Cystic fibrosis is associated with a defect in epithelial chloride ion transport which is caused by mutations in a membrane protein called CFTR (cystic fibrosis transmembrane conductance regulator). Heterologous expression of CFTR produces cyclicAMP-sensitive Cl(-)-channel activity. Deletion of phenylalanine at amino-acid position 508 in CFTR (delta F508 CFTR) is the most common mutation in cystic fibrosis. It has been proposed that this mutation prevents glycoprotein maturation and its transport to its normal cellular location. We have expressed both CFTR and delta F508 CFTR in Vero cells using recombinant vaccinia virus. Although far less delta F508 CFTR reached the plasma membrane than normal CFTR, sufficient delta F508 CFTR was expressed at the plasma membrane to permit functional analysis. delta F508 CFTR expression induced a reduced activity of the cAMP-activated Cl- channel, with conductance, anion selectivity and open-time kinetics similar to those of CFTR, but with much greater closed times, resulting in a large decrease of open probability. The delta F508 mutation thus seems to have two major consequences, an abnormal translocation of the CFTR protein which limits membrane insertion, and an abnormal function in mediating Cl- transport.

The respiratory epithelium is a potential site for somatic gene therapy for the common hereditary disorders alpha 1-antitrypsin (alpha 1AT) deficiency and cystic fibrosis. A replication-deficient adenoviral vector (Ad-alpha 1AT) containing an adenovirus major late promoter and a recombinant human alpha 1AT gene was used to infect epithelial cells of the cotton rat respiratory tract in vitro and in vivo. Freshly isolated tracheobronchial epithelial cells infected with Ad-alpha 1AT contained human alpha 1AT messenger RNA transcripts and synthesized and secreted human alpha 1AT. After in vivo intratracheal administration of Ad-alpha 1AT to these rats, human alpha 1AT messenger RNA was observed in the respiratory epithelium, human alpha 1AT was synthesized and secreted by lung tissue, and human alpha 1AT was detected in the epithelial lining fluid for at least 1 week.

The scrine protease a-thrombin (thrombin) potently stimulates G-protein-coupled signaling pathways and DNA synthesis in CCL39 hamster lung Abroblasts. To clone a thrombin receptor cDNA, selcctivc amplification of mRNA sequences displaying homology to the transmembrane domains of G-protein-coupled receptor genes was performed by polymerasc chain reaction. Using rcverae transcribed poly(A)+ RNA from CCL39 cells and degenerate primers corresponding to conserved regions of several phospholipase C-coupled receptors, three novel putative receptor sequences were identified. One corresponds to an mRNA transcript of 3.4 kb in CCL39 cells and a relatively abundant cDNA. Microinjection of RNA transcribed in vitro from this cDNA in Xenopus oocytcs leads to the expression of a functional thrombin receptor. The hamster thrornbin receptor consists of 427 amino acid residues with 8 hydrophobic domains, in&ding one at the extreme N-terminus that is likely to represent a signal peptide. A thrombin consensus cleavage site is present in the N-terminal extracellular region of the receptor squcnce followed by a negatively charged cluster of residues present in a number of proteins that interact with the anion-binding exositc of thrombin.u-Thrombin receptor; G-protein; Phospholipase C; Polymerase chain reaction; Oocyte expression; Hamster ftbroblast

The predicted protein domains coded by exons 9–12 and 19–23 of the 27 exon cystic fibrosis transmembrane conductance regulator (CFTR) gene contain two putative nucleotide‐binding fold regions. Analysis of CFTR mRNA transcripts in freshly isolated bronchial epithelium from 12 normal adult individuals demonstrated that all had some CFTR mRNA transcripts with exon 9 completely deleted (exon 9‐ mRNA transcripts). In most (9 of 12), the exon 9‐ transcripts represented less than or equal to 25% of the total CFTR transcripts. However, in three individuals, the exon 9‐ transcripts were more abundant, comprising 39, 62 and 66% of all CFTR transcripts. Re‐evaluation of the same individuals 2–4 months later showed the same proportions of exon 9‐ transcripts. Of the 24 CFTR alleles in the 12 individuals, the sequences of the exon‐intron junctions relevant to exon 9 deletion (exon 8‐intron 8, intron 8‐exon 9, exon 9‐intron 9, and intron 9‐exon 10) were identical except for the intron 8‐exon 9 region sequences. Several individuals had varying lengths of a TG repeat in the region between splice branch and splice acceptor consensus sites. Interestingly, one allele in each of the two individuals with 62 and 66% exon 9‐ transcripts had a TT deletion in the splice acceptor site for exon 9. These observations suggest either the unlikely possibility that sequences in exon 9 are not critical for the functioning of the CFTR or that only a minority of the CFTR mRNA transcripts need to contain exon 9 sequences to produce sufficient amounts of a normal CFTR to maintain a normal clinical phenotype.

