Isolation of a catalytically competent phosphorylated tyrosine kinase from Rous sarcoma virus-induced rat tumor by immunoadsorption to and hapten elution from phosphotyrosine binding antibodies.

A procedure has been developed for the isolation of a catalytically competent phosphorylated tyrosine kinase (RSV Y-kinase) from avian sarcoma virus-induced rat tumors. The procedure involves reaction of partially purified RSV Y-kinase with ATP to effect tyrosyl phosphorylation of catalytically competent RSV Y-kinase. Tyrosyl phosphorylated RSV Y-kinase was isolated from the heterogeneous reaction mixture by immunoadsorption on immobilized phosphotyrosyl binding antibodies and elution with the hapten p-nitrophenyl phosphate. Estimation of the phosphate content of the purified phosphorylated RSV Y-kinase indicated that 1-3 tyrosyl groups had been phosphorylated upon reaction with ATP. The specific activity toward histone 2B of the purified phosphorylated RSV Y-kinase was at least 30-fold greater than that estimated for the RSV Y-kinase prepared previously by immunoadsorption on immobilized antiserum from tumor bearing rabbits.

Cardiovascular chemotherapy: anticoagulants.

Anticoagulant therapy has changed dramatically during the past two years. Low molecular weight heparin has substantially replaced unfractionated heparin as the parenteral anticoagulant of choice. The use of warfarin has substantially increased, especially for prevention of stroke in the setting of atrial fibrillation. It has become clear that warfarin cannot be administered effectively in an unmonitored fixed-dose fashion. The parenteral direct thrombin inhibitor desirudin was shown to be efficacious in the prevention of deep vein thrombosis in man. Small thrombin and factor Xa inhibitors with in vivo oral anticoagulant activity have been identified.

Catalytically competent human and bovine zeta-thrombin and chimeras generated from unfolded polypeptide chains.

Human and bovine alpha-thrombin cleaved at the B-chain by chymotrypsin generates catalytically competent zeta-thrombins, which are comprised of two noncovalently linked fragments: a 36-(human) or 49-(bovine) residue A-chain linked by a disulfide to B-chain residues B1-148 (zeta 1-thrombin) and B-chain residues B149-259 (zeta 2-thrombin). Human and bovine D-Phe-Pro-Arg-CH2-zeta- and PhMeSO2-zeta-thrombins were prepared by reaction of the active-site histidine (H-B43) and serine (S-B205) with PPACK and PMSF, respectively. Unfolding and dissociation of the noncovalently linked polypeptide chains of either human or bovine D-Phe-Pro-Arg-CH2-zeta- and PhMeSO2-zeta-thrombins in 4.5 M guanidine-HCl and refolding upon 30-fold dilution in 50 mM sodium phosphate buffer pH 6.5, 750 mM NaCl, 0.1% PEG resulted in biphasic generation of catalytic activity. The slow phase was eliminated in the presence of the competitive inhibitor benzamidine-HCl. Unfolding and refolding mixtures of the appropriate inactive precursors generated the active chimeric thrombins bovine zeta 1-thrombin:human zeta 2-thrombin and human zeta 1-thrombin:bovine zeta 2-thrombin. Human zeta 1-thrombin and zeta 2-thrombin were isolated, and, upon recombining, the isolated fragments refolded to generate catalytically competent zeta-thrombin with an active-site content, specific activity toward Chromozym-TH, and a specificity constant (kcat/Km) for FPA release from fibrinogen that were all within 60% of those of native alpha-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

Demonstration of growth hormone (GH) receptor-associated tyrosine kinase activity in multiple GH-responsive cell types.

