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Glycosaminoglycan synthesis was studied in explant cultures of hamster lungs 15 and 45 days following intratracheal administration of Bleomycin. At both time points, a statistically significant increase in 35S-sulfate incorporation into glycosaminoglycans was seen in the Bleomycin-treated explants compared with that of the controls. Furthermore, the percentage of label associated with dermatan sulfate was significantly higher in the treated explants than in controls at both 15 and 45 days. Conversely, the percentage of labeled heparin and/or heparan sulfate was significantly lower for the treated explants compared to controls at these times. These results indicate that glycosaminoglycan synthesis is altered from normal in this model of interstitial lung disease. Comparison of these data with previous measurements of glycosaminoglycan synthesis in another model of interstitial lung disease, induced by N-nitroso-N-methylurethane, reveals marked similarity in the changes from normal in 35S-labeling.

Amino acid analysis of human fetal lung elastin was undertaken in 49 instances of live-born neonates, ranging from 380 g to full term, and in 3 abortuses of 12-14 wk gestation. The data suggest that formation of the cross-linking agents, desmosine and isodesmosine, occurs early, between 14 and 22 wk. The ratio of neutral to charged amino acids remains low until the 36th wk when it attains adult levels. The composition of elastin was independent of sex and duration of survival. In three neonatal pulmonary diseases (respiratory distress syndrome, atelectasis, and hemorrhage) ratios were significantly lower than those found in nondiseased lungs. This may be a reflection of immaturity or may be a predisposing factor in neonatal lung disease. The latter hypothesis is attractive and receives indirect support from the association of a more polar elastin with other diseases, including adult emphysema and atheromatous aortic change. Our finding of relatively high polarity in elastin from human fetal lung is consistent with previous observations in a variety of fetal organs of other species.

Incompetence of the uterine cervix is a syndrome of painless, progressive dilatation and effacement occurring between the sixteenth and twenty-fourth weeks of gestation that represents abnormal functioning. It may serve as a model to elucidate normal function. Because the incompetent cervix results in painless opening of this organ without uterine contraction before term gestation, it is considered one of the causes of midtrimester spontaneous abortion, habitual spontaneous abortion, and early preterm labor. Untreated, it leads to rapid expulsion and often death of the fetus. We used light microscopy to compare decreased elastic fibers in incompetent cervices with those of normal nonpregnant and pregnant cervices. Morphologic analysis of this difference was extended to biochemical quantification of elastin content in one patient with cervical incompetence. The decrease in elastin suggests that one function of cervical elastin may be to maintain a closed and undilated cervix throughout gestation. There may be a relationship between changes in cross-linked elastin and the incompetent cervix; further studies are therefore indicated.

SummaryGlycosaminoglycan and collagen synthesis were studied in a hamster model of interstitial pulmonary fibrosis, induced by weekly subcutaneous injections of N-nitroso-N-methylurethane (NNNMU) for up to 16 weeks. Experimental and control animals were injected intraperitoneally with 35S or [3H]proline to label glycos-aminoglycans and collagen, respectively. The labeling studies were performed at 1 and at 3 months following completion of NNNMU treatment. Uptake of 35S into lung glycosaminoglycans was higher in diseased animals than in controls at both 1 and 3 months post-NNNMU. The experimental lungs had a significantly increased percentage of labeled dermatan sulfate and/or chondroitin 4-sulfate compared to controls at both time intervals. Additional studies performed only on the 1-month samples showed that this increase in percentage labeling was primarily in dermatan sulfate. NNNMU-treated lungs also had a significantly lower percentage of labeled heparin and/or heparan sulfate than did controls at 1 ...

A monolayer of a clone of endothelial cells derived from rat lung cells (RLE) was overlaid with 1 M urea to extract the surface proteins. Hydrolysis and SDS-gel electrophoresis of the urea extracted cell surface proteins (UCSP) yielded four peptides of 350,000, 84,000, 66,000 and 18,500 molecular weight. Of these only the 66,000 and 18,500 molecular weight peptides reacted with antibodies raised in rabbit against rat lung endothelial cells (RLE). The 18,500 mol. wt. antigenic peptide was a serum protein associated with the cell surface, whereas the 66,000 mol. wt. peptide was the surface antigen synthesized and released into the medium by the rat lung endothelial cells. On rocket Immunoelectrophoresis, the 66,000 mol. wt. rat kidney fibroblast surface peptide produced only a single rocket whereas peptides of RLE produced two rockets, suggesting the presence of an additional antigenic peptide which could serve as a marker for endothelial cells.

