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J E Wilson

Publications and source records attributed to J E Wilson.

At least 73 records · Page 4Linked to original sources
PubMed1996

Novel genetic screen for genes involved in posterior body patterning in Drosophila.

Much of our current understanding of how cytoplasmic determinants are localized and activated stems from genetic analyses in Drosophila. Characterization of mutants defective in establishing the initial embryonic body pattern has revealed the importance of mRNA localization and translational control and identified several factors required for these processes. Nevertheless, many additional genes are likely to be involved. Here, we describe a novel genetic screen designed to identify genes that participate in posterior body patterning, an elaborate process involving the sequential use of two localized cytoplasmic determinants, the products of the oskar and nanos genes. From the screen, we recovered new alleles of genes known to be required for posterior body patterning, demonstrating the validity of the approach. In addition, we isolated numerous other mutants. Further characterization of one mutant, P58, revealed that it is a novel allele of bullwinkle. We find that in bullwinkle mutants, oskar mRNA localization is not maintained in the embryo and oskar protein accumulates ectopically and to abnormally high levels. These defects are distinct from previously described perturbations in oskar activity and provide new insights into the regulation of oskar.

Animals↗
PubMed1996

Purification and characterization of the hexokinase from Schistosoma mansoni, expressed in Escherichia coli.

The hexokinase (ATP;D-hexose 6-phosphotransferase, EC 2.7.1.1) of Schistosoma mansoni has been expressed in Escherichia coli as a fusion protein including an N-terminal polyhistidine tag and enterokinase cleavage site. The enzyme was purified by metal chelate chromatography to > 95% homogeneity, based on analysis by SDS-gel electrophoresis and isoelectric focusing. The absorbance at 280 nm was 0.54 for a 1 mg/ml solution (molar extinction coefficient 2.7 x 10(4) cm2 mol). The pI of the S. mansoni hexokinase was 6.0-6.2, slightly more acidic than the rat Type I isozyme (pI 6.35). The S. mansoni enzyme migrated as a single band of activity during nondenaturing cellulose acetate electrophoresis; the mobility was slightly greater than the rat Type I isozyme, consistent with the estimated pI. The Km values for substrates glucose and ATP were 128 +/- 10 and 927 +/- 41 microM, respectively. In accord with a previous report, the S. mansoni hexokinase exhibited moderate sensitivity to inhibition (competitive vs ATP) by the product, glucose 6-phosphate, with a Ki approximately 150 microM; the product analog, 1,5-anhydroglucitol 6-phosphate, was somewhat less effective as an inhibitor, with Ki approximately 500 microM. These kinetic properties were not altered by removal of the N-terminal fusion partner by enterokinase treatment. Immunological crossreactivity between the rat Type I isozyme and the S. mansoni hexokinase was demonstrated by immunoblotting, but this was markedly dependent on the preparation of antiserum used. The activity of the enzyme is apparently highly dependent on maintenance of free sulfhydryl groups. Activity was maintained during storage in the presence of monothioglycerol; activity lost during storage in the absence of monothioglycerol could be partially restored by treatment with this reagent.

Adenosine Triphosphate↗
PubMed1996

DNA polymerase activity of hepatitis B virus particles: differential inhibition by L-enantiomers of nucleotide analogs.

DNA polymerase activity was assayed in hepatitis B virus (HBV) and core particles isolated from chronic producer lines. The particle-associated DNA polymerase activity, which was found to be limited to incorporation of only a few nucleotides, was inhibited by the 5'-triphosphates of nucleoside analogs. The 1-beta-L (1S,4R) and 1-beta-D (1R,4S) enantiomers of antiviral nucleoside analogs were compared for the ability to inhibit incorporation of natural nucleoside triphosphates into the viral DNA. Previously, both enantiomers of several analogs were found to be substrates for human immunodeficiency virus type 1 reverse transcriptase (HIV RT); the 1-beta-D enantiomers of some pairs were preferred as substrates. In contrast, the 1-beta-L enantiomers of all pairs tested were the more potent inhibitors of labeled substrate incorporation into hepatitis B virus DNA; the concentration required to inhibit the incorporation of the natural substrate by 50% was 6-fold to several hundred-fold lower than the concentration of the 1-beta-D enantiomer required for the same inhibitory effect. This preference for the 1-beta-L enantiomers was observed for both RNA-directed synthesis in core particles and DNA-directed synthesis in viral particles. The observed antiviral effect of the nucleoside analogs in cell culture seemed to be limited chiefly by their phosphorylation in cells.

