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Biomedical subjects

R A Roth

Publications and source records attributed to R A Roth.

At least 127 records · Page 7Linked to original sources
PubMed1994

Cellular distribution of insulin-degrading enzyme gene expression. Comparison with insulin and insulin-like growth factor receptors.

Insulin-degrading enzyme (IDE) hydrolyzes both insulin and IGFs and has been proposed to play a role in signal termination after binding of these peptides to their receptors. In situ hybridization was used to investigate the cellular distribution of IDE mRNA and to compare it with insulin receptor (IR) and IGF-I receptor (IGFR) gene expression in serial thin sections from a variety of tissues in embryonic and adult rats. IDE mRNA is highly abundant in kidney and liver, tissues known to play a role in insulin degradation. IDE and IR mRNAs are highly coexpressed in brown fat and liver. The highest level IDE gene expression, on a per cell basis, is found in germinal epithelium. IDE and IGFR mRNAs are colocalized in oocytes, while IDE is colocalized with the IGF-II receptor in spermatocytes, suggesting that IDE may be involved with degradation of IGF-II in the testis. In summary, IDE expression demonstrates significant anatomical correlation with insulin/IGF receptors. These data are compatible with a role for IDE in degrading insulin and IGFs after they bind to and are internalized with their respective receptors and may also suggest a novel role for IDE in germ cells.

Animals↗
PubMed1994

Activation of protein kinase C alpha inhibits insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1.

Chinese hamster ovary (CHO) cells were transfected with a cDNA encoding protein kinase C alpha (PKC) and a cell line (CHO-PKC alpha) expressing approximately 7-fold greater amounts of PKC as the parental cells were isolated. Activation of PKC by 12-O-tetradecanoylphorbol-13-acetate in the CHO-PKC alpha cells inhibited by approximately 75% the: 1) insulin-stimulated increase in antiphosphotyrosine precipitable phosphatidylinositol 3-kinase activity in these cells; 2) insulin-stimulated increase in PI 3-kinase activity associated with insulin receptor substrate-1; and 3) tyrosine phosphorylation of the endogenous substrate, insulin receptor substrate-1. In contrast, 12-O-tetradecanoylphorbol-13-acetate treatment did not inhibit any of these responses in the parental CHO cells. These results indicate that excessive PKC activity can interfere in a very early step in insulin receptor signaling and are consistent with the hypothesis that excessive PKC activity may contribute to some states of insulin resistance.

Animals↗
PubMed1994

Insulin receptor signaling in Madin-Darby canine kidney cells overexpressing the human insulin receptor.

We have developed and characterized a line of Madin-Darby canine kidney (MDCK) cells overexpressing the human insulin receptor. The expressed receptor was found to be processed normally, and its intrinsic tyrosine kinase was determined to be functional from both in vitro and in vivo phosphorylation studies. The expressed receptor was able to mediate an insulin-stimulated increase in both anti-phosphotyrosine-precipitable and anti-insulin receptor substrate 1-precipitable phosphatidylinositol 3-kinase activity. Moreover, insulin-induced glycogen synthase activity was greater and more sensitive to insulin in the transfected cells than in the parental cells. Interestingly, insulin promoted tubule-like growth in cells overexpressing the insulin receptor but not in the parental cells. Another advantage of this cell system lies in its ability to polarize into distinct basolateral and apical membrane compartments. With the use of biotinylation and Western analysis, the expressed insulin receptor was found to be preferentially expressed in the basolateral membrane (fivefold greater) in comparison with the apical membrane. Therefore, MDCK cells overexpressing the insulin receptor represent a novel system to study not only the pathway of insulin signaling, but also this pathway in the context of cell polarity.

Animals↗
PubMed1994

Selective coexpression of insulin receptor-related receptor (IRR) and TRK in NGF-sensitive neurons.

