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Biomedical subjects

D A Roth

Publications and source records attributed to D A Roth.

At least 19 recordsLinked to original sources
PubMed1992

A substantial proportion of cardiac Gs is not associated with the plasma membrane.

The precise interactions between the subunits of Gs (alpha s, beta, gamma) and the plasma membrane remain to be established. If alpha s is associated loosely with the inner membrane, is labile during activation, or is always present to some extent in the cytoplasm, then it should fractionate to the supernatant of a high-speed centrifugation. We identified abundant alpha s (52-66% of total cellular) in the supernatant fraction of right atrial and left ventricular membrane preparations of porcine heart as shown by two distinct measures of alpha s (immunoblotting and ADP ribosylation by cholera toxin). However, functional assays utilizing reconstitution of cardiac alpha s with cyc- S49 membranes revealed that the supernatant fraction contained approximately 16% of total cellular alpha s activity. The alpha s present in the supernatant fraction did not result from contamination by sarcolemmal membrane fragments. We conclude that traditional methods for quantifying alpha s which utilize only detergent extracts from high-speed pellets do not account for a sizable proportion of total cellular alpha s, but that the majority of this population of cardiac alpha s may not be functional, at least with respect to adenylyl cyclase activation.

Adenosine Diphosphate Ribose↗
PubMed1992

Computer-generated bone models in the planning of osteotomy of multidirectional distal radius malunions.

Computer-assisted design and manufacturing technology has been used to create solid models of five unusually complex, multidirectional malunions of distal radius fractures. Preoperative planning was dramatically enhanced by the ability to perform the surgical procedure on these models, with a model of the uninjured limb used for comparison. All five patients had significant malunions, with malrotation in the horizontal plane in five and an impacted articular fragment in two. A satisfactory outcome was achieved in each case.

Adult↗
PubMed1992

Myocardial beta-adrenergic receptor expression and signal transduction after chronic volume-overload hypertrophy and circulatory congestion.

BACKGROUND: The volume-overload, high-output state induced by aortocaval fistula is unique because it is not generally associated with marked abnormalities of contractile function. Thus, changes in beta-adrenergic receptor (beta AR) expression should reflect more directly the influence of neurohumoral adrenergic tone, clarifying the manner in which peripheral (neurohumoral) versus primary myocardial factors are operative in decreased beta AR-dependent signal transduction. METHODS AND RESULTS: We examined the beta-adrenergic receptor-responsive adenylyl cyclase pathway in hearts from pigs subjected to volume-overload hypertrophy with circulatory congestion. Nine pigs underwent initial pharmacological and hemodynamic studies, and, 5 weeks after aortocaval fistula placement, when signs of circulatory congestion were evident, these measurements were repeated. Biochemical analyses of plasma and myocardium from these animals and seven normal animals were compared. Experimental animals showed signs of circulatory congestion (tachypnea, weight gain, pulmonary rales) within 3-4 weeks of fistula placement. Necropsy showed ascites and biventricular cardiac hypertrophy, but no fibrosis or inflammation was present on histological inspection. Heart rate responsiveness to beta AR stimulation was blunted, with ED50, for isoproterenol increased 133% (p less than 0.001) after development of circulatory congestion. Biochemical analyses of the beta AR-responsive adenylyl cyclase pathway showed uniform decreases in beta AR number in right atrium, right ventricle, and left ventricle (36-41% decreases, p less than 0.005). Downregulation was selective for beta 1-receptors, and remaining receptors in the right and left ventricles showed low-affinity agonist binding, suggesting an uncoupling from Gs. All measures of adenylyl cyclase activity were diminished significantly in membrane homogenates from the right atrium (mean reduction, 50 +/- 10%) and left ventricle (mean reduction, 44 +/- 8%) after volume overload. Finally, we found that amounts of cardiac Gs, as measured in reconstitution assays, were decreased in both the right atrium (p less than 0.02) and the left ventricle (p less than 0.01) of volume-overloaded animals but that levels of pertussis toxin substrate were unchanged. CONCLUSIONS: Biochemical findings occurred in the absence of myocardial inflammation or fibrosis and without pharmacological interventions, suggesting that circulatory congestion, with attendant elevation in plasma norepinephrine, may be a sufficient stimulus to induce such changes. The data are compatible with a catecholamine-driven beta AR pathway desensitization. Thus, a primary defect in intrinsic contractile function is not a necessary component for abnormalities of the myocardial beta AR-responsive adenylyl cyclase pathway.

Adenylyl Cyclases↗
PubMed1992

Myocardial adrenergic denervation supersensitivity depends on a postreceptor mechanism not linked with increased cAMP production.