Three human basement membranes, glomerular basement membrane (GBM) from renal cortex, alveolar basement membrane (ABM) from lung parenchyma and trophoblast basement membrane (TBM) from the terminal villi of placenta have been isolated by sieving and sonication techniques. Canine GBM and ABM were also prepared. There were marked differences among the membranes from human tissues. Compared to GBM, TBM had very little collagen but contained high concentrations of charged amino acids. ABM was intermediate in composition between GBM and TBM and contained desmosine and isodesmosine indicative of the presence of elastin. Canine ABM (c-ABM) did not contain desmosine or isodesmosine. In the canine system an antigen was detected in ABM which was not present in GBM. The membrane preparations were analyzed for fibronectin content using a specific antiserum to fibronectin. This glycoprotein could not be detected in GBM whereas it was present in ABM in amounts up to 0.8% and in TBM in amounts as high as 7.2%. All the membranes induced the formation of precipitating antibodies in rabbits. Soluble material obtained from the membranes by alkali extraction, reduction of disulfide bonds, enzymatic digestion with elastase, plasmin or collagenase provided immunologically reactive fragments. These soluble fragments gave reactions of identity among the three basement membranes in immunodiffusion reactions in gels with antisera raised to all three BMs. The finding that plasmin digests basement membranes suggests that it may play a role in connective tissue remodeling. The fact that elastase degrades basement membranes provides an endogenous system for injury which may be triggered by infections.

A sensitive, reproducible, and rapid radioimmunoassay was developed to determine clastin peptide content of human serum in nanogram amounts. Significant differences have been determined between normal volunteers, both smokers and nonsmokers, and patients wth chronic obstructive pulmonary disease. The average concentration of clastin peptides in 9 normal nonsmokers was 19.88 ± 10.46 μg/ml serum, indistinguishable from that in 7 normal smokers which was 17.64 ± 8.54 μg/ml. Average concentration of clastin peptides in 17 patients with lung disease was 53.38 ± 22.46 μg/ml, significantly different (P

Fibronectin (Fn), a high molecular weight glycoprotein, was found to constitute 0.43% of the normal adult beagle dog lung. The tissue Fn (TFn) was solubilized by sequential chemical extractions and quantified by ELISA (enzyme-linked immunoabsorbent assay). Subsequent plasmin digestion did not appear to solubilize significantly more Fn. Since 70% of the lung tissue was solubilized by the extractions and plasmin digestions, the TFn quantified represented the bulk of lung Fn. The TFn was identical to plasma fibronectin in the ELISA and one can infer that the Fn molecule is not significantly altered as it is incorporated into the lung connective tissue matrix. Lungs from beagles in which fibrosis had been induced with bleomycin contained 0.99% Fn, more than a twofold increase over normal. In the ELISA TFn from fibrotic lungs gave an inhibition curve of the same shape as did TFn from normal lungs. Thus, Fn from fibrotic lungs is not different qualitatively from Fn from normal lungs in any way detectable with this antiserum. The TFn content of plasmin digests of intact lung was less than that of extracts, which was the converse of results obtained on placenta (B. A. Bray (1985) Biochem. J. 226, 811–815). This difference between lung TFn and placental TFn may be due to differences in degree of glycosylation, which determines susceptibility to proteases.

Endotoxin-induced lung injury has previously been shown to produce lesions that resemble emphysema morphologically and biochemically as demonstrated by the reduction in the content of lung elastin. The purpose of this study was to define the changes in one other connective tissue component, glycosaminoglycans, during the acute phase of the lung injury. Intravenous administration of a single dose of endotoxin in rats resulted in an increase in the total synthesis of glycosaminoglycans by the pulmonary parenchyma. There was a significant increase in the proportion of dermatan sulfate synthesized during the first 48 hr and a concomitant decrease in heparin/heparan sulfate synthesis. At 48 hr the increased synthesis of dermatan sulfate had reached 7.3 times control values and began to decline, whereas the synthesis of chondroitin-4-sulfate rose from 4.1 to 10.7 times control values between 48 and 72 hr. Analysis of the rates of synthesis revealed that the total amount of heparin/heparan sulfate remained constant while the synthesis of chondroitin-6-sulfate increased proportionally to the overall synthesis of glycosaminoglycans. These findings indicate that dramatic changes in glycosaminoglycan synthesis are an integral part of endotoxin lung injury.

