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U Kucich

Publications and source records attributed to U Kucich.

At least 37 records · Page 2Linked to original sources
PubMed1994

Microfibril-associated glycoprotein: characterization of the bovine gene and of the recombinantly expressed protein.

Microfibrils having a diameter of 10-12 nm, found either in association with elastin or independently, are an important component of the extracellular matrix of many tissues. Because isolation of native proteins composing the microfibrils has proven difficult, information on structure/function relationships is limited. In order to extend our understanding of the 31-kDa microfibril-associated glycoprotein (MAGP), the bovine gene has been cloned and characterized and the protein has been expressed in a eukaryotic system. The compact coding portion of the gene is contained in 4.5 kbp of genomic DNA and does not appear to share any domain motifs with other known proteins. The size, amino acid composition, and sequence of the amino terminus of the secreted recombinant protein (rMAGP) all agree with values predicted by the nucleotide sequence of the cDNA used in the expression vector. The rMAGP reacts with a monospecific antibody prepared against a defined amino acid sequence of the natural molecule and reacts specifically with recombinantly produced tropoelastin, suggesting that rMAGP will be a useful reagent with which to study its interaction with other extracellular matrix components.

Amino Acid Sequence↗
PubMed1994

Alpha 1-antitrypsin Pittsburgh (Met358-->Arg) inhibits the contact pathway of intrinsic coagulation and alters the release of human neutrophil elastase during simulated extracorporeal circulation.

Cardiopulmonary bypass prolongs bleeding time, increases postoperative blood loss, and triggers activation of plasma proteolytic enzyme systems and blood cells referred to as the "whole body inflammatory response". Contact of blood with synthetic surfaces leads to qualitative and quantitative alterations in platelets, neutrophils, contact and complement systems. Contact and complement pathway proteins both induce neutrophil activation. alpha 1-antitrypsin Pittsburgh (Met358-->Arg), a mutant of alpha 1-antitrypsin, is a potent inhibitor of plasma kallikrein and thrombin. We investigated whether this recombinant mutant protein inhibited platelet activation, as well as contact and/or complement-induced neutrophil activation during simulated extracorporeal circulation. Arg358 alpha 1-antitrypsin did not prevent the 34% drop in platelet count at 5 min of recirculation, did not block the 50% decrease in ADP-induced platelet aggregation at 120 min of recirculation, nor inhibit the release of 6.06 +/- 1.07 micrograms/ml beta-thromboglobulin at 120 min of recirculation suggesting that the inhibitor had little effect on platelet activation. However, Arg358 alpha 1-antitrypsin totally blocked kallikrein-C1-inhibitor complex formation but not C1-C1-inhibitor complex formation. Most importantly, Arg358 alpha 1-antitrypsin decreased the release of 1.11 +/- 0.16 micrograms/ml human neutrophil elastase by 43%. The attenuation of neutrophil activation in the absence of an effect on complement activation via the classical pathway, supports the concept that kallikrein is a major mediator of neutrophil degranulation during cardiopulmonary bypass.

Arginine↗
PubMed1993

Aprotinin inhibits the contact, neutrophil, and platelet activation systems during simulated extracorporeal perfusion.

