Cellular association of antiproteases in lavages from ventilated preterm human neonates.

Lung antiprotease activity is routinely assayed in the supernatant of bronchoalveolar lavage fluid (BALF). In this study the cellular fraction of lavages was also analyzed. Functionally active acid-resistant inhibitors with molecular masses characteristic of the mucus proteinase inhibitor (MPI, 14 kDa) and elastase-specific inhibitor (ESI, 7 kDa) were demonstrated by gel chromatography. Immunocytochemical studies of cells obtained at various postnatal time points from lavages of 10 premature infants with chronic lung disease showed that the inhibitors were confined to neutrophils and macrophages. At each time point, about 70% and 21% of these cells, respectively, stained positively. The polyclonal antibodies usually used to detect MPI did not distinguish between MPI and ESI. Because of this cross reactivity, it was not possible to differentiate between MPI and ESI. Analysis with reverse transcriptase-polymerase chain reaction (RT-PCR) of cells from lavages and of nucleated cells isolated from the peripheral blood showed the production of ESI only, but not of MPI. Nevertheless, MPI can associate with neutrophils and macrophages, as was shown in binding studies with the recombinant protein. These data suggest that when assaying bronchoalveolar lavages (BALs) for these antiproteases in the supernatant only, the total pool of inhibitors may be underestimated.

Isolation and characterization of trypsin-like and chymotrypsin-like proteinases from human cholesteatoma.

The mast-cell-specific proteolytic enzymes tryptase and chymase were identified in and isolated from cholesteatoma in a ratio similar to that found in human skin. We assume that this ratio reflects a similar distribution of tryptase-containing and tryptase/chymase-containing mast cells in both these tissues. It seems conceivable that mechanisms able to trigger excessive and/or continuous mast cell degranulation in the middle ear might be causative for the formation of cholesteatoma either directly or via primed chronic inflammatory reactions. By their ability to amplify degranulation of mast cells, mast cell proteinases, in particular chymase, may contribute to the chain of events leading to the formation of cholesteatoma.

Amino acid sequences of mammalian kazal-type proteinase inhibitors from salivary glands.

1. The amino acid sequences of bikazins (the double-headed Kazal-type proteinase inhibitors from submandibular glands) isolated from the snow leopard (Unica unica), the European mink (Mustela lutreola), and the European pine marten (Martes martes) were determined. 2. N-terminal domains of bikazins are characterized by a cysteine residue spacing that differs from that of C-terminal domains of bikazins and other Kazal-type proteinase inhibitor domains. 3. N-terminal sequences of bikazins seem to be specific for, and highly conserved within, each Carnivora family.

Tryptase in nasal fluid is a useful marker of allergic rhinitis.

Tryptase is a mast cell-specific marker of degranulation. To investigate the possible diagnostic value of tryptase in allergic rhinitis, we measured the levels in both serum and native nasal fluid with a sandwich RIA-assay (Pharmacia). Twenty-three allergic patients and five patients with chronic ethmoidal sinusitis were included. Eighteen of the 23 allergic patients were tested within the pollen season or had perennial rhinitis; the remainder were tested at least 1 month out of the pollen season. None of the patients had detectable serum tryptase (> 0.1 ng/ml). Also patients with chronic ethmoidal sinusitis showed no tryptase in nasal fluid. One of seven allergic patients tested out of season had slightly increased nasal tryptase of 1.8 ng/ml. In patients with active nasal allergy, the tryptase in nasal fluid ranged from 6.4 ng/ml to 640 ng/ml with a mean of 101 ng/ml and SD 173. These results show a clear distinction between active and non-active nasal allergy and other non-mast-cell-related nasal disease. Further, nasal tryptase release by natural allergen exposure is even higher than that observed in allergen challenge tests.

Interaction of human mast cell tryptase and chymase with low-molecular-mass serine proteinase inhibitors from the human respiratory tract.

Mast cell degranulation results in the release of serine class proteinases with trypsin- and chymotrypsin-like specificity. While looking for natural protein inhibitors of these enzymes, we studied their reactions with the double-headed Kunitz-type inhibitor, bikunin, and the human bronchial secretion inhibitor (BSI), which are the only known low-molecular-mass proteinase inhibitors of the human respiratory tract. Both trypsin and chymotrypsin can be inhibited by these inhibitors. However, human BSI is unable to inhibit human tryptase and is the physiological counterpart of chymase in the upper respiratory tract. Human bikunin is unable to inhibit human chymase and human tryptase. Furthermore, human tryptase is also not inhibited by a fragment that consists only of the trypsin-specific C-terminal inhibitor domain of human bikunin. This finding contradicts reports that claim the occurrence of a tryptase-specific proteinase inhibitor in rat mast cells.