DNA, Viral↗
PubMed1996

Cryofixing single cells and multicellular specimens enhances structure and immunocytochemistry for light microscopy.

Cryofixation is widely held to be superior to chemical fixation for preserving cell structure; however, the use of cryofixation has been limited chiefly to electron microscopy. To see if cryofixation would improve sample structure or antigenicity as observed through the light microscope, we cryofixed Nicotiana alata and Lilium longiflorum pollen tubes and Tradescantia virginina stamen hairs by plunge freezing. After freeze-substitution, and embedding in butylmethylmethacrylate, we found using the light microscope that the superiority of cryofixation over chemical fixation was obvious. Cryofixation, unlike chemical fixation, did not distort cell morphology and preserved microtubule and actin arrays in a form closely resembling that of living cells. Additionally, to test further the usefulness of cryofixation for light microscopy, we studied the appearance of cells and the retention of antigenicity in plunge-frozen multicellular organ. Roots of Arabidopsis thaliana were either chemically fixed or plunge frozen, and then embedded in the removable methacrylate resin used above. We found that plunge freezing preserved cell morphology far better than did chemical fixation, and likewise improved the appearance of both actin and microtubule arrays. Plunge-frozen roots also had cells with more life-like cytoplasm than those of chemically fixed roots, as assessed with toluidine-blue staining or high-resolution Nomarski optics. Damage from ice crystal formation could not be resolved through the light microscope, even in the interior of the root, 40-75 microns from the surface. We suggest that plunge freezing would enhance many investigations at the light microscope level, including those of multicellular organs, where damage from ice crystals may be less severe than artefacts from chemical fixation.

Arabidopsis↗
PubMed1996

Functional interaction of hexokinase with ATP requires participation by both small and large lobes of the enzyme: implications for other proteins using the actin fold as a nucleotide binding motif.

The actin fold is a structural motif involved in binding of ATP to an otherwise diverse family of proteins that includes hexokinase, actin, and the 70 kDa heat shock protein. Previous analyses have focused attention on a hydrophobic pocket within the large lobe of hexokinase (and analogous regions in other proteins possessing the actin fold motif) as the-site for binding of the adenine moiety. However, there is reason to believe that binding of the adenine moiety also involves a beta-sheet region located in the small lobe of hexokinase. It is postulated that functional interaction with ATP, required for catalysis, involves interactions with both regions. The expected structural consequences provide a basis for explaining the kinetic mechanism suggested for this enzyme, i.e., although both substrates can bind independently, the preferred kinetic pathway is not random but ordered, with glucose binding first. The structural features postulated to be involved in binding of ATP to hexokinase are also found in other proteins possessing the actin fold motif. Interaction of the nucleotide with the beta-sheet region, analogous to that found in the "small lobe" of hexokinase, may induce functionally important conformational changes in other proteins employing the actin fold as a nucleotide binding motif.

Actins↗
PubMed1996

The pathogenesis of cardiac allograft vasculopathy.

There has been an intensive effort in the past year to identify immunologic and nonimmunologic factors in the pathogenesis of cardiac allograft vasculopathy. Significant progress has been made regarding cell proliferation and cell death, with particular focus on cell growth factors, cell death factors, and their receptors. Endothelial cell injury and endothelin expression and production have been implicated in the development of cardiac allograft vasculopathy. A significant clinical association between dyslipidemia and cardiac allograft vasculopathy has been detected by intracoronary ultrasonography. Furthermore, proteoglycans are postulated to play an important role through lipoprotein-proteoglycan interactions. The role of cytomegalovirus infection remains controversial. Immunosuppressive agents such as rapamycin and FK506 may contribute to a reduction of vasculopathy.

Animals↗
PubMed1996

Direct interactions of coxsackievirus B3 with immune cells in the splenic compartment of mice susceptible or resistant to myocarditis.