The insulin receptor-related receptor (IRR) has recently been identified as a member of the insulin receptor tyrosine kinase family; however, its endogenous ligand and biological function are still unknown. In contrast to the very widespread pattern of expression of the homologous insulin and IGF-I receptors, IRR demonstrates a very restricted cellular distribution. Using in situ hybridization and immunohistochemistry, we now show that the expression of this receptor is selectively concentrated in a subset of neurons where its appearance is closely associated with that of the NGF receptor TRK. IRR and TRK demonstrate synchronized patterns of coexpression in neural crest-derived sensory and sympathetic neurons and in non-neural crest basal forebrain and striatal neurons. Both appear early in the embryonic development of dorsal root and trigeminal neurons and somewhat later, near the time of birth, in sympathetic neurons. Expression of both IRR and TRK appears perinatally in basal forebrain neurons, reaching maximal levels about postnatal day 20. This association is highly selective, since TRK mRNA is not detected anywhere in the developing nervous system in the absence of coordinate IRR expression, and the same is true for IRR expression with respect to TRK. In the adult rat, the majority of TRK-positive sensory neurons still express IRR mRNA, and coexpression in sympathetic and forebrain neurons continues without evidence of diminution. These findings are consistent with a functional linkage of the IRR and TRK receptors in NGF-sensitive neurons.

Animals↗
PubMed1994

Activated neutrophils from rat injured isolated hepatocytes.

BACKGROUND: Activated neutrophils (PMNs) release cytotoxic agents that can damage surrounding tissue. These studies were performed to determine whether activated PMNs from rat could injure isolated, rat hepatic parenchymal cells (HCs) in vitro. EXPERIMENTAL DESIGN: HCs were cocultured with unstimulated rat PMNs or with PMNs activated with either f-met-leu-phe (FMLP) or phorbol myristate acetate (PMA), that stimulate predominantly degranulation or superoxide production, respectively. Toxicity to HCs was evaluated from release of alanine aminotransferase into the medium. RESULTS: Alanine aminotransferase release was greater in HCs cocultured with FMLP- or PMA-stimulated PMNs compared with unstimulated PMNs. Toxicity was observed by 16 hours after stimulation of PMNs. To test the possible involvement of a soluble mediator released by activated PMNs, HCs were incubated with conditioned medium from PMNs. Compared with unstimulated PMNs, toxicity to HCs was greater in the presence of conditioned medium from FMLP-stimulated PMNs, but not conditioned medium from PMA-activated PMNs. Reactive oxygen species do not appear to be involved in the mechanism by which activated PMNs damage HCs since superoxide dismutase, catalase, superoxide dismutase+catalase, or desferrioxamine failed to prevent the injury. Furthermore, less superoxide anion was detected in PMA-stimulated PMNs when either HCs or HC-conditioned medium was present. Proteolytic enzymes released by stimulated PMNs may play a role in HC damage since an inhibitor of proteases diminished injury due to PMNs activated by either FMLP or PMA. CONCLUSIONS: These results indicate that activated, rat PMNs damage HCs in culture. The data suggest that reactive oxygen species are not involved in the mechanism, but that release of proteolytic enzymes may play a role in the toxic response.

Alanine Transaminase↗
PubMed1993

Identification of glutamate-169 as the third zinc-binding residue in proteinase III, a member of the family of insulin-degrading enzymes.

A novel active site has been identified in a family of zinc-dependent metalloendopeptidases that includes bacterial proteinase III, the human and Drosophila insulin-degrading enzymes, and the processing-enhancing protein subunit of the mitochondrial processing proteinase. None of these enzymes contains the conserved active site described in most other metalloendopeptidases, HEXXH; instead, all four contain an inversion of this motif, HXXEH. Prior mutagenesis studies of proteinase III indicate that the two histidines are essential for co-ordinating the zinc atom, while all three residues are required for enzyme activity. To identify the third zinc-binding residue in this protein family, three glutamates downstream from the active site were mutated to glutamine in proteinase III. The mutant proteins were expressed and their ability to degrade insulin was compared with the wild-type enzyme. The glutamate-204 mutant was as active as the wild-type protein, the glutamate-162 mutant retained 20% of the activity of the wild-type enzyme and the glutamate-169 mutant was completely devoid of insulin-degrading activity. The purified wild-type and glutamate-204 mutant enzymes were found to contain nearly stoichiometric levels of zinc by atomic absorption spectrophotometry, whereas the glutamate-162 mutant had a slight reduction in the level of zinc, and the glutamate-169 mutant retained less than 0.3 mol of zinc/mol of enzyme. These findings are consistent with glutamate-169 being the third zinc-binding residue in proteinase III.

Amino Acid Sequence↗
PubMed1993

Identification of SHC as a substrate of the insulin receptor kinase distinct from the GAP-associated 62 kDa tyrosine phosphoprotein.