Aprotinin reduces blood loss after cardiac operations and decreases the bleeding time. The mechanism of action of aprotinin that produces these effects is not clear. During simulated extracorporeal circulation the contact and complement systems, platelets, and neutrophils are activated. We investigated the effect of aprotinin on kallikrein-C1-inhibitor complex and C1-C1-inhibitor complex formation, neutrophil degranulation, and platelet release and aggregation during simulated extracorporeal circulation. Fresh heparinized human blood was recirculated at 37 degrees C for 2 hours in a spiral coil membrane oxygenator-roller pump perfusion circuit. Changes in platelet count, leukocyte count, platelet response to adenosine diphosphate, and plasma levels of beta-thromboglobulin, kallikrein-C1-inhibitor complexes, C1-C1-inhibitor complexes, and neutrophil elastase were measured before and at 5, 30, 60, and 120 minutes of recirculation at 0, 0.015, 0.03, 0.06, and 0.12 mg/ml doses of aprotinin. Platelet counts decreased to 36% +/- 12% of control values at 5 minutes and increased to 56% +/- 13% at 120 minutes without aprotinin. Aprotinin did not affect platelet counts, but it did prevent the decrease in sensitivity of platelets to adenosine diphosphate and it attenuated beta-thromboglobulin release. In the absence of aprotinin, kallikrein-C1-inhibitor and C1-C1-inhibitor complexes increased progressively to 0.53 +/- 0.14 U/ml and 2.38 +/- 0.33 U/ml, respectively, at 120 minutes. Kallikrein-C1-inhibitor complexes were completely inhibited and C1-C1-inhibitor complexes were partially inhibited at aprotinin concentrations of 0.03 mg/ml or greater. Release of neutrophil elastase was partially but not completely inhibited at the highest dose of aprotinin and was 50% inhibited at a dose of 0.03 mg/ml. Because activation of the fibrinolytic system does not occur in this system, the changes were independent of the inhibition of plasmin. We conclude that aprotinin in high doses completely inhibited kallikrein-induced activation of neutrophils and partially inhibited complement-induced activation. Aprotinin did not directly affect platelet adhesion or aggregation, but it indirectly preserved platelet sensitivity to agonists and also attenuated release of alpha-granule contents. The data indicate that in the presence of aprotinin platelet function was partially preserved, kallikrein production was totally inhibited, complement activation was partially inhibited, and neutrophil release was partially inhibited, thus attenuating the "whole body inflammatory response" associated with cardiopulmonary bypass.

Aprotinin↗
PubMed1992

Specificity and sensitivity of the assay for elastin-derived peptides in chronic obstructive pulmonary disease.

One prediction of the protease-antiprotease hypothesis of chronic obstructive pulmonary disease (COPD) pathogenesis is the appearance of elastin-derived degradation products in the plasmas of affected patients that are due to the breakdown of alveolar interstitial elastin by an excess of elastolytic activity in the lung. We previously demonstrated a significant elevation of plasma elastin-derived peptides (EDP) in subjects with COPD in comparison with asymptomatic smokers with normal spirometry or normal nonsmokers. To better determine the selectivity of the assay for EDP as a marker of COPD, we measured plasma EDP levels in different patient populations. These included subjects with COPD, subjects with diseases that may involve accelerated elastolysis (pneumonia, atherosclerotic vascular disease, and inflammatory arthritis), subjects with diseases hypothesized to involve pulmonary inflammation without elastolysis (asthma and acute tracheobronchitis), asymptomatic smokers with normal spirometry, and healthy, nonsmoking subjects. Mean plasma EDP levels in subjects with COPD were elevated above those in all other subjects (p less than 0.01). The prospective analyses of specificity and sensitivity of plasma EDP levels as markers of COPD gave values of 91 and 65%, respectively. Utilizing receiver operating characteristic curve analysis to assess the diagnostic and screening performance of plasma EDP as a test for COPD (perfect test equals an area under the curve of 1.0), the area under the curve was 0.87, which compares favorably with many widely used clinical tests. These data demonstrate that the assay for plasma EDP is a quantitative, easily measured, and highly specific marker for subjects with COPD.

Adult↗
PubMed1991

Size distribution of human lung elastin-derived peptide antigens generated in vitro and in vivo.

The protease-antiprotease hypothesis of emphysema development suggests that degradation of elastin in the lung interstitium may give rise to abnormal quantities of circulating elastin-derived peptides (EDP) during periods of inflammation. Recent studies have shown a relationship between emphysema and high levels of EDP in human plasma. This report characterizes elastin digests on the basis of antigenicity, size, and method of preparation, as well as the size distribution of EDP found in the plasmas of nonsmokers, smokers, and emphysema patients. Gel filtration of elastin digests prepared by hydrolysis of human lung elastin using a low (1:500) ratio of neutrophil elastase to elastin generated a broad protein peak of approximately 70,000 daltons. In contrast, a high (1:25) ratio of neutrophil elastase to human lung elastin gave a broad protein peak, with a size distribution in the 10,000 to 30,000 dalton range. This digest showed distinct immunochemical properties. A polyclonal antibody directed against the low-ratio digest showed a minimum detection of 2 ng/ml for the homologous antigen but required 1,000 ng/ml of the high-ratio digest for detectable inhibition in an indirect ELISA assay. Gel filtration of plasmas from normal nonsmokers and the majority of normal smokers revealed a single immunoreactive EDP fraction of approximately 70,000 daltons. Plasmas from selected normal smokers and emphysema patients with high levels of circulating EDP (greater than 90 ng/ml) fractionated into a complex pattern of peptides in which the 70,000 dalton component represented 50% of the immunoreactive material and several lower molecular weight peptides represented the remaining circulating elastin antigens.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens↗
PubMed1991

A controlled trial of colchicine to reduce the elastase load in the lungs of ex-cigarette smokers with chronic obstructive pulmonary disease.

