Gene Literature Dashboard

Protein tyrosine phosphorylation is regulated by both protein tyrosine kinases and protein tyrosine phosphatases (PTPases). Recently, the structures of a family of PTPases have been described. In order to study the structure-function relationships of receptor-linked PTPases, we analyzed the effects of deletion and point mutations within the cytoplasmic region of the receptor-linked PTPases, LCA and LAR. We show that the first of the two domains has enzyme activity by itself, and that one cysteine residue in the first domain of both LCA and LAR is absolutely required for activity. The second PTPase like domains do not have detectable catalytic activity using a variety of substrates, but sequences within the second domains influence substrate specificity. The functional significance of a stretch of 10 highly conserved amino acid residues surrounding the critical cysteine residue located in the first domain of LAR was assessed. At most positions, any substitution severely reduced enzyme activity, while missense mutations at the other positions tested could be tolerated to varying degrees depending on the amino acid substitution. It is suggested that this stretch of amino acids may be part of the catalytic center of PTPases.

Serine protease inhibitors ("serpins") are highly homologous proteins which inhibit selected "target" serine proteases by acting as a pseudo-substrate. Their specificity is primarily determined by the amino acid sequence around the carboxyl-terminally located reactive center (P1-P1'). In addition, the association rate constant between a serpin and a serine protease can be dramatically increased by non-protein cofactors, such as heparin in the case of thrombin inhibition by antithrombin III. In an attempt to alter the specificity of PAI-1 from an inhibitor of the fibrinolytic system to an inhibitor of coagulation, we replaced P1-P1' or P3 through P3' of the reactive center of PAI-1 by the corresponding residues of antithrombin III and assessed whether the mutant proteins, purified from lysates of transformed Escherichia coli cells, had acquired thrombin inhibitory properties. The experiments were performed in the presence and absence of vitronectin, a multifunctional protein which has been shown to bind PAI-1 in plasma and in the matrix of endothelial cells. The second-order rate constants for t-PA inhibition of "wild-type" PAI-1 and PAI P1-P1'ATIII, irrespective of the presence of vitronectin, were similar, whereas replacing P3-P3' resulted in a 40-fold decrease of the second-order rate constant towards t-PA, again independent of vitronectin. In the absence of vitronectin, reactivity of PAI-1 and its "antithrombin III-like" variants towards thrombin was slow; however, PAI-1 P3-P3' ATIII had a 10-fold higher k1 than wild-type PAI-1 (1.3 x 10(4) M-1 s-1 versus 1.1 x 10(3) M-1 s-1). In contrast, in the presence of vitronectin, PAI-1 and even more rapidly PAI-1 P3-P3'ATIII were found to be effective thrombin inhibitors, with k1 values of 2.2 x 10(5) M-1s-1 and 1.8 x 10(6) M-1 s-1, respectively. Thus, in the presence of vitronectin, PAI-1 P3-P3'ATIII displays a 3-fold higher k1 with thrombin than with t-PA. It is shown that vitronectin enhances, in a dose-dependent manner, the formation of sodium dodecyl sulfate-resistant complexes between PAI-1 or mutants thereof and thrombin. Therefore, vitronectin is the first protein described to function as a cofactor for serpin specificity. PAI-1 is proposed to be a versatile inhibitor which, in the presence of vitronectin, can modulate both coagulation and fibrinolysis.

Non-nucleosidic phosphoramidite linker units suitable for use on commercial DNA synthesis machines have been designed for the direct incorporation of biotin and a new reporter group, phosphotyrosine, at multiple sites on synthetic oligonucleotides. The units are based on a 3-carbon glyceryl backbone where the reporter group is attached to the 2-O-position through a 3-aminopropyl spacer. 17-mer oligonucleotides were synthesized carrying at the 5'-end 1, 2, 4 or 8 biotinyl units or 1, 2, 4 or 8 phosphotyrosinyl units respectively and used for the detection of DNA on nitrocellulose filters by hybridization. Subsequent incubation of the filters with a monoclonal antibody to the reporter group followed by secondary detection using enhanced chemiluminescence (ECL) resulted in amplification of signal strengths as the number of reporter groups was increased. The results were quantitated by use of a charge couple device (CCD) camera. Spacing of biotin moieties by thymidyl residues resulted in further improvements in signal strengths, whereas similar spacing of phosphotyrosinyl units did not.