Insulin stimulated tyrosine phosphorylation of SHC, a SH2 containing protein, was demonstrated in Chinese hamster ovary cells overexpressing the insulin receptor by immunoblotting with antiphosphotyrosine antibodies and in vivo labeling. Insulin induced tyrosine phosphorylation of SHC occurred very rapidly (within 1 min) with a dose curve which paralleled the autophosphorylation of the insulin receptor. Phosphorylation of SHC appeared to occur to a high stoichiometry since insulin induced the majority of SHC to shift to a higher molecular weight. The tyrosine phosphorylated SHC was not bound by the GTPase activating protein of Ras although a distinct 62 kDa tyrosine phosphorylated protein was found to be associated in the same experiments. It also was not bound to the insulin receptor, phosphatidylinositol 3-kinase or insulin receptor substrate-1.

Animals↗
PubMed1993

Overexpression of protein kinase C isoenzymes alpha, beta I, gamma, and epsilon in cells overexpressing the insulin receptor. Effects on receptor phosphorylation and signaling.

Chinese hamster ovary cells overexpressing the human insulin receptor were transfected with cDNAs encoding protein kinase C isoenzymes alpha, beta I, gamma, and epsilon as well as an inactive alpha. Overexpression of these protein kinase Cs did not affect expression of the insulin receptor or insulin-stimulated tyrosine phosphorylation of the receptor. However, in response to phorbol esters, cells overexpressing isoenzymes alpha, beta I, and gamma, but not epsilon or inactive alpha, exhibited 3-4-fold higher levels of insulin receptor phosphorylation. This increased phosphorylation occurred exclusively on serines and threonine. Tryptic peptide maps indicated that this phosphorylation was primarily on serines 1305/1306 and threonine 1348 as well as several other unidentified sites. This phorbol ester-stimulated phosphorylation did not inhibit activation of the insulin receptor kinase when the receptor was activated in situ but assayed in vitro. However, in cells overexpressing protein kinase C alpha, it did inhibit an in vivo monitor of the activation of the insulin receptor kinase, the insulin-stimulated increase in anti-phosphotyrosine-precipitable phosphatidylinositol 3-kinase activity. These results indicate that increased protein kinase C alpha activity can inhibit insulin-stimulated responses and support the hypothesis that excessive protein kinase C is involved in the insulin resistance observed in non-insulin-dependent diabetics.

Animals↗
PubMed1993

Effects of monocrotaline pyrrole on cultured rat pulmonary endothelium.

Administration of monocrotaline pyrrole (MCTP), a putative toxic metabolite of the pyrrolizidine alkaloid, monocrotaline (MCT), results in delayed and progressive pneumotoxicity in the rat. It has been suggested that the lung injury caused by this compound may be initiated by an interaction between MCTP and cells of the pulmonary vasculature. A likely site for initial binding of this reactive electrophile is the pulmonary endothelium. MCTP causes direct toxicity to cultured bovine and porcine pulmonary artery endothelial cells (BECs and PECs, respectively), but there exist species differences both in whole-animal response to the parent alkaloid and in cellular response to direct application of MCTP. In this study, the changes in cultured rat pulmonary vascular endothelial cells (RECs) after a single administration of MCTP were characterized in order to compare these with changes previously identified in this species in vivo. Studies with RECs have also provided an additional model for examination of species-related differences in response to this toxicant. MCTP caused a delayed and progressive release of lactate dehydrogenase from REC monolayers. Progressive cell detachment was evident and remaining cells became enlarged, with morphologic changes comparable to those reported previously in BECs, including cytoplasmic vacuolization and nuclear enlargement. MCTP inhibited cell proliferation at concentrations of 0.05 micrograms MCTP/ml or greater, and DNA crosslinking was evident at 24 and 48 hr post-treatment. These results suggest that MCTP is directly toxic to cultured RECs, and the development of changes is reminiscent of that seen in the rat in vivo. The cytostatic nature of the compound, in combination with its cytolytic effect on RECs, could contribute to the development of pulmonary edema and other lung vascular changes seen in rats treated with MCT or MCTP.

Animals↗
PubMed1993

Fibrinolytic activity in blood and lungs of rats treated with monocrotaline pyrrole.

