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Biomedical subjects

R A Roth

Publications and source records attributed to R A Roth.

At least 145 records · Page 8Linked to original sources
PubMed1993

Characterization of monocrotaline pyrrole-induced DNA cross-linking in pulmonary artery endothelium.

Monocrotaline pyrrole (MCTP) is a putative, toxic metabolite of the pyrrolizidine alkaloid, monocrotaline (MCT). When given intravenously to rats, it produces a delayed and progressive injury to the vasculature of the lungs that results in pulmonary hypertension and right heart hypertrophy. Dysfunctional endothelium and vascular leak are early signs of overt injury to the lung. When administered to endothelial cell cultures, MCTP causes cell enlargement, delayed and progressive cytotoxicity, and inhibition of proliferation in surviving cells. MCTP is a bifunctional alkylating agent which binds to DNA and other macromolecules. To examine DNA-MCTP interactions in endothelium, MCTP-induced DNA cross-linking was characterized in cultures of porcine endothelial cells (PECs) derived from pulmonary artery. MCTP caused DNA cross-linking in a dose-dependent manner that was consistent with its ability to inhibit cell proliferation. PECs exposed to MCTP for 48 h developed cross-linking that was maximal at 2 days and remained significant through 10 days. Increasing the duration of PEC exposure to the medium to which MCTP had been added was associated with increased DNA cross-linking. These results indicate that MCTP causes DNA cross-linking, which may explain the inhibition of cell proliferation observed in pulmonary endothelial cells in vitro. The long-lasting nature of DNA cross-linking and its dose relatedness are consistent with the delayed and progressive effects of MCTP on endothelial cells in vitro and on pulmonary vasculature in vivo.

Animals↗
PubMed1993

Insulin receptor-related receptor messenger ribonucleic acid is focally expressed in sympathetic and sensory neurons and renal distal tubule cells.

The insulin receptor-related receptor (IRR) demonstrates striking structural homology to the insulin receptor (IR) and insulin-like growth factor-I receptor (IGFR), suggesting that IRR is a member of the IR family. However, the endogenous ligand and biological role for this "orphan" receptor are unknown. To identify potential sites of action for the IRR, in situ hybridization was employed to reveal cellular patterns of IRR gene expression in the developing and adult rat and in the adult human kidney. From embryonic days 15-20, IRR mRNA is most abundant in sensory neurons of the trigeminal and dorsal root ganglia and, to a lesser extent, neurons of the autonomic system. IRR gene expression diminishes in the majority of sensory neurons postnatally, but remains abundant in a subpopulation of adult rat trigeminal and dorsal root ganglion neurons. IRR mRNA is localized in peripheral autonomic ganglia localized in the adrenal medulla and renal hilum in the adult. From birth to maturity, IRR mRNA is abundant in renal epithelial cells focally localized in the distal tubule in both rat and human kidney. The specificity of this pattern of IRR gene expression was demonstrated by hybridization of serial tissue sections with two different nonoverlapping cRNA probes. Nonspecific signal, as measured by IRR sense probe hybridization, was negligible. This highly restricted pattern of IRR gene expression is in marked contrast to the very widespread pattern of gene expression demonstrated by the IR and IGFR. This study showed that IRR, IR, and IGFR mRNAs were colocalized in some sensory neurons, suggesting the possibility for hybrid receptor formation in these cells. In summary, IRR gene expression is focally localized in sensory and autonomic neurons and renal distal tubule cells. These observations suggest that the IRR, in contrast to the related IR and IGFR, serves a narrow cell-specific role.

Animals↗
PubMed1993

Characterization of acute 4,4'-methylene dianiline hepatotoxicity in the rat.

