Cellular consequences of the initiation of blood coagulation.
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Animals↗
Biomedical subjects
H Prydz
Publications and source records attributed to H Prydz.
Tissue factor and biotechnology.
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Animals↗
Constitutive and interferon-gamma-induced expression of the human proteasome subunit multicatalytic endopeptidase complex-like 1.
Proteasomes generate peptides from intracellular endogenous and viral proteins for presentation by MHC class I molecules. During viral infection, interferon-gamma (IFN-gamma) acts as a cytokine altering the catalytic specificity of proteasomes by inducing the synthesis of the three proteasome subunits, low molecular weight protein (LMP) 2, LMP7 and multicatalytic endopeptidase complex-like 1 (MECL1). LMP2 and LMP7 have been shown to favour the presentation of certain antigenic peptides. These subunits are constitutively expressed in cell lines related to the immune system and IFN-gamma-inducible in other cell lines. Less is known about MECL1. To reveal the extent of constitutive and IFN-gamma-induced expression of MECL1, we studied MECL1 in different cell lines by Northern and Western blotting. The two B cell lines IM9 and Reh showed high constitutive expression of MECL1, only slightly induced by IFN-gamma stimulation. The B cell line Daudi and the monocyte cell line THP-1 expressed MECL1 constitutively at an intermediate level. The MECL1 protein level in the THP-1 cells increased markedly in response to IFN-gamma. In cells unrelated to the immune system, a very low constitutive expression of MECL1 was detected, highly inducible by IFN-gamma. These results indicate that, similar to LMP2 and LMP7, MECL1 is constitutively expressed at high levels only in certain cell lines and can be induced by IFN-gamma in other cell lines. The differential expression of MECL1 may be of importance for which antigenic peptides are presented by different cells as well as by the same cells at different IFN-gamma levels.
Adenocarcinoma↗
Extensive intimal apolipoprotein A1-derived amyloid deposits in a patient with an apolipoprotein A1 mutation.
In the aortic intima amyloid deposits are often associated with atherosclerotic plaques. In a recent study of one patient with aortic intimal amyloid the major fibril protein was an N-terminal fragment of apolipoprotein A1 (apoA1) consisting of 69 amino acid residues. In the present study, we have screened the apoA1 gene for mutations in autopsy cases with aortic intimal amyloid immunohistochemically positive for apoA1, using single stranded conformational polymorphism (SSCP) analysis and DNA sequencing. All cases except one had a normal apoA1 gene sequence. One case of exceptionally severe atherosclerosis combined with extensive intimal amyloid deposits showed an apoA1 deletion corresponding to Lys 107. Thus, wild type apoA1 is amyloidogenic but our findings suggest that the expression of a mutant apoA1-form may be associated with enhanced amyloidogenicity.
Amyloid↗
Interaction of the CNC-bZIP factor TCF11/LCR-F1/Nrf1 with MafG: binding-site selection and regulation of transcription.
We have previously shown that the widely expressed human transcription factor TCF11/LCR-F1/Nrf1 interacts with small Maf proteins and binds to a subclass of AP1-sites. Such sites are required for beta-globin 5' DNase I hypersensitive site 2 enhancer activity, erythroid porphobilinogen deaminase inducibility, hemin responsiveness by heme-oxygenase 1 and expression of the gene NAD(P)H:quinone oxidoreductase1. Here we report the optimal DNA-binding sequences for TCF11/LCR-F1/Nrf1 alone and as a heterodimer with MafG, identified by using binding-site selection. The heterodimer recognises a 5'-TGCTgaGTCAT-3' binding-site that is identical to the established NF-E2-site, the antioxidant response element and the heme-responsive element while the binding specificity of the homomer is less stringent. To investigate the activity of TCF11 through this selected site, both alone and in the presence of MafG, we have used a transient transfection assay. TCF11 alone activates transcription while MafG alone acts as a repressor. When co-expressed, MafG interferes with TCF11 transactivation in a dose dependent manner. This indicates that MafG protein, which heterodimerises efficiently with TCF11 in vitro (the heterodimer having a higher affinity for DNA than TCF11 alone), does not co-operate with TCF11 in transactivating transcription. We propose that since both these factors are widely expressed, they may act together to contribute to the negative regulation of this specific target site. Efficient positive regulation by TCF11 may require alternative partners with perhaps more restricted expression patterns.
