Decreased fibrinolytic capacity has been sug-

The regulation of type 1 plasminogen activator inhibitor (PAI-1) gene expression was studied in vivo employing a murine model system. Nuclease protection analysis revealed relatively high concentrations of PAT-i mRNA in the aorta, adipose tissue, heart, and lungs of untreated CB6 (BalbC X C57B16) mice. Treatment of CB6 mice with LPS, TNF-a, or transforming growth factor-,@ (TGF-ft) increased the steady-state levels of PAM-i mRNA within 3 h in all tissues examined. However, the greatest responses to TGF-ft were observed in adipose tissue and the kidney, while LPS and TNF-a strongly stimulated PAM-i gene expression in the liver, kidney, lung, and adrenals.

In previous work, the direct injection of 50 micrograms of a plasmid DNA vector encoding firefly luciferase (VR1205) into murine quadriceps muscle produced an average of 6.5 ng of luciferase per muscle at 7 days postinjection. In this report, various elements of the VR1205 vector were modified to increase gene expression levels or to eliminate undesired viral sequences. Expression of the modified vectors was then compared to VR1205 using the intramuscular injection assay. In general, modifications to promoter, enhancer, and intronic sequences either decreased luciferase expression levels or had no effect. However, modifications to the polyadenylation and transcriptional termination sequences, plasmid backbone elements, and the luciferase gene itself each increased luciferase expression levels. The best-expressing vector, designated VR1255, contained a combination of these incrementally beneficial changes. A single intramuscular injection of 50 micrograms of VR1255 produced 300 ng of luciferase at 7 days postinjection, an expression level 46-fold higher than the VR1205 vector (or 22-fold higher, excluding modifications to the luciferase gene) and 154-fold higher than a commercially available luciferase expression vector. Thus, VR1255 represents an improved plasmid DNA vector that may be useful for gene therapy applications.

A Xgtll expression library containing cDNA inserts prepared from human placental mRNA was screened immunologically using an antibody probe developed against the ,8-migrating plasminogen activator inhibitor (p-PAI) purified from cultured bovine aortic endothelial cells. Thirty-four positive clones were isolated after screening 7 x i0-phages. Three clones (X1.2, X3, and X9.2) were randomly picked and further characterized. These contained inserts 1.9, 3.0, and 1.9 kilobases (kb) long, respectively. Escherichia coli lysogenic for A9.2, but not for Xgtll, produced a fusion protein of 180 kDa that was recognized by afflinity-purified antibodies against the bovine aortic endothelial cell fl-PAI and had fl-PAI activity when analyzed by reverse fibrin autography. The largest cDNA insert was sequenced and shown to be 2944 base pairs (bp) long. It has a large 3' untranslated region [1788 bp, excluding the poly(A) tail] and contains the entire coding region of the mature protein but lacks the initiation codon and part of the signal peptide coding region at the 5' terminus. The two clones carrying the 1.9-kb cDNA inserts were partially sequenced and shown to be identical to the 3.0-kb cDNA except that they were truncated, lacking much of the 3' untranslated region. Blot hybridization analysis of electrophoretically fractionated RNA from the human fibrosarcoma cell line HT-1080 was performed using the 3.0-kb cDNA as hybridization probe. Two distinct transcripts, 2.2 and 3.0 kb, were detected, suggesting that the 1.9-kb cDNA may have been copied from the shorter RNA transcript. The amino acid sequence deduced from the cDNA was aligned with the NH2-terminal sequence of the human fl-PAL. Based on this alignment, the mature human P-PAI is 379 amino acids long and contains an NH2-terminal valine. The deduced amino acid sequence has extensive (30%) homology with ao-antitrypsin and antithrombin III, indicating that the fl-PAI is a member of the serine proteinase inhibitor (serpin) superfamily.The generation of plasmin from plasminogen provides an important source of proteolytic activity in cells, tissues, and biological fluids (1, 2). Precise regulation of plasminogen activator (PA) activity may thus constitute a critical feature of many biological systems (3). Such control may be at the level of the formation and resolution of fibrin itself (4) To facilitate the precise biochemical characterization of P-PAI, and to eventually understand the nature of factors regulating 3-PAI gene expression, we have undertaken the molecular cloning of the gene. Here we describe the isolation of P-PAI cDNA from a human placental expression library and demonstrate that the (3-PAI is a member of the serine protease inhibitor (serpin) superfamily (23, 24).