A great deal of experimental evidence suggests that emphysema in smokers is caused by the action of neutrophil elastase on lung elastin. In order to test this hypothesis, it is necessary to find a drug that reduces the load of neutrophil elastase in the lungs of patients with emphysema. In a previous study we treated smokers with emphysema with colchicine, a drug that prevents neutrophil elastase secretion, to determine if it would reduce the elastase burden in the lungs. Colchicine was unable to reduce the elastase load while the stimulus of smoking continued. In this study we treated ex-smokers with emphysema to determine if colchicine could reduce the elastase burden in the lungs. The objective of the study was to determine if colchicine can reduce the elastase load and putative indicators of elastase load in the lungs of patients with chronic obstructive pulmonary disease. The study was a prospective, double-blind, randomized, and placebo-controlled clinical trial. The subjects were outpatients seeking treatment at the University of Texas Health Center at Tyler. We studied 16 ex-cigarette smokers between 45 and 75 yr of age with lung disease defined by FEV1 less than 70% of predicted but greater than 1.2 L whose airflow obstruction was less than 20% reversible with bronchodilators. Colchicine or placebo was taken by mouth in disguised capsules, 0.6 mg three times per day. Volunteers were placed on a baseline bronchodilator regimen of theodur by mouth and albuterol by inhalation. Blood, urine, and bronchoalveolar lavage fluids were obtained after 1 wk of stabilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1991

Effects of simulated extracorporeal circulation on human leukocyte elastase release, superoxide generation, and procoagulant activity.

Activated leukocytes are thought to contribute to respiratory dysfunction, alterations in microvascular permeability, disseminated intravascular coagulation, and thrombosis, all of which can complicate extracorporeal circulation. The purpose of this work was to determine the effects of extracorporeal circulation on leukocyte functions likely to mediate organ damage. White blood cell counts in the bubble circuits (n = 5) fell to 51% +/- 7% (mean +/- standard error of the mean; p less than 0.05) of initial levels within 2 hours of recirculation. In contrast, counts from both the spiral coil (n = 5) and hollow-fiber (n = 5) groups remained at 91% +/- 12% and 100%, respectively. Plasma levels of human neutrophil elastase rose from 0.28 +/- 0.06 micrograms/ml to 3.14 +/- 0.36 micrograms/ml (p less than 0.05) and 0.20 +/- 0.02 micrograms/ml to 1.61 +/- 0.35 micrograms/ml (p less than 0.05) in bubble and spiral coil circuits, respectively, but from only 0.20 +/- 0.03 micrograms/ml to 0.96 +/- 0.42 micrograms/ml in the hollow-fiber circuit despite 2 hours of recirculation. Consistently, in response to N-formyl-L-methionyl-L-leucyl-L-phenylalanine, a chemotactic peptide, cells from spiral coil and bubble circuits released and generated significantly less elastase and superoxide anion, respectively. In contrast, neutrophils from the hollow-fiber circuits demonstrated enhancement of N-formyl-L-methionyl-L-leucyl-L-phenylalanine-induced elastase release and superoxide generation. Finally, mixed leukocytes from all circuits expressed procoagulant activity reaching statistical significance in bubble circuits. In conclusion, extracorporeal circulation has pronounced effects on neutrophil elastase release, superoxide anion generation, and leukocyte procoagulant activity. Spiral coil and bubble oxygenators cause granule release and, subsequently, reduced sensitivity to soluble agonists. In contrast, hollow-fiber oxygenators "prime" cells, actually enhancing reactivity. Recirculation through all circuits induces leukocyte procoagulant activity that is likely to contribute to surface-induced thromboses and excessive bleeding.

Blood Coagulation↗
PubMed1990

Release of lactoferrin and elastase in human allergic skin reactions.

