Secretory leukocyte protease inhibitor, but not alpha-1 protease inhibitor, blocks tryptase-induced bronchoconstriction

Alpha-1-protease inhibitor (alpha(1)-PI) and secretory leukocyte protease inhibitor (SLPI) are two natural airway serine protease inhibitors. While inhibition of neutrophil elastase is a function common to both alpha(1)-PI and SLPI, we showed previously that they exhibit different patterns of protection against antigen-induced changes in airway function in allergic sheep. Specifically, the protective effect seen with SLPI was similar to the profile of action of synthetic tryptase inhibitors in the model. Based on these data, and the fact that tryptase is a serine protease, we hypothesized that SLPI, but not alpha(1)-PI, would block tryptase-induced bronchoconstriction. To test this, we compared the responses to inhaled tryptase in five sheep without treatment or after treatment with either aerosol alpha(1)-PI (10 mg) or aerosol SLPI (50 mg). The doses of alpha(1)-PI and SLPI selected had been shown to be effective in previous antigen-provocation studies. Treatments were given 30 min before aerosol challenge with tryptase (500 ng). Tryptase alone increased (mean+/-SEM) pulmonary resistance (R(L)) 142 +/- 24% over baseline. Pretreatment with alpha(1)-PI had no effect on the tryptase response (R(L)increased 122 +/- 20%). Pretreatment with SLPI, however, blocked the tryptase-induced response (R(L) increased only 40 +/- 4% P<0.05 vs. tryptase). These are the first studies comparing the inhibitory activity of SLPI and alpha(1)-PI on inhaled tryptase-induced bronchoconstriction. We conclude that, in vivo, SLPI, but not alpha(1)-PI, can block tryptase-induced bronchoconstriction and that this activity may explain the differential effects of these two serine protease inhibitors on antigen-induced airway responses in allergic sheep. Copyright Academic Press.     

Unusual evolutionary conservation and frequent DNA segment exchange in class I genes of the major histocompatibility complex.

From comparisons of homologous DNA sequences for many different genes, it was shown that the silent positions of protein-encoding regions and introns evolve at high and remarkably similar rates for different genes. In addition, both silent positions and introns behave like clocks; they accumulated base substitutions at approximately constant rates with respect to geological time. The rates of evolution were estimated to be 5.5 X 10(-9), 3.7 X 10(-9), and 5.3 X 10(-9) per site per year for silent positions, short introns (less than approximately equal to 300 base pairs), and long introns (more than approximately equal to 500 base pairs), respectively. Contrary to expectation from the evolutionary clocks, DNA sequence comparison between pHLA 12.4 (a cloned HLA sequence) of man and Ld together with other H-2 genes of mouse, the class I genes of the major histocompatibility complex, revealed a surprisingly small amount of base substitution for both the introns and the silent positions; the degree of divergence is only about 60% of that of standard genes in the same species comparison. Furthermore, several segmental homologies have been observed between the class I genes of mouse, suggesting the frequent occurrence of gene conversion or double unequal crossing-over in evolution. Interrelations between the extreme polymorphism of the class I genes, the low evolutionary drift of the introns and the silent positions, and the frequent gene conversion or unequal crossing-over within the mouse genes are discussed.     

Nucleotide sequences of immunoglobulin epsilon genes of chimpanzee and orangutan: DNA molecular clock and hominoid evolution.

To determine the phylogenetic relationships among hominoids and the dates of their divergence, the complete nucleotide sequences of the constant region of the immunoglobulin epsilon-chain (C epsilon 1) genes from chimpanzee and orangutan have been determined. These sequences were compared with the human epsilon-chain constant-region sequence. A molecular clock (silent molecular clock), measured by the degree of sequence divergence at the synonymous (silent) positions of protein-encoding regions, was introduced for the present study. From the comparison of nucleotide sequences of alpha1-antitrypsin and beta- and delta-globin genes between humans and Old World monkeys, the silent molecular clock was calibrated: the mean evolutionary rate of silent substitution was determined to be 1.56 X 10(-9) substitutions per site per year. Using the silent molecular clock, the mean divergence dates of chimpanzee and orangutan from the human lineage were estimated as 6.4 +/- 2.6 million years and 17.3 +/- 4.5 million years, respectively. It was also shown that the evolutionary rate of primate genes is considerably slower than those of other mammalian genes.     

