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W Kisiel

Publications and source records attributed to W Kisiel.

At least 37 records · Page 2Linked to original sources
PubMed2000

Sesquiterpenoids from roots of Taraxacum laevigatum and Taraxacum disseminatum.

Chromatographic separation of ethanolic root extracts of Taraxacum laevigatum and Taraxacum disseminatum afforded a total of eight germacrane- and eudesmane-type sesquiterpenoids. including new compounds, 1beta,3beta,6alpha-trihydroxy-4alpha( 15)-dihydrocostic acid methyl ester and its 1-O-beta-glucopyranoside. Their structures were established by spectroscopic analyses. In addition, the structure of 4alpha(15), 11beta(13)-tetrahydroridentin B-1-O-beta-glucopyranoside was elucidated by extensive NMR studies.

Asteraceae↗
PubMed2000

Hancokinol from Vincetoxicum officinale.

Hancokinol (1), a member of migrated lupane triterpenoids, was isolated from the aerial parts of Vincetoxicum officinale, along with known plant sterols and long chain alkanols.

Humans↗
PubMed2000

Further sesquiterpenoids and phenolics from Taraxacum officinale.

Five germacrane- and guaiane-type sesquiterpene lactones, including two previously described taraxinic acid derivatives, were isolated from the roots of Taraxacum officinale, together with benzyl glucoside, dihydroconiferin, syringin and dihydrosyringin. The other three lactones were identified as 11beta, 13-dihydrolactucin, ixerin D and ainslioside. Moreover, the stereochemistry at C-11 in dihydrotaraxinic acid was assigned.

Asteraceae↗
PubMed2000

Tissue factor expression and angiogenesis in human prostate carcinoma.

In tumors, the switch to the angiogenic phenotype is thought to be controlled by a balance of positive and negative angiogenic factors. Tissue factor (TF) produced by tumor cells has been implicated in the regulation of this "angiogenic switch" through its ability to concurrently induce the expression of angiogenic molecules such as vascular endothelial cell growth factor (VEGF), while inhibiting the expression of anti-angiogenic molecules such as thrombospondin 2. We have examined TF expression and its relationship to angiogenesis and tumor progression in human prostate carcinomas. Most of the prostate carcinoma specimens examined (73%; n = 67) express high levels of TF. Immunohistochemical analysis localized TF expression to the epithelial cells of malignant glands. TF expression was significantly correlated with tumor angiogenesis as measured by the microvessel density (MVD). In addition, TF expression was correlated with the preoperative PSA level, a strong predictor of recurrence in prostate carcinomas. Our findings show that TF expression by the malignant glands in prostate cancer is common and suggest a role for this molecule in regulating prostate cancer progression and angiogenesis.

Adenocarcinoma↗
PubMed2000

Pulmonary expression of early growth response-1: biphasic time course and effect of oxygen concentration.

Hypoxia induces complex adaptive responses. In this report, induction of early growth response-1 (Egr-1) transcripts in lungs of mice subjected to hypoxia is shown to be dose and time dependent. Within 30 min of hypoxia, Egr-1 transcripts were approximately 20-fold elevated in 6% oxygen, approximately 5.2-fold increased by 10% oxygen, and returned to the normoxic baseline by 12% oxygen. Time course studies up to 48 h showed a biphasic profile with an initial steep rise in Egr-1 transcripts after 0.5 h of hypoxia and a second elevation beginning after 20-24 h. Hypoxic induction of Egr-1 was paralleled by enhanced expression of the downstream target gene tissue factor. Egr-1 and tissue factor antigen were visualized in bronchial and vascular smooth muscle and in alveolar macrophages. Egr-1 has the capacity to modulate expression of genes involved in the remodeling of the extracellular matrix and properties of smooth muscle, thus possibly contributing to the pulmonary response to chronic hypoxia.

Animals↗
PubMed2000

Nucleotide sequence of the gene encoding murine tissue factor pathway inhibitor-2.

Tissue factor pathway inhibitor-2 (TFPI-2), also known as placental protein 5, is a 32 kDa extracellular matrix-associated serine proteinase inhibitor consisting of three tandemly-arranged Kunitz-type domains. Two overlapping genomic clones containing sequences encoding murine TFPI-2 were isolated from a lambda FIXII 129 SVJ mouse genomic library, and the complete nucleotide sequence of the gene was determined. The murine TFPI-2 gene spans approximately 9.3 kilobases and consists of five exons and four introns. The nucleotide sequences surrounding all the exon-intron boundaries are highly conserved and obey the GT-AG rule. Each Kunitz-type domain is encoded by a single exon, similar to that observed for other Kunitz-type proteinase inhibitors. A total of 1,577 bp of the 3'-flanking region contains a probable polyadenylation site (ATTAAA) at +5,759 and an apparent cleavage or termination site (CATTG) at +6,170. The 5'-flanking region of the murine TFPI-2 gene contains a prototypical TATA box, a GC box and two CAAT boxes. In addition, several candidate transcription factor binding sites responsible for placenta-, endothelial cell-, and smooth muscle cell-specific expression of the TFPI-2 gene were also identified.