BACKGROUND: Two major hypotheses have been proposed to explain catecholamine supersensitivity after myocardial denervation, but neither sufficiently explains certain features of the phenomenon. In addition, a nonsurgical method for long-term myocardial adrenergic denervation is desirable but has not been accomplished or described with respect to catecholamine supersensitivity. METHODS AND RESULTS: We have accomplished chronic myocardial adrenergic denervation by using 6-hydroxydopamine (6-OHDA). Sixteen weeks after 6-OHDA administration to newborn pigs, we found substantial myocardial adrenergic denervation associated with beta-adrenergic receptor (beta AR) downregulation. Despite decreased beta AR number, the dose of isoproterenol yielding 50% maximal heart rate change (ED50) was decreased, and heart rates during exercise showed increased responsiveness despite decreased circulating catecholamines. Thus, stimulation of fewer receptors yielded an increased response, implying improved signal transduction efficiency. Competitive binding studies with isoproterenol showed an increased proportion of beta AR with high-affinity binding in myocardial membranes from 6-OHDA pigs, suggesting that interaction between beta AR and cardiac G, may contribute to improved signal transduction efficiency. However, measures of adenylyl cyclase activity indicated marked reduction in beta AR-dependent and G2-dependent cAMP production in myocardial membranes from denervated animals despite a normal amount of cardiac Gs and decreased Gi. CONCLUSIONS: We have demonstrated that substantial, long-term myocardial adrenergic denervation is possible using 6-OHDA. Denervation supersensitivity in this model does not depend on enhanced cAMP stimulation but rather depends on postreceptor elements in the beta AR-responsive pathway that may be independent of Gs-activated adenylyl cyclase activity. In this model of adrenergic denervation supersensitivity, beta-receptors, through Gs, may be linked to an alternative effector that drives heart rate responsiveness.

Adenylyl Cyclases↗
PubMed1991

The sarcolemmal lactate transporter: transmembrane determinants of lactate flux.

Lactic acid is produced and consumed under various metabolic and pathogenic conditions in numerous cell types of mammalian tissues. The movement of lactic acid and lactate anions within and between tissues depends, ultimately, on flux rates between intracellular and extracellular compartments. Flux rates themselves are dependent on several variables, including (but not limited to) exchange surface area, flow through the tissues of interest, lactate and proton concentration gradients across the cell membranes, and proton concentration gradients across the cell membranes, and permeability of the membranes to these ions. This review focuses on the selective permeability characteristics of the principal cells of lactate metabolism: skeletal muscle, heart, liver, kidney, and erythrocytes. Special attention will be paid to lactate uptake and release in skeletal muscle, the premier tissue of lactate production and consumption, and the regulator of whole body lactate dynamics during exercise and recovery from exercise.

Biological Transport↗
PubMed1990

Lactate transport is mediated by a membrane-bound carrier in rat skeletal muscle sarcolemmal vesicles.

To study the kinetics of lactate transport in an isolated, nonmetabolizing system, skeletal muscle sarcolemmal membrane vesicles were purified from 22 female Sprague-Dawley rats. L(+)-[U-14C] Lactate at 10 concentrations demonstrated saturation kinetics with a Vmax of 139.4 nmol/mg/min, and an apparent Km of 40.1 mM. Threefold higher initial rates of L(+)-lactate uptake were seen at 37 degrees C than at 25 degrees C, indicating temperature sensitivity. Transport was stereospecific for the L(+) isomer: isotopic D(-) uptake rates remained linear at concentrations from 1 to 200 mM, and 1 mM D(-) remained 6-fold lower in net uptake after 60 min than the L(+) isomer. Furthermore, unlabeled 10 mM D(-)-lactate in the external medium could only inhibit 1 mM isotopic (L(+) uptake by 12%, whereas unlabeled 10 mM L(+)-lactate and pyruvate inhibited 82 and 71%, respectively. Additionally, 10 mM beta-hydroxybutyrate and acetoacetate could moderately inhibit (27 and 32%, respectively) 1 mM L(+)-lactate transport, but the unsubstituted aliphatic monocarboxylates (formate, acetate, propionate), tricarboxylic acid cycle intermediates (malate, succinate, oxaloacetate, alpha-ketoglutyrate, citrate), amino acids (alanine, aspartate, glutamate), and palmitate or adenosine in 10-fold excess could not effectively inhibit 1 mM L(+)lactate uptake under cis-transport conditions. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid could inhibit L(+)-lactate transport by only 13%, so that lactate transport does not appear to be affected directly by Cl- or HCO3- fluxes. It was demonstrated that KCl could not evoke a membrane potential-induced overshoot of lactate uptake in the presence or absence of valinomycin. Moreover, gluconate could substitute for Cl-, indicating that Cl- flux does not contribute to a membrane potential-dependent component of the transport mechanism, suggesting an electroneutral translocation process. Protein-modifying reagents significantly inhibited 1 mM L(+)-lactate transport during pH-stimulated conditions (p-chloromercuriphenyl-sulfonic acid, 83%; N-ethylmaleimide, 86%; HgCl2, 56%; mersalyl, 63% inhibition). We conclude that the skeletal muscle lactate transporter is a membrane-bound protein, specifically associated with the sarcolemma, that demonstrates saturation kinetics, competition, stereospecificity, and sensitivity to temperature as well as various ionic cis-inhibitors. The lactate transporter is a potentially important regulator of lactate flux across skeletal muscle, and may help to regulate intracellular pH and intermediary metabolism during lactic acidosis.