Base Sequence↗
Hammerhead ribozyme design and application.
The emerging knowledge about RNA-based enzymes has already had great impact on our concept of evolutionary history, making the 'RNA world' more likely. It may well have an equally important impact on the diagnostic and therapeutic practices of human and veterinary medicine in the next decade. We are not quite there yet. This review addresses the design and application of hammerhead ribozymes, two aspects of a conserved and most commonly studied and used enzymatically active entity among the RNA enzymes. The emerging picture is one of great diversity. There is at this stage no general cell model nor a clearly preferable ribozyme structure. Each and every cell line (and tissue) may be unique in that they vary with respect to structural requirements for optimal uptake, activity and stability of ribozymes. We may have seen only the tip of the iceberg when it comes to RNA-based enzymes and their roles in biology and medicine.
Catalysis↗
Characterization of a factor VII molecule carrying a mutation in the second epidermal growth factor-like domain.
A missense mutation at codon 100 in the second epidermal growth factor-like domain, resulting in Gln100-->Arg, was detected in 19 out of 21 available severely factor VII (FVII) deficient patients in Norway. Seventeen patients were homozygous, and the two remaining were compound heterozygotes. In the homozygous patients, FVII antigen was measured to 10-28%, and activity to 0.6-6.5% of that in normal pooled plasma. Recombinant FVII containing the mutation was expressed transiently in CHO cells to a mean antigen level of 57% of the wild type FVII protein, and with a specific activity of 6% of wild type. The mutant protein had a 14-fold reduction in affinity for tissue factor (TF), whereas binding of FX seemed unaffected. In line with the experimental data, molecular modelling of the mutation based on the coordinates of the tissue factor/FVIIa complex showed that substituting arginine for glutamine disrupts the interface between the catalytic and second epidermal growth factor-like domains.
Adolescent↗
[Cloning of animals and humans].
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Animals↗
A novel human chymotrypsin-like digestive enzyme.
The gene of a novel chymotrypsin-like serine protease has been cloned from human pancreas. The chymotrypsin-like enzyme-1 gene is located on chromosome 16q22.1 in a tight cluster with four unrelated genes. The gene has seven exons with the signal and activation peptide and the three main catalytic residues forming the active site encoded by separate exons. Northern blots of pancreatic mRNA showed a major transcript of 1.0 kilobases and a minor transcript of 1.3 kilobases due to alternative polyadenylation. No transcript was found in other tissues. Its presence in pancreatic tissue, duodenal juice, and urine was demonstrated with antisera raised against synthetic peptides from the derived amino acid sequence of the gene. The peptide sequences were chosen for being most dissimilar to chymotrypsin, and the antisera obtained did not react with purified human chymotrypsin. The proteolytically active CTRL-1 has been identified in pancreatic homogenate, duodenal juice, and urine, and a recombinant CTRL-1 has been characterized. Increased pancreatic secretion of CTRL-1 was induced by protease inhibitors indicating that the enzyme is secreted from pancreas upon feedback stimulation. Both native and recombinant CTRL-1 displayed chymotrypsin- and elastase-2-like activities and hydrolyzed the amide bonds of substrates having tyrosine, phenylalanine, or leucine residues at the P1 position. The enzyme was active over a broad pH range (6.5-9. 0), with a maximum at pH 8.0-8.5. CTRL-1 was produced as a zymogen of 264 amino acids as deduced from the gene sequence, with a sequence identity of 54% with human chymotrypsin B. The number and location of intron/exon junctions as well as the sequence similarity to chymotrypsin both at the DNA and protein level and the presence in duodenal juice indicate that this is a novel digestive enzyme of the chymotrypsin superfamily, albeit one with distinct physiological and biochemical features.