Amino Acid Sequence↗
PubMed1999

Inhibition of neutrophil elastase by recombinant human proteinase inhibitor 9.

Proteinase inhibitor PI9 (PI9) is an intracellular 42-kDa member of the ovalbumin family of serpins that is found primarily in placenta, lung and lymphocytes. PI9 has been shown to be a fast-acting inhibitor of granzyme B in vitro, presumably through the utilization of Glu(340) as the P(1) inhibitory residue in its reactive site loop. In this report, we describe the inhibition of human neutrophil elastase by recombinant human PI9. Inhibition occurred with an overall K(i)' of 221 pM and a second-order association rate constant of 1.5 x 10(5) M(-1) s(-1), indicating that PI9 is a potent inhibitor of this serine proteinase in vitro. In addition, incubation of recombinant PI9 with native neutrophil elastase resulted in the formation of an SDS-resistant 62-kDa complex. Amino-terminal sequence analyses provided evidence that inhibition of elastase occurred through the use of Cys(342) as the reactive P(1) amino acid residue in the PI9 reactive site loop. Thus, PI9 joins its close relatives PI6 and PI8 as having the ability to utilize multiple reactive site loop residues as the inhibitory P(1) residue to expand its inhibitory spectrum.

Binding Sites↗
PubMed1999

Caspase-1 (interleukin-1beta-converting enzyme) is inhibited by the human serpin analogue proteinase inhibitor 9.

The regulation of caspases, cysteine proteinases that cleave their substrates after aspartic residues, is poorly understood, even though they are involved in tightly regulated cellular processes. The recently discovered serpin analogue proteinase inhibitor 9 (PI9) is unique among human serpin analogues in that it has an acidic residue in the putative specificity-determining position of the reactive-site loop. We measured the ability of PI9 to inhibit the amidolytic activity of several caspases. The hydrolysis of peptide substrates by caspase-1 (interleukin-1beta-converting enzyme), caspase-4 and caspase-8 is inhibited by PI9 in a time-dependent manner. The rate of reaction of caspase-1 with PI9, as well as the rate of substrate hydrolysis of the initial caspase-PI9 complex, shows a hyperbolic dependence on the concentration of PI9, indicative of a two-step kinetic mechanism for inhibition with an apparent second-order rate constant of 7x10(2) M(-1).s(-1). The hydrolysis of a tetrapeptide substrate by caspase-3 is not inhibited by PI9. The complexes of caspase-1 and caspase-4 with PI9 can be immunoprecipitated but no complex with caspase-3 can be detected. No complex can be immunoprecipitated if the active site of the caspase is blocked with a covalent inhibitor. These results show that PI9 is an inhibitor of caspase-1 and to a smaller extent caspase-4 and caspase-8, but not of the more distantly related caspase-3. PI9 is the first example of a human serpin analogue that inhibits members of this class of cysteine proteinases.

Caspase 3↗
PubMed1999

Targeted inhibition of intrinsic coagulation limits cerebral injury in stroke without increasing intracerebral hemorrhage.

Agents that restore vascular patency in stroke also increase the risk of intracerebral hemorrhage (ICH). As Factor IXa is a key intermediary in the intrinsic pathway of coagulation, targeted inhibition of Factor IXa-dependent coagulation might inhibit microvascular thrombosis in stroke without impairing extrinsic hemostatic mechanisms that limit ICH. A competitive inhibitor of native Factor IXa for assembly into the intrinsic Factor X activation complex, Factor IXai, was prepared by covalent modification of the Factor IXa active site. In a modified cephalin clotting time assay, in vivo administration of Factor IXai caused a dose-dependent increase in time to clot formation (3.6-fold increase at the 300 micrograms/kg dose compared with vehicle-treated control animals, P < 0.05). Mice given Factor IXai and subjected to middle cerebral artery occlusion and reperfusion demonstrated reduced microvascular fibrin accumulation by immunoblotting and immunostaining, reduced 111In-labeled platelet deposition (42% decrease, P < 0.05), increased cerebral perfusion (2.6-fold increase in ipsilateral blood flow by laser doppler, P < 0.05), and smaller cerebral infarcts than vehicle-treated controls (70% reduction, P < 0.05) based on triphenyl tetrazolium chloride staining of serial cerebral sections. At therapeutically effective doses, Factor IXai was not associated with increased ICH, as opposed to tissue plasminogen activator (tPA) or heparin, both of which significantly increased ICH. Factor IXai was cerebroprotective even when given after the onset of stroke, indicating that microvascular thrombosis continues to evolve (and may be inhibited) even after primary occlusion of a major cerebrovascular tributary.