Methylene dianiline (DDM) is a chemical intermediate in the production of isocyanates and other industrial chemicals, and it is hepatotoxic in humans. The acute hepatotoxicity of orally administered DDM was characterized in rats. Rats receiving DDM (25-225 mg/kg, per os) demonstrated a dose-dependent elevation in serum alanine aminotransferase activity, g-glutamyltransferase activity, and serum bilirubin concentration. DDM also caused a decrease in bile flow and an elevation in liver weight. Significant changes in these markers of liver injury occurred between 8 and 12 hr after a single, oral administration of DDM. Histologically, DDM caused multifocal, necrotizing hepatitis with neutrophil infiltration. Changes in the portal regions consisted of bile ductular necrosis, portal edema, neutrophil infiltration, mild fibrin exudation, and segmental necrotizing vasculitis. The role of cytochrome P450 monooxygenase (MO)-mediated metabolism in DDM hepatotoxicity was evaluated using the MO inhibitors, aminobenzotriazole and SKF-525A and the MO inducers phenobarbital and beta-naphthoflavone. Aminobenzotriazole provided protection from DDM-induced hepatotoxicity, whereas SKF-525A had no effect. The effect of phenobarbital pretreatment depended on the dose of DDM administered. At a dose of DDM that produced a maximal hepatotoxic response, phenobarbital did not influence hepatotoxicity. However, phenobarbital pretreatment provided protection against the hepatotoxic effects of a lower dose of DDM. beta-naphthoflavone pretreatment had a more modest effect on DDM-induced hepatic insult. These results demonstrate that DDM causes acute hepatotoxicity in the rat that is dose and time dependent. Results using inducers and inhibitors of MO suggest that DDM requires bioactivation to exert toxicity; however, the relationship between metabolism and toxicity may be complex.

Administration, Oral↗
PubMed1993

Neutrophil function after exposure to polychlorinated biphenyls in vitro.

Polychlorinated biphenyls (PCBs) are known to be immunotoxic, yet the effects on neutrophil (PMN) function are not well characterized. We incubated PMNs isolated from rat peritoneum with a mixture of PCB congeners, Aroclor 1242, in the absence or presence of either phorbol myristate acetate (PMA) to stimulate generation of superoxide anion (O2-) or N-formyl-methionyl-leucyl-phenylalanine (fMLP) to induce degranulation (measured as release of beta-glucuronidase). Aroclor 1242 alone stimulated O2- production at a concentration of 10 micrograms/ml. Significant cytotoxicity was not observed under these conditions. This concentration of Aroclor 1242 also increased O2- generation in PMNs activated with 20 ng PMA/ml. In the presence of a concentration of PMA (2 ng/ml) that by itself did not stimulate production of O2-, 1 microgram Aroclor 1242/ml caused significant generation of O2-, indicating synergy between Aroclor 1242 and PMA. Aroclor 1242 caused release of beta-glucuronidase from quiescent PMNs; however, in PMNs stimulated with fMLP to undergo degranulation, Aroclor 1242 inhibited release of beta-glucuronidase. The effects of two PCB congeners, one that binds to the Ah receptor (3,3', 4,4'-tetrachlorobiphenyl) and one that has little affinity for this receptor (2,2', 4,4'-tetrachlorobiphenyl) were examined. 3,3', 4,4'-Tetrachlorobiphenyl had no effect on PMN function in vitro, whereas 2,2', 4,4'-tetrachlorobiphenyl had effects similar to those observed with Aroclor 1242. These results indicate that PCBs affect PMN function in vitro in a complex manner, stimulating or inhibiting function under different conditions. These effects are apparently not mediated through the Ah receptor.

Animals↗
PubMed1993

Extracorporeal shock wave lithotripsy and percutaneous nephrostolithotomy for urinary calculi: comparison of immediate and long-term effects.