Naturally occurring sequence polymorphisms within HIV type 1 group O protease.

Mutations within the protease gene associated with reduced susceptibility to protease inhibitors have been well documented for HIV-1 group M subtype B strains. In contrast, limited genotypic and phenotypic information is available for the genetically diverse HIV-1 group O strains. Preexisting resistance-associated polymorphisms have the potential to contribute to a poor virological response to antiviral drug treatment in group O-infected patients. In the present study, the protease genes of 28 protease inhibitor-naive HIV-1 group O-infected patients were analyzed to identify any naturally occurring amino acid polymorphisms associated with drug resistance. Comparison of the consensus group O protease sequence with subtype B of group M indicated that both groups have almost identical sequences in the protease active site, the flap and the substrate-binding site. Analysis of the 28 individual protease sequences revealed polymorphisms at 34% of the positions within the protease gene, but no primary mutations associated with protease inhibitor resistance. In contrast, each of the strains harbored multiple secondary or accessory mutations associated with resistance to protease inhibitors in group M viruses. Residues 10I, 15V, 36I, 41K, 62V, 63T/A/K/I, 64V, 71V, and 93L were identified in most strains. The presence of multiple natural sequence polymorphisms associated with drug resistance in the protease gene of group O viruses may contribute to a more rapid emergence of drug resistance phenotype and treatment failure in group O-infected patients.     

The orangutan adult alpha-globin gene locus: duplicated functional genes and a newly detected member of the primate alpha-globin gene family.

We have cloned and sequenced the complete alpha 1- and alpha 2-globin genes of the orangutan, and here we compare them to the homologous genes of the human. The pattern of similarity apparent among the genes is most consistent with a model of gene correction operating on the primate alpha-globin cluster. This correction breaks down in both human and orangutan in the 3'-untranslated region at 14 base pairs downstream from the termination codon. The unit evolutionary period values calculated for either the replacement substitution or the silent substitutions are only slightly higher than the previously established molecular clock predicts. The 7-base-pair insertion in intron 2 of the human alpha 1-globin gene is not present in either orangutan gene, suggesting that this insertion is not the cause of the sequence divergence in the 3'-untranslated regions of primate alpha 2- and alpha 1-globin genes. Finally, blotting hybridization and partial DNA sequencing reveal a newly detected member of the primate alpha-globin gene family, which is located downstream from the duplicated adult alpha-globin genes.     

Identification of genes from pattern formation, tyrosine kinase, and potassium channel families by DNA amplification.

The study of gene family members has been aided by the isolation of related genes on the basis of DNA homology. We have adapted the polymerase chain reaction to screen animal genomes very rapidly and reliably for likely gene family members. Using conserved amino acid sequences to design degenerate oligonucleotide primers, we have shown that the genome of the nematode Caenorhabditis elegans contains sequences homologous to many Drosophila genes involved in pattern formation, including the segment polarity gene wingless (vertebrate int-1), and homeobox sequences characteristic of the Antennapedia, engrailed, and paired families. In addition, we have used this method to show that C. elegans contains at least five different sequences homologous to genes in the tyrosine kinase family. Lastly, we have isolated six potassium channel sequences from humans, a result that validates the utility of the method with large genomes and suggests that human potassium channel gene diversity may be extensive.     