Our previous skin chamber studies have shown prominent accumulation of viable neutrophils in human allergic skin reaction sites. To determine whether such neutrophils release components that may be pathogenic in allergic reactions, we have compared the patterns of release of five components: 1) lactoferrin, present in specific granules; 2) and 3) elastase and myeloperoxidase, present mainly in azurophilic granules; 4) lactic dehydrogenase, a cytosolic component generally released during cell damage; 5) histamine, present in mast cells and basophils but not in neutrophils. In 13 pollen-sensitive subjects we found that continuous antigen challenge for 5 h lead to a peak of histamine release into overlying skin chambers during the 1st h, followed by a plateau of low level histamine release over the succeeding 4 h. In contrast, there was no significantly increased released of lactoferrin or elastase during the first h, but significantly increased accumulation of these components at Ag challenge sites over the next 4 h. There was no significant difference at Ag vs buffer control sites in the levels of either myeloperoxidase or lactic dehydrogenase. The increased levels of lactoferrin and elastase at antigen challenge sites in the 2nd to 5th h were not simply a reflection of the greater numbers of neutrophils present in such sites because the levels of these components did not correlate significantly with the number of neutrophils in chamber fluids obtained from individual sites. However, such lactoferrin levels did correlate significantly with the amount of histamine released earlier during the 1st h of Ag challenge at individual sites. These findings suggest a selective in vivo release of neutrophil components in IgE-mediated human allergic skin reactions, possibly related in degree to earlier mast cell activation. Inasmuch as lactoferrin likely plays a role in reactive oxidants effects and elastase is a potent nonspecific protease, release of these agents could play a pathogenic role in late phase allergic reactions.

Adult↗
PubMed1990

Production of recombinant human tropoelastin: characterization and demonstration of immunologic and chemotactic activity.

Tropoelastin cannot readily be prepared in quantity from natural sources and this has limited research in several important areas including structure/function relationships and fiber assembly. In order to eliminate this limitation, human tropoelastin has been expressed in a recombinant bacterial system and the protein has been highly purified. The size, amino acid composition, and sequence of the amino terminus of the recombinant tropoelastin (rTE) all agree with values predicted by the nucleotide sequence of the cDNA used in the expression vector. The rTE exhibits cross-reactivity with antibodies directed against a mixture of peptides derived from human elastin as well as antibody against a defined peptide located at the carboxy terminus of the protein. In addition, the rTE is chemotactic for fetal calf ligament fibroblasts. These results suggest that rTE could be a useful reagent for many types of studies.

Animals↗
PubMed1990

A controlled trial of colchicine to reduce the elastase load in the lungs of cigarette smokers with chronic obstructive pulmonary disease.

Current data suggest that emphysema in smokers is caused at least in part by the unrestrained action of neutrophil elastase on pulmonary tissues. Since colchicine reduces the secretion of enzymes from stimulated neutrophils, we designed a clinical trial to determine if colchicine could reduce the elastase load in the lungs or several putative indicators of elastin destruction. We carried out a prospective, double-blind, randomized, and placebo-controlled clinical trial. Outpatients seeking treatment for chronic obstructive pulmonary disease at the University of Texas Health Center at Tyler who met specific criteria were recruited into the study. A group of 46 cigarette smokers between 45 and 75 yr of age with chronic obstructive pulmonary disease (COPD) were studied. Colchicine or placebo was given orally in disguised capsules, 0.6 mg three times per day. Volunteers were placed on a baseline bronchodilator regimen of Theodur orally and albuterol by inhalation. Blood, urine, and bronchoalveolar lavage fluids were obtained after 1 wk of stabilization. The patients were then randomized and treated for 14 days with colchicine, and the measurements were repeated. Modifications in plasma elastin peptides and neutrophil elastase-generated fibrinopeptide A, urinary desmosines, and bronchoalveolar lavage fluid neutrophils or neutrophil elastase were the indicators of success or failure of the treatment. Pre- and posttreatment measurements in each patient and the difference between colchicine-treated and placebo-treated groups were compared. There were no statistically significant differences in either of the two types of analyses in any of the variables. We conclude that variables related to elastase load in the lungs were not modified by colchicine treatment. If a drug can be identified that is successful in modifying one of these variables, it would then have to be tested in a large-scale clinical trial in which the rate of decline in the FEV1.0 or mortality would be measured. The data presented here may provide useful information about the variability of key measurements of elastase load in the lungs and the breakdown of elastin and may aid investigators in designing similar trials in the future.