Characterization of a trypsin inhibitor from equine urine.

A trypsin inhibitor was isolated from pregnant mares' urine by adsorption on bentonite and elution with aqueous pyridine followed by batch DEAE-cellulose treatment and column chromatography. Final purification to an electrophoretically homogenous glycoprotein was achieved by gel permeation chromatography. This equine urinary trypsin inhibitor (E-UTI) is acid- and heat-stable, has a molecular weight of 22 to 23 kDa, an isoelectric point of 4.55, forms a 1:1 molar complex with trypsin and has serine as its N-terminal amino acid. The N-terminal amino acid sequence of this protein is almost identical with that of EI-14, the inhibitor obtained from horse serum by tryptic treatment, except for two extra amino acid residues, Ser-Lys- on the N-terminal end of E-UTI. In its isoelectric point E-UTI differs from EI-14 and the inhibitor from human urine.

Complementary DNA and deduced amino acid sequences of procine alpha 1-microglobulin and bikunin.

Analysis of complementary DNA for porcine alpha 1-microglobulin and bikunin indicates that both proteins result from proteolytic processing of a common precursor similar to that found in man. Complete primary structures of these proteins are deduced from the nucleic acid sequence and partially confirmed by peptide sequencing.

The amino-acid sequence of the double-headed proteinase inhibitor from badger (Meles meles) submandibular glands.

Badger submandibular glands contain a double-headed secretory proteinase inhibitor. Its amino acid sequence was determined. Extensive homologies were found between this inhibitor and the corresponding inhibitors of fox, dog, lion and cat in both domains. As in fox and dog inhibitor, the trypsin-inhibiting domain of badger inhibitor contains an Arg residue in the reactive site in contrast to a Lys residue in the inhibitors of lion and cat. Domains I and II of badger inhibitor are structurally related both to the sequenced inhibitors of fox, dog, lion and cat and to the sequenced monovalent secretory pancreatic trypsin inhibitors. The sequence of the badger inhibitor is N-terminally extended by four amino acids in comparison to fox and dog inhibitors and extended by eight amino acids in comparison to lion and cat inhibitors. Furthermore, the badger inhibitor is C-terminally extended by two amino acids in comparison to the lion inhibitor and by three amino acids in comparison to all other sequenced inhibitors.

Primary structure of a proteinase inhibitor released from goat serum inter-alpha-trypsin inhibitor.

An acid-resistant trypsin inhibitor was released from goat serum inter-alpha-trypsin inhibitor and isolated by affinity chromatography. The primary structure of the inhibitor was established and the inhibitory properties were estimated. The inhibitor designed gIK-14 was characterized as a serine proteinase inhibitor from the family of the double-headed Kunitz-type inhibitors as suggested.

Sequence of a human keratin 13 specific cDNA encompassing coil 1B through the 3' end.

An expression library established in lambda gt11 with cDNA from squamous epithelium of the human upper digestive tract was screened with an antibody raised against keratin 13. A 1.2 kb fragment from the most strongly reacting plaque was sequenced and compared to known type I keratin sequences. The highest degree of homology was detected with the murine 47K type I keratin, which we consider to be the counterpart of human keratin 13. Tryptic peptides of keratin 13 were separated on a HPLC column and one peptide was sequenced. The amino acid sequence obtained supports the identity of the cDNA. An eight codon motif has been tandemly repeated in the C-domain of keratin 13. In spite of substantial divergence by point mutations and deletions, the remaining sequence homologies suggest that the C-domains of both the human keratin 13 and the orthologous murine protein have originated from a common ancestor.

Two out of the three kinds of subunits of inter-alpha-trypsin inhibitor are structurally related.

