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Biomedical subjects

J E Wilson

Publications and source records attributed to J E Wilson.

At least 19 recordsLinked to original sources
PubMed1998

Product release is the major contributor to kcat for the hepatitis C virus helicase-catalyzed strand separation of short duplex DNA.

Hepatitis C virus (HCV) helicase catalyzes the ATP-dependent strand separation of duplex RNA and DNA containing a 3' single-stranded tail. Equilibrium and velocity sedimentation centrifugation experiments demonstrated that the enzyme was monomeric in the presence of DNA and ATP analogues. Steady-state and pre-steady-state kinetics for helicase activity were monitored by the fluorescence changes associated with strand separation of F21:HF31 that was formed from a 5'-hexachlorofluorescein-tagged 31-mer (HF31) and a complementary 3'-fluorescein-tagged 21-mer (F21). kcat for this reaction was 0.12 s-1. The fluorescence change associated with strand separation of F21:HF31 by excess enzyme and ATP was a biphasic process. The time course of the early phase (duplex unwinding) suggested only a few base pairs ( approximately 2) were disrupted concertedly. The maximal value of the rate constant (keff) describing the late phase of the reaction (strand separation) was 0. 5 s-1, which was 4-fold greater than kcat. Release of HF31 from E. HF31 in the presence of ATP (0.21 s-1) was the major contributor to kcat. At saturating ATP and competitor DNA concentrations, the enzyme unwound 44% of F21:HF31 that was initially bound to the enzyme (low processivity). These results are consistent with a passive mechanism for strand separation of F21:HF31 by HCV helicase.

Adenosine Triphosphate↗
PubMed1998

Further studies on the coupling of mitochondrially bound hexokinase to intramitochondrially compartmented ATP, generated by oxidative phosphorylation.

Hexokinase, bound to nonphosphorylating rat brain mitochondria, exhibits Michaelis-Menten kinetic behavior, with an apparent K(m) for ATP of 0.44 +/- 0.08 mM. After initiation of oxidative phosphorylation, a steady-state rate of Glc phosphorylation is maintained despite the fact that extramitochondrial [ATP] continues to increase but remains well below saturating levels (i.e., < 0.4 mM). This independence from extramitochondrial [ATP] is taken to indicate that hexokinase is not utilizing extramitochondrial ATP as substrate, but rather draws substrate ATP from an intramitochondrial compartment supplied by oxidative phosphorylation. The steady-state rate of Glc phosphorylation by hexokinase bound to phosphorylating mitochondria is not altered by increase in total rate of ATP production resulting from addition of hexokinase-depleted mitochondria to the system. In contrast, the steady-state rate of Glc phosphorylation by yeast hexokinase, which does not bind to mitochondria, is directly related to the total rate of ATP production in the system. These results are also consistent with the view that, during oxidative phosphorylation, mitochondrially bound hexokinase is selectively using intramitochondrially compartmented ATP; such substrate selectivity would be expected to require physical association of hexokinase with the mitochondria and be dependent solely on the oxidative phosphorylation activity of the hexokinase-bearing organelles. The K(m) for Glc is only modestly affected by the binding of hexokinase to mitochondria and not further altered upon induction of active oxidative phosphorylation, suggesting that neither binding nor oxidative phosphorylation greatly affects the conformation of the Glc binding site. The reliance on intramitochondrial ATP is suggested to result from oxidative phosphorylation-dependent changes in the interaction between the mitochondrial surface and the regions of the hexokinase molecule involved in binding ATP.

Adenosine Diphosphate↗
PubMed1998

Induction of virus-induced IDDM in virus resistant mice without lymphocyte maturation.

The role of lymphocytes in the pathogenesis of viral-induced insulin dependent diabetes mellitus (IDDM) is controversial. To better understand how a virus-induced IDDM depends on the infiltrating lymphocytes, encephalomyocarditis virus (EMCV) was inoculated intraperitoneally into three kinds of mice; virus-susceptible C57BL/6, virus-resistant 129/SV and recombination activity gene-2 (Rag2) knockout 129/SV mice. Pancreatic inflammation and beta cell necrosis were evaluated after EMCV, D variant (10(3) pfu/mouse) inoculation. On post-inoculation day 14, the lethal rates of C57BL/6, 129/SV and Rag2 knockout mice were 52, 10 and 100%, respectively. The blood glucose in Rag2KO mice on day 8 was significantly elevated as compared with 129SV mice (231 +/- 49 vs 169 +/- 32 mg/dl, P<0.05). In situ hybridization demonstrated the EMCV genome in the pancreas of Rag2 knockout and C57BL/6 mice, but not in 129/SV mice. Beta cell necrosis were more severe in Rag-2 knockout mice than in wild type 129/SV mice, but lymphocyte infiltration was less severe than C57BL/6. Pancreas in Rag2 knockout mice infected with virus were affected more severely than the virus-resistant strain of mice. Diabetogenic virus induced IDDM in virus-resistant mice without mature lymphocytes.