Mature, crosslinked elastin has been isolated from 4 human and 12 monkey uterine cervices. A modification of previous methods for determination of elastin content was devised to quantitate the low amounts of elastin in the crude connective tissue of uterine cervices. The percentage of elastin was found to range between 0.9 and 2.4% and did not appear to change at various stages of gestation.

A peptide of 6,700–8,000 daltons has been isolated from an ethanolic KOH digest of bovine ligamentum nuchae elastin by a combination of ion exchange chromatography and gel filtration techniques. The peptide is non-polar and is composed almost entirely of glycine, valine, and proline (or hydroxyproline) in the ratio 2:2:1. Edman degradation of the first 17 residues gave the sequence: NH 2 -Gly-Phe-Pro-Gly-Val-Gly-Val-Pro-Gly-Val-Gly-Val-Pro-Gly-Val-Gly-Val. These results suggest that the peptide is made up of repeating pentapeptide units with the sequence: Pro-Gly-Val-Gly-Val.

We have determined the microanatomy of the cervix in relation to elastic fibers by serial dissection of human cervix in three tissue planes: cross, sagittal, and frontal sections. Analysis suggests that elastin is localized to specific regions of the uterine cervix and not dispersed throughout the connective tissue stroma. By Musto stain the majority of elastic fibers are noted to be oriented from the external os to the periphery and from there in a band upward toward the internal os where they become sparse in the area of the cervix with the greatest amount of smooth muscle just below the internal os. Elastic fibers were noted to be sparsely distributed in the cervical stroma.

At 5, 15, and 45 days following induction of interstitial pulmonary fibrosis by intratracheal administration of bleomycin in hamsters, glycosaminoglycan synthesis was measured, using [35S]sulfate. Total labeled sulfate incorporation into lung glycosaminoglycans was maximally increased over that of saline-instilled controls at 5 days (P less than or equal to 0.05), declined markedly at 15 days, and returned to control values at 45 days. Separation of the various labeled glycosaminoglycans by chondroitinase digestion and chromatography revealed a transient rise from controls (P less than or equal to 0.05) in the proportion of labeled chondroitin 4-sulfate at 5 days, followed by an increase from controls (P less than or equal to 0.05) in proportionate labeling of dermatan sulfate at 15 and 45 days postbleomycin. Autoradiography, using [35S]sulfate, performed at 21 days postbleomycin, revealed an increase from controls in film grain formation in areas of interstitial reaction. Grain formation was greatly reduced by pretreatment of the slide sections with hyaluronidase and chondroitinase, demonstrating the specificity of the label for glycosaminoglycans. The results indicate that glycosaminoglycan synthesis is significantly altered from normal in this model of interstitial lung disease and that dermatan sulfate is preferentially synthesized during the fibrotic phase of the lung reaction.

The ability of soluble salts of ATP to solubilize numerous inorganic and organic compounds in neutral or slightly alkaline medium and to keep these compounds in solution is demonstrated. The latter can be natural products or synthetic substances. Difficulty soluble salts of ATP themselves are solubilized at physiological conditions of pH and temperature by other solubilizing agents. Among the latter are normal constituents of cells and products of intermediary metabolism. To these phenomena connected in different ways with ATP might be attributed essentiality as ATP is an omnicellular substance and plays a part in the course of many important biological processes.

Alpha-1 antitrypsin, alpha-2 macroglobulin, and total trypsin inhibitory capacity levels were significantly lower in the umbilical cord serum of newborn infants with idiopathic respiratory distress syndrome (IRDS) than in weight-matched control subjects. Levels were unrelated to gestational age or sex. Sequential serum samples were drawn in 17 infants with IRDS; there was a tendency for levels of total trypsin inhibitory capacity to increase in 6 of 9 survivors. In contrast, 7 of the 8 infants who died had persistently low levels and some had further decreases in concentration. In amniotic fluid obtained at 12 to 22 weeks' gestation in cases not related to IRDS, alpha-1 antitrypsin and total trypsin inhibitory capacity could usually be detected but alpha-2 macroglobulin was not. Serum enzyme inhibitor levels may be of diagnostic significance in IRDS.