Animals↗
PubMed1999

Inhibitory properties of human recombinant Arg24-->Gln type-2 tissue factor pathway inhibitor (R24Q TFPI-2).

Human type-2 tissue factor pathway inhibitor (TFPI-2), also known as placental protein 5, is a 32-kDa serine proteinase inhibitor consisting of three tandemly arranged Kunitz-type domains homologous to tissue factor pathway inhibitor. TFPI-2 inhibits a variety of serine proteinases involved in coagulation and fibrinolysis through an arginine residue (R24) in its first Kunitz-type domain, which constitutes a putative P1 residue for the substrate recognition sites of these proteinases. As recent studies have shown that this P1 residue to be a glutamine in murine TFPI-2, we constructed, expressed, and purified a human TFPI-2 mutant with glutamine substituted for arginine at position 24 (R24Q TFPI-2). R24Q TFPI-2 lost approximately 90% of its inhibitory activity towards bovine trypsin and virtually all inhibitory activity towards human plasmin and the factor VIIa-tissue factor complex, emphasizing the importance of the P1 Arg24 residue in the inhibition of these serine proteinases. However, whereas wild-type TFPI-2 is a relatively weak inhibitor of human factor Xa amidolytic activity (IC50 approximately 1 microM), R24Q TFPI-2 exhibited enhanced inhibitory activity towards the amidolytic and coagulant activities of this proteinase with a Ki of 18 nM. While the molecular basis for the enhanced inhibition of human factor Xa by R24Q TFPI-2 is unknown, these data provide suggestive evidence that murine TFPI-2 may function as a serine proteinase inhibitor in spite of the absence of a P1 Arg or Lys residue.

Amino Acid Sequence↗
PubMed1999

Hypoxia/Hypoxemia-Induced activation of the procoagulant pathways and the pathogenesis of ischemia-associated thrombosis.

Although oxygen deprivation has long been associated with triggering of the procoagulant pathway and venous thrombosis, blood hypoxemia and stasis by themselves do not lead to fibrin formation. A pathway is outlined through which diminished levels of oxygen activate the transcription factor early growth response-1 (Egr-1) leading to de novo transcription/translation of tissue factor in mononuclear phagocytes and smooth muscle cells, which eventuates in vascular fibrin deposition. The procoagulant response is magnified by concomitant suppression of fibrinolysis by hypoxia-mediated upregulation of plasminogen activator inhibitor-1. These data add a new facet to the biology of thrombosis associated with hypoxemia/stasis and imply that interference with mechanisms causing Egr-1 activation in response to oxygen deprivation might prevent vascular fibrin deposition occurring in ischemia without directly interfering with other pro/anticoagulant pathways.

Animals↗
PubMed1999

Expression of tissue factor and tissue factor pathway inhibitor in situ in laryngeal carcinoma.

The blood coagulation mechanism may support tumor progression by several mechanisms including promotion of cell proliferation and angiogenesis. Immunohistochemical procedures were applied to AMeX-fixed sections of twelve cases of squamous cell carcinoma of the larynx obtained at surgical resection to determine the presence and distribution of tissue factor (TF), tissue factor pathway inhibitor (TFPI), other coagulation factors, fibrinogen, and fibrin in situ. TF antigen was present in normal squamous epithelial cells and tumor cells, predominantly in immature tumor cells in the vicinity of the host-tumor interface. Tumor cells stained also for factors VII and X. Staining for TFPI antigen was demonstrated in the connective tissue stroma adjacent to the tumor, in microvascular endothelial cells, and in normal squamous epithelial cells. Fibrinogen and factor XIIIa were distributed throughout the tumor connective tissue stroma. Fibrin (thrombin-cleaved fibrinogen) was detected at the host-tumor interface and along the margins of tumor nodules. Tumor cells in carcinoma of the larynx express a functional, TF-initiated pathway of blood coagulation. Interpretation of these findings together with the results of clinical trials of inhibitors of TF-induced coagulation activation versus effects of inhibitors of TF expression suggest novel approaches to the experimental therapy of laryngeal carcinoma.