Animals↗
PubMed1998

The structure of mammalian hexokinase-1.

We have determined the structures of the glucose-6-phosphate (G6P)-inhibitable 100,000 Mr Type I hexokinase from rat and the G6P-sensitive 50,000 Mr hexokinase from Schistosoma mansoni at a resolution of 2.8 and 2.6 A respectively. The structures define the glucose and G6P binding sites in these enzymes, suggest the mechanisms of intradomain G6P inhibition and activity loss in the Type I hexokinase N-terminal half, and reveal the structure of the membrane targeting motif that integrates the Type I hexokinase into the outer mitochondrial membrane.

Animals↗
PubMed1998

Caspase activation and specific cleavage of substrates after coxsackievirus B3-induced cytopathic effect in HeLa cells.

Coxsackievirus B3 (CVB3), an enterovirus in the family Picornaviridae, induces cytopathic changes in cell culture systems and directly injures multiple susceptible organs and tissues in vivo, including the myocardium, early after infection. Biochemical analysis of the cell death pathway in CVB3-infected HeLa cells demonstrated that the 32-kDa proform of caspase 3 is cleaved subsequent to the degenerative morphological changes seen in infected HeLa cells. Caspase activation assays confirm that the cleaved caspase 3 is proteolytically active. The caspase 3 substrates poly(ADP-ribose) polymerase, a DNA repair enzyme, and DNA fragmentation factor, a cytoplasmic inhibitor of an endonuclease responsible for DNA fragmentation, were degraded at 9 h following infection, yielding their characteristic cleavage fragments. Inhibition of caspase activation by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (ZVAD.fmk) did not inhibit the virus-induced cytopathic effect, while inhibition of caspase activation by ZVAD.fmk in control apoptotic cells induced by treatment with the porphyrin photosensitizer benzoporphyrin derivative monoacid ring A and visible light inhibited the apoptotic phenotype. Caspase activation and cleavage of substrates may not be responsible for the characteristic cytopathic effect produced by picornavirus infection yet may be related to late-stage alterations of cellular homeostatic processes and structural integrity.

Amino Acid Chloromethyl Ketones↗
PubMed1998

Distinguishing the type I and type II isozymes of hexokinase: the need for a reexamination of past practice.

The type I and type II isozymes of hexokinase coexist in insulin-sensitive tissues, such as cardiac and skeletal muscle and adipose tissue. Based on an early report that the purified type I isozyme was stable at 45 degrees C whereas the purified type II isozyme was not, investigators in a number of studies have used heat lability as a criterion for distinguishing these isozymes in crude tissue extracts or subcellular fractions; that is, activity lost after incubation at 45 degrees C was believed to be type II while remaining activity was considered type I. This extrapolation is dangerous because thermal lability can be markedly affected by the solvent environment, including the presence or absence of other proteins. In the present study, the rate of thermal inactivation of the type I isozyme has been shown to vary by at least an order of magnitude in soluble and particulate fractions prepared from rat heart and brain. Thus, the use of thermal stability as a general criterion for identifying the type I isozyme is invalid, and conclusions based on thermal inactivation as a means for distinguishing the type I and type II isozymes need to be reconsidered.

Animals↗
PubMed1997

Two Sp sites are important cis elements regulating the upstream promoter region of the gene for rat type I hexokinase.