Two methods are described for the purification of Clostridium histolyticum collagenase. One method involves gel filtration on columns of Sephadex G-200, the other, ion-exchange chromatography on DEAE Sephadex A-50. In both cases, calcium-containing buffers were found necessary for good recoveries of enzyme. Yields of collagenase activity up to 94%, free of nonspecific proteolytic activities, were consistently obtained.

Bovine ligamentum nuchae elastin was partially hydrolyzed by ethanolic KOH. Fractionation of the dialyzed product on a column of SE-Sephadex yielded eight fractions which were characterized by amino acid analysis as well as hexose and lipid contents. The approximate molecular weight or size range of each of the fractions was determined by Sephadex gel filtration. Study of the amino acid distribution among these fractions revealed the presence of three classes of peptide. The first peak eluted from the SE-Sephadex was found to be a mixture of acidic glyco-peptides, possibly derived from a structural glycoprotein component of elastin. A second type of peptide was represented by the second and third peaks which contained almost exclusively the neutral amino acids glycine, valine and proline. Fraction II was a monodisperse peptide of about 10,000 molecular weight, containing these three amino acids in the approximate ratio of 2:2:1. The third general group of peptide isolated, fractions IV through VIII, was a...

A modification of the Bratton-Marshall test (10) developed by Goldbarg and Rutenburg (9) for the assay of amino peptidase has been adapted to the determination of naphthylamine released from the specific trypsin substrate benzoylarginine-β-naphthylamide (3). Advantages of this method over others used in similar procedures include specificity, sensitivity, good reproducibility, easy colorimetric assay, lack of interference by contaminants present in crude enzyme preparations, tissues, and fluids, and the possibility of using the same substrate for parallel histochemical tests. The procedure has been applied to the determination of trypsin itself, other enzymes with similar specificity for arginine side chains, and the trypsin-like cathepsin B enzymes of liver, kidney, spleen, muscle, and other tissues. It has also been used to assay trypsin inhibitors, in particular those found in tissues.

The enzymatic degradation of purified blood group substances represents an increasingly fruitful approach to the elucidation of their immunochemical specificity, and progress to date has recently been reviewed (Kabat, 1956). A number of enzymes from different microorganisms have been described which split one or more of the blood group substances with resultant changes in serological specificity. Stack and Morgan (1949) have investigated enzymes, first studied by Schiff (1935), of Clostridium welchii, which decompose the A, B and O(H) substances. Watkins (1953) described enzymes in cell free extracts of the protozoan Trichomonas foetus which inactivated the A, B, O(H), and Lewis blood group substances; and Watkins and Morgan (1954) have shown that partially purified enzyme preparations from the same source destroyed the O(H), M and N substances on intact erythrocytes without affecting the A, B, P or S antigens. Besides inactivating the specific antigens mentioned, the enzymes of T. foetus rendered all erythrocytes nonspecifically agglutinable (panagglutinable), rendered D-positive cells agglutinable by incomplete anti-D, and inactivated the receptor sites for the hemagglutinin of influenza virus type A (PR8). Cell free extracts of Lactobacillus bifidus (var. penn) have been shown by Gy6rgy, et al. (1952) to possess enzymatic activity against human and animal blood group A, B, and 0(H) substances, being most active against the latter. Iseki and Tsunoda (1952) have reported that cell free extracts of Bacillu-s fulminans, an aerobic spore bearer, show enzymatic

A bacterial elastase has been isolated from a flavobacterium species and freed from contaminating peptidase and unspecific proteinase activity. Some properties of this new enzyme, such as pH, stability, effect of metals and other potential inhibitors, kinetics, and specificity are described. The bacterial enzyme is more specific than pancreatic elastase, has a lower pH optimum, and is not affected by 1% NaCl and serum inhibitors.

