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Query: UNIPROT:P00750 (PLA)
 16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A recombinant plasminogen activator with high fibrin affinity and specificity was expressed by transfecting hybridoma cells with a plasmid that combines sequence coding for low molecular mass (32 kDa) single-chain urokinase-type plasminogen activator [scuPA(32kDa)] and anti-fibrin monoclonal antibody 59D8. The expression of the recombinant molecule [r-scuPA(32kDa)-59D8] was optimized by replacing the 3' untranslated region (initially that of high molecular mass scuPA) in the plasmid with the 3' untranslated region of either beta-globin or mouse immunoglobulin. This modification resulted in a greater than 100-fold improvement in the level of protein expression. The 103-kDa r-scuPA(32kDa)-59D8 protein displayed catalytic activity indistinguishable from that of high molecular mass scuPA and fibrin binding comparable to that of native antibody 59D8. r-scuPA(32kDa)-59D8 was 6 times more potent than high molecular mass scuPA in lysing a human plasma clot in vitro and was 20 times more potent than high molecular mass scuPA in the rabbit jugular vein model of thrombolysis. Molecules of this type may serve as prototypes for highly specific, antibody-targeted enzymes suitable for human use.
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PMID:A recombinant chimeric plasminogen activator with high affinity for fibrin has increased thrombolytic potency in vitro and in vivo. 194 53

We report the development of a novel in vivo transcription assay for trans-acting factors regulating the human gamma- and beta-globin genes. A cDNA coding for the human tissue-type plasminogen activator (t-PA) was inserted into the globin genes. Simian virus 40 small T-antigen splice and polyadenylation signals were included to produce a mature transcript coding for t-PA, whose activity can be detected in single cells by a fibrin-agarose plaque assay. Stable murine L-cell transfectants of the gamma.t-PA and beta.t-PA hybrid genes were fused to various cell lines to show that t-PA expression is increased specifically by erythroid MEL, HEL, and K562 cell fusion. The analogous H-2Kb.t-PA construct was not inducible under the same conditions. Interestingly, uninduced MEL cells increased beta.t-PA expression to the same extent as induced MEL cells. Chemiosmotic permeabilization of the beta-globin tester cell line and exposure to nuclear extracts were used to assay for trans-acting factors capable of stimulating beta.t-PA expression. Such factors were shown to be present in the nuclei of uninduced MEL cells.
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PMID:A novel in vivo transcription assay demonstrates the presence of globin-inducing trans-acting factors in uninduced murine erythroleukemia cells. 312 20

Development of a CHO cell-based production system for the hybrid plasminogen activator K2tu-PA is described. Using the major immediate-early promoter of mouse cytomegalovirus (MCMV) transient and stable expression levels were 3-10-fold higher than those obtained with several other strong promoters. Splicing and polyadenylation signals from the rabbit beta-globin gene were used downstream of the DNA segment coding for K2tu-PA. The strong enhancer moiety of the MCMV promoter also stimulated strongly the promoter of the dihydrofolate reductase (DHFR) gene, placed adjacently for selection/gene amplification purposes. One construct with opposing K2tu-PA and DHFR RNA transcripts yielded the highest expression level with a single copy of the plasmid, but K2tu-PA expression was consistently lost after amplification of such genes, possibly as a result of the formation of antisense RNA. With other constructs, K2tu-PA production leveled off at 6.5 micrograms per million cells per day despite a high gene copy number. This was due to a combination of inefficient mRNA translation and mRNA instability, caused by elements from the untranslated portions of tissue-type and urokinase-type plasminogen activator cDNA which were included in the expression vector. After elimination of these inhibitory DNA segments, 4-5-times higher expression levels were reached.
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PMID:Production of the chimerical plasminogen activator K2tu-PA in CHO cells. 757 41