Blood Coagulation↗
PubMed1999

Synthesis and expression of tissue factor pathway inhibitor by serum-stimulated fibroblasts, vascular smooth muscle cells and cardiac myocytes.

Tissue factor pathway inhibitor (TFPI) plays an important role in regulating tissue factor (TF)-initiated blood coagulation. Since serum stimulation of fibroblasts, vascular smooth muscle cells and cardiac myocytes in culture increases the expression of TF mRNA and antigen (Ag) in these cells, we hypothesized that serum may also induce increased synthesis of TFPI in these cells to regulate the TF-induced extravascular clotting at an injury site. To test this concept, we used primary isolates of the following human cell types - fetal and adult lung fibroblasts, pulmonary and aortic smooth muscle cells, and cardiac myocytes. Serum-stimulation of these cells resulted in an increased expression of TF mRNA and Ag (8 to 10-fold). Upon serum stimulation, expression of TFPI mRNA and Ag was also increased in these cells. However, the increase in TFPI-Ag (6 to 8-fold) was significantly greater than the TFPI mRNA (2 to 3-fold). Notably, increased expression of TFPI persisted after the TF expression had declined. Further, increased synthesis of TFPI initially led to the saturation of heparin-releasable binding sites. TFPI-Ag was detected by Western blotting, 35S-metabolic labeling and activity assays on the conditioned media, heparin-released material from cells, and in cell lysates. TFPI-Ag was also detected by immunofluorescence staining of cells. Actinomycin D partially whereas cycloheximide completely prevented the serum-induced increased expression of TFPI synthesis by these cells, suggesting control primarily at the translational but some at the transcriptional level as well. The Mr of undegraded TFPI in all cases was approximately 45 kDa and was of full length. TFPI synthesized locally by fibroblasts, vascular smooth muscle cells and cardiac myocytes could play a significant role in regulating TF-initiated extravascular clotting especially since plasma TFPI that may be available at the injury site lacks a portion of the carboxyl segment and is a less efficient inhibitor.

Adult↗
PubMed1998

Tissue factor transcription driven by Egr-1 is a critical mechanism of murine pulmonary fibrin deposition in hypoxia.

Local hypoxemia and stasis trigger thrombosis. We have demonstrated previously that in a murine model of normobaric hypoxia pulmonary fibrin deposition is a result of expression of tissue factor, especially in oxygen-deprived mononuclear phagocytes (MPs). We now show that transcription factor early-growth-response gene product (Egr-1) is rapidly activated in hypoxia, both in vitro and in vivo, and is responsible for transcription and expression of tissue factor in hypoxic lung. MPs and HeLa cells subjected to hypoxia (pO2 approximately 13 torr) had increased levels of tissue factor transcripts (approximately 18-fold) and an increased rate of transcription (approximately 15-fold), based on nuclear run-on analysis. Gel-shift analysis of nuclear extracts from hypoxic MPs and HeLa cells demonstrated increased DNA-binding activity at the serum response region (SRR; -111/+14 bp) of the tissue factor promoter at Egr-1 motifs. Using 32P-labeled Egr consensus oligonucleotide, we observed induction of DNA-binding activity in nuclear extracts from hypoxic lung and HeLa cells because of activation of Egr-1, by means of supershift analysis. Transient transfection of HeLa cells with chimeric plasmids containing wild-type or mutant SRR from the tissue factor promoter showed that intact Sp1 sites are necessary for basal promoter activity, whereas the integrity of Egr-1 sites was required for hypoxia-enhanced expression. A central role for Egr-1 in hypoxia-mediated tissue factor expression was confirmed by experiments with homozygous Egr-1 null mice; wild-type mice subjected to oxygen deprivation expressed tissue factor and showed fibrin deposition, but hypoxic homozygous Egr-1 null mice displayed neither tissue factor nor fibrin. These data delineate a novel biology for hypoxia-induced fibrin deposition, in which oxygen deprivation-induced activation of Egr-1, resulting in expression of tissue factor, has an unexpected and central role.

Animals↗
PubMed1998

HT-1080 fibrosarcoma cell matrix degradation and invasion are inhibited by the matrix-associated serine protease inhibitor TFPI-2/33 kDa MSPI.