Multiple transcriptional start sites have been identified for the gene encoding the rat Type I isozyme of hexokinase (White, J.A., Liu, W., and Wilson, J. E., Arch. Biochem. Biophys. 335, 161-172, 1996); these are clustered at positions approximately -460, -300, and -100 relative to the translational start codon (ATG, with A being +1). PC12 cells and H9c2 cells were transfected with luciferase reporter constructs containing genomic sequence between positions -3366 and -171. Marked (85%) decrease in promoter activity was associated with deletion of sequence between -742 and -516. In DNase I footprinting experiments, two regions, called P1 (-552 to -529) and P2 (-480 to -458) boxes, were protected by proteins present in nuclear extracts from PC12 cells. Mutation or deletion of the P2 box had no effect on promoter activity; protection in this region, which includes the most upstream cluster of transcriptional start sites, is attributed to binding of RNA polymerase II or associated factors. In contrast, mutations or deletions in the P1 box had markedly detrimental effects on promoter activity and on binding of proteins in PC12 cell nuclear extracts. Maintenance of a consensus Sp1 binding site centrally located in the P1 box was critical for both promoter activity and binding. A second Sp1 site (-570), just upstream from the P1 box, was also shown to be functionally important but no protection of this region was detected in footprinting experiments, presumably reflecting lower affinity at this site under the conditions used. Supershift experiments demonstrated the involvement of Sp1, Sp3, and Sp4 in formation of complexes with the P1 box region and implicate these transcription factors in regulating promoter activity associated with this region. Another series of reporter constructs, including sequence between -171 and -1, permitted detection of an additional promoter activity downstream from -364. While not yet extensively characterized, it is already evident that the cis elements influencing the downstream promoter activity are distinct from the Sp factors determined to be important in expression from the upstream promoter region.

Animals↗
PubMed1997

Structural determinants for the intracellular localization of the isozymes of mammalian hexokinase: intracellular localization of fusion constructs incorporating structural elements from the hexokinase isozymes and the green fluorescent protein.

Fusion constructs incorporating structural elements from mammalian isozymes of hexokinase, Types I-IV, in frame with sequence encoding the green fluorescent protein (GFP) have been made and expressed in hexokinase-deficient M + R 42 cells. Fusion proteins incorporating catalytically active regions from the Type II isozyme, or the entire Type IV sequence, were expressed in catalytically active form. The intracellular localization of the fusion proteins was determined using confocal microscopy. Fusion proteins including the N-terminal halves of the Type I or Type II isozymes were targeted to mitochondria, while the N-terminal half of the Type III isozyme did not confer mitochondrial targeting. The mitochondrial targeting signal was represented by the hydrophobic sequence at the extreme N-termini ("binding domain") of the Type I and Type II isozymes. Inclusion of the binding domain from the Type I isozyme was sufficient to confer mitochondrial binding on GFP itself as well as on constructs including the N-terminal half of Type III hexokinase. However, the Type I hexokinase binding domain was not sufficient to cause mitochondrial targeting of a construct containing the Type IV sequence. These results suggest that, although the binding domain is critical for mitochondrial targeting, other interactions involving an adjacent structure might also play a role. Fusion proteins including the N-terminal half of Type I hexokinase became dissociated from mitochondria under conditions favorable for accumulation of intracellular Glc-6-P. The 2-deoxy analog was much less effective than Glc in causing mitochondrial dissociation of the fusion construct, in accord with previous studies showing 2-deoxy-Glc-6-P to be much less effective than Glc-6-P at promoting release of Type I hexokinase from mitochondria. Dissociation, induced by formation of Glc-6-P or 2-deoxy-Glc-6-P, did not occur with the fusion protein including only the binding domain of Type I hexokinase. This is consistent with previous studies indicating that Glc-6-P-dependent dissociation results from binding of this ligand to a site in the N-terminal half of the enzyme, but which is not likely to be present in the small segment represented by the binding domain. These studies demonstrate the usefulness of this approach in defining structural elements involved in targeting hexokinase isozymes to specific subcellular locations and modulation of that intracellular location by perturbations of metabolic status.

Animals↗
PubMed1997

Structure of the gene for type I hexokinase from rat.

Based on presumed analogy with the previously characterized gene encoding the Type II isozyme of rat hexokinase (Printz, R.L., Koch, S., Potter, L.R., O'Dougherty, R.M., Tiesinga, J.J., Moritz, S., and Granner, D. K., J. Biol. Chem. 268, 5209-5219, 1993), the locations of splice sites in the gene encoding the rat Type I isozyme of hexokinase have been determined by PCR amplification of intronic DNA. Sequences at the splice sites conform to the consensus sequence, with GT and AG being found at 5' and 3' ends of the introns, respectively. Sizes of exons 1 and 2 were determined directly while others were estimated based on identified splice sites and the previously determined cDNA sequence. These exon sizes were confirmed by PCR amplification, which gave products having sizes consistent with those of introns and exons predicted to be within the amplified sequence. Thus, it is unlikely that the gene encoding the Type I isozyme contains any introns not having analogs in the gene for Type II hexokinase. The deduced structure for the rat Type I hexokinase gene is therefore identical to that for the rat Type II isozyme, and spans over 51 kb. Six tandem repeat sequences of (AC/GT)n have been identified in the 5' flanking region and in introns 10, 11, 12, and 16; this is an unusually high frequency of tandem repeat sequences.

