Impact of FK 506 on myocarditis in the enteroviral murine model.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
J E Wilson
Publications and source records attributed to J E Wilson.
Preferential endocardial residence of B-cells in the "Quilty effect" of human heart allografts: immunohistochemical distinction from rejection.
Endocardial infiltrates (EI) are a common and often problematic observation in endomyocardial biopsy specimens (EMBs) from patients receiving cyclosporine immunosuppression following cardiac transplant. Histologic and immunohistologic findings in 23 EMBs from 19 patients and 15 autopsy or explanted allografts demonstrated EIs to be rich in B lymphocytes (871/mm2) compared to T-lymphocytes (803/mm2). Macrophages also demonstrated an endocardial preference over deeper myocardium. In contrast, T-lymphocytes outnumbered B-lymphocytes in deeper myocardium (mean 44/mm2 versus 22/mm2) especially when rejection was present. In allograft specimens, the overall number of typical nodular EIs or percent length of endocardial involvement by EI did not correlate with the presence or absence of myocardial rejection at autopsy or explant but were related to implant duration (r = +0.63, p less than 0.01) and number of previous rejection episodes. The number of thin, nondiscrete endocardial infiltrates was greater in hearts with any myocardial rejection or inflammation present. No relationship was observed between EIs present in either EMB or allografts and the cumulative or mean dose or mean serum level of cyclosporine. Thus, a distinct morphologic and immunohistologic profile distinguishes EIs from acute rejection.
Adolescent↗
Effect of ligand-induced conformational changes on the reactivity of specific sulfhydryl residues in rat brain hexokinase.
Rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) contains 21 cysteine residues. On the basis of the amino acid sequence of the enzyme, these are predicted to be distributed among 14 peptides produced by tryptic digestion. Ten of these peptides, containing cysteine residues derivatized by reaction with the specific sulfhydryl reagent 2-bromoacetamido-4-nitrophenol have been identified in HPLC peptide maps; the four missing peptides are predicted to be relatively large and hydrophobic in character, properties that may have prevented their detection under the present conditions. The sequences encompassed by the 10 identified peptides include 12 of the 21 cysteine residues in the enzyme. The relative reactivity of these sulfhydryl groups with 2-bromoacetamido-4-nitrophenol has been assessed, and is in general accord with what might be predicted on the basis of their accessibility in the previously proposed structure for this enzyme. The effect of various ligands on reactivity of identified sulfhydryl groups has been determined; unique patterns of altered reactivity, resulting from ligand-induced conformational changes, have been observed. Biphasic effects were observed with increasing concentrations of either glucose 6-phosphate (Glc-6-P) or Pi. In both cases, decreased reactivity of sulfhydryls in the N-terminal half of the molecule was observed at low concentrations of the ligand, while further increase in ligand concentration resulted in decreased reactivity of sulfhydryl groups in the C-terminal half. In contrast, sulfhydryls in both N- and C-terminal halves were protected concomitantly by increasing concentrations of Glc. These results are consistent with previous studies that indicated (a) the existence of two sites for binding of Glc-6-P or Pi, a high affinity site in the N-terminal half and a site with lower affinity in the C-terminal half of the brain hexokinase molecule, and (b) binding of Glc to a single site located in the C-terminal half but evoking conformational effects throughout the molecule; the glucose analog, N-acetylglucosamine, previously shown to have more limited effects on conformation, affected reactivity of sulfhydryl groups only in the C-terminal half of the molecule. As reflected by effects on reactivity of sulfhydryl groups, conformational changes induced by binding of nucleotides depends markedly on the specific nature of the purine or pyrimidine base as well as the length and chelation status of the polyphosphate side chain. These results focus attention on specific regions of the molecule (the immediate environment of the sulfhydryl groups) that are affected by the binding of these ligands.
Acetamides↗
Binding of nucleoside triphosphates, inorganic phosphate, and other polyanionic ligands to the N-terminal region of rat brain hexokinase: relationship to regulation of hexokinase activity by antagonistic interactions between glucose 6-phosphate and inorganic phosphate.