Extracorporeal shock wave lithotripsy and percutaneous nephrostolithotomy have become widely used technologies for removal of urinary calculi, despite a lack of adequate evaluative studies. The possibility of long-term adverse effects of these treatments has been raised, particularly an increase in stone recurrence and development of hypertension following extracorporeal shock wave lithotripsy. We conducted a retrospective study of 1,000 patients treated with extracorporeal shock wave lithotripsy or percutaneous nephrostolithotomy for upper tract stone disease to assess immediate effectiveness and complications, with prospective follow-up to assess stone recurrence and development of hypertension. The effectiveness of each treatment (defined in terms of patients rendered stone-free or having only fragments at discharge) was similar for most stone types. While stone recurrence rates in the first 2 years after treatment were similar in the two groups, cumulative recurrence at 3 years was higher following extracorporeal shock wave lithotripsy compared to percutaneous nephrostolithotomy (39% vs. 23%, p = 0.04). However, logistic regression and Kaplan-Meier analyses showed no significant difference in clinically evident recurrence for patients treated with extracorporeal shock wave lithotripsy. Cumulative incidence of clinically evident stone recurrence did not differ significantly between patients with fragments at discharge (20%) and patients rendered stone-free (15%) (p = 0.24). There was no difference in the development of new hypertension requiring medical treatment. Our findings indicate that extracorporeal shock wave lithotripsy is effective and associated with lower short-term morbidity than percutaneous nephrostolithotomy. Stone fragments left behind by lithotripsy do not appear to confer significant risk of early stone recurrence. Further study is necessary to define long-term risk of recurrence following extracorporeal shock wave lithotripsy.

Follow-Up Studies↗
PubMed1993

Correlation between insulin receptor occupancy and tyrosine kinase activity at low insulin concentrations and effect of major histocompatibility complex class I-derived peptide.

In rat adipocytes, insulin dose-response curves were determined for the following effects in the same cells under the same conditions: glucose uptake, binding to insulin receptors (IR), IR autophosphorylation in vivo and in vitro, IR tyrosine kinase activity and insulin-stimulated phosphatidylinositol (PI) kinase. All the EC50 values were essentially the same (mean +/- S.E.M. was 7 +/- 1 nM), except for glucose uptake, which was 170 pM. Using an improved method, we were able to measure PI kinase activity at picomolar concentrations of insulin (> 30 pM) corresponding to the EC50 for glucose uptake. These experiments showed that insulin-stimulated increase in glucose uptake was associated with an increase in antiphosphotyrosine antibody precipitable PI kinase activity, consistent with the view that IR tyrosine kinase activity may be involved in insulin-mediated signaling of glucose uptake. Small peptides (17-25 residues long) derived from major histocompatibility complex class I have previously been shown to inhibit IR internalization without any effect on the affinity of insulin to the receptor. It is now shown that the peptide-mediated inhibition of internalization, which doubles the number of insulin-occupied receptors at an insulin concentration of 70 pM, also results in a corresponding enhancement of PI kinase activity and glucose uptake. Thus, the receptors arrested on the cell surface by the peptide are biologically active.

1-Phosphatidylinositol 4-Kinase↗
PubMed1992

Characterization of insulin-stimulated protein serine/threonine kinases in CHO cells expressing human insulin receptors with point and deletion mutations.

The activation of insulin-stimulated protein-serine/threonine kinases has been investigated in CHO cell lines transfected with cDNAs encoding either wild-type or mutant human insulin receptors. (1) Insulin treatment of CHO cells over-expressing wild-type insulin receptors resulted in the rapid and substantial (5-10-fold) activation of cytosolic protein kinases which phosphorylated myelin basic protein, Kemptide and two peptide substrates based on sites phosphorylated on ribosomal protein S6 in vivo. (2) Further fractionation of cytosolic extracts by MonoQ chromatography revealed two peaks of insulin-stimulated myelin basic protein kinase activity which were highly related to the previously described mitogen-activated protein (MAP) kinases ERK1 and ERK2. In addition, at least two major peaks of S6 kinase activity were resolved, which exhibited properties similar to the 70 kDa and 90 kDa S6 kinases described by others; the predominant effect of insulin was on the activity of the 90 kDa enzyme and was in excess of 10-fold. (3) MonoQ fractionation of extracts from parental CHO cells, or cells expressing kinase-deficient receptors, showed all insulin-stimulated peaks of activity to be almost completely absent. (4) Further studies demonstrated that substitution of tyrosine residues 1162 and 1163 (or 1162 alone) with phenylalanine led to a substantial reduction in the ability of insulin to stimulate these protein kinase activities when assayed in cytosolic extracts. In contrast, deletion of 69 amino acids from the C-terminus of the insulin receptor beta-subunit caused a leftward shift in the insulin dose-response curve of the MAP kinase activity, but apparently not in that of the 90 kDa S6 kinase activity.

