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Biomedical subjects

R A Roth

Publications and source records attributed to R A Roth.

At least 163 records · Page 9Linked to original sources
PubMed1991

Involvement of glutathione in 1-naphthylisothiocyanate (ANIT) metabolism and toxicity to isolated hepatocytes.

1-Naphthylisothiocyanate (ANIT) is a model compound which causes cholestasis in laboratory animals. Various biochemical and morphological changes including biliary epithelial and parenchymal cell necrosis occur in the liver of animals treated with ANIT. Although the mechanism(s) for these effects is not understood, a role for glutathione (GSH) in toxicity has been implicated. The possible role of GSH in hepatocellular toxicity caused by ANIT was investigated in this study. Treatment of freshly isolated rat hepatocytes with ANIT caused a concentration- and time-dependent depletion of cellular GSH that preceded lactate dehydrogenase (LDH) leakage. Analysis of the incubation medium indicated that the majority of the cellular GSH which was lost was present extracellularly as GSH or as a GSH-releasing compound. Mixing ANIT with GSH at pH 7.5 yielded a compound that was characterized by HPLC and fast atom bombardment-mass spectrometry (FAB-MS) S-(N-naphthyl-thiocarbamoyl)-L-glutathione (GS-ANIT). When dissolved in aqueous solutions at neutral pH, 95% of GS-ANIT dissociated to yield free ANIT and GSH. Under conditions designed to maximize formation and stability of GS-ANIT, GS-ANIT was found in the extracellular medium of hepatocytes treated with ANIT. Treatment of hepatocytes with the GS-ANIT caused GSH depletion and LDH leakage similar to that observed with equimolar amounts of ANIT. These data suggest that ANIT depletes hepatocytes of GSH through a reversible conjugation process. Such a process may play a role in the toxicity of ANIT.

1-Naphthylisothiocyanate↗
PubMed1991

A region of the insulin receptor important for ligand binding (residues 450-601) is recognized by patients' autoimmune antibodies and inhibitory monoclonal antibodies.

Chimeric receptors containing different portions of the homologous human insulin receptor, insulin-like growth factor I receptor, and insulin receptor-related receptor were utilized to identify the epitopes recognized by various anti-insulin receptor antibodies. The antibodies studied included 12 monoclonal antibodies to the extracellular domain of the human insulin receptor as well as 15 patients' sera with autoimmune anti-insulin receptor antibodies. All of the patients' sera and all 8 monoclonal antibodies that inhibit insulin binding were found to recognize an epitope contained within residues 450-601 of the alpha subunit of the receptor. In contrast, 2 monoclonal antibodies that do not inhibit insulin binding were found to recognize the cysteine-rich region of the alpha subunit. Chimeric insulin receptors that had residues 450-601 replaced with the homologous residues of the insulin-like growth factor I receptor exhibited a decreased ability to bind insulin. In contrast, insulin-like growth factor I receptors that have had the comparable region replaced with that of the insulin receptor showed no decrease in their ability to bind ligand. These results indicate that residues 450-601 of the insulin receptor are important for insulin binding and include the major site for recognition by inhibitory monoclonal antibodies and patients' autoimmune anti-receptor antibodies.

Antibodies, Monoclonal↗
PubMed1991

Evidence for hybrid rodent and human insulin receptors in transfected cells.

Chinese hamster ovary cells and NIH 3T3 cells overexpressing mutant human insulin receptors were examined for the presence of hybrid receptors composed of human and rodent insulin receptors. In the present studies, most of the endogenous rodent receptors were found to be immunoprecipitated from the transfected cells but not the parental cells with a monoclonal antibody specific for human receptor. These data indicate that in these transfected cells, most of the endogenous rodent receptors are in a hybrid complex with the overexpressed human receptor. These results together with the in vitro studies of Treadway et al. (Treadway, J.L., Morrison, B.D., Soos, M.A., Siddle, K., Olefsky, J., Ullrich, A., McClain, D.A., and Pessin, J.E. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 214-218) showing that hybrid receptors exhibit transdominant inhibition explain the prior finding indicating that overexpression of defective insulin receptors interferes with the normal signaling of endogenous receptors.

