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Biomedical subjects

R A Roth

Publications and source records attributed to R A Roth.

At least 199 records · Page 11Linked to original sources
PubMed1990

Sympathetic reactivity of cerebral arteries in developing fetal lamb and adult sheep.

The response of cerebral arteries to norepinephrine was examined in vivo in six dated preterm fetal (94-121 days gestation), eight term fetal (127-141 days gestation), five newborn (7-14 days), and five adult sheep, anesthetized and equipped with a closed cranial window. Norepinephrine (10(-8)-10(-4) M in cerebrospinal fluid) caused a dose-dependent decrease in pial arteriolar diameter in fetal and newborn lambs; however, preterm fetuses were 7- and 14-fold more sensitive to norepinephrine than term fetuses and newborn lambs, respectively. The effective concentration of norepinephrine inducing a 15% decrease in diameter (EC15) was 4.6 +/- 1.8, 33 +/- 11, and 64 +/- 23 microM, for the respective ages. Adult cerebral arterioles did not contract to norepinephrine. In preterm and term fetuses, the contractile response to norepinephrine was blocked by alpha 1-antagonist, prazosin (3 mg iv), and was enhanced by cocaine (10(-5) M; EC15 = 0.086 +/- 0.04 and 1.84 +/- 1.20 microM, respectively) indicating that alpha 1-adrenoceptors mediate the response and that the decrease in sensitivity is not caused by development of neuronal uptake processes. In seven fetuses (111-141 days; mean 123 days gestation), electrical stimulation of the superior cervical sympathetic ganglion constricted pial arterioles by 21 +/- 5%; this contractile response was also blocked by prazosin. The cerebral arterioles of the fetus in utero possess a functional sympathetic innervation capable of influencing cerebrovascular resistance. There is a loss of responsiveness of cerebral arterioles to norepinephrine during fetal and postnatal development, suggesting that the contribution of neuroadrenergic mechanisms to cerebrovascular regulation may be relatively unique to the immature brain.

Aging↗
PubMed1990

Insulin-degrading enzyme: stable expression of the human complementary DNA, characterization of its protein product, and chromosomal mapping of the human and mouse genes.

We have recently described the isolation of a cDNA encoding an enzyme thought to be involved in the degradation of insulin by insulin-responsive tissues. This enzyme, referred to as insulin-degrading enzyme (IDE), is a cytosolic proteinase of 110,000 mol wt which shares structural and functional homology with bacterial protease III. The enzyme may function in the termination of the insulin response. We report here the mapping of the human and mouse IDE genes to human chromosome 10 and mouse chromosome 19, respectively, and evidence for the existence of a single complex IDE gene. We also describe the stable transfection of Chinese hamster ovary cells with a plasmid containing the IDE cDNA under the transcriptional control of the SR alpha promoter. The recombinant protein synthesized by these cells is indistinguishable from the isolated human enzyme in both its size and immunoreactivity and degrades insulin with a specific activity similar to that of the purified proteinase. Overexpression of IDE using this system should allow for a functional test of the role of IDE in insulin action. In addition, expression of various site-directed mutants of IDE will aid in identifying the residues of IDE and protease III that are essential to the activity of this unique family of proteinases.

Amino Acid Sequence↗
PubMed1990

The cholestatic agent, alpha-naphthylisothiocyanate, stimulates superoxide release by rat neutrophils in vitro.