Continuing our studies of the antigenicity of papillary cystadenocarcinoma of the ovary we have investigated the production of antibodies in rabbits immunized with a tissue lyophilisate. Ouchterlony double diffusion showed 4 precipitin lines, of which one cross-reacted with normal ovarian tissue, one was identical with the line developed with tissue culture cell sonicate antigen, and 2 additional lines were found only with the tumor homogenate. Immunoelectrophoresis indicated that one antigenic component had a mobility corresponding to α-globulin and one line appeared in the β-globulin region. Gel filtration on Sephadex G-200 yielded 6 fractions of which Fraction 1 and Fraction 3 were tumor specific. Fraction 1 gave one precipitin line against nonadsorbed serum to unfractionated tumor homogenate, Fraction 3 gave 2 nonidentical and distinct lines.

SUMMARY: Oakley & Warrack (1950) established the presence of two serologically distinct proteolytic enzymes in Clostridium histolyticum filtrates, a β-toxin digesting native collagen and a cysteine-activated γ-toxin attacking azocoll and other protein substrates. In the present paper we give evidence of another proteolytic enzyme, not activated by cysteine and serologically different from those previously described. This we designate as δ-toxin. We also indicate the possibility of the presence of a fourth enzyme more stable than the others and active against various synthetic substrates in conjunction with cysteine. This enzyme could not be inhibited by antisera. Furthermore peptidases have been detected and will be described in detail elsewhere. Some of the differences between these enzyme systems are discussed.

Purified insoluble native Achilles tendon collagen was digested with Clostridium histolyticum collagenase. The N-terminal sequence of the mixed peptides to position 3 was determined by a modification of the Edman degradation procedure, C-terminal amino acids by hydrazinolysis. Experimental losses and non-specific cleavage of the peptides were estimated from model compounds and appropriate corrections made. At the N-terminus 182 residues out of 200 were glycine, 10 were alanine, while isoleucine, phenylalanine, and valine accounted for the remainder. In the second position 77 residues out of 200 were proline, but significant amounts of other amino acids were present showing that the specificity requirement of the enzyme is less rigorous than originally suspected. Ten residues of hydroxyproline in this position were unexpected. In the third N-terminal position the distribution was even more heterogenous, but hydroxyproline and alanine, mostly in tripeptides, predominated. C-terminally, 58 residues were hydroxyproline, 42 alanine, 29 arginine, with other amino acids in considerably lower amounts. No glycine was detected at the C-terminus. Attempts were made to deduce the distribution of third position residues between tripeptides and longer chain peptides and comparisons made with results obtained by others with different collagens.

Bovine ligamentum nuchae elastin, after solubilization by pancreatic elastase, was dialyzed and fractionated on a column of DEAE-Sephadex A-50. The resulting fractions were analyzed for their component amino acids. The first fraction eluted had the greatest average peptide chain length and 87% of its residues as the three nonpolar amino acids-glycine, valine, and proline in the ratio 2:2:1, with insignificant amounts of the polar amino acids and cross-linking agents. It was found not to have any antigenic sites. Another fraction which was the major component of the digest contained the cross-links in an amount three times that found in complete elastin but was only weakly antigenic. The fraction with the greatest antigenicity was found to be enriched in polar amino acids and tyrosine; it also contained 7% carbohydrate as glucose and 50% lipid.

This paper describes a test for elastase which employs oxalic acid-solubilized elastin as the substrate. The test described is more sensitive than those using insoluble elastin-dye complexes and more specific than the recently introduced tests using synthetic peptide-esters. It can be used in the presence of other proteins, including serum, and is especially suited for the study of serum elastase inhibitors in health and disease.

Concentrations of α1-antitrypsin were measured in 80 specimens of amniotic fluid collected at various stages of pregnancy. Levels remained relatively unchanged throughout gestation, with a median concentration of 10.0 mg/100 ml at 12–24 weeks, 18.7 mg/100 ml at 34–37 weeks, and 16.2 mg/100 ml, after the 37th week. In three instances where the infant developed RDS, the median concentration was 12.4 mg/100 ml.

Extraction of fibronectin from two human tissues, lung parenchyma and placental villi, was facilitated by the incorporation of heparin into extraction media. The effect of heparin was additive to the effect of urea which is known to extract fibronectin. These experiments provide further evidence that fibronectin and glycosaminoglycans are associated in connective tissues and the use of heparin forms the basis for a simple method for extraction and quantitation of tissue fibronectin.

A decreased inhibition of pancreatic elastase has been detected in the serums of six patients with alpha(1)-antitrypsin deficiency. Five have severe clinical and physiological pulmonary emphysema. This observation extends the defect of inhibition by serum to a second, biologically active proteolytic enzyme in this form of familial emphysema.