Thrombin was acetylated by treatment with acetic anhydride, and the potency for protein C activation and the fibrinogen clotting activity of the resultant acetylthrombin were investigated in vitro and in vivo. The acetylthrombin retained an amidolytic activity to the synthetic substrate, S-2238, and reduced both of the potency for protein C activation and the clotting activity. The potency for protein C activation (0.47% of that of thrombin) was retained more than the clotting activity (0.015% of that of thrombin). The enzymatic activities of acetylthrombin were in inverse proportion to the molecular weights of the substrates, S-2238, protein C, and fibrinogen. Similarly, the inhibitory activity on acetylthrombin was dependent on the molecular weights of the inhibitors, Thromstop, I-2581, hirudin, and antithrombin-III. When acetylthrombin (5000 units/kg body weight) was infused into rabbits, the activated partial thromboplastin time was prolonged to the same extent as that following infusion of thrombin (125 units/kg), but the fibrinogen level was not decreased in contrast to the large decrease following infusion of thrombin. It is suggested that acetylthrombin activates protein C without clotting fibrinogen in vivo.

The authors have studied HLA class I polymorphism in an attempt to identify a genomic marker of the hemochromatosis gene. Five enzymes were used (HindIII, EcoRI, EcoRV, PvuII, and HincII) in 47-106 unrelated patients and 71-91 controls. Both populations were HLA-typed. The relationship between the restriction fragments and HLA-A antigens was considered. Comparison of the two groups revealed a significantly high frequency of one 14 kb EcoRI fragment among the HLA-A3 hemochromatosis patients. The fragment hybridizes to an HLA-A specific probe but not to an HLA-B specific probe, and is associated with the hemochromatosis gene, which is itself associated with HLA-A3.

The present study examines the role of antithrombin III (ATIII) and heparin in the inactivation of thrombin on rabbit aortae in vitro. Thrombin on the endothelium was functionally assayed with a synthetic chromogenic substrate (thrombins) or with fibrinogen (thrombinf), measured as the liberation of fibrinopeptide A. After incubation with thrombin, residual amounts were found on the endothelium. Surface-bound thrombinf constituted approximately one-half of surface-bound thrombins. Displacement of thrombin activity from the thrombin loaded endothelial surface was achieved with Polybrene. The thrombin could be quantitatively recovered in the Polybrene solution and equal amounts of thrombins and thrombinf were found. Incubation of endothelial segments with ATIII resulted in only a minimal uptake of ATIII on the surface but in a significantly increased capacity to inactivate thrombin, whereas incubation of the segments with heparin alone had the opposite effect. The effect of ATIII could be inhibited by prior incubation with Polybrene or by a subsequent incubation with heparin. It is concluded that the vessel wall content of ATIII is one determinant for the capacity to inhibit thrombin and that ATIII is bound to glycosaminoglycans on the vessel wall since it could be displaced by heparin.

Six patients with advanced renal cell carcinoma was treated with a new chemo-endocrine regimen consisting of Tegafur, Adriamycin, Methotrexate and Tamoxifen. Estrogen receptor was measured in four cases from renal or metastatic tumors by DCC method, presenting 14.7, 9.7, 1.0 and 0 f moles/mg protein respectively. The patients were medicated with 800-1,200 mg of Tegafur and 20 mg of Tamoxifen daily po, and 20 mg of Adriamycin and 10 mg of Methotrexate intermittently for two weeks interval iv. According to a criteria of Japan Society for Cancer Therapy, two were regarded as CR, one as PR, one as NC and two as PD. The one out of two cases with and without estrogen receptor responded favourably to this therapy. Side effects observed in the treatment were mild gastrointestinal disorders including nausea and vomiting, slight degree of leukopenia, stomatitis, pigmentation and liver dysfunction. The patients were found to be in good quality of life during the treatment because of less toxicity. This therapy can be regarded as a good modality for a treatment of advanced renal cell carcinoma. This is a first report of combined chemo-endocrine therapy with Tegafur, Adriamycin, Methotrexate and Tamoxifen for renal cell carcinoma in the world.