Animals↗
PubMed1991

Protection against alpha-naphthylisothiocyanate-induced liver injury by decreased hepatic non-protein sulfhydryl content.

alpha-Naphthylisothiocyanate (ANIT) injures bile duct epithelium and hepatic parenchymal cells in rats. It is commonly believed that ANIT must undergo bioactivation by hepatic, cytochrome P450-dependent mixed-function oxidases (MFO), since agents which are inducers or inhibitors of hepatic MFO activity enhance or attenuate, respectively, the liver injury associated with ANIT. Several of these agents also affect hepatic glutathione (GSH) content and/or GSH S-transferase activity in a manner to suggest a causal role for GSH in ANIT-induced hepatotoxicity. To determine whether GSH might be involved in the mechanism of injury, buthionine sulfoximine (BSO), diethyl maleate (DEM), or phorone was used to reduce hepatic non-protein sulfhydryl (NPSH) content, an indicator of GSH content. Twenty-four hours after ANIT treatment, rats exhibited cholestasis and elevations in serum of total bilirubin concentration, total bile acid concentration, aspartate aminotransferase (AST) activity, and gamma-glutamyltransferase activity. Cotreatment of rats with BSO decreased NPSH content by 70% at 24 hr and prevented the cholestasis and elevations in serum markers of liver injury caused by ANIT. Likewise, cotreatment of rats with DEM afforded protection against markers of liver injury. Phorone treatment attenuated ANIT-induced elevations in serum total bilirubin concentration and AST activity. Although BSO treatment afforded protection against ANIT-induced liver injury at 24 hr, the injury was evident at 48 hr, and it appeared to coincide with a return of hepatic NPSH content. These results suggest that GSH plays a causal or permissive role in the liver injury caused by ANIT.

1-Naphthylisothiocyanate↗
PubMed1991

Relationship between alpha-naphthylisothiocyanate-induced liver injury and elevations in hepatic non-protein sulfhydryl content.

Acute administration of alpha-naphthylisothiocyanate (ANIT) to rats has been used as a model of intrahepatic cholestasis. The mechanism of toxicity of ANIT is unknown, although recent evidence suggests a causal or permissive role for glutathione (GSH) (Dahm LJ and Roth RA, Biochem Pharmacol 42: 1181-1188, 1991). In these studies, ANIT treatment elevated hepatic non-protein sulfhydryl (NPSH) content, an indicator of GSH content, when liver injury was evident. The purpose of the present study was to characterize the effects of ANIT on hepatic NPSH content and to relate these changes to the development of liver injury. In rats fasted for 24 hr, administration of ANIT (100 mg/kg, per os [p.o.]) did not change hepatic NPSH content, bile flow, or serum measurements of total bilirubin concentration, alanine aminotransferase (ALT) activity, or gamma-glutamyltransferase (GGT) activity by 12 hr post-treatment relative to corn oil vehicle controls. However, by 24 hr after ANIT treatment, rats exhibited cholestasis and elevations in serum markers of liver injury. These markers were associated temporally with an increase in hepatic NPSH content, which consisted entirely of GSH. To determine whether the cholestasis caused by ANIT treatment might have caused the elevation of hepatic NPSH content, an extrahepatic cholestasis in rats was produced by ligation of the common bile duct. Bile duct ligation elevated hepatic NPSH content between 6 and 12 hr after ligation. Administration to rats of a non-hepatotoxic analog of ANIT, beta-naphthylisothiocyanate, also elevated hepatic NPSH content 24 hr after treatment. Taken together, these results indicate that the elevation in hepatic NPSH content after ANIT treatment is associated temporally with the onset of liver injury, but this elevation does not appear to participate causally in the mechanism of injury.

1-Naphthylisothiocyanate↗
PubMed1991

Early indications of monocrotaline pyrrole-induced lung injury in rats.

Monocrotaline pyrrole (MCTP), a putative metabolite of the naturally occurring plant toxin, monocrotaline (MCT), causes lung injury, pulmonary hypertension, and right cardioventricular hypertrophy when administered intravenously to rats. The lesions caused by MCTP administration are similar to those caused by MCT but appear to occur on a slightly accelerated time course. To explore the onset and development of lung lesions, male Sprague-Dawley rats were treated with a single, intravenous injection of MCTP, and several markers of lung injury were evaluated at early times after administration. Rats received 3.5 mg MCTP/kg or an equal volume of the vehicle, N,N-dimethylformamide (DMF), via the tail vein at time 0 and were killed at 4, 12, 24, 48, 72, or 120 hr after treatment. Beginning at 4 hr, MCTP-treated rats had increased wet lung-to-body-weight ratios (LW/BW). The LW/BW remained elevated at 12 hr, returned to baseline at 24 hr, then increased steadily over the next few days. At 24 hr, the protein concentration of cell-free bronchoalveolar lavage fluid (BALF) was slightly elevated. Lactate dehydrogenase activity in cell-free BALF samples was moderately increased 48 hr after MCTP. Changes in these markers were modest initially but became much more pronounced by 120 hr. Total nucleated cell counts in BALF were variable but were moderately elevated at 120 hr. Cytologic examination of the BALF samples revealed small but significant infiltrates of segmented neutrophils at 4 hr and relatively large infiltrates of segmented neutrophils and small lymphocytes at 120 hr post-treatment. Mature neutrophils had degenerate cytomorphologic characteristics at both 4 and 120 hr. These results confirm that pronounced lung injury is delayed for several days after a single, intravenous administration of MCTP, but they also indicate that subtle lung injury can be detected using quantitative markers quite early after MCTP administration.

