Recently, we demonstrated that tissue plasminogen activator directly releases fibrinopeptides A and B (FPA and FPB) from fibrinogen. The purpose of this study was to determine whether urokinase has similar activity. Incubation of urokinase with fibrinogen or heparinized plasma results in concentration-dependent FPB release unaccompanied by FPA cleavage. For equivalent amidolytic activity, high molecular weight urokinase releases twofold more FPB than the low molecular weight species. In contrast, prourokinase does not release FPB until activated to urokinase. Contaminating thrombin or plasma is not responsible for urokinase-mediated FPB release because this activity is unaccompanied by FPA or B beta 1-42 cleavage, and is unaffected by heparin, hirudin, a monospecific antibody against thrombin, aprotinin, or alpha 2-antiplasmin. FPB release reflects a direct action of urokinase on fibrinogen because release is completely inhibited by a monospecific antibody against the enzyme. Further, urokinase releases FPB from the FPB-containing substrate B beta 1-42, thus confirming its specificity for the B beta 14 (Arg)-B beta 15 (Gly) bond. In addition to FPB release, SDS-PAGE analysis of the time course of urokinase-mediated fibrinogenolysis indicates progressive proteolysis of both the A alpha- and B beta-chains of fibrinogen that occurs after FPB release is completed. As a consequence of urokinase-mediated fibrinogenolysis, there is progressive prolongation of the thrombin clotting time. These studies indicate that urokinase has direct catalytic activity against fibrinogen. By releasing FPB, a potent chemoattractant, and by rendering fibrinogen less clottable by thrombin, urokinase may participate in processes extending beyond fibrinolysis.
J I Weitz, B Leslie
Cytochrome P450 content and activities are increased in the kidneys of spontaneously hypertensive rats (SHR) as compared with those of normotensive, Wistar-Kyoto (WKY), control rats during the period of rapid elevation of blood pressure. We studied the effect of heme arginate, a potent inducer of heme oxygenase (EC 1.14.99.3), on microsomal cytochrome P450 levels and activities and blood pressure in SHR at 7 wk of age. Administration of heme arginate (15 mg/kg body weight for 4 d) resulted in a marked decrease in blood pressure from 156.3 +/- 4.7 to 129.8 +/- 4.5 mm Hg (P less than 0.001), whereas blood pressure in SHR receiving the vehicle control was not affected. The blood pressure of age-matched WKY was not affected by heme arginate. Heme oxygenase activity increased in both hepatic and renal microsomes of SHR and WKY by two- to four-fold after treatment with heme arginate. Maximal increase of heme oxygenase mRNA occurred 5-7 h after the last injection of heme arginate and returned to control levels after 24 h. The increase in heme oxygenase activity was associated with a parallel decrease in cytochrome P450 content and in the activity of cytochrome P450 omega/omega-1 arachidonate hydroxylases in kidneys of SHR. It is postulated that heme arginate treatment resulted in induction of heme oxygenase which consequently led to a diminution of cytochrome P450, especially the arachidonate omega/omega-1 hydroxylases leading to a marked decrease in 19-hydroxyeicosatetraenoic acid (HETE) and 20-HETE. The effect of heme arginate on blood pressure may be mediated via these biochemical events inasmuch as both 19-HETE and 20-HETE produced by the kidney may promote hypertension by causing vasoconstriction and sodium retention.
R D Levere, P Martasek, B Escalante, M L Schwartzman, N G Abraham
A 55-kD organic anion binding protein (OABP) was identified previously in liver cell plasma membrane sinusoidal subfractions. Although this protein was localized to the surface of hepatocytes by immunofluorescence, immunoblot analysis revealed reactivity toward both plasma membrane and mitochondrial fractions. To clarify these findings, an immunoreactive clone from a rat liver cDNA expression library was isolated, the 1,500-base pair cDNA insert was sequenced, and the corresponding beta-galactosidase fusion protein was expressed and purified. The resulting sequence corresponded to that of the rat mitochondrial F1-adenosine triphosphatase (F1-ATPase) beta-subunit. This protein and OABP are of similar size and are mutually immunologically cross-reactive. That the antigen was present on the cell surface as well as in mitochondria was suggested from studies of immunoprecipitation after cell-surface iodination, and light- and electron-microscopic immunocytochemistry. Photoaffinity labeling of bovine F1-ATPase with high-specific-activity [35S]sulfobromophthalein revealed binding only to the beta-subunit. Hepatocyte uptake of bilirubin and sulfobromophthalein requires cellular ATP and mitochondria also transport these organic anions, which at high doses inhibit respiration. The presence of an organic anion binding site on the F1-ATPase beta-subunit suggests that it may play a role in these processes.