Studies in rats indicate that neutrophils (polymorphonuclear leukocytes (PMNs] are associated with areas of tissue damage after treatment with the hepatotoxicant, alpha-naphthylisothiocyanate (ANIT). Several synthetic and naturally occurring substances stimulate PMNs to release cytotoxic mediators, such as superoxide (O2-). The purpose of the present study was to test the hypothesis that ANIT stimulates the release of O2- from isolated rat PMNs. PMNs derived from rat peritoneum were treated with ANIT in vitro and tested for release of O2-. ANIT caused the release of O2- from PMNs in a concentration-dependent manner. Maximal O2- release (10 +/- 1 nmoles/30 minutes/2 x 10(6) cells) was achieved by an ANIT concentration of 110 microM. This ANIT-induced O2- release was significantly reduced or blocked completely by preincubation of PMNs for 10 minutes with 10 microM or 100 microM SKF 525A, respectively. The beta-isomer of ANIT, which does not cause cholestasis in vivo, did not stimulate O2- release. ANIT-stimulated O2- production decreased sharply after 5 minutes of incubation with ANIT and ceased entirely between 10 to 15 minutes. Shortly after this decrease in O2- production was an increase in the extracellular activity of lactate dehydrogenase. PMNs exposed to ANIT also failed to exclude trypan blue dye, either in the presence or in the absence of superoxide dismutase and catalase, suggesting a direct, oxygen radical-independent, cytotoxic effect of ANIT on PMNs. Release of the lysosomal enzyme, beta-glucuronidase, occurred within 5 minutes of incubation of isolated PMNs with ANIT (110 microM). These results indicate that exposure of rat PMNs to the hepatotoxicant, ANIT, causes the release of cytotoxic agents, whereas its less hepatotoxic beta-isomer does not.

1-Naphthylisothiocyanate↗
PubMed1989

Effects of altered platelet number on pulmonary hypertension and platelet sequestration in monocrotaline pyrrole-treated rats.

To study the role of platelets in monocrotaline pyrrole (MCTP)-induced pulmonary hypertension, pulmonary sequestration of 111In-labeled platelets in rats treated with MCTP and anti-rat platelet serum (PAS) was examined. Lung injury from a single, intravenous injection of MCTP (3.5 mg/kg) at Day 8 was evident as elevated lung weight and lavage fluid protein and lactate dehydrogenase activity. Additionally, right ventricular hypertrophy and elevated pulmonary arterial pressures (PAP) occurred. Treatment with PAS on Days 6-8 did not affect the lung injury but resulted in an attenuation of the pulmonary hypertensive response. Pulmonary platelet sequestration was also decreased in PAS-treated rats, yet the sequestration in the lungs of MCTP-treated rats that received PAS was significantly higher than that in the lungs of N,N-dimethylformamide (DMF) controls. MCTP-treated rats receiving control serum (CS) tended to sequester more 111In-labeled platelets than respective DMF controls, but this was not statistically significant. Blood platelet half-life was unaltered in rats receiving CS. When rats were treated similarly with MCTP and PAS and were killed at 18 days, the attenuation of the pulmonary hypertensive response previously described was not observed, and lung injury was more extensive than when CS was given. Apparently, platelet depletion delayed the development of the pulmonary hypertensive response. Supranormal platelet numbers produced by splenectomy did not affect MCTP-induced lung injury or the elevation in PAP. These results support the hypothesis that the development of MCTP-induced pulmonary hypertension is mediated in part by platelets.

Animals↗
PubMed1989

Interactions of recombinant and platelet transforming growth factor-beta 1 precursor with the insulin-like growth factor II/mannose 6-phosphate receptor.

Recombinant transforming growth factor (TGF)-beta 1 precursor was recently found to contain mannose 6-phosphate (Purchio et al., 1988, J. Biol. Chem. 263, 14211-14215). In the present study, recombinant TGF-beta 1 precursor was shown to bind to the insulin-like growth factor (IGF)-II/mannose 6-phosphate (man6P) receptor on the plasma membrane of cells since: 1) Insulin, which induces an increase in cell surface IGF-II/man6P receptors on adipocytes, caused a 2.7-fold increase in TGF-beta 1 precursor binding to adipocytes; 2) Chinese hamster ovary cells selected for overexpression of the IGF-II/man6P receptor exhibited an increased binding of TGF-beta 1 precursor in comparison to the parental cells; and 3) the binding of 125I-TGF-beta 1 precursor to these transfected cells and adipocytes was largely inhibited by man6P. After 15 minutes at 37 degrees C, 75% of the recombinant TGF-beta 1 precursor was found to be internalized in the transfected cells. Additional studies with latent TGF-beta 1 isolated from platelets indicated that this material could also bind to the isolated IGF-II/man6P receptor.