Monocrotaline pyrrole (MCTP), a putative toxic metabolite of the pyrrolizidine alkaloid, monocrotaline, causes pulmonary vascular thrombi that are associated with vascular remodeling, pulmonary hypertension, and right cardioventricular hypertrophy in rats. It is possible that such thrombi contribute to the lung injury and pulmonary hypertension in this model. A previous study indicated that rats treated with MCTP did not have excessive procoagulant activity in the peripheral blood. Since thrombosis may also result from insufficient fibrinolytic activity in the systemic circulation, we evaluated the fibrinolytic system of rats given MCTP. Male Sprague-Dawley rats were given a single injection of MCTP (3.5 mg/kg) or an equal volume of N,N-dimethylformamide vehicle in the tail vein and were killed at 1, 3, 5, 8, 11, or 14 days after toxin administration. Several markers of lung injury and fibrinolysis were measured. Lung injury was evident 3 days after administration of MCTP and became more pronounced with time. In MCTP-treated rats, right heart hypertrophy was observed at 11 days and became more pronounced at 14 days. There was no change in the plasminogen concentration or in the activities of tissue plasminogen activator or alpha 2-antiplasmin in blood throughout the time course. Beginning at Day 8 and continuing through Day 14, there was an increase in the activity of plasminogen activator inhibitor in blood of rats that received MCTP. In addition, we evaluated the fibrinolytic activity of lung tissue slices. Rats treated with MCTP had a significant decrease in fibrinolytic activity of lung tissue at Day 3. A more pronounced decrease was evident by Day 8, and this continued through Day 14. In summary, MCTP treatment of rats decreases the fibrinolytic activity of lung tissue relatively early after exposure to the toxicant and before the onset of pulmonary hypertension. The change is reflected slightly later in plasma. These alterations in fibrinolytic activity may explain why fibrin thrombi form in the lungs of rats treated with MCTP.

Animals↗
PubMed1993

Procoagulant and fibrinolytic properties of bovine endothelial cells treated with monocrotaline pyrrole.

Administration to rats of monocrotaline pyrrole (MCTP), a putative metabolite of the pyrrolizidine alkaloid, monocrotaline, causes pulmonary microvascular thrombosis that is associated with vascular remodeling and pulmonary hypertension. It is possible that vascular thrombi contribute to the lesions that occur after MCTP treatment. Since MCTP is toxic to pulmonary endothelial cells and because the pulmonary endothelium is in a unique position to influence procoagulant and fibrinolytic reactions in the lungs, we examined changes in the procoagulant and fibrinolytic properties of cultured pulmonary artery endothelial cells exposed to MCTP to see if they might favor thrombosis. Monolayers of confluent bovine pulmonary arterial endothelial cells received a single administration of MCTP or N,N-dimethylformamide vehicle, and the media or lysates were examined at 4, 24, 48, or 120 hr after treatment. MCTP caused a time-dependent cell detachment and an increase in release of lactate dehydrogenase into culture medium. Although cell numbers decreased dramatically with time, the protein concentration of cell monolayer lysates was unchanged by treatment. MCTP treatment caused no change in the amount of tissue factor activity/micrograms cellular protein in bovine endothelial cell lysates and only a small increase in the activity of Factor V in the culture medium at 120 hr. In addition, the medium from bovine endothelial cells treated with MCTP had a time-dependent increase in the activity of plasminogen activators and a decrease in the activity of plasminogen activator inhibitors. Thus bovine endothelial cells exposed to MCTP in vitro do not produce changes in these procoagulant or fibrinolytic properties that would explain the thrombosis observed in vivo.

Animals↗
PubMed1993

Transurethral ultrasound-guided laser-induced prostatectomy: National Human Cooperative Study results.

Between November 1990 and March 1992, 150 patients at 10 United States institutions were treated with transurethral ultrasound-guided laser-induced prostatectomy (TULIP) for the relief of bladder outlet obstruction secondary to benign prostatic hypertrophy. The TULIP system incorporates ultrasound visualization with a 90-degree angle, side-firing laser to effect coagulation necrosis of prostate tissue. The overall preoperative prostate volume in this TULIP study was 40 cc and all types of prostatic enlargement, including median lobe obstruction, were treated. There were no intraoperative complications, with no hemorrhage or post-transurethral resection syndrome, and no blood transfusions were required. Hospital stay averaged 1.7 days and 83% of the patients went home after a 1-night stay. We evaluated 63 patients at 6 months after the TULIP procedure. Mean symptom scores decreased from 18.8 to 6.1, for a 68% improvement. The mean peak flow increased from 6.7 ml. per second preoperatively to 11.9 ml. per second, for a 78% improvement. Overall, 87% of the patients exhibited at least 50% improvement in either the symptom score or peak flow parameter, while 49% of the patients demonstrated at least a 50% improvement in both parameters.