Inter-alpha-trypsin inhibitor is a 240-kDa plasma-protein complex of three different types of glycoproteins. Their stoichiometric relation in the complex is not yet known. One subunit results from proteolytic processing of a precursor protein composed of alpha 1-microglobulin and a double-headed Kunitz-type proteinase inhibitor protein. From this, only the inhibitor protein becomes part of the inter-alpha-trypsin inhibitor complex. Another subunit whose function is not yet understood is structurally unrelated to the first one as well as to other proteins of various data collections. Now we have obtained a cDNA clone coding for 837 amino acid residues of a precursor protein of the third subunit. Its primary structure is 40% identical to that of the completely known second-subunit precursor. Peptide sequences obtained from isolated inter-alpha-trypsin inhibitor represent a distinct part of only about two thirds of the predicted polypeptide precursor, suggesting that its maturation is very similar to that of the second subunit. Therefore, we conclude that the deduced primary structure covers about 98% of the mature third subunit.

The diagnosis of CSF fistulae on the basis of detection of beta 2-transferrin by polyacrylamide gel electrophoresis and immunoblotting.

A sensitive method is described for the detection of beta 2-transferrin, a transferrin-variant found only in cerebrospinal fluid (CSF). The determination of beta 2-transferrin, whose presence is characteristic of CSF-admixtures in secretions, is performed in three steps. The proteins of the secretion are separated by polyacrylamide gel electrophoresis and then transferred electrophoretically onto a nitrocellulose sheet. Finally, the transferrins on the nitrocellulose sheet are specifically detected by an antibody reaction. The bands are visualized either by using antibodies conjugated with peroxidase or by protein A gold. With the exception of certain cases (Ritchie, R. F. & Smith, R. (1976) Clin. Chem. 22, 497-499; Görg, A. et al. (1983) Human Genetics 64, 222-226) beta 2-transferrin is found only in cerebrospinal fluid, and not in other body fluids. Therefore the detection of beta 2-transferrin can be used for the diagnosis of rhino- and otoliquorrhea. The advantage of this method is that beta 2-transferrin can be unequivocally identified by the use of a relatively small amount of technical equipment. CSF can therefore be clearly identified in secretions. An additional advantage of the method is its high sensitivity.

[Protease inhibitors as tumor-associated growth factors]. / Proteaseninhibitoren als tumorassoziierte Wachstumsfaktoren.

Two apparent endothelial cell growth factors were isolated and characterized from serum free cell culture medium of hepatoma cells by McKeehan et al. The factors were identified as proteinase inhibitors with known primary structure. They are the pancreatic secretory trypsin inhibitor (HPSTI) and the double headed inhibitor HI-30, the inhibitory active part of the inter-alpha-trypsin inhibitor complex. We were able to isolate acid resistant inhibitory active material from serum free culture medium of 4 out of 11 tumor cell lines, which we have analyzed. The cell lines were not derived from liver cells. The inhibitory active material was identified as the inhibitor HI-30 by N-terminal amino acid sequence analysis. The results indicate that HI-30 is a real growth factor, since it is expressed as well in tumor cells which are not derived from liver cells.

Complementary DNA and derived amino acid sequence of the precursor of one of the three protein components of the inter-alpha-trypsin inhibitor complex.

Inter-alpha-trypsin inhibitor is composed of three distinct protein components. These protein components stem from independently encoded and proteolytically processed precursor proteins. Only the structure of the protein component responsible for the inhibitory activity has been established so far. We now present the complete amino acid sequence of the precursor of the second protein component derived from cloned cDNA. The precursor molecule includes both a signal peptide and a propeptide sequence and seems to be further processed prior to the assembly of the inter-alpha-trypsin inhibitor complex.

The amino-acid sequence of the double-headed proteinase inhibitor from fox (Vulpes vulpes) submandibular glands.

Fox submandibular glands contain a double-headed secretory proteinase inhibitor. Its amino acid sequence was determined. Extensive homologies were found between this inhibitor and the corresponding inhibitors of cat, lion and dog in both domains. As in dog inhibitor the trypsin-inhibiting domain of fox inhibitor contains an Arg residue in the reactive site in contrast to a Lys residue in the inhibitors of cat and lion. Domains I and II of fox inhibitor are structurally related both to the sequenced inhibitors of cat, lion and dog and to the sequenced monovalent secretory pancreatic trypsin inhibitors. In comparison to cat and lion inhibitors the N-terminally extended sequences of fox and dog inhibitors seem to be characteristic for the inhibitor of Canidae.