Mg2(+)-chelates of several nucleoside triphosphates were shown to increase the inactivation of rat brain hexokinase (ATP:D-hexose-6-phosphotransferase, EC 2.7.1.1) by 0.6 M guanidine hydrochloride, with ATP-Mg2+ having the greatest effect; unchelated forms did not significantly affect inactivation. Since catalytic activity has been associated with the C-terminal half of the molecule, these results were interpreted as indicating a destabilization of this C-terminal region by binding of these chelates to the substrate nucleotide sites, with the particular effectiveness of ATP-Mg2+ reflecting the specificity for this species as a phosphoryl donor. These compounds were also shown to bind to the N-terminal half of the enzyme, as judged by their ability to protect against denaturation by guanidine hydrochloride and subsequent digestion with trypsin. Both free and Mg2(+)-chelated forms afforded protection, with the unchelated nucleotides being most effective; a preference for ATP was seen only with the chelated forms. Thus, it was concluded that the N-terminal half of hexokinase contains a relatively nonspecific nucleotide binding site, distinct from the substrate nucleotide site previously shown to reside in the C-terminal half. On the basis of this same ability to protect the N-terminal half against denaturation and proteolysis, several other polyanionic ligands were shown to bind to this region of the molecule. These included inorganic phosphate, its analogs, sulfate and arsenate, and its homologs, pyrophosphate and tripolyphosphate. All of these anionic ligands were also shown to antagonize inhibition by the glucose 6-phosphate (Glc-6-P) analog, 1,5-anhydroglucitol 6-phosphate. The allosteric site for binding of Glc-6-P has previously been shown to reside in the N-terminal half of the molecule, and it is suggested that the antagonism of inhibition by Glc-6-P (or its analog) by these anionic ligands results from interaction with an anion binding site for which the 6-phosphate group of inhibitory hexose 6-phosphates must compete. A model depicting possible relationships between ligand binding sites on brain hexokinase, and how their interactions might lead to observed regulatory properties, is developed based on these and previous studies of ligand binding as well as evidence that mammalian hexokinases (Mr 100,000) have evolved by duplication and fusion of a gene coding for an ancestral hexokinase with Mr 50,000 and which, like the mammalian enzyme, was sensitive to inhibition by Glc-6-P.
Animals↗
Differential scanning calorimetric study of rat brain hexokinase: domain structure and stability.
Rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) has been studied by differential scanning calorimetry. In "high-ionic-strength" buffer (50 mM Tris-Cl, 0.5 mM EDTA, 10 mM monothioglycerol, pH 8.5), and assuming two-state behavior with calorimetric enthalpy equal to van't Hoff enthalpy, the endotherm could be deconvoluted into two transitions with Tm values of about 48 and 51 degrees C and enthalpies of about 109 and 112 kcal/mol, respectively. A similar endotherm was seen when glucose or glucose 6-phosphate was present, except that Tm values for both transitions were increased. The glucose analog, N-acetylglucosamine, had no observable effect on the endotherm, which is in agreement with previous studies indicating that this ligand, unlike glucose and glucose 6-phosphate, does not induce conformational changes that lead to increased stability of the enzyme. In "low-ionic-strength" buffer (5 mM Tris-Cl, 0.5 mM EDTA, 10 mM monothioglycerol, pH 8.5), the transitions were partially resolved even in the absence of ligands, with Tm values of about 49 and 55 degrees C. Due to difficulties with erratic baseline behavior under the low-ionic-strength conditions, enthalpies were not routinely determined, but these appeared to be similar to those seen in high-ionic-strength buffer. Also similar was the increase in stability, as reflected by the increase in Tm for both transitions, when glucose or glucose 6-phosphate was present. Correlation of these transitions with specific regions of the molecule was established by analysis of enzyme in which the domain corresponding to the first transition was selectively denatured by a partial scan in the calorimeter. Subsequent rescanning of these samples showed only the second transition, confirming the selective denaturation of the domain corresponding to the first transition and retention of the folded structure of the second domain. Discrimination between denatured (first transition) and undenatured (second transition) domains was based on the markedly increased susceptibility of the denatured region to tryptic digestion; regions of the molecule that retained their folded structure and resistance to proteolysis were identified by immunoblotting techniques using monoclonal antibodies recognizing epitopes having defined locations within the overall sequence. Based on this analysis, the first transition corresponds to unfolding of the C-terminal half of the molecule, with the second transition resulting from unfolding of the more stable N-terminal half. The order of unfolding could be reversed in the presence of ATP-Mg2+ and N-acetylglucosamine, conditions which have been shown to result in selective stabilization of the C-terminal domain.
Animals↗
Tetrameric structure of mitochondrially bound rat brain hexokinase: a crosslinking study.
