PubMed Health⌕ Search

Biomedical subjects

T Orfeo

Publications and source records attributed to T Orfeo.

15 recordsLinked to original sources
PubMed2006

Peptidomimetic inhibitors for activated protein C: implications for hemophilia management.

BACKGROUND: Several clinical studies and experiments with transgenic mice have suggested that the severity of the bleeding phenotype in hemophilic patients is substantially reduced in association with impaired inactivation of factor (F) Va by activated protein C (APC) in the presence of the FV Leiden mutation. Experiments using a synthetic coagulation proteome model showed that the presence of FV Leiden significantly increased thrombin generation in the absence of FVIII or FIX. OBJECTIVE: To test the effect of APC inhibition on thrombin generation in hemophilia. METHODS: Prothrombinase and a synthetic coagulation proteome model of tissue factor-triggered thrombin generation were used. RESULTS: Peptide-based APC inhibitors, which mimic the P4-P4' residues surrounding the APC cleavage site at Arg306 of FVa, were synthesized. These compounds are specific and reversible inhibitors of APC, with Ki values as low as 1-2 microM; most have insignificant affinity for FXa or thrombin. The affinity for APC is dependent upon the location and character of the protecting groups. Representatives of this group of compounds inhibit FVa inactivation by APC and prolong FVa functional activity in the prothrombinase complex. When evaluated in a synthetic coagulation proteome model, one inhibitor partially compensated for the absence of FVIII. CONCLUSIONS: Synthetic APC inhibitors may be useful as adjuvants for hemophilia treatment.

Activated Protein C Resistance↗
PubMed2000

Homocysteine inhibits inactivation of factor Va by activated protein C.

We report the effect of homocysteine on the inactivation of factor Va by activated protein C (APC) using clotting assays, immunoblotting, and radiolabeling experiments. Homocysteine, cysteine, or homocysteine thiolactone have no effect on factor V activation by alpha-thrombin. Factor Va derived from homocysteine-treated factor V was inactivated by APC at a reduced rate. The inactivation impairment increased with increasing homocysteine concentration (pseudo first order rate k = 1.2, 0.9, 0.7, 0.4 min(-1) at 0, 0.03, 0.1, 1 mm homocysteine, respectively). Neither cysteine nor homocysteine thiolactone treatment of factor V affected APC inactivation of derived factor Va. Western blot analyses of APC inactivation of homocysteine-modified factor Va are consistent with the results of clotting assays. Factor Va, derived from factor V treated with 1 mm beta-mercaptoethanol was inactivated more rapidly than the untreated protein sample. Factor V incubated with [(35)S]homocysteine (10-450 micrometer) incorporated label within 5 min, which was found only in those fragments that contained free sulfhydryl groups: the light chain (Cys-1960, Cys-2113), the B region (Cys-1085), and the 26/28-kDa (residues 507-709) APC cleavage products of the heavy chain (Cys-539, Cys-585). Treatment with beta-mercaptoethanol removed all radiolabel. Plasma of patients assessed to be hyperhomocysteinemic showed APC resistance in a clot-based assay. Our results indicate that homocysteine rapidly incorporates into factor V and that the prothrombotic tendency in hyperhomocysteinemia may be related to impaired inactivation of factor Va by APC due to homocysteinylation of the cofactor by modification of free cysteine(s).

Blotting, Western↗
PubMed1999

Distamycin A selectively inhibits Acanthamoeba RNA synthesis and differentiation.

