The protein components of biomineralized structures (matrix proteins) are believed to modulate crystal nucleation and growth, and thereby influence the shape and strength of the final structure. The chicken eggshell contains a complex array of distinct matrix proteins. The most abundant of these was purified to homogeneity by a combination of anionic exchange and hydroxyapatite chromatographies. Antibodies to this protein were raised in rabbit, and utilized for Western blotting and immunohistochemistry. These studies indicated that the 17 kDa antigen (ovocleidin 17, OC-17) is found in the shell gland mucosa, and that only the tubular gland cells were positive. Immunohistochemistry with decalcified shell indicated that OC-17 is uniformly distributed throughout the shell matrix, but concentrated in the mammillary bodies. Our results indicate that this protein is secreted during shell formation and becomes incorporated into this structure. It may therefore play a role in the crystallization process and influence the properties of the resulting eggshell.

Cloned cDNAs have been isolated that encode a variant of hirudin, a potent thrombin inhibitor that is secreted by the salivary glands of the medicinal leech, Hirudo medicinals. This variant probably corresponds to a form that has been purified from leech heads but differs in amino acid sequence from the hirudin purified from whole leeches. There are at least three hirudin transcripts detectable in leech RNAs that are different in size, site of synthesis, inducibility by starvation, and relationship to hirudin activity. The new hirudin variant predicted by the cDNA and the heterodisperse transcription products suggest a hirudin protein family. The hirudin cDNA was expressed in Esckerichia coli under the control of the bacteriophage X PL promoter. The recombinant product is biologically active, inhibiting the cleavage by thrombin of fibrinogen and a synthetic tripeptide substrate.Leech hirudin is the most potent natural inhibitor of coagulation known (1-4). A very stable noncovalent 1:1 complex is rapidly and specifically formed with a-thrombin, thereby abolishing its ability to cleave fibrinogen (4). To date there is no evidence that it can interact with other components of the human coagulation cascade (5, 6).Hirudin is a polypeptide of 65 amino acids that is stable to extremes of pH and heat (4). It contains six cysteine residues grouped in the NH2-terminal half of the protein, an acidic COOH-terminal half, and one sulfated tyrosine (7). A hirudin form with isoleucine at the NH2 terminus was first purified from leech heads (H. medicinalis) (4, 8) in which activity was found to be concentrated in the salivary glands. Subsequently, Bagdy et al. (9) adopted new purification schemes using whole leeches instead of heads, yielding a form with Val-Val as the first two NH2-terminal amino acids. The amino acid sequence of the "whole body form" has been determined by independent groups (8, 10), and valine residues at positions 1 and 2 have been confirmed. Both forms had a specific activity of around 8000-10,000 antithrombin units/mg. However, more recently, Baskova et al. (11) Hirudin Activity. Antithrombin activity in leech or bacterial extracts was measured in a clotting assay (4) using citrated human platelet-poor plasma as a fibrinogen source or in a colorimetric assay using the thrombin chromogenic substrate Tos-Gly-Pro-Arg-p-nitroanilide (Chromozym TH, Boehringer Mannheim; Tos = tosyl) (7). Standard curves Abbreviation: kb, kilobases. tPresent address:

To evaluate the contribution of mononuclear phagocytes, and particularly alveolar macrophages, to alpha-l-antitrypsin (alAT) production in normal and alAT-deficient individuals, Northern analysis with a human alAT complementary DNA was used to demonstrate that alAT messenger RNA (mRNA) can be detected in liver, blood monocytes, and alveolar macrophages.Quantification of alAT mRNA expression demonstrated that: (a) type PiMM monocytes and alveolar macrophages expressed, respectively, 200-fold and 70-fold less alAT mRNA per cell than the liver, (b) the level of expression of the alAT gene was increased during the in vitro maturation of blood monocytes; and (c) blood monocyte and alveolar macrophage levels of expression of the alAT gene were the same in PiMM and PiZZ individuals. However, the amount of newly synthesized alAT secreted by ZZ alveolar macrophages was 10 times lower than that secreted by MM alveolar macrophages. Thus, mononuclear phagocytes of PiZZ individuals express a secretory defect in alAT in a fashion similar to hepatocytes. Not only do mononuclear phagocytes provide a readily accessible cell to evaluate the regulation of alAT gene expression, but these cells may contribute to the levels of alAT present in the lower respiratory tract in the nornul and ZZ states.

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.

Two low molecular weight (LMW) apoproteins were isolated from human pulmonary surfactant. SDS polyacrylamide gel analysis showed one protein (SP 18) to have an apparent molecular weight of 18,000 when unreduced and 9,000 D after reduction. The second protein (SP 9) migrated at -9,000 D in the presence or absence of reducing agents. Both proteins contain a high number of hydrophobic amino acids. The NH2-terminal sequence of SP 18 was determined to be: NH2-phe-proile-pro-leu-pro-tyr-. A cDNA clone isolated from a human adult lung cDNA library contained a long open reading frame encoding at an internal position the human SP 18 amino-terminal sequence.Mixtures of phospholipids (PL) and SP 9 and SP 18 were assessed for their capacity to reduce surface tensions on a pulsating bubble surfactometer. The addition of 1% apoprotein resulted in a reduction of surface tension after 15 s from 42.9 dyn/cm for PL alone to 16.7 and 6.3 dyn/cm for preparations containing SP 9 and SP 18, respectively. In vivo assessment of reconstituted surfactant activity was performed in fetal rabbits. Reconstituted surfactant consisting of PL + 0.5% SP 18 instilled intratracheally at delivery resulted in a marked increase in lung compliance, while the incorporation of 0.5% SP 9 yielded a moderate increase. These data show the ability to produce biologically active surfactant by the addition of isolated LMW apoproteins to defined PL.