This two-plasmid system for transient gene expression can be used in a wide variety of primate cells. It consists of a cDNA expression vector and a helper plasmid. The cDNA cloned in the expression vector is transcribed by the powerful major immediate-early promoter of murine cytomegalovirus. A segment of the rabbit beta-globin gene placed downstream of the cDNA provides signals for splicing and polyadenylation. The helper plasmid provides SV40 T antigen and adenovirus VA RNA. The T antigen induces replication of the expression vector, which contains an SV40 origin of replication, and VA RNA enhances translation of the transcribed mRNA. In monkey kidney cells, with human tissue-type plasminogen activator (t-PA) cDNA as reporter gene, the helper plasmid boosted t-PA production 30-fold and up to 500 ng/ml t-PA accumulated in the medium in the 5 days following transfection of the two plasmids. In nonprimate cells the helper plasmid stimulated expression 3- to 5-fold.
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PMID:A two-plasmid system for transient expression of cDNAs in primate cells. 838 92

We show that the mouse gamma 2b heavy chain or human beta-globin 3' untranslated region can greatly enhance protein expression in myeloma cells transfected by genes coding for antibody-plasminogen activator fusion proteins. Expression plasmids were constructed containing a cloned genomic heavy chain variable region from fibrin-specific monoclonal antibody 59D8, a cloned genomic constant region of the mouse gamma 2b heavy chain, and DNA sequence coding for either tissue-type plasminogen activator (tPA) or a segment of urokinase (UK) and their respective 3' untranslated sequences. Cell lines transfected with these constructs, pSVtPA (tPA) and pSVUKG(UK), produced extremely low levels of mRNA and protein (0.008-0.06 micrograms/ml) in comparison with the parental 59D8 myeloma cell line (7.6-10 micrograms/ml). In vitro nuclear run-off analysis indicated that the low steady-state levels of mRNA encoded by pSVUKG(UK) did not result from a lower rate of transcription of the transfected gene (relative to the rate of transcription of the endogenous heavy chain gene in the 59D8 parent cells). In an attempt to increase protein secretion, we assembled the expression plasmids pSVtPA(Ig), pSVUKG(Ig), and pSVUKG(beta), in which the 3' untranslated region of the mouse gamma 2b heavy chain or human beta-globin gene was substituted for the 3' untranslated region of the plasminogen activator gene. Analysis of supernatant media from cell lines transfected with these constructs showed an increase in recombinant protein secretion of 68 to 100 fold in comparison with that from cell lines transfected with pSVtPA(tPA) or pSVUKG(UK).
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PMID:High-level expression of antibody-plasminogen activator fusion proteins in hybridoma cells. 844 52

Type-1 plasminogen activator-inhibitor (PAI-1) is a major physiologic inhibitor of plasminogen activation. Incubation of HTC rat hepatoma cells with the cyclic nucleotide analogue, 8-bromo-cAMP, causes a dramatic increase in tissue-type plasminogen activator activity secondary to a 90% decrease in PAI-1 mRNA. Although 8-bromo-cAMP causes a modest decrease in PAI-1 transcription, regulation is primarily the result of a 3-fold increase in the rate of PAI-1 mRNA degradation. To determine the cis-acting sequences required for cyclic nucleotide regulation, we have stably transfected HTC cells with chimeric genes containing sequences from the rat PAI-1 cDNA and the mouse beta-globin gene and examined the effect of cyclic nucleotides on the decay rate of these transcripts. The mRNA transcribed from the beta-globin gene is stable and not cyclic nucleotide-regulated, whereas the transcript from a construct containing the beta-globin coding region and the PAI-1 3'-untranslated region (UTR) is destabilized in the presence of 8-bromo-cAMP, suggesting that this response is mediated by sequences in the PAI-1 3'-UTR. Analyses by deletion of sequences from this chimeric construct indicate that, whereas more than one region of the PAI-1 3'-UTR can confer cyclic nucleotide responsiveness, the 3'-most 134-nucleotide sequence alone is sufficient to do so. Insertion of PAI-1 sequences within the beta-globin 3'-UTR confirms that the 3'-most 134 nucleotides of PAI-1 mRNA can confer cyclic nucleotide regulation of stability on a heterologous transcript, suggesting that this sequence may play a major role in hormonal regulation of PAI-1 mRNA stability.
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PMID:Cyclic nucleotide regulation of type-1 plasminogen activator-inhibitor mRNA stability in rat hepatoma cells. Identification of cis-acting sequences. 960 32