In vitro replication of coxsackievirus B3 (CVB3) in cells of the immune system derived from uninfected adolescent A/J and C57BL/6J mice and replication of CVB3 in and association with immune cells from spleens of infected animals in vivo were assessed. Nonstimulated or mitogen-stimulated spleen cells were minimally permissive for viral replication during an 8-h period. Three days postinfection (p.i.), CVB3 RNA was localized in vivo to B cells and follicular dendritic cells of germinal centers in both A/J and C57BL/6J mice; however, extrafollicular localization was greater in C57BL/6J mice (P = 0.0054). Although the pattern of CVB3 RNA localization was different, the total load of infections virus (PFU per milligram of tissue) was not different. Splenic CVB3 titers (PFU per milligram of tissue) in both strains were maximal at day 3 or 4 p.i. and were back to baseline by day 7 p.i., with most infectious virus being non-cell associated. CVB3 titers (PFU per milligram of tissue) correlated directly with in situ hybridization positivity in splenic follicles and extrafollicular regions in both murine strains; however, follicular hybridization intensity was greater in A/J mice at day 5 p.i. (P = 0.021). Flow cytometric analysis demonstrated that 50.4% of total spleen cells positive for CVB3 antigen were B cells and 69.6% of positive splenic lymphocytes were B cells. Myocardial virus load in C57BL/6J mice was significantly lower than that in A/J mice at days 4 and 5 p.i. These data indicate that CVB3 replicates in murine splenocytes in vitro and in B cells and extrafollicular cells in vivo.

Animals↗
PubMed1996

aubergine enhances oskar translation in the Drosophila ovary.

Although translational regulation of maternal mRNA is important for proper development of the Drosophila embryo, few genes involved in this process have been identified. In this report, we describe the role of aubergine in oskar translation. Previously, aubergine has been implicated in dorsoventral patterning, as eggs from aubergine mutant mothers are ventralized and seldom fertilized (Schupbach, T. and Wieschaus, E. (1991) Genetics 129, 1119-1136). We have isolated two new alleles of aubergine in a novel genetic screen and have shown that aubergine is also required for posterior body patterning, as the small fraction of eggs from aubergine- mothers that are fertilized develop into embryos which lack abdominal segmentation. Although aubergine mutations do not appear to affect the stability of either oskar mRNA or protein, the level of oskar protein is significantly reduced in aubergine mutants. Thus, aubergine is required to enhance oskar translation. While aubergine-dependence is conferred upon oskar mRNA by sequences in the oskar 3' UTR, aubergine may influence oskar translation through an interaction with sequences upstream of the oskar 3' UTR.

Animals↗
PubMed1996

Human transplant coronary artery disease: pathological evidence for Fas-mediated apoptotic cytotoxicity in allograft arteriopathy.

Cytotoxicity mediated by cytolytic T cells occurs exclusively through Fas- and perforin-based pathways. Such mechanisms may be important in transplant coronary artery disease (TxCAD). We studied Fas and perforin expression and apoptosis using immunohistochemical methods, in situ terminal deoxyribonucleotide transferase-mediated dUTP nick-end labeling (TUNEL), and morphologic analysis of vessels with TxCAD taken from 12 transplant patients, which we compared with vessels of 10 patients with native coronary artery disease (CAD) and vessels from 14 patients with normal coronary arteries. Fas was detected in all TxCAD specimens. Fas-positive cells were mainly endothelial cells (EC); 100% of EC and approximately one-third of T cells and macrophages were positive for Fas. Almost all TUNEL-positive cells were Fas-expressing cells. Of the T cells and macrophages that expressed Fas, 18% appeared apoptotic in the mild and moderate-to-severe TxCAD group as compared with 78% in severe TxCAD patients (p = 0.0124). By contrast, EC damage was less evident in the vessels with greater intimal disease severity: 10% in severe TxCAD versus 75% in the mild and moderate-to-severe TxCAD group (p = 0.0122). Perforin was positive in 5% of the total intimal T cells in 3 of 12 arteriopathy specimens. Fas and perforin were virtually negative in vessels taken from CAD patients. TUNEL was diffusely positive in CD68-positive foam cells in the lipid-rich core, but Fas was negative in all CAD specimens. In normal arteries, 8 of 14 specimens contained a few TUNEL-positive and Fas-positive EC and T cells. Perforin was negative. We conclude that EC damage in TxCAD seems to be brought about through a Fas-based apoptotic pathway and that CD4-positive cytolytic T cells may be the major lytic cells involved in TxCAD.

Adolescent↗
PubMed1996

Prominence of apolipoproteins B, (a), and E in the intimae of coronary arteries in transplanted human hearts: geographic relationship to vessel wall proteoglycans.