T Goeser, R Nakata, L F Braly, A Sosiak, C G Campbell, R Dermietzel, P M Novikoff, R J Stockert, R D Burk, A W Wolkoff
The effect of hypercholesterolemia on vascular function was studied in humans. To eliminate the potential confounding effects of atherosclerosis, vascular reactivity was measured in the forearm resistance vessels of 11 normal subjects (serum LDL cholesterol = 111 +/- 7 mg/dl) and 13 patients with hypercholesterolemia (serum LDL cholesterol = 211 +/- 19 mg/dl, P less than 0.05). Each subject received intrabrachial artery infusions of methacholine, which releases endothelium-derived relaxant factor, and nitroprusside which directly stimulates guanylate cyclase in vascular smooth muscle. Maximal vasodilatory potential was determined during reactive hyperemia. Vasoconstrictive responsiveness was examined during intra-arterial phenylephrine infusion. Forearm blood flow was determined by venous occlusion plethysmography. Basal forearm blood flow in normal and hypercholesterolemic subjects was comparable. Similarly, reactive hyperemic blood flow did not differ between the two groups. In contrast, the maximal forearm blood flow response to methacholine in hypercholesterolemic subjects was less than that observed in normal subjects. In addition, the forearm blood flow response to nitroprusside was less in hypercholesterolemic subjects. There was no difference in the forearm vasoconstrictive response to phenylephrine in the two groups. Thus, the vasodilator responses to methacholine and nitroprusside were blunted in patients with hypercholesterolemia. We conclude that in humans with hypercholesterolemia, there is a decreased effect of nitrovasodilators, including endothelium-derived relaxing factor, on the vascular smooth muscle of resistance vessels.
M A Creager, J P Cooke, M E Mendelsohn, S J Gallagher, S M Coleman, J Loscalzo, V J Dzau
Only one herpes simplex virus type 1 (HSV-1) gene is expressed in sensory neurons of latently infected animals and humans, yielding two RNAs, called latency-associated transcripts (LATs). The LATs appear to modulate virus reactivation. In mice and rabbits the 5' origins, kinetics of synthesis, and splicing pattern of the LATs are well established. Because these details of LAT structure and expression have not been defined in humans, we sought to do so. Using primer extension and Northern hybridization analyses, we demonstrate that in human trigeminal ganglia, the smaller (1.35 kb) HSV-1 transcript differs from the larger (1.85 kb) LAT by excision of an intron near its 5' end; they are otherwise colinear, and 5' coterminal. In infected cells only the 1.85 kb LAT is detected. Its expression is inhibited by cycloheximide or acyclovir, indicating this LAT is synthesized late in the viral replicative cycle. All of these features of the LATs in humans are consistent with those reported in rabbits and mice and further validate the animal models of human HSV-1 infection.
P R Krause, K D Croen, J M Ostrove, S E Straus
A defect in the E1 beta subunit of the branched chain alpha-ketoacid dehydrogenase (BCKDH) complex is one cause of maple syrup urine disease (MSUD). In an attempt to elucidate the molecular basis of MSUD, we isolated and characterized a 1.35 kbp cDNA clone encoding the entire precursor of the E1 beta subunit of BCKDH complex from a human placental cDNA library. Nucleotide sequence analysis revealed that the isolated cDNA clone (lambda hBE1 beta-1) contained a 5'-untranslated sequence of four nucleotides, the translated sequence of 1,176 nucleotides and the 3'-untranslated sequence of 169 nucleotides. Comparison of the amino acid sequence predicted from the nucleotide sequence of the cDNA insert of the clone with the NH2-terminal amino acid sequence of the purified mature bovine BCKDH-E1 beta subunit showed that the cDNA insert encodes for a 342-amino acid subunit with a Mr = 37,585. The subunit is synthesized as the precursor with a leader sequence of 50 amino acids and is processed at the NH2 terminus. A search for protein homology revealed that the primary structure of human BCKDH-E1 beta was similar to the bovine BCKDH-E1 beta and to the E1 beta subunit of human pyruvate dehydrogenase complex, in all regions. The structures and functions of mammalian alpha-ketoacid dehydrogenase complexes are apparently highly conserved. Genomic DNA from lymphoblastoid cell lines derived from normal and five MSUD patients, in whom E1 beta was not detected by immunoblot analysis, gave the same restriction maps on Southern blot analysis. The gene has at least 80 kbp.
Y Nobukuni, H Mitsubuchi, F Endo, I Akaboshi, J Asaka, I Matsuda
Decreased sweating, especially of feet and legs, occurs in human diabetic neuropathy. It might be studied in experimental diabetes to characterize it, elucidate its mechanisms, and determine whether it can be prevented or treated. The pilocarpine-induced sweat responses (SR) in the hind foot pads of groups of control and streptozocin diabetic rats, in good (GC) and in poor (PC) glycemic control and with a crossover design after 20 wk of diabetes, were evaluated with the silicone mold sweat test to determine the number of sweat droplets per group of foot pads. The SR was dose dependent and reproducible. The SR disappeared with denervation and reappeared with reinnervation; denervation hypersensitivity did not develop. In the GC group, euglycemia was achieved by regulating the caloric intake and using multiple daily injections of Ultralente insulin. The SR was not different from that of the control group for up to 136 d. In the PC group, the SR became abnormal (P less than 0.005) at 16 d and progressively worsened: 40% of baseline values at 14 wk (P less than 0.001). After restoring euglycemia in the PC group, a normal SR occurred at 12 d. These results show that one human neuropathic deficit, failure of sweating, can be prevented or ameliorated by good glycemic control.