Yield improvement for manufacture of alpha-proteinase inhibitor

BACKGROUND AND OBJECTIVES: The aim of this study was to increase the yield of active alpha(1)-proteinase inhibitor (alpha(1)-PI) from Cohn fraction IV-1 paste during the manufacture of this therapeutic protein and to investigate the molecular mechanism for this yield increase. MATERIALS AND METHODS: Dissolution experiments with IV-1 paste investigated the impact of different variables on the yield of alpha(1)-PI activity. Solutions of IV-1 paste prepared under different conditions were assayed for evidence of protease activity by Western blots of alpha(1)-PI following SDS-PAGE, by azocaseinolytic and amidolytic (S-2288) assays, and by zymography, and for the extent of alpha(1)-PI oligomerization by Western blots following nondenaturing PAGE. RESULTS: Minor modification of the manufacturing process by combining dissolution of IV-1 paste with the subsequent pH adjustment (to 9.25-9.50 with NaOH), achieved by addition of a standard concentration of NaOH to the 10-mm Tris base dissolvent for IV-1 paste, was found to give a highly reproducible 9.4 +/- 0.9% increase in yield of active alpha(1)-PI. Solutions of IV-1 paste prepared with this combined dissolvent contained reduced amounts of low molecular weight fragments of alpha(1)-PI, reduced protease activity, and reduced amounts of oligomers of alpha(1)-PI. Addition of the protease inhibitor leupeptin to the 10-mm Tris base dissolvent for IV-1 paste also caused an increase in the yield of alpha(1)-PI activity. CONCLUSIONS: Dissolution of IV-1 paste in a more alkaline medium gave a significant increase in the yield of active alpha(1)-PI. This yield increase was attributed to a reduction both in protease activity and in the extent of oligomerization of alpha(1)-PI.     

Serum protease inhibitor capacity for elastase and the severity of pancreatitis.

To clarify the relationship between the diminution of the serum protease inhibitor capacity and the severity of pancreatitis, the binding capacity of serum protease inhibitors for exogenous elastase 1 (E1) was investigated by gel filtration, the elastase activity of the alpha 2-macroglobulin (alpha 2-M)-elastase complex was measured, and the relationship between these findings and the severity of pancreatitis was studied in 13 patients with pancreatic disease and 6 healthy subjects. When 125I-labeled E1 was added to the sera of healthy subjects, it bound to alpha 2-M and alpha 1-protease inhibitor (alpha 1-PI) with a mean ratio of 72:28. In mild acute pancreatitis (n = 5), the binding capacity of alpha 2-M was less than that in healthy subjects. In severe pancreatitis (n = 4), most of the exogenous E1 bound to alpha 1-PI (alpha 2-M vs. alpha 1-PI, 13:87). This diminution in the binding capacity of alpha 2-M correlated well with the severity of acute pancreatitis. In the sera of patients (n = 4) with pancreatic cancer containing much immunoreactive E1, the proportion of exogenous E1 bound by alpha 2-M and alpha 1-PI (25:75) was similar to that seen in severe acute pancreatitis. A significant inverse relationship between the binding capacity of alpha 2-M and the activity of the endogenous elastase bound to alpha 2-M was seen in various pancreatic diseases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence and evolution of the human T-cell antigen receptor beta-chain genes.

We present the nucleotide sequences of the two genomic constant (C)-region gene segments, C beta 1 and C beta 2, encoding the beta chain of the human T-cell antigen receptor. The two C beta genes are organized identically to each other and to the corresponding mouse genes, both having four exons, whose boundaries were confirmed from the sequence of a C beta 2 cDNA clone from the T-cell line MOLT-4. The predicted amino acid sequences of human C beta 1 and C beta 2 differ at only five positions, which suggests that the proteins have very similar functions. This similarity is the result of strong nucleotide-sequence conservation in protein-coding regions, which extends to silent positions. A quantitative analysis of an alignment of the nucleotide sequences of the two human genes shows that whereas the 5' ends (including the first exon) are extremely homologous, the 3' ends are widely divergent, with other regions having intermediate levels of homology. Analysis of published data [Gascoigne, N.R.J., Chien, Y., Becker, D.M., Kavaler, J. & Davis, M.M. (1984) Nature (London) 310, 387-391] shows that the mouse C beta 1 and C beta 2 genes are also virtually identical in their first exons but more divergent in the remaining coding regions. Therefore, partial gene conversion events may have occurred during the evolution of both human and mouse C beta genes.     

Effect of Recombinant Human DNase on a1-Proteinase Inhibitor Function: An Experimental Approach to the Combined Clinical Use of rhDNase and a1-PI in CF Patients