The effects of distamycin A on Acanthamoeba transcription, growth and differentiation were determined. Distamycin A inhibits transcription both in vitro and in vivo and can displace from DNA the transcription activator TATA binding protein promoter binding factor (TPBF). Inhibition in vivo is surprisingly selective for large rRNA precursors, 5S rRNA, profilin, S-adenosylmethionine synthetase, and extendin. Transcription from the TATA binding protein (TBP), TPBF, protein disulfide isomerase, tubulin and RNA polymerase II large subunit genes is only slightly inhibited. Moreover the rate of 5S rRNA transcription eventually recovers and exceeds that of untreated cells, while profilin transcription remains inhibited. Distamycin A inhibition is accompanied by a complex pattern of alterations to steady state levels of mRNAs. Actin, profilin and S-adenosylmethionine synthetase mRNAs are degraded, whereas mRNA encoding TBP is increased slightly in abundance. Transcription inhibition is accompanied by cessation of growth and severe morphological changes to Acanthamoeba, which are consistent with loss of production of mRNA encoding cytoskeletal proteins. Distamycin A also prevents starvation-induced differentiation of Acanthamoeba, in part due to complete prevention of cellulose production and cell wall formation.

Acanthamoeba↗
PubMed1998

Transcription by RNA polymerase II during Acanthamoeba differentiation.

The rates of transcription of several protein coding genes during Acanthamoeba differentiation have been examined by nuclear run-on and RNase protection assays. During early encystment, transcription by RNA polymerase II increases approximately 4-fold, whereas transcription by RNA polymerases I and III is decreased, as previously described. The rates of transcription from a wide variety of individual genes are only slightly affected during the first 16 h of encystment, although profilin gene expression is markedly increased. The levels of mRNAs encoding TPBF, TATA binding protein, cyclin-dependent kinase, protein disulfide isomerase, profilin, myosin II heavy chain, ubiquitin and extendin are stable during mature cyst formation, whereas mRNAs encoding actin, S-adenosyl methionine synthase and tubulin are substantially decreased in abundance within 16 h of starvation-induced encystment. We conclude that in contrast to the negative regulation of large rRNA and 5S rRNA synthesis during differentiation, the RNA polymerase II transcription apparatus is not negatively regulated. Control of Acanthamoeba differentiation is likely to be mediated by positive regulation of genes necessary for cyst maturation.

Acanthamoeba↗
PubMed1995

Alpha-thrombin stimulates urokinase production and DNA synthesis in cultured human cerebral microvascular endothelial cells.

alpha-Thrombin regulation of endothelial cell (EC) fibrinolysis has been documented by using endothelia derived from a number of anatomic locations but not with those derived from the human cerebral vasculature. In the present study, the fibrinolytic properties of human cerebral microvascular ECs and their regulation by alpha-thrombin are delineated and contrasted with those of human umbilical vein and foreskin microvascular ECs. In cerebral ECs, alpha-thrombin elicited a unique dose-dependent increase in urokinase production and DNA synthesis. Maximal stimulation, observed with 10 nmol/L alpha-thrombin, resulted in a 30- to 50-fold increase in urokinase production and a concomitant fourfold increase in DNA synthesis; the increase in urokinase was reflected in higher steady-state levels of urokinase mRNA. The major urokinase product secreted is the single-chain form of the enzyme. No effect was observed with the addition of other proteases or catalytically inactive variants of alpha-thrombin. A thrombin receptor agonist peptide upregulated urokinase production but had no effect on DNA synthesis, suggesting that fibrinolysis is mediated by the thrombin receptor but that proliferation is regulated by a different pathway. These findings suggest the possibility that the cerebral microvasculature may be a specialized region of the vascular system in which urokinase-type plasminogen activator, not tissue-type plasminogen activator, is the key catalyst of fibrin lysis when the brain responds to thrombotic events and that alpha-thrombin may regulate repair of the cerebral microvascular system.

Blotting, Northern↗
PubMed1994

Osteonectin in matrix remodeling. A plasminogen-osteonectin-collagen complex.