Highly purified GH-receptor preparations from 3T3-F442A fibroblasts, whose differentiation into adipocytes is promoted by GH, have been shown to contain a tyrosine kinase capable of phosphorylating GH receptors. In the current work, characteristics of the tyrosine kinase responsible for the in vitro phosphorylation of GH receptors from cultured 3T3-F442A fibroblasts were examined, and the presence of this GH receptor-associated tyrosine kinase activity was demonstrated in multiple cell types. GH-receptor complexes from GH-treated cells were partially purified by immunoprecipitation using anti-GH antibodies and then incubated as an immune complex with [gamma 32P] ATP. Incorporation of 32P into the GH receptor from 3T3-F442A fibroblasts was apparent within 1 min at 30 C after the addition of [gamma 32P]ATP (5-10 microM). A divalent cation was requisite for the phosphorylation; Mn2+ was significantly more effective than Mg2+ and Co2+; Ba2+, Ca2+, or Zn2+ had no effect. Excess unlabeled ATP, but not cytosine triphosphate, GTP, or uridine triphosphate, abolished 32P incorporation into the GH receptor and [gamma 32P]GTP could not replace [gamma 32P]ATP as a source of 32P. At 5.5 mM Mn2+, phosphorylation exhibited a biphasic dose response to ATP, with maximal phosphorylation occurring at a concentration of 10 microM ATP. At more physiological concentrations of ATP (1 mM), phosphorylation of the GH receptor was also stimulated by lower concentrations of Mn2+ (as low as 500 nM). Optimal reaction conditions determined for the phosphorylation reaction in 3T3-F442A fibroblasts were used to demonstrate incorporation of 32P from [gamma 32P]ATP into partially purified GH receptors from cultured human IM-9 lymphocytes, murine 3T3-F442A adipocytes, rat H-35 hepatoma cells, and freshly isolated rat adipocytes. The 32P was shown to be incorporated into tyrosyl residues in receptors from the two cell types tested (IM-9 lymphocytes and rat adipocytes). Cross-linked [125I] hGH-receptor complexes solubilized from the four cell types (IM-9 lymphocytes, 3T3-F442A adipocytes, H-35 hepatoma cells, and freshly isolated rat adipocytes) bound to and could be eluted from phosphotyrosyl antibody, suggesting that tyrosyl phosphorylation of GH receptors in all of these cells occurs in vivo. The presence of tyrosine kinase activity associated with GH receptors in multiple cell types from different species is consistent with tyrosine kinase activity playing a role in the actions of GH.

Stimulation by growth hormone (GH) of GH receptor-associated tyrosine kinase activity.

We have shown previously that GH receptors (GHRs) become phosphorylated on tyrosyl residues when GH-responsive cells are exposed to GH. In this work we investigate the molecular mechanism by which GH binding stimulates tyrosyl phosphorylation of GHR. To test whether in the presence of GH, GHR and the tyrosine kinase responsible for GHR phosphorylation are tightly associated in a complex or form a transient enzyme-substrate complex, the rate of in vitro phosphorylation of GHR was determined as a function of receptor concentration. GH-GHR complexes were purified from 3T3-F442A fibroblasts by immunoadsorption to and elution from immobilized phosphotyrosyl-binding antibody. The rate of in vitro tyrosyl phosphorylation of GH-GHR complexes was not substantially reduced when the GHR preparation was diluted 1:10 or 1:100 before phosphorylation, consistent with a tight association between kinase and substrate (GHR). When cells were labeled metabolically with 35S, and GHRs containing phosphorylated tyrosyl residues were isolated by immunoadsorption to and elution from phosphotyrosyl-binding antibody, the ability to immunoprecipitate 35S-labeled GHR with GHR antibodies was only evident when the cells had been incubated with GH. This indicates that in vivo, GHRs are phosphorylated on tyrosyl residues only when GH is bound. Additionally, when anti-GHR antibodies were used to immunoprecipitate GHR from solubilized cells, only GHRs isolated from GH-treated cells were phosphorylated when subjected to an in vitro kinase assay. The increased tyrosyl phosphorylation of GHR detected after incubation of cells with GH is consistent either with GH increasing tyrosine kinase activity associated with the GHR or with GH inducing a conformational change in GHR that renders it susceptible to tyrosyl phosphorylation. To discern whether GH binding increased GHR-associated kinase activity, we tested the ability of anti-GHR antibody immunoprecipitates to phosphorylate the synthetic substrate poly(Glu4,Tyr). GH treatment of cells resulted in a 2-fold increase in the rate of phosphorylation of poly(Glu4,Tyr). The increase in kinase activity was dose dependent, with half-maximal stimulation between 15-20 ng/ml GH. These results provide strong evidence that GH actually increases tyrosine kinase activity associated with the GHR. This would be consistent with GH-dependent complex formation between a constitutively activated kinase and GHR and/or activation of a constitutively associated or intrinsic kinase.

Discovery of a novel, selective, and orally bioavailable class of thrombin inhibitors incorporating aminopyridyl moieties at the P1 position.

A novel class of thrombin inhibitors incorporating aminopyridyl moieties at the P1 position has been discovered. Four of these thrombin inhibitors (13b,c,e and 14d) showed nanomolar potency (Ki 0.8-12 nM), 300-1500-fold selectivity for thrombin compared with trypsin, and good oral bioavailability (F = 40-76%) in rats or dogs. The neutral P1 was expected to increase metabolic stability and oral absorption. Identification of this novel aminopyridyl group at P1 was a key step in our search for a clinical candidate.

Efficacious, orally bioavailable thrombin inhibitors based on 3-aminopyridinone or 3-aminopyrazinone acetamide peptidomimetic templates.