Chronic inflammation in cystic fibrosis (CF) airways leads to high concentrations of deoxyribonucleic acid (DNA) and neutrophil elastase (NE). Both play a major role in CF lung pathophysiology and are aims of new therapeutic approaches: rhDNase degrades highly viscosic DNA and alpha1-proteinase inhibitor (alpha1-PI) inhibits NE activity and thereby pulmonary inflammation and hypersecretion. Given the reports on increased sputum NE concentrations upon rhDNase inhalation, there is a rationale for a combined rhDNase/alpha1-PI treatment. With the question of whether a combined therapy is feasible, we first investigated in vitro whether incubation of CF sputum with rhDNase changes proteolytic and secretagogue activity of sputum supernatants and its inhibition by alpha1-PI. Next, we studied whether incubation of alpha1-PI with rhDNase impairs the inhibitory effect of alpha1-PI on proteolytic activity of NE and the inhibitory effect of alpha1-PI on NE-induced secretion from a human mucoepidermoid cell line. Incubation of CF sputum with rhDNase led to a twofold increase in sputum NE activity. Correspondingly, the inhibitory effect of alpha1-PI on sputum NE activity and on secretion induced by these sputum samples was significantly reduced by rhDNase. Preincubation of alpha1-PI with rhDNase significantly reduced the inhibitory effect of alpha1-PI on purified NE activity and on NE-induced secretion. However, this effect was limited to alpha1-PI concentrations lower than those achievable after inhalation. Therefore, impairment of alpha1-PI function by rhDNase is not likely to be relevant in vivo, provided that a sufficient dosage of alpha1-PI is inhaled.     

Information transfer between duplicated chromosomal sequences in mammalian cells involves contiguous regions of DNA.

We have investigated the nature of information transfer that appears to occur nonreciprocally between duplicated chromosomal sequences in cultured mouse L cells. We have studied gene conversion between two different defective thymidine kinase genes derived from two closely related strains of type 1 herpes simplex virus and that share a silent restriction site polymorphism. Our results demonstrate that this silent site can be coconverted along with the selected mutant sites. The findings are consistent with a mechanism of gene conversion that involves contiguous blocks of DNA differing in length, position, or both. An additional finding is that the products of coconversion events involving the silent site are unequally recovered although the rates of conversion observed at four different selected sites are similar.     

Synthesis and secretion of alpha 2-plasmin inhibitor by established human liver cell lines.

The site of synthesis of alpha 2-plasmin inhibitor (alpha 2-PI), a physiologic inhibitor of plasmin, is not known with certainty. We have studied the production and secretion of alpha 2-PI by three established human liver cell lines derived from hepatocellular carcinoma and hepatoblastoma (Hep G2, Hep 3B, and PLC/PRF/5). As measured by a specific radioimmunoassay, the titer of alpha 2-PI increased in the medium of Hep G2 and Hep 3B cells with time, but no significant amount of alpha 2-PI was found in the medium of PLC/PRF/5. There was no evidence for a significant intracellular pool of this protein. On immunodiffusion against anti-alpha 2-PI serum, alpha 2-PI secreted by Hep G2 (G2 alpha 2-PI) formed a simple precipitin line of complete identity with the alpha 2-PI present in plasma (plasma alpha 2-PI). G2 alpha 2-PI behaved similarly to plasma alpha 2-PI in Sephadex G-150 gel filtration, sucrose density-gradient centrifugation, and crossed immunoelectrophoresis. G2 alpha 2-PI inhibited plasmin activity instantaneously in a functional assay and formed a complex with plasmin demonstrable by crossed immunoelectrophoresis. De novo synthesis of alpha 2-PI was shown by the presence of specific immunoprecipitable radioactivity in the medium after 5 hr of labeling of the cells with [35S]methionine. Analysis of the immunoprecipitates by NaDodSO4/polyacrylamide gel electrophoresis showed a single peak of radioactivity corresponding to Mr 68,000. These results indicate that the liver is a site of alpha 2-PI production.     

Neurexin III alpha: extensive alternative splicing generates membrane-bound and soluble forms.

The structure of neurexin III alpha was elucidated from overlapping cDNA clones. Neurexin III alpha is highly homologous to neurexins I alpha and II alpha and shares with them a distinctive domain structure that resembles a cell surface receptor. cDNA cloning and PCR experiments revealed alternative splicing at four positions in the mRNA for neurexin III alpha. Alternative splicing was previously observed at the same positions in either neurexin I alpha or neurexin II alpha or both, suggesting that the three neurexins are subject to extensive alternative splicing. This results in hundreds of different neurexins with variations in small sequences at similar positions in the proteins. The most extensive alternative splicing of neurexin III alpha was detected at its C-terminal site, which exhibits a minimum of 12 variants. Some of the alternatively spliced sequences at this position contain in-frame stop codons, suggesting the synthesis of secreted proteins. None of the sequences of the other splice sites in this or the other two neurexins include stop codons. RNA blot analysis demonstrate that neurexin III alpha is expressed in a brain-specific pattern. Our results suggest that the neurexins constitute a large family of polymorphic cell surface proteins that includes secreted variants, indicating a possible role as signaling molecules.     