Osteonectin is an adhesive glycoprotein synthesized constitutively by osteoblasts, endothelial cells, and megakaryocytes. Bone-derived and platelet-derived osteonectins differ in their electrophoretic mobility and carbohydrate content, and each displays different affinities for collagen matrices. Both types of osteonectin bind to plasminogen (Kd(app), of 4.7 +/- 1.0 x 10(-8) M for bone osteonectin and 1.2 +/- 0.1 x 10(-7) M for platelet osteonectin). The osteonectin-plasminogen interaction is inhibited by alpha 2-antiplasmin and epsilon-aminocaproic acid, suggesting that the interaction is mediated through the kringle 1 region of plasminogen. Both osteonectins enhance the rate of plasmin generation by tissue-type plasminogen activator to approximately the same extent as fibrinogen. Equilibrium binding measurements conducted using total internal reflection fluorescence spectroscopy indicate that plasminogen binds to collagen in the presence of bone osteonectin (Kd = 1.30 +/- 0.1 x 10(-7) M). No binding of plasminogen to collagen matrix was detected in the presence of platelet osteonectin or in the absence of bone osteonectin. Bone osteonectin-dependent binding of plasminogen to collagen matrix is reversed by the addition of epsilon-aminocaproic acid. The ability of both types of osteonectin to bind to and influence plasminogen activation and of bone osteonectin to anchor plasminogen on collagen matrices suggests that osteonectin may play a role in directing extracellular matrix proteolysis.

Aminocaproic Acid↗
PubMed1994

Sublethal percussion trauma in vitro causes a persisting derangement in the nonthrombogenic properties of brain endothelial cells.

The delivery of a blow to the head represents a transfer of energy, part of which manifests itself as a short-lived pressure change within the skull. An in vitro model was developed to test whether cerebral endothelial cell hemostatic function is altered with exposure to this type of pressure event. Human cerebral microvascular endothelium (HCME) cells were subjected to rapid (2-5 msec) changes in pressure (delta atmosphere = 1.2-10), the sublethal range defined (delta atmosphere < or = 6.5), and the nonthrombogenic status of sublethally percussed HCME cells assessed using the adherence of alpha-thrombin activated platelets as an indicator. The HCME cells had lost their normal capacity to suppress adherence of activated platelets when evaluated 1 hour or 24 hours after percussion. Adherence of activated platelets to percussed HCME cells was blocked by the addition of PGI2, an inhibitor of platelet adherence, when evaluated at 1 hour but not 24 hours after percussion, indicating that percussed HCME cells were undergoing further derangement of their nonthrombogenic mechanisms. Percussed HCME cells cultured for 24 hours in medium containing scavengers of oxygen free radicals recovered their capacity to block platelet adherence. We conclude that sublethal percussion immediately compromises the nonthrombogenic character of HCME cells and initiates the development of a persisting prothrombotic state in HCME cells. This derangement appears linked to increased production of reactive oxygen species by percussed HCME cells.

Brain↗
PubMed1992

Aminonaphthalenesulfonamides, a new class of modifiable fluorescent detecting groups and their use in substrates for serine protease enzymes.