We have addressed the key deficiency of noncovalent pyridinone acetamide thrombin inhibitor L-374,087 (1), namely, its modest half-lives in animals, by making a chemically stable 3-alkylaminopyrazinone bioisostere for its 3-sulfonylaminopyridinone core. Compound 3 (L-375,378), the closest aminopyrazinone analogue of 1, has comparable selectivity and slightly decreased efficacy but significantly improved pharmacokinetics in rats, dogs, and monkeys to 1. We have developed an efficient and versatile synthesis of 3, and this compound has been chosen for further preclinical and clinical development.

A kinetic method for characterization of heterogenous fibrinogen and its application to fibrinogen Grand Rapids, a congenital dysfibrinogenemia.

A kinetic analysis was developed to determine the steady state kinetic parameter Kcat/KM for the thrombin-catalyzed release of FPA from abnormal and normal fibrinogen in mixtures of the two. Such mixtures are likely to comprise the fibrinogen of individuals with congenital dysfibrinogenemia. The analysis was used to characterize fibrinogen Grand Rapids a new congenital dysfibrinogenemia. It indicated that fibrinogen from affected individuals was composed of normal and abnormal fibrinogen in roughly equal amounts, and that the value of kcat/KM for the thrombin-catalyzed release of FPA from the fibrinogen variant was 77-fold lower than that for the release of FPA from the normal fibrinogen. In separate studies, fibrinogen Grand Rapids was found to exhibit a reduced clottability. Additionally, affected individuals appeared to have plasma fibrinogen concentrations which were about one-third the normal value.

A facile system for construction of HSV-1 variants: site directed mutation of the UL26 protease gene in HSV-1.

A 2-plasmid/4-cosmid-based system of mutagenesis is described for construction of herpes simplex virus type 1 (HSV-1) variants with point mutations in the protease gene. The system was used to reconstruct a mutant virus (V701) with Tyr30 to Phe and Ala48 to Val mutations in HSV-1 protease that exhibits the temperature sensitive phenotype of the previously characterized temperature sensitive HSV-1 mutant, ts1201. The 2-plasmid/4-cosmid system of mutagenesis was further validated by using it to construct a virus wherein the active site Ser129 of HSV-1 protease was mutated to Ala. The resulting virus mutant (V713) grew only on the Vero host range cell line PHS-23. In V713 infected Vero cells, the processing of Pra to N(O) was almost completely blocked, and B capsids accumulated in the nucleus in crystal-like aggregates, suggesting that protease activity is required for emergence of monodispersed capsids from these aggregates. Back mutation of Ala129 to Ser using the V713 viral DNA as template for PCR mutagenesis restored the wild-type phenotype verifying that the replicative incompetence of V713 reflected only the effect of the Ser to Ala mutation. The 2-plasmid/4-cosmid system of mutagenesis (and modifications thereof) should facilitate production of new mutant viruses for delineating interactions of domains of HSV-1 protease (as well as other HSV-1 proteins) important for virus replication.

alpha-Thrombin within fibrin clots: inactivation of thrombin by antithrombin-III.

To demonstrate that human alpha-thrombin is effectively inactivated by human antithrombin III (AT) during the production of a fibrin clot we measured the amount of alpha-thrombin activity which can be recovered from a clot generated from purified human proteins. We discovered that 0.05-0.07% of the original alpha-thrombin activity is recovered from a fibrin clot produced from a reaction mixture where the initial concentrations of AT and alpha-thrombin were chosen at a ratio (17.5) to allow complete conversion of fibrinogen to fibrin. These results indicated that alpha-thrombin is successfully inactivated by AT during the production of a fibrin clot. Further, when an amount of alpha-thrombin equal to that recovered from a fibrin clot is introduced into a solution of fibrinogen and AT identical to that utilized to produce the clot only 4% of the fibrinogen is converted to fibrin. These results suggest that i) when a fibrin clot is dissolved during fibrinolytic therapy little active alpha-thrombin should be released from the clot and ii) this amount of thrombin is insufficient to catalyze rethrombosis without proposing de novo production of thrombin. The action on factors XI, VIII, and V of the small amount of thrombin released upon thrombolysis, however, may provide the stimulus for de novo production of sufficient thrombin to catalyze rethrombosis.

A thrombin assay based upon the release of fibrinopeptide A from fibrinogen: definition of a new thrombin unit.