The two yeast histone H2A genes encode similar protein subtypes.

The sequences of the two histones H2A genes in the yeast Saccharomyces cerevisiae have been determined. These genes encode two histone H2A subtypes which are 131 amino acids in length but differ at 2 amino acid positions: an Ala leads to Thr and a Thr leads to Ala change at positions 124 and 125. Thus, the two histone H2A subtypes have identical amino acid compositions. The coding regions of the two H2A genes are homologous at 369 of 393 bases (94%), with all but 2 of the 24 changes being silent. There is only 30% homology in the 5' flanking sequences of the two H2A genes. Like other eukaryotic histone genes, the yeast H2A genes are not interrupted by intervening sequences. When the yeast H2A histones are compared to those from other eukaryotes, there is at least 80% homology in amino acid sequence.     

Serum antiproteases in smokers and nonsmokers. Relationships to smoking status and pulmonary function

In this study of 50 relatively young male smokers and an equal number of age- and race-matched male nonsmokers, smokers had a 13.3% (p = 0.007) increase in mean serum alpha 1-proteinase inhibitor (alpha 1-Pl) concentration. This increase in serum alpha 1-Pl concentration was accompanied by increases in both the serum trypsin inhibitory capacity (TIC) (9.9%, p = 0.002) and the elastase inhibitory capacity (EIC) (12.4%, p = 0.001). That cigarette smoking increases serum alpha 1-Pl concentration and total protease inhibitory capacity was further supported by a significant association of alpha 1-PI, TIC, and EIC with increased pack-years smoking history, plasma nicotine, and plasma cotinine concentrations. Pulmonary function did not correlate with serum alpha 1-PI concentration. However, higher serum TIC and EIC did correlate with tests of small airways dysfunction. Highly significant correlations (r greater than or equal to 0.6, p = 0.001) were observed between TIC (or EIC) and alpha 1-PI concentrations. The linear relationships between TIC (or EIC) and serum alpha 1-PI concentration were not significantly different in smokers and nonsmokers. Further, no significant differential effect of smoking on either the TIC or EIC could be demonstrated. A decreased apparent functional activity of alpha 1-PI (i.e., nanomoles of protease inhibited per nanomole of alpha 1-PI) was associated with its higher serum concentration, a phenomenon observed in both smokers and nonsmokers. Thus, although cigarette smoking increases serum alpha 1-PI concentration and total protease inhibitory capacity, no evidence was obtained to suggest that the functional activity of serum alpha 1-PI (against either trypsin or elastase) was directly affected by smoking.     

Proteolytic inactivation of alpha 1-proteinase inhibitor in infected bronchial secretions from patients with cystic fibrosis

The chronic, progressively destructive bronchitis of patients with cystic fibrosis (CF) is characterized by an important imbalance between tissue destroying granulocyte proteases such as granulocyte elastase (GE) and its physiological inhibitors in bronchial secretions. Recent in vitro studies suggest, that proteases derived from bacteria or endogenous proteases may contribute to inactivation of physiological inhibitors of GE. Since only trypsin-unreactive alpha 1-proteinase inhibitor (alpha 1-PI) was detected in CF bronchial secretions, we attempted to identify the mechanism of inactivation of alpha 1-PI. We found a heat stable, serine protease-like enzymatic activity capable of degrading 125I-labelled alpha 1-PI extensively in 22 infected but not in one non-infected CF bronchial secretion. In infected secretions, only degraded alpha 1-PI, which did not migrate like oxidized alpha 1-PI in tandem-crossed immunoelectrophoresis, was detectable. We conclude, that free GE in excess as well as GE bound to bronchial mucosal inhibitor may partly account for the alpha 1-PI-cleaving activity, but that other yet unknown bacterial or host serine proteases also contribute to alpha 1-PI inactivation.