A series of new compounds, 6-amino-1-naphthalenesulfonamides (ANSN), were used as fluorescent detecting groups for substrates of amidases. These compounds have a high quantum fluorescent yield, and the sulfonyl moiety permits a large range of chemical modification. Fifteen ANSN substrates with the structure (N alpha-Z)Arg-ANSNR1R2 were synthesized and evaluated for their reactivity with 8 proteases involved in blood coagulation and fibrinolysis. Thrombin, activated protein C, and urokinase rapidly hydrolyzed substrates with monosubstituted sulfonamide moieties (R1 = H). The maximum rate of substrate homologue). The hydrolysis rates for substrates with branched substituents were slower than their linear analogues. Monosubstituted (N alpha-Z)Arg-ANSNR1R2 possessing cyclohexyl or benzyl groups in the sulfonamide moiety were hydrolyzed by these three enzymes at rates similar to that of the n-butyl homologue (except the cyclohexyl compound for u-PA). Factor Xa rapidly hydrolyzed substrates with short alkyl chains, especially when R1 = R2 = CH3 or C2H5. Lys-plasmin and rt-PA demonstrated low activity with these compounds, and the best results were accomplished for monosubstituted compounds when R2 = benzyl (for both enzymes). Factor VIIa and factor IXa beta exhibited no activity with these substrates. A series of 14 peptidyl ANSN substrates were synthesized, and their reactivity for the same 8 enzymes was evaluated. Thrombin, factor Xa, APC, and Lys-plasmin hydrolyzed all of the substrates investigated. Urokinase, rt-PA, and factor IXa beta exhibited reactivity with a more limited group of substrates, and factor VIIa hydrolyzed only one compound (MesD-LGR-ANSN(C2H5)2). The substrate ZGGRR-ANSNH (cyclo-C6H11) showed considerable specificity for APC in comparison with other enzymes (kcat/KM = 19,300 M-1 s-1 for APC, 1560 for factor IIa, and 180 for factor Xa). This kinetic advantage in substrate hydrolysis was utilized to evaluate the activation of protein C by thrombin in a continuous assay format. Substrate (D-LPR-ANSNHC3H7) was used to evaluate factor IX activation by the factor VIIa/tissue factor enzymatic complex in a discontinuous assay. A comparison between the commercially available substrate chromozyme TH (p-nitroanilide) and the ANSN substrate with the same peptide sequence (TosGPR) demonstrated that aminonaphthalenesulfonamide increased the specificity (kcat/KM) of substrate hydrolysis by thrombin more than 30 times, with respect to factor Xa substrate hydrolysis.

Chemical Phenomena↗
PubMed1992

Modulation of the fibrinolytic response of cultured human vascular endothelium by extracellularly generated oxygen radicals.

Clinical and experimental data indicate that activated oxygen species interfere with vascular endothelial cell function. Here, the impact of extracellular oxidant injury on the fibrinolytic response of cultured human umbilical vein endothelial (HUVE) cells was investigated at the protein and mRNA levels. Xanthine (50 microM) and xanthine oxidase (100 milliunits), which produces the superoxide anion radical (O2-) and hydrogen peroxide (H2O2), was used to sublethally injure HUVE cells. Following a 15-min exposure, washed cells were incubated for up to 24 h in serum-free culture medium. Tissue-type plasminogen activator (t-PA) antigen, plasminogen activator inhibitor-1 (PAI-1) antigen, and PAI-1 activity were determined in 1.25 ml of conditioned medium and t-PA and PAI-1 mRNA in the cell extracts of 2 x 10(6) HUVE cells. Control cells secreted 3.9 +/- 1.3 ng/ml (mean +/- S.D., n = 12) within 24 h. Treatment with xanthine/xanthine oxidase for 15 min induced a 2.8 +/- 0.4-fold increase (n = 12, p less than 0.05) of t-PA antigen secretion after 24 h. The t-PA antigen was recovered predominantly in complex with PAI-1. The oxidant injury caused a 3.0 +/- 0.8-fold increase (n = 9, p less than 0.05) in t-PA mRNA within 2 h. Total protein synthesis was unaltered by xanthine/xanthine oxidase. The oxidant scavengers superoxide dismutase and catalase, in combination, abolished the effect of xanthine/xanthine oxidase on t-PA secretion and t-PA mRNA synthesis. Xanthine/xanthine oxidase treatment of HUVE cells did not affect the PAI-1 secretion in conditioned medium nor the PAI-1 mRNA levels in cell extracts. Thus extracellular oxidant injury induces t-PA but not PAI-1 synthesis in HUVE cells.

Antigens↗
PubMed1990

Cardiac and skeletal muscle adaptations to training in systemic hypertension and effect of beta blockade (metoprolol or propranolol).