Adipose Tissue↗
PubMed1989

Internalization and degradation of insulin by a human insulin receptor-v-ros hybrid in Chinese hamster ovary cells.

Chinese hamster ovary cell lines expressing either the wild-type human insulin receptor or a hybrid molecule in which the tyrosine kinase domain of the insulin receptor is replaced with that of the oncogene, v-ros were examined for their ability to internalize and degrade insulin. Cells expressing the hybrid receptor were found to internalize and degrade insulin at approximately half the rate of cells expressing the native insulin receptor. Moreover, insulin was incapable of inducing the internalization of the cell-surface hybrid molecule. In contrast, the constitutive rate of receptor internalization was found to be the same for the hybrid and wild-type receptors. These results obtained were similar to those with cells expressing either wild-type or mutant receptors lacking kinase activity. In conclusion, the substitution of the specificity of tyrosine kinase of the insulin receptor with that of the v-ros oncogene product results in defective internalization and degradation of insulin, and loss of ligand-induced receptor internalization.

Animals↗
PubMed1989

Experimental study of hollow, absorbable polyglycolic acid tube as stent for vasovasostomy.

Bilateral vasovasostomy in 16 rats permitted evaluation of a microsurgical technique using an original absorbable hollow stent made of polyglycolic acid. Advantages of this stent included ease of anastomosis, maintenance of luminal patency, satisfactory approximation of vasal ends, and reduction of perivasal inflammation possibly due to reduction of extravasation of sperm. Absorbable stents were eliminated within four weeks of placement, and patency was confirmed by histologic studies performed two, four, and six weeks after vasovasostomy.

Animals↗
PubMed1989

Inhibition of insulin receptor phosphorylation by peptides derived from major histocompatibility complex class I antigens.

Peptides from the alpha 1 region (residues 61-85) of the D and K molecules of the major histocompatibility complex class I antigens inhibit insulin-induced tyrosine kinase activity of the purified human insulin receptors (IRs) as measured both by autophosphorylation and IR-mediated substrate [poly(Glu,Tyr)] phosphorylation. Half-maximal effect of the Dk-(61-85) peptide on IR autophosphorylation is obtained at 1.2 microM, and almost complete inhibition of IR kinase activity is obtained at 10 microM peptide. The corresponding K kappa-(61-85) peptide has a significantly weaker effect on autophosphorylation. No such effects are observed with nine peptides of similar length, but unrelated to major histocompatibility complex class I antigens. Neither of the major histocompatibility complex class I-derived peptides has any effect on the constitutively active kinase of a genetically engineered cytoplasmic IR domain. Further, insulin binding to IR is unaltered in the presence of the major histocompatibility complex class I-derived peptides. The inhibitory activity of the peptides on insulin-induced IR phosphorylation facilitated the observation that IRs require insulin to become substrate for an independent tyrosine kinase. In the presence of an inhibitory peptide, the constitutively active cytoplasmic IR kinase domain only phosphorylates the intact IR in the presence of insulin. We conclude that the tyrosine kinase activity of IRs may be altered by peptide interaction at an allosteric site and, moreover, IRs require insulin to assume a conformation permitting phosphorylation by an independent kinase.

Allosteric Site↗
PubMed1989

Dilation of benign ureteral strictures.

Balloon dilation of benign ureteral strictures was performed in 33 patients in a percutaneous antegrade (25 patients) or retrograde (eight patients) fashion followed by placement of a 7-10-F stent for 4-8 weeks. Dilation was possible in all patients. During the follow-up period of 1-40 months the overall success rate was 76%. The success rate in primary strictures at the ureteropelvic junction was 86%. Among 10 patients with postsurgical (pyeloplasty, ureteroenterostomy, and ureteroneocystostomy) anastomotic strictures, dilation was successful in 50%. Of six patients with postoperative strictures after calculus removal by ureterolithotomy or pyelolithotomy, four (67%) had good results. All patients with iatrogenic postureteroscopic strictures (five patients) or accidental ureteral ligation (two patients) had a normal pyelogram at follow-up. The success rate in acute strictures of less than 3 months duration was 88%; in strictures of more than 3 months duration the success rate was 67%. Cause, length, and duration of stricture are the prime determinants of success. Early intervention with balloon dilation is suggested.