Amino Acid Sequence↗
Sodium butyrate inhibits the expression of the human lecithin: cholesterol acyltransferase gene in HepG2 cells by a post-transcriptional mechanism.
We have previously demonstrated that LCAT is downregulated by TGF-beta and that the regulation is post-transcriptional and involves an increased rate of RNA degradation. Sodium butyrate affects the expression of several liver-specific genes including some whose levels are altered during an acute-phase response. We have investigated the effect of sodium butyrate on LCAT activity and mRNA levels in HepG2 cells. Both the LCAT mRNA level and activity were reduced in a dose- and time-dependent manner. The reduction of LCAT mRNA levels was not, however, due to an increased degradation of processed mRNA. The transcriptional activity of the LCAT gene as seen in run-on experiments was not affected by sodium butyrate, whereas the total level of LCAT transcripts was reduced. Thus, LCAT activity and mRNA level in HepG2 cells are decreased by sodium butyrate treatment by a post-transcriptional mechanism, most likely involving increased degradation of pre-mRNA.
Butyrates↗
Synchronized Ca2+ oscillations induced in Madin Darby canine kidney cells by bradykinin and thrombin but not by ATP.
In an earlier report, we described synchronous Ca2+ oscillations in globally stimulated, subconfluent MDCK cells [Røttingen J-A, Enden T., Camerer E., Iversen J-G., Prydz H. Binding of human factor VIIa to tissue factor induces cytosolic Ca2+ signals in J82 cells, transfected COS-1 cells, Madin-Darby canine kidney cells and in human endothelial cells induced to synthesize tissue factor. J Biol Chem 1995; 270: 4650-4660]. In order to elucidate the mechanisms behind these oscillations, we have analyzed the fluctuations in cytosolic Ca2+ in single, Fura-2 loaded, MDCK cells grown to subconfluence, after stimulation with bradykinin, thrombin and ATP. All three agonists gave rise to an initial Ca2+ spike followed by oscillations or transients. Both the initial and subsequent spikes appeared to be due mainly to release of Ca2+ from internal stores, since they remained after Ca2+ influx was impeded by either La3+ or by chelation of extracellular Ca2+ with EGTA. The secondary spikes were apparently synchronized when the cells were (permanently and globally) stimulated with bradykinin or thrombin, but each cell seemed to oscillate independently when stimulated in the same way with ATP. Synchronized secondary spikes arose with a constant frequency and amplitude, independent of agonist concentration in contrast to most Ca2+ oscillations observed. Pretreatment of the cells with octanol to block gap junctions, or with EGTA or La3+ to inhibit Ca2+ influx, abolished the synchronization induced by bradykinin or thrombin. We observed that in the MDCK cell layer there are some "pacemaker' cells and hypothesize that these have a higher sensitivity for the agonists than their neighboring cells. From these pacemakers, an intercellular Ca2+ wave can be seen to spread to adjacent cells in the presence of intact gap junctions, thereby initiating concurrent transients in all cells. The Ca2+ wave is amplified by release from internal stores, probably owing to the bell-shaped Ca2+ activation curve of the IP3 receptor and by subsequent Ca2+ influx through Ca2+ release activated channels.
Adenosine Triphosphate↗
Mice overexpressing human lecithin: cholesterol acyltransferase are not protected against diet-induced atherosclerosis.