Current concepts relating to the pathogenesis of emphysema associated with cigarette smoking is that an imbalance exists within the lower respiratory tract between neutrophil elastase and the local anti-neutrophil elastase screen, enabling uninhibited neutrophil elastase to destroy the alveolar structures over time. The possible role of alveolar macrophages in contributing to this imbalance was investigated by evaluating the ability of cigarette smokers' alveolar macrophages to inactivate alpha 1-antitrypsin (alAT), the major anti-neutrophil elastase of the human lower respiratory tract. In vitro, alveolar macrophages of smokers spontaneously released 2.5-fold more superoxide anion and eightfold more H202 than macrophages of nonsmokers (P < 0.01, both comparisons). Using a model system that reproduced the relative amounts of alveolar macrophages and aIAT found in the epithelial lining fluid of the lower respiratory tract, we observed that smokers' macrophages caused a 60±5% reduction in the ability of alAT to inhibit neutrophil elastase. In marked contrast, under the same conditions, nonsmokers' macrophages had no effect upon the anti-neutrophil elastase function of alAT. Addition of superoxide dismutase, catalase, mannitol, and methionine prevented inactivation of alAT by smokers' macrophages, implying that the release of oxidants mediated the inactivation of alAT. In addition, by utilizing a recombinant DNA produced modified form of alAT containing an active site substitution (met" -* val), the inactivation of alAT by smokers' alveolar macrophages was prevented, suggesting that the smokers' macrophages inactivate alAT by oxidizing the active site of the alAT molecule. These results suggest that in cigarette smokers, the alveolar macrophage can modulate the activity of alAT as an inhibitor of neutrophil elastase and thus play a role in the pathogenesis of emphysema associated with cigarette smoking.

The primary function of alpha 1-antitrypsin (alpha 1-AT), an antiprotease produced by the liver, is the inhibition of neutrophil elastase, a protease capable of hydrolysing most connective tissue components. The importance of alpha 1-AT is demonstrated by the high incidence of early-onset emphysema in individuals with hereditary alpha 1-AT deficiency (Type PiZZ), in whom serum levels of alpha 1-AT are 10-20% of normal. Oxidants in tobacco smoke can inactivate alpha 1-AT in vitro, and studies have shown that alpha 1-AT from the lungs of individuals who smoke cigarettes may also be partially inactivated, perhaps explaining the high incidence of emphysema associated with cigarette smoking. Oxidative inactivation is probably due to modification of the Met residue (Met358) at the P1 subsite position of the elastase binding site of the protein. To study the possibility of modulating the biological properties of alpha 1-AT, we have introduced selected sequence modifications at the reactive site by in vitro mutation of a cloned alpha 1-AT complementary DNA. We describe here the characterization of two alpha 1-AT analogues produced in Escherichia coli. The first, alpha 1-AT(Met385----Val), is not only fully active as an elastase inhibitor but is also resistant to oxidative inactivation. The other, alpha 1-AT(Met358----Arg), no longer inhibits elastase but is an efficient thrombin inhibitor. The active site of the latter is identical to that of the alpha 1-AT (Pittsburgh) variant, which was associated with a fatal bleeding disorder.

Transgenic mice were generated in which 5 kb of the 5′ flanking promoter region of the human Factor IX (FIX) gene fused to various FIX constructs (gene, minigene and cDNA) were stably integrated in the germ line. Several transgenic mouse lines expressed high circulating levels of active and correctly processed recombinant human FIX. The presence of at least one FIX intron had a positive effect on the expression. The FIX transgenes were expressed in a tissue‐specific manner in the liver of transgenic mice. By crossing transgenic mice synthesizing FIX with others prone to develop hepatoma, progeny which co‐express the transgenes in hepatocytes were obtained. Hepatoma‐derived cell lines were shown to have a differentiated phenotype and secrete active human FIX for many generations.

In normal plasma, the serine protease inhibitor a,-antitrypsin (al-AT) plays little or no role in the control of plasma kallikrein or activated Factor XII fragment (Factor XIIf), this function being performed by Cl-inhibitor. Recently, an al-AT variant was described with a Met -Arg mutation at the reactive center PI residue (position 358) which altered the specificity of inhibition from the Met-or Val-specific protease neutrophil elastase to thrombin, an Arg-specific protease. We have now examined the inhibition of plasma kallikrein and Factor XIIf, both Arg-specific enzymes, with recombinant a1-AT(Mete--p Arg) produced by an Escherichia coli strain carrying a mutated human a,-AT gene.The engineered protein was a very efficient inhibitor of both enzymes. It was more effective than Cl-inhibitor by a factor of 4.1 for kallikrein and 11.5 for Factor XIIf. These results suggest that recombinant a1-AT(Met3m -Arg) has therapeutic potential for disease states where activation of the plasma kinin-forming system is observed, for example in hereditary angioedema or septic shock.