Animals↗
PubMed1991

Binding properties of chimeric insulin receptors containing the cysteine-rich domain of either the insulin-like growth factor I receptor or the insulin receptor related receptor.

We constructed and expressed chimeric receptor cDNAs with insulin receptor exon 3 (residues 191-297 of the cysteine-rich region) replaced with either the comparable region of the insulin-like growth factor I receptor (IGF-IR) or the insulin receptor related receptor (IRR). Both chimeric receptors still could bind insulin with as high affinity as the wild-type receptor. In addition, chimeric receptors containing exon 3 of the IGF-IR could also bind with high affinity both IGF-I and IGF-II. In contrast, chimeric receptors containing exon 3 of IRR did not bind either IGF-I, IGF-II, or relaxin. These results indicate that (1) the high affinity of binding of insulin to its receptor can occur in the absence of insulin receptor specific residues encoded by exon 3, the cysteine-rich region; (2) the cysteine-rich region of the IGF-I receptor can confer high-affinity binding to both IGF-I and IGF-II; and 3) the IRR is unlikely to be a receptor for either IGF-I, IGF-II, or relaxin.

Base Sequence↗
PubMed1991

Molecular basis of the activation of the tumorigenic potential of Gag-insulin receptor chimeras.

A previous study showed that the human insulin receptor (IR) could be activated by insertion of a 3' portion of the cDNA encoding the beta subunit into a retrovirus genome to form a Gag-IR fusion protein. While capable of transforming cells in culture, this IR cDNA-containing virus, called UIR, was not able to induce tumors in animals. Subsequently, we isolated a spontaneous sarcomagenic variant called UIR19t from the parental UIR. UIR19t was molecularly cloned, sequenced, and found to harbor two mutations. A 44-amino acid deletion immediately upstream from the transmembrane domain of the Gag-IR fusion protein removes all the extracellular sequence of the IR remaining in the original UIR construct. In addition, a single nucleotide deletion at the 3' end results in truncation and replacement of the carboxyl-terminal 12 amino acids by 4 new amino acids. The specific kinase activity of UIR19t is 4- to 5-fold higher than that of the parental UIR. However, no new cellular substrates were detected in UIR19t-transformed cells as compared to UIR cells. Viruses containing either the 5' or the 3' deletion mutation were constructed and assessed for their biological function. Our data indicate that the 5' deletion alone is sufficient to confer tumorigenic ability. We conclude that sequence immediately upstream from the transmembrane domain imposes a negative effect on the transforming and tumorigenic potential of the Gag-IR fusion protein.

Amino Acid Sequence↗
PubMed1991

The regulatory role of known tyrosine autophosphorylation sites of the insulin receptor kinase domain. An assessment by replacement with neutral and negatively charged amino acids.

Autophosphorylation of the insulin receptor has been previously documented to activate the phosphotransferase activity of the receptor from 20- to 200-fold. Biochemical studies have correlated activation of the receptor kinase with the autophosphorylation of tyrosines residues 1158, 1162, and 1163. To further assess the role of these 3 tyrosines in the activation process, we have studied the effect of their substitution with either the neutral amino acids phenylalanine or alanine or with the negatively charged amino acids aspartate and glutamate. In several other proteins, it has been shown that substitution of phosphorylated residues with negatively charged amino acids can mimic the phosphorylation state of the protein. In agreement with previous studies, tyrosines at positions 1162 and 1163 were found to be crucial in the kinase activation process. In contrast, mutant receptors with tyrosine 1158 changed to either phenylalanine or aspartate were still activated to the same extent as the wild-type receptor. An increased basal exogenous kinase activity was observed upon replacement of tyrosines 1162 and 1163 with, in increasing order of potency, aspartate = glutamate less than alanine = phenylalanine. These results indicate that phosphorylation of tyrosines 1162/1163 but not 1158 play a critical role in the activation of the receptor kinase and that the mechanism of activation of the receptor kinase by autophosphorylation is more complex than just an introduction of a cluster of negative charges in this region of the receptor. In addition, the finding of an increased basal kinase activity in receptors lacking tyrosines 1162 and 1163 could explain the reported ability of this receptor to mediate certain biological responses.