Lecithin: cholesterol acyltransferase (LCAT) (EC 2.3.1.43) is generally assumed to participate in reverse cholesterol transport, i.e., cholesterol transport from peripheral tissues to the liver. LCAT is secreted by the liver and transported in plasma mostly associated with high density lipoprotein. It catalyzes the esterification of cholesterol, mainly high density lipoprotein cholesterol, and produces cholesteryl ester and lysolecithin. Transgenic mice overexpression human LCAT on a C57BL/6 background have elevated high density lipoprotein cholesterol and markedly reduced low and very low density lipoprotein cholesterol and triglyceride levels in plasma, suggesting that such mice may be less susceptible to diet-induced atherosclerosis than isogenic nontransgenic controls. To determine if the apparent anti-atherogenic lipoprotein profile of the LCAT transgenics reduced their susceptibility to atherogenesis, the atherosclerotic lesions developing in transgenic LCAT mice and controls when fed an atherogenic diet were compared by histology and morphometry. Histological examination of the aortas from mice fed a high fat diet for 12, 17 and 22 weeks revealed that the aortic lesions were no smaller or less developed in the transgenic LCAT mice than in the C57BL/6 controls. After 17 weeks there were significantly more "fatty streaks" in the transgenic mice than in the controls. Thus, overexpression of human LCAT in transgenic mice, in spite of their very favourable blood lipoprotein and lipid profile, does not protect against development of atherosclerosis.
Animals↗
Functional and clinical aspects of the myelomonocyte protein calprotectin.
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Antifungal Agents↗
The gene cluster containing the LCAT gene is conserved between human and pig.
A pooled DNA probe from P1 artificial chromosome clones (PACs) containing the human lecithin:cholesterol acyl transferase (LCAT) gene cluster was used in fluorescence in situ hybridization (FISH) experiments assigning the genes to pig chromosome 6p13. In addition, probes derived from the coding regions in the human gene cluster were used in long range mapping experiments to show that the overall structures of the human and porcine LCAT gene clusters are identical. Both the linear order and the close physical distance of five apparently unrelated genes have been maintained throughout 90 million years of divergent evolution between human and pig. The extremely dense clustering of the genes in the LCAT gene cluster suggests that this gene organization has biological significance. The conservation of the gene cluster between human and pig supports this suggestion.
Animals↗
Contribution of factor VII genotype to activated FVII levels. Differences in genotype frequencies between northern and southern European populations.
The relationship between coagulation factor VII (FVII) levels in plasma and FVII genotypes, determined by three polymorphisms (5'F7, IVS7, and 353R/Q), were studied in 500 control subjects enrolled in European multicenter study. The selection of particular FVII genotypes and the analysis of variance clearly indicated the independent contribution of a single 5'F7 insertion (A2) or 353Q (M2) allele to lowering plasma levels of activated FVII (FVIIa) (by a mean 25%). The M2 allele alone was found to make a major contribution to the genetically determined component of the FVIIa levels. Genotypes associated with low FVII levels were significantly rarer in the northern part of Europe (Oslo) than in the southern part (Rome, Murcia). The contribution made by the FVII genotype to the total variance of FVIIa levels was higher (30%) than that made to either FVII activity (25%) or FVII antigen (12%). Subjects with different FVII genotypes showed up to fivefold differences in mean FVIIa values, thus allowing attribution of a substantial part of the considerable interindividual variation to genetic variation, which may be of assistance in the interpretation of FVIIa levels on an individual basis. When FVII levels were adjusted by age and by triglyceride levels, the contribution of FVII genotypes to the FVII phenotypic variance was virtually unchanged. Taken together, these data indicate that in healthy control subjects the FVII genotype is a major predictor of plasma FVIIa levels and would support further study on the role of FVII genetic components in the development of cardiovascular disease.
Adult↗
Coagulation factors VII and X induce Ca2+ oscillations in Madin-Darby canine kidney cells only when proteolytically active.