Rat brain hexokinase (ATP:D-hexose-6-phosphotransferase; EC 2.7.1.1) was derivatized with sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)ethyl-1,3'-dithiopro pionate (SAND), a photosensitive and cleavable crosslinking agent. The catalytic activity and mitochondrial binding properties of the enzyme were only marginally affected by reaction with SAND. When the derivatized enzyme was bound to liver mitochondria, photolysis resulted in extensive formation of a single crosslinked species with estimated molecular mass 460 kDa. This was determined to contain only hexokinase and thus represents a tetramer of the 116 kDa (apparent molecular mass in gel system used) monomeric enzyme. Although small amounts of tetramer were detected after photolysis of relatively high concentrations of derivatized enzyme in free solution, tetramer formation was greatly enhanced when the enzyme was bound to mitochondria. No evidence of dimeric or trimeric structures was seen even when only a small fraction of the available binding sites on the mitochondrial membrane were occupied. It is thus concluded that tetramer formation is closely linked with binding of the enzyme to the outer mitochondrial membrane and, more specifically, to the pore structure through which metabolites traverse this membrane. It is speculated that a tetrameric structure surrounding the mitochondrial pores may facilitate interactions between the hexokinase reaction and oxidative phosphorylation, mediated by the adenine nucleotides which are common intermediates in these reactions.
Animals↗
Lipid phenotypes, apolipoprotein genotypes and cardiovascular risk in nonagenarians.
Despite great interest in the role of lipids in overall and disease-free survival, virtually no information is available on the lipids, lipoproteins and apolipoproteins of persons over 90 years of age. Furthermore, the genetic underpinnings of atherosclerosis and the particular genetic factors responsible for protection against coronary artery disease remain speculative. In Bloomfield, Nebraska, we studied 41 nonagenarians (10 males, 31 females), with a mean age of 92.7 years, in whom lipids, lipoproteins, apolipoproteins and restriction fragment length polymorphisms (RFLPs) of genes for apolipoprotein B (apo B), aop AI and apo CIII were assessed. Nearly complete historical, physical and laboratory data were obtained on 39 subjects. The mean diastolic and systolic blood pressures for this group were nonhypertensive, body mass indices (weight/height2) had a mean of 23.9 and triceps skinfold thickness measurements an overall mean of 14.8 mm. The mean total serum cholesterol was 5.42 mmol/l. HDL-cholesterol levels in females persisted to be higher when compared to males (P less than 0.013). The allele frequencies for apo AI (MspI and PstI), apo CIII (Sst) and apo B (XbaI) gene RFLPs were typical for larger population studies. In these preliminary studies, we did not identify a distinctive phenotype, genotype, or phenotype-genotype relationship. Diversity of cardiovascular risk was the hallmark of these nonagenarians.
Aged↗
Lunar phases and crisis center telephone calls.
The lunar hypothesis, that is, the notion that lunar phases can directly affect human behavior, was tested by time-series analysis of 4,575 crisis center telephone calls (all calls recorded for a 6-month interval). As expected, the lunar hypothesis was not supported. The 28-day lunar cycle accounted for less than 1% of the variance of the frequency of crisis center calls. Also, as hypothesized from an attribution theory framework, crisis center workers reported significantly greater belief in lunar effects than a non-crisis-center-worker comparison group.
Adult↗
Isolation and characterization of the discrete N- and C-terminal halves of rat brain hexokinase: retention of full catalytic activity in the isolated C-terminal half.
Selective stabilization of either the N- or C-terminal half (by ligands binding to these regions) of rat brain hexokinase against partial denaturation with guanidine hydrochloride and subsequent digestion with trypsin has provided a means for isolating these regions, referred to as N fragment and C fragment, respectively, in quantities adequate for characterization. The N fragment (mol wt 52 kDa) is devoid of catalytic activity. In contrast, the C fragment (mol wt 51 kDa) has a specific activity of about 110 U/mg, nearly twice that (60 U/mg) of the intact 100-kDa enzyme, indicating that the kappa cat is virtually identical for both species. Unlike the parent enzyme, the C fragment is quite sensitive to inhibition by Pi (competitive vs ATP, noncompetitive vs Glc); sulfate and arsenate, but not acetate, inhibit with effectiveness similar to that seen with Pi. The Glc-6-P analog, 1,5-anhydroglucitol-6-P, also inhibits the C fragment (competitive vs ATP, uncompetitive vs Glc). Both N and C fragments bind to Affi-Gel Blue, an affinity matrix bearing a covalently attached analog of ATP, and are eluted by hexose 6-phosphates competitive with nucleotide binding to the parent enzyme. Based on the ability of various hexoses and hexose 6-phosphates (and analogs) to protect against guanidine-induced denaturation and subsequent proteolysis it is concluded that both fragments contain discrete sites for hexoses and hexose 6-phosphates, with specificities resembling those seen for the binding of these ligands to the parent enzyme. Synergistic interactions between the hexose and hexose-6-P binding sites, previously seen with the parent enzyme, are also observed with the C fragment but not the N fragment. The existence of binding sites for hexoses and hexose 6-phosphates on both halves conflicts with previous binding studies demonstrating a single hexose binding site and a single hexose 6-phosphate binding site on the intact 100-kDa enzyme, leading to the conclusion that one of each pair of sites must be latent in the intact enzyme, becoming manifest only in the isolated discrete halves. Several investigators have previously suggested that the 100-kDa mammalian hexokinases evolved by duplication and fusion of a gene encoding an ancestral 50-kDa Glc-6-P-insensitive hexokinase, similar to the present-day yeast enzyme, with sensitivity to Glc-6-P resulting from evolution of a duplicated catalytic site into a regulatory site.(ABSTRACT TRUNCATED AT 400 WORDS)
Amino Acid Sequence↗
Progress in lipid reporting practices and reliability of blood cholesterol measurement in clinical laboratories in Nebraska. Efforts to align results with the Centers for Disease Control, and feasibility of meeting National Cholesterol Education Program Guidelines.