Amino Acids↗
PubMed1991

Time interval gating for analysis of cell function using flow cytometry.

We propose a method which significantly shortens the time required for both the collection and analysis of data derived from multiple sample, flow cytometric kinetic assays. We have defined the term Time Interval Gating (TIG) to describe this method. TIG effectively allows one flow cytometer to concurrently monitor several samples over the course of a kinetic assay. Data for all samples are stored in a single FCS 2.0 compatible listmode data file which we refer to as the TIG data file. TIG is adaptable to most commerical flow cytometers. Standard listmode analysis software can be used to analyze the TIG data files and correlate any combination of tubes and/or time intervals from the assay. Results for the entire assay can be displayed on a single two parameter plot. This paper describes how TIG is applied to neutrophil oxidative burst measurement using a standard EPICS Elite flow cytometer. In this assay, 11 samples were each monitored for 30 min to identify the extent to which volatile organic chemicals (VOCs) inhibited the oxidation of DCFH in stimulated neutrophils. TIG makes the oxidative burst assay practical for high volume screening by reducing the overall flow cytometer and analysis time required by a factor of ten. In addition, TIG provides an organized approach to managing data acquisition on instruments equipped with automated sampling systems.

Flow Cytometry↗
PubMed1991

Lung vascular injury from monocrotaline pyrrole, a putative hepatic metabolite.

The pyrrolizidine alkaloid, monocrotaline (MCT), is a plant toxin that causes injury to the vasculature of the lungs and pulmonary hypertension in animals. To produce lung injury, MCT is bioactivated in the liver by cytochrome P450 monooxygenases to pyrrolic metabolites which travel via the circulation to the lungs, where they cause injury by unknown mechanisms. One putative metabolite of MCT is monocrotaline pyrrole (dehydromonocrotaline, MCTP), a moderately reactive, bifunctional alkylating agent. A single, iv injection of chemically synthesized MCTP into rats causes delayed and progressive lung vascular injury and pulmonary hypertension similar to that caused by MCT itself. Since pulmonary vascular endothelium is likely an important target of MCTP in vivo, the effects of MCTP on cultured endothelium were studied. A single application of MCTP to confluent monolayers of cultured endothelium from bovine pulmonary artery results in release of lactate dehydrogenase, some cell detachment from the growth surface and markedly altered morphology of remaining viable cells. These effects are dose-dependent and, as in vivo, are delayed in onset (1-2 days) and progressive. In endothelial cells of porcine origin, these particular responses to MCTP are also apparent but much less pronounced. Inhibition of proliferation of cells plated at low density occurred in both cell types at nominal MCTP concentrations (0.5 micrograms/ml) that were not overtly cytotoxic. These results indicate that MCTP causes a direct, dose-dependent injury to pulmonary vascular endothelium in culture that is delayed and progressive and suggest a mechanism by which MCT may act in vivo to cause lung injury and pulmonary hypertension.

Animals↗
PubMed1991

An evaluation of procoagulant activity in the peripheral blood of rats treated with monocrotaline pyrrole.