The urokinase-urokinase receptor system plays a dominant role in the degradation and invasion of extracellular matrix (ECM) by tumor cells. In this system, urokinase bound to its cell receptor converts plasminogen to plasmin, a broad-spectrum serine protease that participates in the degradation and invasion of connective tissues by tumor cells. In this study, we evaluated whether these activities of plasmin are inhibited by a newly characterized human 32 kDa recombinant serine protease inhibitor called trypsin/tissue factor pathway inhibitor-2 (rTFPI-2). We found that rTFPI-2 dose-dependently inhibited fluid-phase plasmin as well as plasmin generated on the ECM and/or the cell surface of HT-1080 fibrosarcoma cells. The degradation of radiolabeled matrix as well as Matrigel invasion by these tumor cells is also inhibited by rTFPI-2 in a dose-dependent fashion. We have reported that rTFPI-2 is identical to 33 kDa extracellular matrix-associated serine protease inhibitor (33 kDa MSPI), whereas the 31 and 27 kDa MSPIs are under-glycosylated forms of the 33 kDa MSPI. We therefore evaluated the ability of MSPIs from the ECM of dermal fibroblasts to inhibit plasmin and found that the plasmin activity was effectively blocked by the MSPIs. We have also evaluated whether the HT-1080 cells synthesize and secrete the MSPIs and found that the synthesis and secretion of the MSPIs was undetectable in these cells. Collectively, our results suggest that rTFPI-2/33 kDa MSPI inhibits plasmin on the tumor cell and ECM surfaces as well as the degradation and invasion of matrix by HT-1080 fibrosarcoma cells.

Antineoplastic Agents↗
PubMed1998

Functional consequences of mutations in Ser-52 and Ser-60 in human blood coagulation factor VII.

Human blood coagulation factor VII has unique carbohydrate moieties O-glycosidically linked to serine 52 and serine 60 residues in its first epidermal growth factor-like domain. To study the functional role of these glycosyl moieties in factor VII, we constructed, expressed, and purified site-specific recombinant mutants of human factor VII in which serine 52 and serine 60 were conservatively replaced with alanine residues. S52A factor VIIa (Ser-52-->Ala), S60A factor VIIa (Ser-60-->Ala), and S52,60A factor VIIa (Ser-52, Ser-60-->Ala) exhibited 56, 73, and 44%, respectively, of the clotting activity of wild-type factor VIIa using human brain thromboplastin as a source of tissue factor/phospholipids and 32, 43, and 14% of wild-type factor VIIa using a mixture of recombinant soluble tissue factor and mixed brain phospholipids. The tissue factor-dependent and -independent amidolytic activities of these mutants were essentially indistinguishable from that of wild-type factor VIIa. In addition, equilibrium dialysis experiments indicated that the profiles of 45Ca2+ binding to these mutants were identical with that of wild-type factor VII. In the presence of either Ca2+ or EGTA, the Kd values for the interaction of the three factor VIIa mutants to full-length tissue factor were 2- to 5-fold higher than that of wild-type factor VIIa, while the Kd values for the interaction of these mutants to soluble tissue factor were 4- to 15-fold higher than that of wild-type factor VIIa. Measurement of the association and dissociation rate constants for factor VIIa binding to relipidated tissue factor apoprotein revealed that the association rate constants of the three factor VII mutants were decreased in comparison with that of wild-type factor VIIa, while the dissociation rate constants of these three mutants were virtually identical to that of wild-type factor VIIa. These findings strongly suggest that glycosyl moieties attached to Ser-52 and Ser-60 in factor VII/VIIa provide unique structural elements that are important for the rapid association of factor VII/VIIa with its cellular receptor and cofactor.

Blood Coagulation↗
PubMed1998

Manipulation of the membrane binding site of vitamin K-dependent proteins: enhanced biological function of human factor VII.

Recent studies suggested that modification of the membrane contact site of vitamin K-dependent proteins may enhance the membrane affinity and function of members of this protein family. The properties of a factor VII mutant, factor VII-Q10E32, relative to wild-type factor VII (VII, containing P10K32), have been compared. Membrane affinity of VII-Q10E32 was about 20-fold higher than that of wild-type factor VII. The rate of autoactivation VII-Q10E32 with soluble tissue factor was 100-fold faster than wild-type VII and its rate of activation by factor Xa was 30 times greater than that of wild-type factor VII. When combined with soluble tissue factor and phospholipid, activated factor VII-Q10E32 displayed increased activation of factor X. Its coagulant activity was enhanced in all types of plasma and with all sources of tissue factor tested. This difference in activity (maximum 50-fold) was greatest when coagulation conditions were minimal, such as limiting levels of tissue factor and/or phospholipid. Because of its enhanced activity, factor VII-Q10E32 and its derivatives may provide important reagents for research and may be more effective in treatment of bleeding and/or clotting disorders.

Animals↗