The National Cholesterol Education Program has recommended that all laboratories be consistent, precise, and accurate in the reporting and measurement of blood cholesterol levels. In a follow-up to a 1984 survey study, we assessed the changes in reporting procedures for measurements of blood lipid levels in 16 clinical laboratories in Nebraska. Using human serum reference materials of known cholesterol concentrations provided by the Centers for Disease Control, we also assessed the precision and accuracy of measurement of blood cholesterol levels in clinical laboratories in Nebraska. Fourteen of the 16 laboratories restudied in 1987 had altered the reference range for total serum cholesterol since 1984, 86% of whom lowered the upper limit of the reference range. Eleven of 16 laboratories expressed reference ranges for total serum cholesterol in terms of patient age in 1987, while only 7 of 20 did in 1984. Gender-based reference ranges increased from 0 to 5 from 1984 to 1987. Similar trends were seen in the reporting of high-density lipoprotein cholesterol and triglyceride concentrations. Reporting procedures varied greatly; only 1 laboratory used National Cholesterol Education Program risk levels for measuring total serum cholesterol levels. Fifteen laboratories met the National Cholesterol Education Program recommendation for precision (coefficient of variation, less than or equal to 5%) and 78% of laboratories obtained results that satisfied the current recommendation for accuracy (within 5% of "true value," as determined by the Centers for Disease Control).
Adult↗
Rat brain hexokinase: further studies on the specificity of the hexose and hexose 6-phosphate binding sites.
Based on the lack of correlation between the ability of various hexoses to serve as substrate and the ability of the corresponding hexose 6-phosphates to inhibit brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1), R. K. Crane and A. Sols (1954, J. Biol. Chem. 210, 597-606) proposed that this enzyme possesses two discrete sites capable of binding hexose moieties, one serving as the substrate binding site and a second, regulatory in function, to which inhibitory 6-phosphates bind. Subsequent work has provided further experimental support for this proposal. The pioneering work by Crane and Sols focused primarily on the specificity of these sites with respect to requirements for orientation of hydroxyl substituents at the various positions of the pyranose ring. The present study explores additional aspects of the specificity of these sites, namely, the effect of substitution of a sulfur atom in place of the oxygen in the pyranose ring on ability to serve as substrate or inhibitor, and the effect of modification in charge of the substituent at the 6-position on inhibitory effectiveness. 5-Thioglucose is a linear competitive (versus glucose) inhibitor of rat brain hexokinase, with a Ki of about 0.2 mM, and is a linear mixed inhibitor (versus ATP), with Ki values in this same range. 5-Thioglucose is not, however, readily phosphorylated by brain hexokinase. Thus, although 5-thioglucose binds with moderate affinity to the glucose binding site, it is not effectively used as a substrate of the enzyme. Inhibition of brain hexokinase by glucose 6-phosphate or its analogs has been found to require a dianionic substituent at the 6-position. The 6-fluorophosphate derivative and glucose 6-sulfate are poor inhibitors of the enzyme, and the Ki for inhibition by 1,5-anhydroglucitol 6-phosphate increases markedly at pH values below the pK of the 6-phosphate group, indicating that the monoanionic form is ineffective as an inhibitor. In contrast to the detrimental effect that substitution of the oxygen atom in the pyranose ring with a sulfur has on ability to serve as substrate, 5-thio analogs are considerably more effective as inhibitors, the Ki for inhibition by 5-thioglucose 6-phosphate being 10-fold lower than that seen with glucose 6-phosphate. This effect of the heteroatom substitution can partially offset the decreased inhibition resulting from monoanionic character at the 6-position, but the 6-fluorophosphate derivative of 5-thioglucose 6-phosphate still inhibits with a Ki about 1000-fold greater than that seen with 5-thioglucose 6-phosphate.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Age-related changes in naive and memory CD4+ T cells in healthy human children.