Ancient septicemic plague epidemics were reported to have killed millions of people for 2 millenniums. However, confident diagnosis of ancient septicemia solely on the basis of historical clinical observations is not possible. The lack of suitable infected material has prevented direct demonstration of ancient septicemia; thus, the history of most infections such as plague remains hypothetical. The durability of dental pulp, together with its natural sterility, makes it a suitable material on which to base such research. We hypothesized that it would be a lasting refuge for Yersinia pestis, the plague agent. DNA extracts were made from the dental pulp of 12 unerupted teeth extracted from skeletons excavated from 16th and 18th century French graves of persons thought to have died of plague ("plague teeth") and from 7 ancient negative control teeth. PCRs incorporating ancient DNA extracts and primers specific for the human beta-globin gene demonstrated the absence of inhibitors in these preparations. The incorporation of primers specific for Y. pestis rpoB (the RNA polymerase beta-subunit-encoding gene) and the recognized virulence-associated pla (the plasminogen activator-encoding gene) repeatedly yielded products that had a nucleotide sequence indistinguishable from that of modern day isolates of the bacterium. The specific pla sequence was obtained from 6 of 12 plague skeleton teeth but 0 of 7 negative controls (P < 0.034, Fisher exact test). A nucleic acid-based confirmation of ancient plague was achieved for historically identified victims, and we have confirmed the presence of the disease at the end of 16th century in France. Dental pulp is an attractive target in the quest to determine the etiology of septicemic illnesses detected in ancient corpses. Molecular techniques could be applied to this material to resolve historical outbreaks.
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PMID:Detection of 400-year-old Yersinia pestis DNA in human dental pulp: an approach to the diagnosis of ancient septicemia. 977 May 38

Incubation of HTC rat hepatoma cells with the cyclic nucleotide analogue 8-bromo-cAMP results in a 3-fold increase in the rate of degradation of type-1 plasminogen activator-inhibitor (PAI-1) mRNA. Previous studies utilizing HTC cells stably transfected with beta-globin:PAI-1 chimeric constructs demonstrated that at least two regions within the PAI-1 3'-untranslated region mediate the cyclic nucleotide-induced destabilization of PAI-1 mRNA; one of these regions is the 3'-most 134 nucleotides (nt) of the PAI-1 mRNA (Heaton, J. H., Tillmann-Bogush, M., Leff, N. S., and Gelehrter, T. D. (1998) J. Biol. Chem. 273, 14261-14268). In the present study, ultraviolet cross-linking analyses of this region demonstrate HTC cell cytosolic mRNA-binding proteins ranging from 38 to 76 kDa, with a major complex migrating at approximately 50 kDa. RNA electrophoretic mobility shift analyses demonstrate high molecular weight multiprotein complexes that specifically interact with the 134-nt cyclic nucleotide-responsive sequence. The 50, 61, and 76 kDa and multiprotein complexes form with an A-rich sequence at the 3' end of the cyclic nucleotide-responsive region; a 38-kDa complex forms with a U-rich region at the 5' end of the 134 nt sequence. Mutation of the A-rich region prevents both the binding of the 50-, 61-, and 76-kDa proteins and formation of the multiprotein complexes, as well as cyclic nucleotide-regulated degradation of chimeric globin:PAI-1 transcripts in HTC cells. These data suggest that the proteins identified in this report play an important role in the cyclic nucleotide regulation of PAI-1 mRNA stability.
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PMID:Cyclic nucleotide regulation of PAI-1 mRNA stability. Identification of cytosolic proteins that interact with an a-rich sequence. 987 66