Equipment Design↗
PubMed1993

Monocrotaline pyrrole-induced changes in angiotensin-converting enzyme activity of cultured pulmonary artery endothelial cells.

1. Changes in the structural and functional integrity of endothelium have been recognized as relatively early features of delayed and progressive pulmonary vascular injury caused by the pyrrolizidine alkaloid, monocrotaline (MCT). Although a number of investigators have evaluated angiotensin-converting enzyme (ACE) activity in the lungs of rats treated with MCT, the exact nature of changes in activity of this enzyme and the role they may play in MCT pneumotoxicity remain controversial. 2. We examined the direct effects of monocrotaline pyrrole (MCTP), a toxic metabolite of MCT, on cultured endothelial cell ACE activity. Post-confluent monolayers of porcine or bovine pulmonary artery endothelial cells (PECs or BECs, respectively) were treated with a single administration of MCTP at time 0; then they were examined for their ability to degrade the synthetic peptide, [3H]-benzoyl-Phe-Ala-Pro. 3. In PECs, which are relatively insensitive to the direct cytolytic effects of MCTP, monolayer ACE activity was unchanged initially but gradually decreased within 4 days after treatment with a high concentration of MCTP (150 microM). This decrease was transient, and PEC monolayer ACE activity returned to the control value by 10 days post treatment. 4. BEC monolayer ACE activity was also unchanged initially but rapidly declined within 4 days after MCTP treatment and remained depressed throughout the post treatment period. BECs were quite sensitive to the cytolytic effects of MCTP and the decline in ACE activity occurred coincident with the decrease in monolayer cellularity and appearance of marked cytotoxicity. 5. We conclude that high concentrations of MCTP decrease endothelial ACE activity. The decline in ACE activity is delayed and the magnitude and duration of the decrease corresponds to the degree ofMCTP-induced cytotoxicity. This suggests that altered endothelial ACE activity is unlikely to be a direct effect of MCTP on the enzyme but may reflect the delayed cell injury which results from exposure to this compound.

Acetylcholinesterase↗
PubMed1993

Effect of extracorporeal shock wave lithotripsy on implantable cardioverter defibrillator.

Extracorporeal shock wave lithotripsy (ESWL) is frequently used for nephrolithiasis of the upper urinary tract. Because of the powerful shock wave and electromechanical forces created, this therapy has been contraindicated in the patient with an implantable cardioverter defibrillator (ICD). To determine whether or not ESWL affects ICD devices, we subjected ten devices to a full course of ESWL. The devices were then returned to the manufacturer to undergo bench analysis, which revealed no abnormalities in function. Additionally, one device was placed in the pathway of the shock wave, resulting in a discharge of the device despite a frequency of ESWL of 100 shocks/min (well below the rate cutoff of that device). Manufacturer analysis of this device, likewise, revealed no abnormalities even though the device had been exposed to the focal point of the shock wave. We conclude that contralateral ESWL is not contraindicated for the patient with an ICD. We do, however, recommend a post-procedure evaluation to ensure appropriate function of the ICD.

Contraindications↗
PubMed1993

Relationship between tumor necrosis factor-alpha and neutrophils in endotoxin-induced liver injury.

Tumor necrosis factor-alpha (TNF-alpha) and blood neutrophils (polymorphonuclear leukocytes; PMNs) have been implicated in the pathogenesis of endotoxin (lipopolysaccharide, LPS) hepatotoxicity. However, the mechanism by which these factors mediate liver injury during LPS exposure is uncertain. The objective of this study was to test the hypothesis that TNF-alpha contributes to LPS hepatotoxicity by an indirect, PMN-dependent mechanism. Pretreatment of rats with an antiserum to TNF-alpha afforded protection against liver injury 6 h after LPS exposure. Pretreatment with pentoxifylline (100 mg/kg i.v.), which attenuated the increase in circulating TNF-alpha concentration 1.5 h after administration of LPS, also afforded protection against liver injury. Neither antiserum to TNF-alpha nor pentoxifylline affected hepatic PMN accumulation 1.5 h after LPS exposure. Depletion of circulating PMNs, which protects against LPS hepatotoxicity, enhanced circulating TNF-alpha concentration compared with control rats 1.5 h after LPS exposure. These results suggest that TNF-alpha contributes to liver injury after LPS exposure, but in the absence of circulating PMNs it is insufficient for full manifestation of liver injury. TNF-alpha apparently contributes to the pathogenesis of LPS-induced liver injury by an indirect, PMN-dependent mechanism.

Animals↗