Amino Acid Sequence↗
PubMed1992

The insulin receptor-related receptor. Tissue expression, ligand binding specificity, and signaling capabilities.

In 1989, Shier and Watt identified a gene which was predicted to encode a new member of the insulin receptor (IR) family, and they called it the insulin receptor-related receptor (IRR) (Shier, P., and Watt, V. M. (1989) J. Biol. Chem. 264, 14605-14608). However, the tissues expressing this receptor, its ligand binding specificity and its signaling capability have remained unknown. In the present studies we report Northern blot analyses and polymerase chain reaction data, which indicate that the IRR mRNA is expressed in a variety of tissues, including the human kidney, heart, skeletal muscle, liver, and pancreas. In order to examine the ligand(s) recognized by IRR, we constructed a chimeric receptor with the extracellular domain of the IR replaced with that of IRR. This chimera was found not to bind radioactively labeled insulin, insulin-like growth factor I (IGF-I), or IGF-II. These ligands and relaxin, the only other known member of the mammalian insulin family, also failed to stimulate the tyrosine kinase activity of this chimeric receptor. A second chimeric receptor with the extracellular domain of IR and the kinase domain of IRR was also constructed and utilized to study the signaling capabilities of the kinase domain of IRR. This chimera exhibited high affinity insulin binding and insulin-stimulated tyrosine kinase activity. The kinase domains of the IR and IRR were found capable of phosphorylating the same spectrum of exogenous and endogenous substrates. However, Chinese hamster ovary (CHO) cells stably overexpressing the kinase domain of IRR exhibited elevated basal thymidine incorporation and 2-deoxyglucose uptake compared with CHO cells and CHO cells overexpressing wild-type IR. We conclude that: 1) IRR is expressed in the human kidney, heart, skeletal muscle, liver, and pancreas, 2) IRR does not appear to be the receptor of any known member of the insulin family, and 3) the tyrosine kinase of IRR appears to be similar to that of IR in both the spectrum of substrates phosphorylated and the biological responses stimulated.

1-Phosphatidylinositol 4-Kinase↗
PubMed1992

A panel of monoclonal antibodies for the type I insulin-like growth factor receptor. Epitope mapping, effects on ligand binding, and biological activity.

We obtained 20 mouse monoclonal antibodies specific for human type I insulin-like growth factor (IGF) receptors, using transfected cells expressing high levels of receptors (IGF-1R/3T3 cells) as immunogen. The antibodies immunoprecipitated receptor.125I-IGF-I complexes and biosynthetically labeled receptors from IGF-1R/3T3 cells but did not react with human insulin receptors or rat type I IGF receptors. Several antibodies stimulated DNA synthesis in IGF-1R/3T3 cells, but the maximum stimulation was only 25% of that produced by IGF-I. The antibodies fell into seven groups recognizing distinct epitopes and with different effects on receptor function. All the antibodies reacted with the extracellular portion of the receptor, and epitopes were localized to specific domains by investigating their reaction with a series of chimeric IGF/insulin receptor constructs. Binding of IGF-I was inhibited up to 90% by antibody 24-60 reacting in the region 184-283, and by antibody 24-57 reacting in the region 440-586. IGF-I binding was stimulated up to 2.5-fold by antibodies 4-52 and 16-13 reacting in the region 62-184, and by antibody 26-3 reacting downstream of 283. The latter two groups of antibodies also dramatically stimulated insulin binding to intact IGF-1R/3T3 cells (by up to 50-fold), and potentiated insulin stimulation of DNA synthesis. Scatchard analysis indicated that in the presence of these antibodies, the affinity of the type I IGF receptor for insulin was comparable with that of the insulin receptor. These data indicate that regions both within and outside the cysteine-rich domain of the receptor alpha-subunit are important in determining the affinity and specificity of ligand binding. These antibodies promise to be valuable tools in resolving issues of IGF-I receptor heterogeneity and in studying the structure and function of classical type I receptors and insulin/IGF receptor hybrids.