Previous studies investigated the effect of heparin fractions on the rates of thrombin inhibition by naturally occurring antithrombin III (ATIII) isoforms differing in affinity for heparin. Heparin with low-affinity for ATIII increased the rate of thrombin inhibition by the higher affinity isoform about 10-fold more effectively than by the other isoform. This paper reports on the effect of a series of high-affinity heparin fractions with decreasing affinity for ATIII. As affinity decreased, the ability of the heparin fractions to increase the rate of the ATIII-thrombin reactions decreased, and these fractions slightly more effectively increased the rate of thrombin inhibition by the higher-affinity ATIII isoform. The effect of the heparin fractions on the ATIII-factor Xa reactions was also investigated. The activity of the fractions in this reaction also showed a dependence on ATIII-affinity. Studies on the competition of isoforms for immobilized heparin showed that the isoform with higher affinity for ATIII effectively competes with its congener for binding to heparin. The results indicate that heterogeneity in high-affinity heparin results in heterogeneity in affinity for ATIII that is significantly correlated with the ability of the heparin to potentiate ATIII-protease reactions. In spite of about equal activation of the ATIII isoforms by high-affinity heparin, the importance of the higher-affinity isoform is indicated by its ability to compete effectively for these heparin species.

A cyclic hexapeptide cyclo(Lys-Gly-Asp-Gln-Leu-Ser-) 10 was synthesized stepwise in solution by acylation of peptide ester trifluoroacetates directly with preactivated Boc-amino acids using the DCC/HOBt method; the final cyclization reaction was performed using the pentafluorophenyl ester method in solution (1-4). This peptide is a cyclic derivative of murine tumor necrosis factor-(127-132) and is designed as a potential antitumor agent. The cyclic peptide 10 displayed weak cytotoxic activity on three of the four human tumor cell lines tested.

Thrombin has been reported to elicit two temporally different effects on neutrophil-endothelial interaction, categorically described as 'neutrophil adhesion': one expressed within a few minutes, the other after several hours of endothelial preincubation. Prolonged activation resulted in often elongated, tightly interacting neutrophils in contact with human umbilical vein endothelial cells (HUVE) mainly at the intercellular region. In contrast, the neutrophil-endothelial interaction due to short-time priming with thrombin was dominated by round, randomly distributed neutrophils, loosely adhering on the endothelial surface. Sheer stress, introduced to characterize these morphological entities in a quantitative manner, clearly defined the neutrophil response due to short time priming as sheer stresslabile compared to the sheer stress-resistant interaction expressed after prolonged preincubation of HUVE with thrombin. Quantitative determination of neutrophil transendothelial migration served to demonstrate the relevance of such differences in neutrophil binding with respect to subsequent layer penetration. Dependent upon endothelial protein synthesis and inhibited by antithrombin-III-heparin and hirudin, neutrophil layer penetration only occurred after prolonged activation of HUVE whereas the neutrophil-endothelial interaction due to short time priming with thrombin was limited to superficial neutrophil adhesion. We conclude that thrombin can be added to the list of activators capable of inducing the neutrophil passage-guiding principle, an effect that has recently been detected as closely related to neutrophil adhesion to endothelial ligands expressed upon activation with interleukin-1 and tumor necrosis factor. This biological activity, which can be separated from superficial attachment of polymorphonuclear leukocytes (PMN) to endothelial cells, is a catalytic site-dependent and late phase-specific effect of thrombin on HUVE.

The expression of the gene for the C3 polypeptide is confined to the ventral prostate of the male rat and is regulated by androgens. To study the mechanism of this tissue-specific and hormone-dependent regulation we have used DNase-I footprinting and band-shift assays to locate binding sites for nuclear proteins isolated from different tissues. In this paper we present evidence that there are tissue-specific differences in the nuclear proteins that are able to bind to a CCAAT motif and a nuclear factor-I consensus that are present in the promoter of the rat C3 gene. Using competition assays and heat denaturation we show that the CAAT box/enhancer binding protein itself may be one of the transcription factors involved.

Levels of oestrogen receptor (ER) and progesterone receptor (PgR) in ovarian cancer tissue were examined with regard to their prognostic importance for survival in 179 patients with primary epithelial ovarian cancer stage III or IV in relation to: FIGO-stage, histological type, histological grade, age, ascites, and postoperative residual tumour. Hormone receptor content was determined with the DCC-method, receptor values higher than 9 fmol/mg protein were considered positive. Response to postoperative chemotherapy was significantly correlated with PgR content (80% responders in the group with PgR positive tumours and only 61% responders in the group with PgR negative tumours). A Cox proportional hazards regression model identified histological grade, residual tumour, age and PgR content as independent prognostic factors for survival in advanced epithelial ovarian carcinoma. PgR content had particularly significant prognostic relevance for patients with postoperative residual tumour mass less than or equal to 2 cm in diameter. Within this group of patients, those who are PgR positive have a 2-years survival probability of 83% compared with only 51% in the PgR-negative group.