Adolescent↗
PubMed1989

The role of cyclooxygenase products in the acute airway obstruction and airway hyperreactivity of ponies with heaves.

Airway obstruction and hyperreactivity are characteristics of human asthma and of "heaves," a naturally occurring respiratory disorder of horses and ponies. To document the role of cyclooxygenase products of arachidonic acid metabolism in the pathogenesis of heaves, we measured plasma and bronchoalveolar lavage (BAL) fluid concentrations of metabolites of thromboxane (TX)A2 and prostaglandins (PG) I2 and D2 in five affected ponies and their age- and gender-matched controls prior to and during acute airway obstruction precipitated by housing the ponies in a barn and exposing them to hay dust. Pulmonary resistance increased significantly and dynamic compliance and arterial oxygen tension decreased significantly in affected ponies that were placed in the barn. At this time, histamine aerosol challenge demonstrated the presence of airway hyperresponsiveness in the affected ponies. Plasma TXB2 was the only metabolite that increased significantly during the acute disease state. In a subsequent experiment, the ponies were treated with flunixin meglumine, a cyclooxygenase inhibitor, to determine if this would alter the onset or development of clinical disease. At a dose of 1.1 mg/kg intramuscularly, 3 times daily, flunixin meglumine inhibited TXB2 production but did not alter the degree of airway obstruction or airway hyperreactivity measured at pasture and in the barn. We conclude that cyclooxygenase products of arachidonic acid metabolism are altered but do not play a role in the airway obstruction and hyperreactivity observed in ponies with heaves.

Airway Resistance↗
PubMed1989

Effect of indomethacin on the regulation of cerebral blood flow during respiratory alkalosis in newborn piglets.

The effect of cyclooxygenase inhibition by indomethacin on regional cerebral blood flow (CBF) during hypocapnia induced by hyperventilation and during hypercapnia induced by CO2 inhalation was examined. CBF was measured in 27 anesthetized, ventilated piglets (2-8 d) using microspheres in control and indomethacin treated animals (5 mg/kg) after hyperventilation or inhalation of 6% CO2. In the control group (n = 6), CBF decreased significantly (p less than 0.05) to all regions of the brain after hyperventilation with a 32% decrease in the cerebral cortex. In the indomethacin-treated group (n = 6), blood flow significantly decreased by 35 to 49% in all regions of the brain, except the cerebral white matter, during normocapnia with no further decrease in flow during subsequent hypocapnia. Although CBF increased significantly after indomethacin treatment during hypercapnia the response was markedly attenuated with blood flow to the cerebral gray matter, hippocampus and pons rising only 42, 25, and 42% in contrast to 108, 75, and 225% in the control group. Since indomethacin decreased resting CBF, unilateral sympathetic nerve stimulation at 15 Hz was used to test the specificity of indomethacin on hypocapnic vasoconstriction (n = 5). Unilateral sympathetic nerve stimulation caused a further statistically significant decrease in CBF on the stimulated side after hyperventilation with indomethacin (12%), which was comparable to that which occurred during normocapnia (16%). The data demonstrate that indomethacin attenuates the cerebrovascular sensitivity to both increases and decreases in CO2/H+ and implicate a possible role for vasoactive prostanoids in mediating the response of CBF to fluctuations in CO2 in newborn piglets.

Alkalosis, Respiratory↗
PubMed1989

A role for the insulin-like growth factor II/mannose-6-phosphate receptor in the insulin-induced inhibition of protein catabolism.