3T3 Cells↗
PubMed1992

An unusual active site identified in a family of zinc metalloendopeptidases.

An unusual active site has been identified in a family of zinc metalloendopeptidases that includes bacterial protease III and the human and Drosophila insulin-degrading enzymes. All of these enzymes have been characterized as metalloendopeptidases and purified protease III has been shown to contain stoichiometric levels of zinc. However, all three proteases lack the consensus sequence (HEXXH) described in the active site of other zinc metalloendopeptidases. Instead, these proteases contain an inversion of this motif, HXXEH. To determine whether this region could represent the active site in these proteins, the two histidines in protease III were individually mutated to arginine and the glutamate was mutated to glutamine. All three mutants were devoid of proteolytic activity toward an exogenous substrate, insulin, as compared to the wild-type protease. Three lines of evidence indicate that this loss of activity in the mutants is not due to distortion of the three-dimensional structure of the protein: (i) the mutants are secreted into the periplasmic space and chromatograph normally; (ii) all three mutants are expressed at levels nearly identical to wild-type protein and do not appear to have an increased susceptibility to proteolysis in the bacteria; and (iii) the mutants compete equally with wild-type protein in a radioimmunoassay. The purified wild-type and glutamate mutants were found to contain stoichiometric amounts of zinc by atomic absorption spectrophotometry, whereas both histidine mutants had negligible zinc signals. These findings are consistent with this region being the active site in this protein, with the histidine residues coordinating the essential zinc atom and the glutamate involved in catalysis.

Amino Acid Sequence↗
PubMed1992

Comparison of the enzymatic and biochemical properties of human insulin-degrading enzyme and Escherichia coli protease III.

The enzymatic and biochemical properties of human insulin-degrading enzyme and Escherichia coli protease III have been compared. Both enzymes were found to degrade insulin in such a way that its receptor binding activity was rapidly lost but its precipitability in trichloracetic acid was only slightly decreased. Both enzymes were also found to be inhibited by chelating agents. The bacterial enzyme, which could be purified in large amounts, was found to contain 0.6 mol of zinc per mol of enzyme but no detectable manganese. The mammalian enzyme but not the bacterial one was inhibited by a sulfhydryl alkylating agent. The two enzymes also differed in substrate specificity. The mammalian enzyme degraded insulin much better than insulin-like growth factor II, whereas the bacterial enzyme degraded them equally. The mammalian enzyme could be labeled by cross-linking to insulin = bombyxin II much greater than insulin-like growth factor I and II much greater than relaxin, while the bacterial enzyme was labeled by insulin-like growth factor II greater than insulin = insulin-like growth factor I much greater than relaxin much greater than bombyxin. Finally, sucrose gradient centrifugation and cross-linking studies both in vitro and in vivo indicated that active human enzyme partially existed as a homo- or heterodimer, whereas the bacterial enzyme was active as a monomer.

Alkylating Agents↗
PubMed1992

Substrates and signalling complexes: the tortured path to insulin action.

In the last few years several potential substrates of the insulin receptor tyrosine kinase have been identified, purified, and their cDNAs isolated. These putative substrates include: 1) pp15, a fatty acid-binding protein; 2) pp120, a plasma membrane ecto-ATPase; 3) pp42, a MAP serine/threonine kinase; 4) pp85, a subunit of the Type 1 phosphatidylinositol kinase; and 5) pp185, a phosphatidylinositol kinase binding protein. Although the tyrosine phosphorylation of several of these substrates correlates with the signalling capabilities of various mutant receptors, the role of these substrates in mediating any one of insulin's many biological responses is still unknown. In addition, recent data indicate that the tyrosine phosphorylation of pp42 may in fact be due to autophosphorylation, thereby removing it from the list of putative substrates of the insulin receptor kinase. Finally, the present review discusses the question of whether signalling occurs as a result of the tyrosine phosphorylation of substrates or via the formation of signalling complexes.