An assay for thrombin is presented wherein thrombin-catalyzed hydrolysis at Arg-A alpha-16 to release fibrinopeptide A (FPA) from fibrinogen is measured using high-performance liquid chromatography (HPLC). In this assay one thrombin unit (TU) is defined as that amount of thrombin that will release half of the FPA in one min from one ml of a solution of greater than 90% clottable normal human fibrinogen (less than or equal to 0.35 microM) at 37 degrees C, pH 7.4, /2 0.15. One TU is equivalent to approximately 0.1 NIH unit of thrombin and approximately 1 pmol of pure human thrombin. At 37 degrees C, pH 7.4, and plasma levels of fibrinogen of 3 mg/ml, one TU will catalyze the release of 3.6 nmol FPA min-1. Variability in fibrinogen samples which produce dramatic differences in clotting time assays with the same sample of thrombin, produce little or no variation in the catalytic assay for TU. The assay for TU obviates the need for maintenance of a thrombin reference standard.

Inhibition of thrombin and other trypsin-like serine proteinases by cyclotheonamide A.

Cyclotheonamide A (CA), a cyclic peptide isolated from the marine sponge of the genus Theonella was shown to be a slow-binding inhibitor of several trypsin-like serine proteinases. Values of 4.6 x 10(4), 4.8 x 10(4), 9.3 x 10(3), 2.1 x 10(3) and 2.7 x 10(2) M-1 s-1 were determined for the second-order rate constants for formation of CA complexes with thrombin, trypsin, plasmin, 2-chain t-PA and factor Xa, respectively. The equilibrium constant (Ki) was measured for dissociation of CA from the CA complex with human thrombin (Ki = 1.0 nM), bovine trypsin (Ki = 0.2 nM), human plasmin (Ki = 12 nM), human factor Xa (Ki = 50 nM) and human 2-chain tissue plasminogen activator (t-PA) (Ki = 40 nM). CA produces dose dependent increases in clotting time assays. The clotting time in the thrombin time, activated partial thromboplastin time and prothrombin time assays, were doubled by 1.5, 0.9 and 48 microM CA, respectively. A model for the binding of CA to the active site of thrombin is proposed.

International trade in bear gall bladders: forensic source inference.

Fresh and desiccated gall bladders of the Ursidae family (bears) obtained as criminal evidence were characterized by analysis of the principal biliary components, mainly ursodeoxycholyl-taurine, cholyl-taurine and chenodeoxycholyl-taurine using TLC and HPLC. This bile acids profile appears to be an Ursidae family characteristic. Results show that of the samples from Asia only 3% were from the Ursidae family and 18% were from "farmed bears." Samples seized in the U.S.A. and Canada showed that 22.6% and 85% respectively, were from Ursids. The remaining samples were consistent with bile from the domestic pig (Suidae).

Winter poisoning of coyotes and raptors with Furadan-laced carcass baits.

Three bald eagles (Haliaeetus leucocephalus), a red-tailed hawk (Buteo jamaicensis), and two coyotes (Canis latrans) found in a field in north-central Kansas (USA) in December 1992 were poisoned by flowable carbofuran (Furadan 4F) placed on sheep (Ovis aries) carcasses to kill coyotes. The carbofuran was placed on the carcasses in October 1992, but the coyotes and raptors apparently were killed in late December. Thus, flowable Furadan can cause direct and secondary deaths of wildlife under some circumstances for at least 60 days following placement.

Outcome of hematopoietic stem cell transplant as salvage therapy for Hodgkin's lymphoma in adolescents and young adults at a single institution.

For patients with relapsed Hodgkin's lymphoma (HL), high dose chemotherapy with stem cell rescue (HDCT-SCT) may improve survival over chemotherapy alone. We assessed the outcomes of HDCT-SCT in 37 consecutive adolescent and young adult patients with relapsed HL whose malignancy was categorized based on sensitivity to chemotherapy. We determined whether current outcomes supported the use of HDCT-SCT in all of our patients or just those patients with lower-risk characteristics such as chemosensitivity. With a median follow-up of 6.5 years, the 2-year overall survival (OS) was 89% (95% CI: 62-97%) for the chemosensitive patients (n = 21), whereas for patients with resistant disease (n = 16), OS was 53% (95% CI: 25-74%). Both autologous and allogeneic transplants were well tolerated, with 100-day treatment-related mortality under 10%. Our data show encouraging outcomes for patients with chemosensitive relapsed HL who receive hematopoietic stem cell transplant (HSCT) and support the value of the procedure even when the disease is chemoresistant.

Blood and marrow morphology in acute leukemia patients receiving chemotherapy: a photo-essay.

Acute leukemia patients are often treated with drugs which have effects upon bone marrow cell production roles and which can alter the morphology of blood cells. In this paper some typical morphology findings in the marrow and blood are illustrated; the causes of the altered cellular appearances are discussed.