Amiodarone, a cardiac antiarrhythmic agent, has been associated with the development of interstitial pulmonary fibrosis in patients receiving prolonged therapy with the drug. To further assess the toxic effects of amiodarone on lung tissue, Syrian hamsters were given a single intratracheal insufflation of the agent and evaluated for histologic evidence of lung injury. Control animals received intratracheal insufflations of the vehicle in which amiodarone was dissolved. After an initial, transient alveolitis in both experimental and control animals, the amiodarone-treated lungs developed increased interstitial thickening due to fibrinous exudates, alveolar epithelial hyperplasia, inflammatory cell infiltrates, and marked deposition of collagen manifested on trichrome staining. Controls, in contrast, showed nearly complete resolution of the initial alveolitis. An unusual feature of the amiodarone-induced lung injury was reemergence of the alveolitis between 5 and 14 days, which included a marked influx of eosinophils into the lung. Although the precise mechanism of the lung injury is not known, the persistence of the acute inflammatory cells as well as the presence of eosinophils suggests a hypersensitivity-type reaction. Furthermore, the progression of lung injury to fibrosis after a single insult with the drug suggests that mere discontinuation of amiodarone therapy in humans may not reverse the disease process, but that corticosteroid therapy may also be required. Amiodarone appears to be a useful agent to induce diffuse fibrotic reactions in the lung that morphologically resemble idiopathic pulmonary fibrosis in humans.

Considered here are recent concepts concerning pathogenetic mechanisms underlying alterations in lung tissue structure and function. Mechanisms of pulmonary injury are described and include (1) the capacity of proteolytic enzymes to induce experimental pulmonary emphysema with particular emphasis on the effects of elastases and collagenases on connective tissue components, (2) the effects on distal lung units of specific chemical toxins, and (3) the capacity of the circulating polymorphonuclear leukocyte to induce pulmonary injury by the action of lysosomal enzymes. Evidence is reviewed which indicates a predominant role for alterations in alveolar elastin as the basis for alveolar tissue destruction along with the delayed effects on lung function and morphology of proteolytic injury. Newer developments in the understanding of the functions of the alpha 1 -globulin and other proteolytic enzyme inhibitors in the respiratory tract are considered. The special characteristics of cell damage induced by noxious agents such as cadmium, nitrogen dioxide, ozone, high oxygen concentrations as well as alpha-napthyl-thiourea, alloxan and paraquat are discussed. Finally, the proteolytic capacity of the polymorphonuclear leukocyte and the links between the leukocyte proteases and the activation of leukokinin and bradykinin generating systems are summarized, and their role in the tissue injury induced by antibody-antigen reactions, shock lung and nonspecific tissue damage is indicated.

Eighteen fetal lambs have been studied at six different gestational ages ranging from 90 days to term. At 90 days gestation, only the microfibrillar component of elastin is present in pulmonary alveolar septal elastic fibers. The amorphous component appears at 110 days and gradually increases in amount, relative to microfibrils. At term (150 days), the composition of the pulmonary alveolar septal elastin resembles that of the adult ewe.

The glycosaminoglycans produced by a mixed cell line of normal adult rat lung and an endothelial clone derived from this line were isolated and examined. Cellulose acetate electrophoresis of media and cells before and after digestion with specific enzymes indicated that all the major glycosaminoglycans except keratan sulfate were synthesized by both cultures. Heparan sulfate and dermatan sulfate were found only in the cell fraction while hyaluronic acid was found in both the medium and the cell fractions. The chondroitin sulfates were isolated from the medium.The endothelial clone produced a 4: 1 ratio of glucosamine to galactosamine in the medium from the fifth through thirteenth months of culture. The medium of the mixed cell line initially contained glycosaminoglycans with a glucosamine to galactosamine ratio of 2:1 but after approximately one year of culture, the ratio had changed to 4.6: 1 suggesting that the culture contained predominantly endothelial cells.

A pleural surface membrane, which could be ablated from frozen human lungs with forceps, has been analyzed chemically. The surface membrane was found to consist primarily of collagen (Type I) and to contain only 2.3–8% elastin. The mechanical properties of this membrane should differ markedly from those of alveolar parenchyma, since the connective tissue of the parenchyma contains 32% elastin.