1-Phosphatidylinositol 4-Kinase↗
PubMed1992

Effect of viscosity and iodine concentration of nonionic radiographic contrast media on coronary arteriography in patients.

To assess the influence of viscosity and iodine concentration, three matched and standardized left coronary arteriograms were obtained in 20 patients using iopamidol (Isovue-370), ioversol (Optiray-320), and iohexol (Omnipaque-350). The order of contrast media was randomized and the administration of contrast was double-blinded. Quantitative densitometric angiographic evaluation of the coronary angiograms was performed in addition to independent operator qualitative assessment. The injection volume of iopamidol (5.4 +/- 1.0 ml) was slightly but significantly less than that of ioversol and iohexol (5.6 +/- 1.0 ml, 5.7 +/- 1.0 ml, both p less than 0.05). The calculated iodine concentration was also lower for ioversol (1.7 +/- 0.32 gm) than for iopamidol (1.98 +/- 0.35 gm) and iohexhol (1.9 +/- 0.35 gm, both p less than 0.05). There were significantly lower contrast syringe injection pressures for ioversol (6.6 +/- 0.8 atm) than for iopamidol (7.5 +/- 0.9 atm) and iohexol (7.2 +/- 1.1 atm, both p less than 0.05). The quantitative densitometric analysis failed to demonstrate significant differences among the contrast media with respect to image density parameters for any individual agent. All coronary angiograms were deemed of diagnostic quality. The data in this study indicated that although differences in iodine concentration exist among the three agents, operator compensation with more rapid contrast delivery (higher volume) and lower viscosity (lower injection pressure) produced equivalent image opacification during coronary angiography. Given the same incidence of adverse hemodynamic and clinical effects, selection of a low viscosity media theoretically provides an advantage during procedures using small diameter catheters or interventional procedures requiring contrast visualization through reduced catheter lumina.

Contrast Media↗
PubMed1992

Monocrotaline pyrrole alters DNA, RNA and protein synthesis in pulmonary artery endothelial cells.

Administration of monocrotaline pyrrole (MCTP) to animals results in pulmonary vascular injury. Pulmonary vascular endothelium is a likely target for this pneumotoxicant. Cultured porcine pulmonary artery endothelial cells (PECs) treated with MCTP remain viable but are unable to divide and exhibit an altered morphology. Such responses raise a question about the extent to which affected cells carry out normal functions such as RNA and protein synthesis. Accordingly, the cellular activity of MCTP-treated PECs was examined in this study. PECs were treated with a single administration of MCTP or vehicle, and determinations of cell number, protein, and DNA content were made at times up to 7 days posttreatment. DNA, RNA, and protein synthesis were quantified by incorporation of [3H]thymidine, [3H]uridine, and [3H]leucine, respectively. Increases in cell number that occurred with time in the control cells were reduced in MCTP-treated cells. At 7 days posttreatment, both protein and DNA content increased above control levels. Synthesis of DNA, RNA, and protein continued in all treatment groups throughout the posttreatment period, but cells treated with high concentrations of MCTP showed less synthetic activity than controls during the initial 48 h posttreatment. By 7 days, MCTP-treated cells were producing significantly more DNA, RNA, and protein. These results indicate that cells treated with MCTP continue to synthesize DNA, resulting in an increased DNA content. In addition, treated cells continue to synthesize RNA and translate RNA into protein. Thus, cellular activity is maintained but altered substantially by MCTP exposure.

Animals↗
PubMed1992

Neutrophil depletion protects against liver injury from bacterial endotoxin.