Animals↗
PubMed1990

Lactate and pyruvate transport is dominated by a pH gradient-sensitive carrier in rat skeletal muscle sarcolemmal vesicles.

The mechanisms of lactate and pyruvate transport across the plasma membrane of rat skeletal muscle under various pH and ionic conditions were studied in skeletal muscle sarcolemmal (SL) membrane vesicles purified from 22 female Sprague-Dawley rats. Transport by SL vesicles was measured as uptake of L(+)-[U-14C] lactate and [U-14C] pyruvate. Lactate (La-) transport is pH-sensitive; stimulations to fivefold overshoot above equilibrium values were observed both directly by a proton gradient directed inward, and indirectly by a monensin- or nigericin-stimulated exchange of Na+ or K+ for H+ across the SL. Isotopic pyruvate could utilize the transporter, and demonstrated pH gradient-stimulated overshoot and cis-inhibition characteristics similar to those of lactate. Overshoot kinetics were also demonstrated by pH gradient formed by manipulation of external media at pH 5.9, 6.6, and 7.4 and intravesicular media at 6.6, 7.4, and 8.0, respectively. Carbonyl cyanide m-chlorophenylhydrazone, an H+ ionophore, was used as a "pH clamp" to return all stimulated uptake courses back to equilibrium values. Lactate uptake was depressed when internal pH was lower than external pH. These data strongly suggest that La- and H+ are either cotransported by the carrier, or transported as the undissociated HLa, and can account for the majority of the lactate uptake at pH 7.4. The mechanism does not require cotransport of either K+ or Na+. However, an inwardly directed Na+ gradient without ionophore in the absence of a pH gradient doubled La- transport; treatment with amiloride, an inhibitor of the Na+/H+ exchanger, abolished this stimulation, suggesting that this transporter may be an important coregulator of intracellular pH, and could disrupt 1:1 H+ and La- efflux stoichiometry in vivo. We conclude that the majority of La- crosses the skeletal muscle SL by a specific carrier-mediated process that is saturable at high La- concentrations, but flux is passively augmented at low intracellular pH by undissociated lactic acid. In addition, a Na+/H+ exchange mechanism was confirmed in skeletal muscle SL, does affect both lactate and proton flux, and is potentially an important coregulator of intracellular pH and thus, cellular metabolism.

Animals↗
PubMed1989

Regions of the rat osteocalcin gene which mediate the effect of 1,25-dihydroxyvitamin D3 on gene transcription.

We have cloned the genomic DNA encoding rat osteocalcin and have isolated fragments in the 5' flanking region which mediate the effects of 1,25-(OH)2D3 (1,25-dihydroxyvitamin D3) on osteocalcin gene transcription. Approximately 3 kilobase pairs of the osteocalcin gene's 5' flanking region, including the promoter and transcription start site, were fused to the reporter gene chloramphenicol acetyltransferase. Transfection into ROS 17/2.8 rat osteosarcoma cells demonstrated low level basal expression of the chloramphenicol acetyltransferase gene. The expression increased markedly in the presence of 1,25-(OH)2D3; induction was observed at doses as low as 10(-11) M 1,25-(OH)2D3. Chloramphenicol acetyltransferase activity increased as early as 16 h after stimulation with 10(-9) M 1,25-(OH)2D3. Basal chloramphenicol acetyltransferase activity in ROS 24/1 and 25/1 cells was much lower than in ROS 17/2.8 cells. In these two cell lines, there was little induction of chloramphenicol acetyltransferase activity in the presence of 10(-9) M 1,25-(OH)2D3. Deletion studies of the 5' flanking region demonstrated two regions that contribute to the induction by 1,25-(OH)2D3. Deletion of a 650-base pair fragment ending 1.4 kilobase pairs upstream from the initiator ATG led to an 80% decrease in responsiveness. Removal of an additional 1.1 kilobase pairs, leaving a 300-base pair promoter containing fragment obliterated responsiveness to 1,25-(OH)2D3.