Cardiovascular and peripheral adaptations to an aerobic conditioning program were studied in 30 hypertensive adults taking either placebo, beta 1-selective beta-adrenergic blocker (metoprolol) or beta 1-nonselective beta-adrenergic blocker (propranolol). The placebo group increased aerobic capacity (VO2max) 24% (p less than 0.002), largely explained by an increased peripheral arteriovenous (AV) oxygen difference with minimal changes in cardiac size and function. Resting blood pressure and total systemic resistance also decreased. The group taking a beta 1-selective beta blocker increased VO2max 8% (p less than 0.05), reduced resting blood pressure but had no significant change of AV oxygen difference or cardiac size or function. The group taking the beta 1-nonselective beta blocker propranolol had no increase in VO2max, no decrease in resting blood pressure and no cardiovascular or peripheral adaptations to the exercise program. Thus, beta 1-selective and beta 1-nonselective beta blockers attenuate conditioning in hypertensive patients to differing degrees, in each case by blocking peripheral mechanisms of conditioning.

Adaptation, Physiological↗
PubMed1980

Twenty-three new human tumor lines established in nude mice.

Subcutaneous inoculation of 87 human tumors into athymic nude mice, including gastrointestinal (non-colon) tumors, germ cell-primitive cell tumors, kidney tumors, lower urinary tract tumors, malignant melanomas and several metastases from unknown primary sites, resulted in growth in primary transplants in 32 cases (36.8%). Take rates varied among the tumor categories, from 56% for malignant melanomas to 13% for lower urinary tract tumors. They also differed among recurrent tumors (50%), metastases (41%) and tumors of primary site (28%). 23 tumor lines were established, including five lines each from renal cell adenocarcinomas and malignant melanomas, two each from adenocarcinoma of the pancreas and Wilms' tumors and only line each from various tumors of the gastrointestinal, germ cell-primitive cell, lower urinary tract and primary site unknown categories. The frequency of line establishment was greater for recurrent tumors and metastases than for primary tumors. Tumor lines have been carried continuously up to passage 17 in one case. Time in primary transplant varied from 3 to 29 weeks for the individual tumors and the average primary transplant time varied from 5.8 to 14.7 weeks for the six tumor categories. The average time in passage varied among the established lines from 2.3 to 10.6 weeks. The average passage time for all tumor lines was 5.3 weeks, with those of recurrent tumor origin showing the shortest average (4.0 weeks) followed by lines of metastatic origin (5.4 weeks) and those of primary tumor origin (5.8 weeks).

Adenocarcinoma↗
PubMed1979

Establishment of human colon carcinoma lines in nude mice.

After subcutaneous inoculation into nude mice of 24 human colon adenocarcinomas, growth, defined as histopathologically confirmed tumor growth which has been passed, was observed in 13 cases (54%). Tumors from metastatic sites showed higher take rates (58%) than tumors from primary sites or recurrent tumors (50%). Nine continuous tumor lines were established (69% of growing tumors) with metastatic tumors establishing more readily (100% of growing tumors) than primary tumors (40%). The average period in primary transplant was shorter for metastasis (8.3 weeks), than for primary tumors (18.5 weeks); total material 10.6 weeks. Average periods between passages were shorter than primary transplant times; these periods were shorter for metastases (6.6 weeks) than for primary tumors (9.4 weeks); total material 7.4 weeks. Of four growing tumors not established as continuous lines, three were primary and one a recurrent tumor, and the loss of tumor growth occurred in very early passages, not later than passage 3. All nude mouse-grown colon tumors were moderately well differentiated.

Adenocarcinoma↗
PubMed1977

One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice.

One hundred and twenty-seven cultured human tumor cell lines produced tumors after sc inoculation of 1-20 million cells into nude mice. They included 56 carcinoma lines, 14 sarcoma lines, and 57 lines from miscellaneous tumors and were all glucose-6-phosphate dehydrogenase type B. Twenty-nine percent of the lines produced tumors of 1 cm3 size within 1 month and 41% in the second month after inoculation. The histopathology correlated with the human tumor of origin in all cases.

Animals↗