse transcriptase.

Base Sequence↗
PubMed1993

Mitochondrial hexokinase in brain of various species: differences in sensitivity to solubilization by glucose 6-phosphate.

Approximately 90% of the hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) activity was solubilized by treatment of rat brain mitochondria with glucose 6-phosphate (Glc-6-P), while only about 20% of the hexokinase could be solubilized from human brain mitochondria. Intermediate amounts of solubilized activity were obtained with brain mitochondria from other species. In contrast, > or = 80% of the activity could be released by 0.5 M potassium thiocyanate (KSCN), regardless of the species from which the mitochondria were obtained. Hexokinase activities solubilized by treatment of bovine brain mitochondria with Glc-6-P (HKG6P) and by a subsequent treatment with KSCN (HKKSCN) were indistinguishable in their isoelectric focusing pattern and molecular weight, and both were inhibited by Glc-6-P with Ki approximately 20 microM. Both HKG6P and HKKSCN could bind to mitochondria from rat liver or brain, and both were again solubilized by a subsequent treatment with Glc-6-P. These results do not suggest any intrinsic molecular difference between HKG6P and HKKSCN. Rather, the difference in susceptibility to release by Glc-6-P is reasonably attributed to discrete types of binding sites for hexokinase on brain mitochondria, with the relative proportion of these varying with species. Bovine brain mitochondria bearing HKG6P and HKKSCN were not resolved by sucrose density gradient fractionation, suggesting that both forms may coexist on the same mitochondrion. Given the probable importance of mitochondrially bound hexokinase in regulating aerobic glycolysis in brain, these differences in hexokinase-mitochondrial interactions may be related to previously documented differences in cerebral energy metabolism of these various species.

Adolescent↗