BACKGROUND: Transplant arteriopathy (TA) is characterized by vessel wall thickening with prominent glycosaminoglycan and lipid deposits. In this light, we have recently demonstrated the distribution of proteoglycans in allograft coronary arteries. The aim of this study is to examine the distribution of apolipoproteins within allograft coronary arteries and to investigate their localization in relation to proteoglycans. METHOD: Particular transverse sections of left and right epicardial coronary arteries from 46 human cardiac allografts, 11 normal hearts, and 11 hearts with native atherosclerosis were stained immunohistochemically for apolipoprotein B, apolipoprotein (a) (apo[a]), apolipoprotein E (apoE), biglycan, decorin, and versican by use of an automated immunostainer. RESULTS: Apo(a) and apoE immunopositivity in TA was much more intense than that in native atherosclerosis, whereas the reverse was true for apoB. Prominent apoE deposits were evident circumferentially in endothelia and extracellularly in superficial intima of mildly diseased TA, as well as in deeper intima of severely diseased TA. Apo(a) had a staining pattern very similar to apoE except for a patchy deposition also seen in TA media. The intimal areas staining prominently with apoE or apo(a) in TA arteries corresponded very closely to the areas with proteoglycan deposits, especially versican. CONCLUSION: The distinctive patterns of apolipoprotein accumulation in TA and native atherosclefosis appear to reflect different pathogenetic processes in the two conditions. The colocalization of proteoglycans and apolipoproteins in TA intima supports the hypothesis that interactions between proteoglycans and apolipoproteins influence lipid retention and overload in allograft coronary arteries.

Apolipoproteins↗
PubMed1996

Biglycan, decorin, and versican protein expression patterns in coronary arteriopathy of human cardiac allograft: distinctness as compared to native atherosclerosis.

BACKGROUND: Histochemical staining has demonstrated previously dramatic deposits of glycosaminoglycans associated with prominent lipid accumulations in thickened vessel walls of allograft coronary arteries. In this study, we characterized the amount, distribution, and types of proteoglycan in the walls of coronary arteries from human cardiac allografts and from native atherosclerotic (NA) controls as part of a strategy to understand the pathogenesis of transplant arteriopathy (TA). METHOD: We used polyclonal rabbit antibodies against human biglycan, decorin, and versican localize the proteoglycan molecules in standardized transverse sections of the proximal left anterior descending and right coronary arteries. Slides were scored in a blinded fashion for intensity of proteoglycan staining (0 to 6+) and for localization in the vessel walls. RESULTS: Unique patterns of proteoglycan distribution were present in TA and NA. Biglycan was particularly prominent in intima and evolving atheromata in severely diseased TA coronary arteries, but not in NA. Decorin was present mainly in adventitia of all vessels and in the intima of NA. Prominent versican accumulation occurred in intima and media of TA coronaries, associated with smooth muscle cells and foam cells. There was a reciprocal pattern of biglycan and decorin staining. Versican colocalized with biglycan. Intimal biglycan and versican deposits were positively correlated to the extent of luminal narrowing in TA. CONCLUSION: The distinctive staining patterns for biglycan, decorin and versican in both native and allograft disease indicate that the synthesis and distribution of these proteoglycans are regulated by different local mechanisms in different atheromatous diseases.

Biglycan↗
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

Cytometric analysis of ventricular myocyte nuclei in idiopathic dilated cardiomyopathy: a tool for evaluation of disease progression?

The progression of idiopathic dilated cardiomyopathy (IDC) is governed by factors that remain obscure. The disease pathway toward cell degeneration or death results in irreversible myocyte change including nuclear cytometric alterations which may be evaluable and ultimately correlated with other measures of disease evolution. Using a novel image cytometry system, we analysed differences in ventricular myocyte nuclear morphology and DNA content and distribution in right and left ventricular free wall myocardium and in ventricular septal myocardium from 11 normal and 13 IDC human autopsy hearts. Nine morphological features of IDC myocyte nuclei differed significantly (P < 0.001) from normal. These were used to establish a classification matrix and cytometry-based assessment and allowed correct categorization of left and right ventricular and ventricular septal myocyte nuclei in concordance with their respective pathological diagnosis (i.e. normal or IDC) 71%, 81% and 77% of the time. Additionally, four photometric features were significantly different (P < 0.005) in IDC versus normal hearts, as were three discrete texture features (P < 0.001). Thus, the spectrum of myocyte nuclei seen in IDC have highly characteristic and measurable morphologic, photometric and texture features. Our findings indicate the potential value of cytometry in the classification of myocytes with regards to a disease continuum and suggest its applicability in both clinical and experimental studies.

Adult↗
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↗