Neutrophil (PMN) infiltration is an early occurrence in the liver after exposure to hepatotoxic doses of endotoxin lipopolysaccharide (LPS). The purpose of this study was to test the hypothesis that PMNs contribute to the pathogenesis of LPS hepatotoxicity. The immunoglobulin fraction from serum of rabbits immunized with rat PMNs (anti-PMN Ig) was administered intravenously to rats 18 and 6 hours before exposure to an hepatotoxic dose of LPS (Escherichia coli 0128:B12). This protocol caused a greater than 95% reduction in circulating PMNs, which was maintained for the duration of the study. The immunoglobulin fraction from nonimmunized rabbits was used as a control (control Ig). Rats pretreated with control Ig exhibited a marked increase in the number of PMNs in the liver 1.5 hours after LPS exposure. This increase in hepatic PMNs was significantly reduced by pretreatment with anti-PMN Ig. Marked elevations in both alanine and aspartate aminotransferase activities (1086 +/- 311 and 880 +/- 183 SF units/ml, respectively) were observed in plasma from control Ig-treated rats 6 hours after intravenous administration of LPS (3.0 mg/kg). The response to LPS was greatly attenuated in animals receiving anti-PMN Ig (145 +/- 111 and 224 +/- 49 SF units/ml alanine and aspartate aminotransferase activities, respectively). Pretreatment of rats with immunoglobulins to rat lymphocytes reduced numbers of circulating lymphocytes but did not afford protection against the hepatotoxic effects of LPS. These results suggest that PMNs contribute to the pathogenesis of LPS hepatotoxicity.

Animals↗
PubMed1992

Thromboxane and prostacyclin production in ponies with colonic volvulus.

Effects of 1 hour of colonic volvulus and 3 hours of reperfusion on concentrations of thromboxane (TXB2) and prostacyclin (6-keto-PGF1 alpha) in portal, pulmonary arterial, and jugular blood were determined by radioimmunoassay to assess the site of production and clearance of these eicosanoids from the circulation in 5 anesthetized ponies. Colonic volvulus had no significant effect on mean arterial pressure or TXB2 concentrations, but significantly (P less than 0.05) increased 6-keto-PGF1 alpha concentrations in all blood samples. Immediately after colonic reperfusion, all eicosanoid concentrations were significantly (P less than 0.05) increased. Then, TXB2 returned to baseline values, whereas 6-keto-PGF1 alpha concentrations remained significantly (P less than 0.05) high for the remainder of the study. Eicosanoid concentrations were significantly (P less than 0.05) greater in portal blood than in pulmonary arterial and jugular blood samples at all periods. This suggests that the splanchnic circulation is the primary site of eicosanoid production during and after colonic volvulus and the liver appears to provide most of the circulatory clearance of thromboxane and prostacyclin.

Animals↗
PubMed1991

Insulin-stimulated serine and threonine phosphorylation of the human insulin receptor. An assessment of the role of serines 1305/1306 and threonine 1348 by their replacement with neutral or negatively charged amino acids.

Insulin promotes insulin receptor beta-subunit phosphorylation on tyrosine, serine, and threonine residues in a variety of cells, including simian COS cells which transiently express human insulin receptors following transfection with a cDNA encoding the wild-type receptor protein. To examine the potential roles of serines 1305 and 1306 and threonine 1348 as sites of insulin-stimulated phosphorylation in these cells, these residues (i.e. either serines 1305 and 1306, or threonine 1348) were replaced with neutral (alanine) or negatively charged (aspartate) amino acids. Following transient expression of each of these mutant receptors in COS cells, two-dimensional phosphopeptide mapping reveals that threonine 1348 is the major, if not the only, insulin-stimulated threonine phosphorylation site. In contrast, while serines 1305 and/or 1306 are phosphorylated in an insulin-dependent manner, these sites comprise only a minor proportion of insulin receptor serine phosphorylation in these cells. Substitution of either serines 1305 and 1306 or threonine 1348 with neutral or negatively charged amino acids has no effect on insulin-stimulated tyrosine autophosphorylation of these mutant receptors in intact cells. Furthermore, insulin-stimulated exogenous protein-tyrosine kinase activity of the mutant receptors is unaffected, as assessed following either phosphorylation of receptors in intact cells or following immunopurification of receptors and their autophosphorylation in vitro.

Amino Acid Sequence↗