Monocrotaline pyrrole (MCTP), a putative toxic metabolite of the naturally occurring pyrrolizidine alkaloid, monocrotaline, causes pulmonary vascular thrombi that are associated with vascular remodeling, pulmonary hypertension, and right cardioventricular hypertrophy in rats. The thrombi are composed of platelets and fibrin and occur in the absence of vascular necrosis. Since thrombosis may result from excessive procoagulant activity in the systemic circulation, we evaluated the hemostatic system of rats treated with MCTP to determine if a hypercoagulable state developed in the peripheral blood. Male Sprague-Dawley rats received a single, bolus injection of MCTP (3.5 mg/kg) or an equal volume of the N,N-dimethylformamide (DMF) vehicle in the tail vein. Rats were killed at 1, 3, 5, 8, 11, or 14 days after toxin administration, and several markers of lung injury and hemostasis were evaluated. The protein concentration of cell-free bronchoalveolar lavage fluid (BALF) from MCTP-treated rats was slightly elevated at 1 and 3 days. At 5 days the elevation had become more pronounced, and values increased markedly thereafter. The wet lung-to-body-weight ratio and lactate dehydrogenase activity of cell-free BALF from rats treated with MCTP were mildly increased at 3 days. Increases in these markers progressed at 5 days and values reached a plateau thereafter. MCTP-treated rats had moderately increased total nucleated cell counts in BALF at 5 days, and counts increased markedly thereafter. Right cardioventricular hypertrophy was first detected at 8 days in MCTP-treated rats and became more pronounced with time. The prothrombin time and modified prothrombin time of MCTP-treated rats were consistently greater than those of controls. Although statistically different, these values never exceeded the range of normal values. The activated partial thromboplastin time of MCTP and control rats was variable. Only at Day 14 did MCTP-treated rats have significantly longer activated partial thromboplastin times than those of controls, and these values were within the normal range. Rats treated with MCTP had statistically significant elevations in antithrombin 3 activity at Day 8 and of fibrinogen concentration and antithrombin 3 activity at Day 11 that exceeded the normal range. Platelet numbers of both MCTP- and DMF-treated rats were greater than normal on Day 1 but quickly returned to baseline. The plasminogen values of rats treated with MCTP were lower than controls on Day 5 only. These results suggest that the pulmonary vascular thrombi induced by administration of MCTP to rats are not mediated by an excess in procoagulant activity in the peripheral blood.

Animals↗
PubMed1991

Transurethral ultrasound-guided laser-induced prostatectomy (TULIP procedure): a canine prostate feasibility study.

We describe the TULIP procedure, a new system to relieve bladder outlet obstruction caused by benign prostatic hyperplasia. This device is composed of a real-time 7.5 MHz ultrasound transducer coupled to a Nd:YAG laser with a 1.064 microns wavelength that fires through an intact intraprostatic balloon. A series of feasibility studies in 21 canine prostate glands was performed with a follow-up time to 3 months. Results indicate that the Nd:YAG laser in the 20 to 40 W range at a pull rate of approximately 1 mm. per second is an effective means of removing substantial amounts of canine benign prostatic hyperplasia. Transurethral ultrasonography was a reliable means of identifying essential landmarks and of controlling the laser. Prostatectomy by laser coagulation necrosis resulted in no bleeding or postoperative obstruction. Intraoperative irrigation fluids were not required, eliminating systemic volume related problems.

Animals↗
PubMed1991

Renal extracorporeal shock wave lithotripsy performed in patient with implantable cardioverter defibrillator.

The effect of extracorporeal shock wave lithotripsy on the automatic implantable cardioverter defibrillator is unknown. To evaluate what effect might occur, a non-implanted automatic implantable cardioverter defibrillator was subjected to a full course of extracorporeal shock wave lithotripsy while inactive. Bench testing by the manufacturer after lithotripsy demonstrated normal function of the device. A patient with an automatic implanted cardioverter defibrillator who required contralateral extracorporeal shock wave lithotripsy then underwent this procedure. The right renal calculus was destroyed successfully with no apparent damage to the automatic implantable cardioverter defibrillator. A test of the automatic implantable cardioverter defibrillator after lithotripsy demonstrated normal sensing and conversion of induced ventricular tachycardia.

Aged↗
PubMed1991

Comparison of response of bovine and porcine pulmonary arterial endothelial cells to monocrotaline pyrrole.

Monocrotaline (MCT)-induced vascular injury in liver and lung may be caused by interaction of MCT metabolites such as monocrotaline pyrrole (MCTP) with vascular cells. Responses of bovine and porcine pulmonary artery endothelial cells (BECs and PECs, respectively) to a single administration of MCTP were compared. MCTP caused a delayed and progressive release of lactate dehydrogenase (LDH) activity from BECs and a gradual decrease in monolayer cellularity. Surviving cells became markedly hypertrophic. PECs were less sensitive to the cytolytic effects of MCTP, showing minimal cell detachment and little release of LDH activity. However, monolayer cellularity, as assessed by PEC enumeration, decreased in a dose-dependent manner. Hypertrophy of surviving PECs was less pronounced than in BECs. MCTP caused enhanced release of prostacyclin from monolayers of BECs and PECs exposed to 10 micrograms MCTP/ml, and concentrations of 0.5 microgram/ml or greater caused equivalent reduction in colony-forming efficiency in both cell types. In summary, whereas BECs were more susceptible to the cytolytic and hypertrophic effects of MCTP, BECs and PECs responded similarly with regard to prostacyclin release and were equally sensitive to the cytostatic effects of this compound.

6-Ketoprostaglandin F1 alpha↗