We have recently reported that the activated serine protease and blood coagulation Factor VII (FVIIa) can induce Ca2+ oscillations in Madin-Darby canine kidney cells. We now demonstrate a similar response by Madin-Darby canine kidney cells to the active coagulation Factor X (FXa), which is also a serine protease and a substrate of the tissue factor (TF).FVIIa complex in the initiation of the coagulation cascade. The phosphatidyl inositol-specific phospholipase C inhibitor U73122 inhibited the signals elicited by both FVIIa and FXa. Lack of sensibility to the tyrosine kinase inhibitors herbimycin A, genistein, and the tyrphostin AG18 and discordance between TF expression and FVIIa responsiveness argued against TF acting as a cytokine-like receptor, with tyrosine kinase-mediated activation by FVIIa. As demonstrated using the protease inhibitor benzamidine and by specific active site inhibition with 1,5-dansyl-Glu-Gly-Arg chloromethyl ketone, both FVIIa and FXa lost their ability to elicit a calcium response when devoid of their proteolytic activity. Consistent with this, the native (zymogen) form of Factor X did not induce Ca2+ transients. Homologous but not heterologous inhibition of FVIIa- and FXa-evoked Ca2+ signals by 1,5-dansyl-Glu-Gly-Arg chloromethyl ketone-inactivated FVIIa and FXa suggested that each factor had its own specific cell surface anchoring receptor. The two coagulation factors did not show homologous desensitization as seen for thrombin stimulation. Studies with hirudin excluded involvement of the established activation pathway through thrombin itself. Lack of desensitization of the response to FVIIa or FXa by thrombin ruled out any involvement of proteinase activated receptor-1 (PAR-1), the thrombin receptor. We speculate that FXa and FVIIa may work via a receptor (possibly common) analogous to PAR-1 or its functional homologue PAR-2. Although TF is essential for the FVIIa-induced signaling event, its role in the phosphatidyl inositol-specific phospholipase C-mediated Ca2+ signal may be in anchoring FVIIa to the cell surface rather than in transmembrane signal mediation.
Animals↗
Small Maf proteins interact with the human transcription factor TCF11/Nrf1/LCR-F1.
The human TCF11 gene encodes a ubiquitously expressed bZIP transcription factor of the cap n' collar (CNC) domain family. It has a high sequence similarity to the erythroid-specific bZIP factor p45 NF-E2 in the CNC domain, which is involved in DNA binding. LCR-F1, a TCF11 isoform, is a more potent transcriptional activator than p45 NF-E2 in erythroid cells. We show here that the TCF11 protein interacts to form heterodimers with small Maf proteins, previously shown to dimerize with p45 NF-E2, ECH and Fos. Such heterodimerization significantly alters the DNA binding characteristics of TCF11. While TCF11 alone binds in vitro to the tandem NF-E2 site derived from 5' DNase hypersensitive site 2 in the beta-globin locus control region and to the single NF-E2 site in the porphobilinogen deaminase gene promoter, stronger binding is detected in the presence of small Maf proteins. Using antibodies, TCF11 isoforms bound to the single NF-E2 site were detected in K562 erythroid cell nuclear extracts. These findings place TCF11 as a good candidate for the proposed widely expressed factor(s) known to interact with small Maf proteins and bind NF-E2 sites in a sequence-specific manner resembling NF-E2.
Avian Proteins↗
Opposite sorting of tissue factor in human umbilical vein endothelial cells and Madin-Darby canine kidney epithelial cells.
Tissue factor (TF) is a 48-kD transmembrane glycoprotein that triggers the extrinsic pathway of blood coagulation by interacting with the plasma coagulation factor VII (FVII). TF is also a true receptor in that a cellular signal is generated when activated FVII (FVIIa) binds to TF. For both of these functions, the cellular surface distribution of TF is important, since FVII is primarily available on the apical side of vascular endothelial cells and on the basolateral side of epithelial cells lining the internal and external surfaces. We show that in endothelial cells, TF (both antigen and procoagulant activity) is sorted to the apical surface, whereas in wild-type and stably transfected Madin-Darby canine kidney epithelial cells (MDCK), which form tight junctions and express TF constitutively, TF antigen is on the basolateral surface. No significant clotting activity is detectable on this surface. Truncated TF (cytoplasmic tail residues 246 to 263 deleted) is sorted as wild-type in MDCK cells.
Amino Acid Sequence↗