Subsets of CD4+ T cells originally identified functionally as suppressor-inducer and helper-inducer populations have recently been reinterpreted as naive and memory maturational states. The subsets can be identified by the surface expression of CD45R and CDw29, respectively. Using two-color flow cytometric analysis, we measured these CD4+ T cell subsets in two samples of cord blood and in 26 healthy children between the ages of 1 and 19 years. As has been reported by others, we observed that the majority of CD4+ T cells in cord blood consist predominantly of the CD45R+ subset. With aging we could demonstrate a gradual acquisition of CDw29+, CD4+ T cells and a concomitant gradual decrease in the percentage of CD45R+, CD4+ T cells. These age-related changes are consistent with the concept of naive (CD45R+) and memory (CDw29+) subsets. Further, because of the dynamic changes, their utilization as prognostic indicators in immunologic disease states cannot be applied to children in the same manner as adults.
Adolescent↗
Oligoanalgesia in the emergency department.
A review of the charts of 198 patients who were admitted through the emergency department with a variety of acutely painful medical and surgical conditions revealed that 56% received no analgesic medication while in the emergency department. In the 44% of patients who received pain medication, 69% waited more than 1 hour while 42% waited more than 2 hours before narcotic analgesia was administered. In addition, 32% initially received less than an optimal equianalgesic dose of narcotic when compared with morphine. This study demonstrates that narcotic misues, in the form of oligoanalgesia, is prevalent and is the shared responsibility of both emergency physicians and housestaff consultants.
Adolescent↗
Complete amino acid sequence of rat brain hexokinase, deduced from the cloned cDNA, and proposed structure of a mammalian hexokinase.
The complete amino acid sequence for the type I isozyme of hexokinase from rat brain has been deduced from the nucleotide sequence of cloned cDNA. The nucleotide sequence of 91 bases in the 5' untranslated region as well as that of the entire 3' untranslated region preceding the poly(A) sequence have also been determined. The N- and C-terminal halves of brain hexokinase show extensive sequence similarity to each other and to yeast hexokinase. These results provide direct support for the proposal that the mammalian hexokinases of approximately 100 kDa have evolved by a process of duplication and fusion of a gene encoding an ancestral hexokinase similar to the yeast enzyme of approximately 50 kDa. Taking this similarity in sequence to indicate basic similarity in structure between the N- and C-terminal regions of brain hexokinase and the yeast enzyme, a proposed structure for the mammalian hexokinase has been developed by fusing two molecules of yeast hexokinase, whose structure has previously been determined by x-ray crystallographic studies. Various features of the model are shown to be consistent with experimental observations bearing on the structure of the brain enzyme.
Amino Acid Sequence↗
Rapid purification of mitochondrial hexokinase from rat brain by a single affinity chromatography step on Affi-Gel blue.
The mitochondrial hexokinase from rat brain, selectively released from mitochondria by the action of glucose 6-phosphate, can be purified to greater than 90% homogeneity by a single affinity chromatography step on Affi-Gel Blue; the Cibacron Blue F3GA ligand bound to this matrix serves as an analog of ATP, the normal substrate for the enzyme, and selective elution is accomplished using glucose 6-phosphate which is a competitive ligand vs. ATP. With this and other modifications to the previously described procedure highly purified enzyme is readily obtained in good yield and with retention of the ability to rebind to mitochondria.
Animals↗
Thromboresistant polymers: a new approach.
Selected antithrombotic agents were found to impart improved thromboresistance to polysilicone tubes when covalently bound to such tubes in pilot studies. Bonding was accomplished by radiation grafting of epoxypropyl acrylate monomer on the polysilicone, followed by reaction of the hydroxyl groups of the antithrombotic agents with the epoxy groups on the grafted monomer chains. The bound antithrombotic agents may act by chelating or complexing calcium ions, which are known to perform several functions in blood coagulation.
Acrylates↗
Vitamin supplements and purported learning enhancement in mentally retarded children.
Dr. Ruth Harrell and her colleagues conducted a study in 1981 in which the administration of high dosages of vitamins were determined to dramatically improve the intellectual functioning of mentally retarded children. This paper documents the efforts of its authors to replicate the Harrell et al. research and, although no significant differences were found between active treatment groups and control groups at study's end, a thorough comparison is made between the two research efforts.
Adolescent↗