Animals↗
PubMed1997

Functional organization of mammalian hexokinases: characterization of the rat type III isozyme and its chimeric forms, constructed with the N- and C-terminal halves of the type I and type II isozymes.

Previous studies have shown that catalytic function is associated with both halves of the Type II isozyme of mammalian hexokinase, while the Type I isozyme is functionally differentiated into a catalytic C-terminal half and regulatory N-terminal half. The Type III isozyme has now been shown to be similar to the Type I isozyme in its functional organization. Chimeras composed of the N-terminal half of Type III hexokinase and the C-terminal half of either Type I or Type II hexokinase have activities that can be attributed to the C-terminal half and are similar in activity to chimeras composed of the C-terminal half of Type III and the intrinsically inactive N-terminal domain of Type I or the inactivated (by site-directed mutation) N-terminal half of Type II hexokinase. Virtually no activity was seen with chimeras constructed with the N-terminal half of the Type III isozyme and catalytically inactive (by site-directed mutation) C-terminal halves of Type I or Type II hexokinase. Substrate inhibition by Glc is seen only with the Type III isozyme and with chimeric forms containing the C-terminal half of Type III hexokinase and the N-terminal half of Type I or Type II isozyme, the latter inactivated by site-directed mutation; this is attributed to conformational changes induced by binding of Glc to a low affinity site in the N-terminal half, with subsequent effect on catalytic activity of the C-terminal half. These results also provide further insight into the role of interactions (or lack of interactions) between the N- and C-terminal halves in the inhibition of the Type I-III isozymes by Glc-6-P, its antagonism by low concentrations of Pi, and the inhibition seen at higher concentrations of Pi.

Adenosine Triphosphate↗
PubMed1997

In vitro mutational and inhibitory analysis of the cis-acting translational elements within the 5' untranslated region of coxsackievirus B3: potential targets for antiviral action of antisense oligomers.

The 5' untranslated region (5'UTR) of coxsackievirus B3 (CVB3) RNA forms a highly ordered secondary structure that has been implicated in controlling initiation of viral translation by internal ribosomal entry. To test this hypothesis, synthetic bicistronic RNAs, with all or part of the 5'UTR in the intercistronic space, were translated in rabbit reticulocyte lysates. In the presence of an upstream cistron, the chloramphenicol acetyltransferase gene, designed to block ribosomal scanning, the CVB3 5'UTR was capable of directing the internal initiation of translation of the downstream reporter gene (P1), confirming the presence of an internal ribosomal entry site (IRES). This finding was further supported by the data on predicted secondary structures within the 5'UTR. Of special note, analysis of various deletion mutants demonstrated that the IRES of CVB3 is located roughly at stem-loops G, H, and I spanning nucleotides (nt) 529 and 630. The region from nt 1 to 63 (stem-loop A) also appears important, and it may be an essential binding site for translation initiation factors. Based on these findings, in vitro translation inhibition assays using RNA fragments of the 5'UTR as inhibitor were performed. Both antisense and sense RNA segments transcribed from these two cis-acting regions and the surrounding sequence of the initiation codon AUG showed strong inhibition of viral protein synthesis. Antisense molecules may inhibit translation by blocking ribosome and initiation factor binding within the 5'UTR via specific hybridization to their viral RNA target sequences, while sense sequences may function by competing with viral RNA for ribosomes and/or translation initiation factors. These cis-acting translational elements may serve as potential targets for the antiviral action of oligomers.

Animals↗
PubMed1997

Atraumatic and expeditious laparoscopic bowel handling using a new endoscopic device.

In laparoscopic colorectal surgery, effective handling of the bowel is mandatory to avoid bowel injury and excessive manipulation and to obtain adequate traction and clear exposure. We have developed a simple laparoscopic tool that permits effective and safe retraction of the small or large intestine and that is especially helpful in taking down the hepatic and splenic flexures and in dissecting the mesorectum.

Colon↗
PubMed1997

Human leukocyte antigen class II associations in patients with idiopathic dilated cardiomyopathy. Myocarditis Treatment Trial Investigators.