Insulin and insulin-like growth factor (IGF)-I inhibit intracellular protein degradation in a variety of different cell types. In the present studies, the IGF-I-induced inhibition of protein metabolism in Chinese hamster ovary (CHO) cells was found to be blocked by polyclonal antibodies to the IGF-II/mannose-6-phosphate phosphate (Man-6-P) receptor, but not by control immunoglobulin. In contrast, these antibodies had no effect on the ability of IGF-I to stimulate glucose uptake in the same cells. The antibodies to the IGF-II/Man-6-P receptor also inhibited the effect of IGF-I and insulin on protein catabolism in human foreskin fibroblasts and human hepatoma cells, respectively. Moreover, CHO cells overexpressing a cDNA coding for the IGF-II/Man-6-P receptor were found to exhibit an increased effect of insulin on protein catabolism. In contrast, the insulin stimulation of glucose uptake is the same in these transfected cells as in the parental CHO cells. These results implicate the IGF-II/Man-6-P receptor in the insulin- and IGF-I-induced inhibition of protein catabolism.

Animals↗
PubMed1989

Cyclooxygenase inhibition in lungs or in neutrophils attenuates neutrophil-dependent edema in rat lungs perfused with phorbol myristate acetate.

Results from previous studies indicate that injury in isolated rat lungs perfused with buffer containing phorbol myristate acetate (PMA) and rat neutrophils (PMNs) is dependent on the production of reactive oxygen species and thromboxane (Tx) A2. The purpose of this study was to determine whether the lung or the PMN was the source of TxA2 required to produce lung injury in this model. Prostanoid synthesis by rat lungs or PMNs was inhibited selectively by pretreatment of either rats or isolated PMNs with aspirin (100 mg/kg p.o. or 100 microM, respectively). Unbound aspirin was removed from the lungs and PMNs before use in experiments. Lungs from vehicle-pretreated rats that were perfused with PMA and untreated PMNs exhibited increases in weight, lavage fluid albumin content and TxB2 production with respect to lungs perfused with PMA but no PMNs. Increases in these markers were prevented when cyclooxygenase from either the lungs or the PMNs was inhibited. These results indicate that TxA2 is produced by both PMNs and by lung cells in this preparation, and that TxA2 production by both of these sources is required for the manifestation of edema.

6-Ketoprostaglandin F1 alpha↗
PubMed1988

Human insulin-degrading enzyme shares structural and functional homologies with E. coli protease III.

A proteinase with high affinity for insulin has been proposed to play a role in the cellular processing of this hormone. A complementary DNA (cDNA) coding for this enzyme has been isolated and sequenced. The deduced amino acid sequence of the enzyme contained the sequences of 13 peptides derived from the isolated protein. The cDNA could be transcribed in vitro to yield a synthetic RNA that in cell-free translations produced a protein that coelectrophoresed with the native proteinase and could be immunoprecipitated with monoclonal antibodies to this enzyme. The deduced sequence of this proteinase did not contain the consensus sequences for any of the known classes of proteinases (that is, metallo, cysteine, aspartic, or serine), but it did show homology to an Escherichia coli proteinase (called protease III), which also cleaves insulin and is present in the periplasmic space. Thus, these two proteins may be members of a family of proteases that are involved in intercellular peptide signaling.

Amino Acid Sequence↗
PubMed1988

Identification of mannose 6-phosphate in two asparagine-linked sugar chains of recombinant transforming growth factor-beta 1 precursor.

Recombinant transforming growth factor-beta 1 (TGF-beta 1) precursor produced and secreted by a clone of Chinese hamster ovary cells was found to be glycosylated and phosphorylated. Treatment of 32P-labeled precursor protein with N-glycanase indicated that phosphate was incorporated into asparagine-linked complex carbohydrate moieties. Fractionation of 32P-labeled glycopeptides followed by amino acid sequence analysis indicated that greater than 95% of the label was incorporated into two out of three glycosylation sites at Asn-82 and Asn-136 of the TGF-beta 1 precursor. Two-dimensional electrophoretic analysis of acid hydrolyzed precursor protein and precursor protein-derived glycopeptides indicated that 32P was incorporated as mannose 6-phosphate. Binding studies with the purified receptor for mannose 6-phosphate indicated that the TGF-beta 1 precursor could bind to this receptor and the binding was specifically inhibited with mannose 6-phosphate.

Amino Acid Sequence↗