C Cardone, P J Dyck
We have studied a patient with a congenital bleeding disorder and phenotypic manifestations typical of Bernard-Soulier syndrome, including giant platelets with absent ristocetin-induced von Willebrand factor binding. Two monoclonal antibodies reacting with distinct epitopes in the amino-terminal domain of the alpha-chain of glycoprotein (GP) Ib were used to estimate the number of GP Ib molecules on the platelet membrane. In the patient, binding of one antibody (LJ-Ib10) was approximately 50% of normal, while binding of the other (LJ-Ib1) was absent. Binding of both antibodies was reduced to approximately 50% of normal in the mother and one sister of the propositus, and their platelets exhibited approximately 70% of normal von Willebrand factor binding. Immunoblotting studies confirmed the presence of GP Ib alpha, as well as GP IX, in patient platelets. Antibody LJ-Ib10, but not LJ-Ib1, could immunoprecipitate the patient's GP Ib alpha from surface-labeled proteins. Thus, platelets from the propositus contained a structurally and functionally altered GP Ib-IX complex lacking a specific antibody epitope and the ability to bind von Willebrand factor. In contrast, the binding of human alpha-thrombin to the patient's platelets was normal, and three classes of binding sites with high, intermediate, and low affinity could be detected. These studies define a distinct variant form of Bernard-Soulier syndrome and provide evidence, based on a naturally occurring mutant molecule, that the amino-terminal region of GP Ib alpha contains a von Willebrand factor-binding domain distinct from the high affinity thrombin-binding site. Use of different monoclonal antibodies with distinct epitope specificities appears to be essential for a correct identification of variant Bernard-Soulier syndrome.
L De Marco, M Mazzucato, F Fabris, D De Roia, P Coser, A Girolami, V Vicente, Z M Ruggeri
The nucleotide sequence was determined for all 22 exons of the insulin receptor gene from three patients with genetic syndromes associated with extreme insulin resistance. In all three patients, insulin resistance was caused by decreased insulin binding to the cell surface. The patient with leprechaunism (leprechaun/Winnipeg) came from a consanguineous pedigree and was homozygous for a missense mutation substituting arginine for His209 in the alpha-subunit of the insulin receptor. The other two patients were both compound heterozygotes with a nonsense mutation in one allele of the insulin receptor gene, and a missense mutation in the other allele. In the patient with the Rabson-Mendenhall syndrome (patient RM-1), the missense mutation substituted lysine for Asn15 in the alpha-subunit. In the patient with type A extreme insulin resistance (patient A-1), the missense mutation substituted serine for Asn462 in the alpha-subunit. Both nonsense mutations markedly reduced the levels of insulin receptor mRNA transcribed from the alleles with the nonsense mutation as compared to the transcripts from the other allele. The reduction in the level of mRNA would be predicted to greatly reduce the rate at which the truncated receptors would be synthesized. Furthermore, the truncated receptors would be severely impaired in their ability to mediate insulin action.
T Kadowaki, H Kadowaki, M M Rechler, M Serrano-Rios, J Roth, P Gorden, S I Taylor
Prednisone treatment causes protein wasting and adds additional risks to a patient, whereas human growth hormone (hGH) treatment causes positive nitrogen balance. To determine whether concomitant administration of hGH prevents the protein catabolic effects of prednisone, four groups of eight healthy volunteers each were studied using isotope dilution and nitrogen balance techniques after 7 d of placebo, hGH alone (0.1 mg.kg-1.d-1), prednisone alone (0.8 mg.kg-1.d-1), or prednisone plus hGH (n = 8 in each group). Whether protein balance was calculated from the leucine kinetic data or nitrogen balance values, prednisone alone induced protein wasting (P less than 0.001), whereas hGH alone resulted in positive (P less than 0.001) protein balance, when compared to the placebo-treated subjects. When hGH was added to prednisone therapy, the glucocorticoid-induced protein catabolism was prevented. Using leucine kinetic data, negative protein balance during prednisone was due to increased (P less than 0.05) proteolysis, whereas hGH had no effect on proteolysis and increased (P less than 0.01) whole body protein synthesis. During combined treatment, estimates of proteolysis and protein synthesis were similar to those observed in the placebo treated control group. In conclusion, human growth hormone may have a distinct role in preventing the protein losses associated with the administration of pharmacologic doses of glucocorticosteroids in humans.
F F Horber, M W Haymond
No posts were found with this tag.