BACKGROUND: Idiopathic dilated cardiomyopathy (IDC) is a disease of unknown etiology for which immune abnormalities, possibly related to viral infections, are suspected but unproven. Previous serologic studies have reported associations between human leukocyte antigen DR4 and IDC. A molecular study of human leukocyte antigen associations was undertaken in patients with IDC to further explore the possibility of susceptibility markers of genetically determined disease. METHODS AND RESULTS: In this study, 36 patients from the Myocarditis Treatment Trial (32 IDC and 4 myocarditis patients) were examined using restriction fragment length polymorphism analysis and polymerase chain reaction amplification with sequence-specific primers to perform class II typing. All 4 myocarditis patients were DQ5 positive and 3 possessed the allele DQB1*0501. In the IDC group, the frequency of human leukocyte antigen DR4 was similar to that reported in the normal population. In addition, there was no excess prevalence of any molecularly defined DR4 alleles (0401-0419). There was an increase in the frequency of DR12 in IDC patients. The frequencies of the alleles DQB1 *0503 and DQB1*0301 and/or *0304 were also increased in IDC patients versus the normal population. CONCLUSION: The molecular studies point to a relationship between the DQ locus and IDC.

Adult↗
PubMed1997

Homologous and heterologous interactions between hexokinase and mitochondrial porin: evolutionary implications.

Binding of the Type I isozyme of mammalian hexokinase to mitochondria is mediated by the porin present in the outer mitochondrial membrane. Type I hexokinase from rat brain is avidly bound by rat liver mitochondria while, under the same conditions, there is no significant binding to mitochondria from S. cerevisiae. Previously published work demonstrates the lack of significant interaction of yeast hexokinase with mitochondria from either liver or yeast. Thus, structural features required for the interaction of porin and hexokinase must have emerged during evolution of the mammalian forms of these proteins. If these structural features serve no functional role other than facilitating this interaction of hexokinase with mitochondria, it seems likely that they evolved in synchrony since operation of selective pressures on the hexokinase-mitochondrial interaction would require the simultaneous presence of hexokinase and porin capable of at least minimal interaction, and be responsive to changes in either partner that affected this interaction. Recent studies have indicated that a second type of binding site, which may or may not involve porin, is present on mammalian mitochondria. There are also reports of hexokinase binding to mitochondria in plant tissues, but the nature of the binding site remains undefined.

Animals↗
PubMed1997

Conducting ecologically valid prevention research: recruiting and retaining a "whole village" in multimethod, multiagent studies.

Many prevention studies are now designed with complementary interventions in different settings. Evaluations of these interventions require assessing the child's behavior in each of these settings. Conducting these studies, therefore, may involve recruiting school districts, principals, classroom teachers, peers, parents, siblings, and in later years, employers and intimate partners. These participants may be considered natural raters or satellite subjects, depending on their degree of involvement. Issues of recruitment and retention thus are magnified in multimethod, multiagent studies. To illustrate these issues, findings are presented for three studies conducted with risk populations in the past decade at the Oregon Social Learning Center: a passive longitudinal study, a selected prevention study, and an indicated prevention study. Findings indicate that achieving high recruitment and retention rates for at-risk and high-risk subjects in multisetting studies is possible, and that a developmental approach should be taken to recruiting risk populations.

Adolescent↗
PubMed1997

Inhibitors of protein kinases and phosphatases alter root morphology and disorganize cortical microtubules.

To investigate molecular mechanisms controlling plant morphogenesis, we examined the morphology of primary roots of Arabidopsis thaliana and the organization of cortical microtubules in response to inhibitors of serine/threonine protein phosphatases and kinases. We found that cantharidin, an inhibitor of types 1 and 2A protein phosphatases, as previously reported for okadaic acid and calyculin A (R.D. Smith, J.E. Wilson, J.C. Walker, T.I. Baskin [1994] Planta 194: 516-524), inhibited elongation and stimulated radial expansion. Of the protein kinase inhibitors tested, chelerythrine, 6-dimethylaminopurine, H-89, K252a, ML-9, and staurosporine all inhibited elongation, but only staurosporine appreciably stimulated radial expansion. To determine the basis for the root swelling, we examined cortical microtubules in semithin sections of material embedded in butyl-methyl-methacrylate. Chelerythrine and 100 nM okadaic acid, which inhibited elongation without causing swelling, did not change the appearance of cortical arrays, but calyculin A, cantharidin, and staurosporine, which caused swelling, disorganized cortical microtubules. The stability of the microtubules in the aberrant arrays was not detectably different from those in control arrays, as judged by similar sensitivity to depolymerization by cold or oryzalin. These results identify protein phosphorylation and dephosphorylation as requirements in one or more steps that organize the cortical array of microtubules.

Arabidopsis↗