Animals↗
PubMed1988

Tobacco mosaic virus infection stimulates the phosphorylation of a plant protein associated with double-stranded RNA-dependent protein kinase activity.

The influence of tobacco mosaic virus (TMV) infection on nucleotide binding and phosphorylation of an Mr 68,000 host-encoded protein (p68) was examined. The phosphorylation of p68 in homogenates from TMV-infected tissues was 4-fold greater than in homogenates from mock inoculated tissues. Phosphorylation of p68 in extracts from mock inoculated tissues was enhanced by the addition of double-stranded (ds) RNA. Nucleotide photoaffinity labeling experiments indicate that p68 contains an ATP binding site with characteristics consistent with protein kinase activity. Antiserum raised against a dsRNA-dependent protein kinase activity. Antiserum raised against a dsRNA-dependent protein kinase from interferon-treated human cells immunoprecipitated p68 from extracts of TMV-infected tissue, and p68-containing immunocomplexes catalyzed the phosphorylation of endogenous p68. These data suggest that p68 may be an autophosphorylating, dsRNA-dependent protein kinase involved in viral pathogenesis. Based upon analogous functions demonstrated for dsRNA-dependent protein kinases in mammalian systems, p68 may have a role in the regulation of protein synthesis and viral replication in infected cells.

Electrophoresis, Polyacrylamide Gel↗
PubMed1988

Viroid-induced phosphorylation of a host protein related to a dsRNA-dependent protein kinase.

Viroids are very small, unencapsidated RNAs that replicate and induce severe disease in plants without encoding for any proteins. The mechanisms by which the viroid RNA regulates these events and interacts with host factors are unknown. An Mr 68,000 host-encoded protein has been identified that is differentially phosphorylated in extracts from viroid-infected and mock-inoculated tissues. This phosphoprotein is immunologically related to a double-stranded (ds) RNA-dependent protein kinase from virus-infected, interferon-treated human cells. Further, nucleotide photoaffinity labeling indicates that the protein has an ATP binding site. This protein is similar to dsRNA-dependent protein kinases implicated in mammalian systems in the regulation of protein synthesis and virus replication.

Molecular Weight↗
PubMed1988

Induced lactacidemia does not affect postexercise O2 consumption.

To study the effects of circulatory occlusion on the time course and magnitude of postexercise O2 consumption (VO2) and blood lactate responses, nine male subjects were studied twice for 50 min on a cycle ergometer. On one occasion, leg blood flow was occluded with surgical thigh cuffs placed below the buttocks and inflated to 200 mmHg. The protocol consisted of a 10-min rest, 12 min of exercise at 40% peak O2 consumption (VO2 peak), and a 28-min resting recovery while respiratory gas exchange was determined breath by breath. Occlusion (OCC) spanned min 6-8 during the 12-min work bout and elicited mean blood lactate of 5.2 +/- 0.8 mM, which was 380% greater than control (CON). During 18 min of recovery, blood lactate after OCC remained significantly above CON values. VO2 was significantly lower during exercise with OCC compared with CON but was significantly higher during the 4 min of exercise after cuff release. VO2 was higher after OCC during the first 4 min of recovery but was not significantly different thereafter. Neither total recovery VO2 (gross recovery VO2 with no base-line subtraction) nor excess postexercise VO2 (net recovery VO2 above an asymptotic base line) was significantly different for OCC and CON conditions (13.71 +/- 0.45 vs. 13.44 +/- 0.61 liters and 4.93 +/- 0.26 vs. 4.17 +/- 0.35 liters, respectively). Manipulation of exercise blood lactate levels had no significant effect on the slow ("lactacid") component of the recovery VO2.

Energy Metabolism↗
PubMed1987

Involvement of renal allograft by Fabry's disease.

A man with Fabry's disease received a renal allograft from a heterozygous sister. Renal allograft dysfunction necessitated an allograft biopsy 5.5 years after transplantation. Extensive accumulation of Fabry's disease deposits in the glomeruli, tubules, blood vessels and interstitium was noted.

Adult↗
PubMed1986

In vitro study of bacterial growth in continuous ambulatory peritoneal dialysis fluids.

We examined the in vitro survival of bacteria in continuous ambulatory peritoneal dialysis effluents of patients with clinical peritonitis and those without peritonitis. Standard strains of coagulase-negative staphylococci (CNS), Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa were inoculated into the fluids, and portions were plated for bacterial counts at 0.5, 4, 24, 48, 72, and 96 h. Unused dialysate fluid was also inoculated simultaneously. Our results show that CNS increased minimally up to 48 h in the noninfected continuous ambulatory peritoneal dialysis effluents and decreased by 96 h, whereas survival was only minimal in the infected effluent. S. aureus showed trends similar to those of CNS, but differences in survival in infected and noninfected effluents were less marked. By contrast, E. coli and P. aeruginosa increased by greater than 1,000-fold in all solutions tested. Based on the above findings, it is likely that a proportionate number of culture-negative cases of peritonitis are due to gram-positive cocci, especially CNS, which are not retrievable by standard culture techniques because of poor survival rate.

Escherichia coli↗
PubMed1985

Photoaffinity labeling of a viral induced protein from tobacco. Characterization of nucleotide-binding properties.

We have used the photoaffinity analogs 8-azidoadenosine 5'-triphosphate (8-N3ATP) and 8-azidoguanosine 5'-triphosphate (8-N3GTP) to investigate the relationship between a viral induced protein (Mr = 120,000) in tobacco mosaic virus (TMV)-infected tobacco and the TMV-induced RNA-dependent RNA polymerase activity. When the radioactive analogs [gamma-32P]8-N3ATP and [gamma-32P]8-N3GTP were incubated with the tobacco tissue homogenate from TMV-infected plants, incorporation of label occurred into the viral induced protein in the presence of UV light. The incorporation was found to be totally dependent on UV-illumination and greatly enhanced by Mg2+. Saturation of photoincorporated label indicates an apparent Kd of 16 microM (+/- 3 microM) and 12 microM (+/- 3 microM) for 8-N3ATP and 8-N3GTP, respectively. Protection against photolabeling by [gamma-32P]8-N3ATP and [gamma-32P]8-N3GTP with various nonradioactive nucleotides and nucleosides suggests that the photolabeled site is protected best by nucleoside triphosphates. At 200 microM both deoxyribonucleoside triphosphates and ribonucleoside triphosphates were very effective at protecting the site from photolabeling. These data suggest that the photolabeled protein may be part of an RNA-dependent RNA polymerase. The utility of nucleotide photoaffinity analogs as a method to study viral induced nucleotide-binding proteins is discussed.

Adenosine Triphosphate↗
PubMed1985

Schönlein-Henoch syndrome in adults.

Nine adult patients, aged from 16 to 70 years, (mean 42 years), with Schönlein-Henoch syndrome were studied during the acute stage of their illness. Each presented with the typical purpuric skin rash, and renal disease was found in every case. Microscopic haematuria and proteinuria were consistently found and red cell casts were seen in urine from five patients. Kidney biopsy performed on nine patients showed focal glomerulonephritis in seven cases and diffuse proliferative nephritis in two. The finding of mesangial deposits of immunoglobulin A (IgA) in seven of eight patients is in agreement with recent reports suggesting that the syndrome is an immune complex disorder. The diffuse proliferative lesion was accompanied by loss of renal function, but there were no fatalities in this series. Corticosteroids administered during the acute stage appear to ameliorate the symptoms, especially the arthralgia and abdominal pain, and may prevent progression of the renal disease in some cases.

Adolescent↗
PubMed1984

Computer-aided nucleic acid secondary structure modeling incorporating enzymatic digestion data.

We present a computer-aided method for determining nucleic acid secondary structure. The method utilizes a program which has the capability to filter matrix diagonal data on the basis of diagonal length, stabilization energy, and chemical and enzymatic data. The program also allows the user to assign selected regions of the structure as uniquely single-stranded or paired, and to filter out "trade-off" structures on the basis of such pairing. In order to demonstrate the utility of the program we present a preliminary secondary structure for the 3' end of alfalfa mosaic virus RNA 4 (AMV-4 RNA). This structure is based on an analysis which includes the use of in vitro partial enzymatic digestion of the RNA.

Base Sequence↗
PubMed1983

Early renal failure in Fabry's disease.

Two young males were observed with renal failure from Fabry's disease. These two patients had progressive decrease of renal function and required hemodialysis at ages 16 and 24. Diagnosis was confirmed by low plasma galactosidase levels and by skin and kidney biopsy. Each has had a successful kidney transplant with good graft function, the 16-year-old patient for five years and the 24-year-old for two years. Although the renal impairment usually does not appear until the fourth or fifth decade of life. Fabry's disease should be considered in the differential diagnosis of renal failure in young males.

Adolescent↗