Aged↗
PubMed1989

Structure of the elastin gene and alternative splicing of elastin mRNA: implications for human disease.

The protein elastin is largely responsible for the elastic properties of vertebrate lungs, large blood vessels, and skin. The structure of the human, bovine, and chick elastin gene and protein monomer, tropoelastin, has recently been elucidated by using techniques of molecular biology. Extensive homology of amino acid sequence exists among the mammalian species and there is in addition strong conservation of nucleotide sequences in the 3' untranslated region of the gene. The translated exons are small and embedded in large expanses of introns. Sequences coding for the hydrophobic regions, responsible for the elastic properties of the molecule, and the alanine-lysine rich regions, responsible for crosslink formation between molecules, reside in separate exons and alternate for the most part in the elastin gene. S1 analyses and sequence analysis of cDNA and genomic clones have indicated that there is substantial alternative splicing of the primary elastin transcript. Variations in the structure of mRNAs resulting from alternative splicing could explain the existence of the multiple forms of tropoelastin observed electrophoretically in several species. Different kinds of splicing patterns could occur in human populations and may contribute to aging and pathological situations in the cardiovascular and pulmonary systems.

Aging↗
PubMed1989

Human neutrophils contain and bind high molecular weight kininogen.

Because plasma kallikrein activates human neutrophils, and in plasma prekallikrein (PK) circulates complexed with high molecular weight kininogen (HMWK), we determined whether HMWK could mediate kallikrein's association with neutrophils. HMWK antigen (237 +/- 61 ng HMWK/10(8) neutrophils) was present in lysates of washed human neutrophils. Little if any plasma HMWK was tightly bound and nonexchangeable with the neutrophil surface. Human neutrophils were found to possess surface membrane-binding sites for HMWK but no internalization was detected at 37 degrees C. 125I-HMWK binding to neutrophils was dependent upon Zn2+. Binding of 125I-HMWK to neutrophils was specific and 90% reversible. 125I-HMWK binding to neutrophils was saturable with an apparent Kd of 9-18 nM and 40,000-70,000 sites per cell. Upon binding to neutrophils, 125I-HMWK was proteolyzed by human neutrophil elastase (HNE) into lower relative molecular mass derivatives. Furthermore, HMWK found in neutrophils also served as a cofactor for HNE secretion because neutrophils deficient in HMWK have reduced HNE secretion when stimulated in plasma deficient in HMWK or with purified kallikrein. These studies indicate that human neutrophils contain a binding site for HMWK that could serve to localize plasma or neutrophil HMWK on their surface to possibly serve as a receptor for kallikrein and to participate in HNE secretion by this enzyme.

Antigens↗
PubMed1988

Development of immunoreagents to ciliary zonules that react with protein components of elastic fiber microfibrils and with elastin-producing cells.

We describe the generation of a monoclonal antibody library to ocular zonule components and the characterization of three monoclonal antibodies: 1) one specific for microfibrillar associated glycoprotein (MAGP), a component of both ocular zonules and microfibrils of elastin fibers, 2) an antibody to an as yet unidentified 70,000 dalton antigen that is present in abundance in the extracellular matrix (ECM) of elastin-producing cells, and 3) an antibody reacting with the 67000 dalton subunit of the elastin receptor. The presence of antigenic determinants common to the ocular zonule and elastic fiber microfibrils suggests that zonules, which can be obtained in relatively pure form, can provide a valuable resource for characterizing proteins common to both microfibrillar structures.

Animals↗
PubMed1988

Fibronectin degradation products containing the cytoadhesive tetrapeptide stimulate human neutrophil degranulation.

We investigated whether adhesive glycoproteins, such as fibronectin or fibrinogen, could function to provide a nidus for neutrophil degranulation. Elastase release in recalcified plasma was normal in afibrinogenemic plasma, but 73% less in plasma depleted of fibronectin. Proteolytic digests of fibronectin, but not intact fibronectin (50-1,000 micrograms/ml), induced a concentration-dependent release of neutrophil elastase and lactoferrin. MAbs N293, which recognized the mid-molecule of fibronectin, N294, which was directed toward the 11-kD cell adhesive fragment, and N295, generated against the amino terminal of the 11-kD fragment, inhibited the release of elastase by 7, 24, and 60%, respectively. The cytoadhesive tetrapeptide portion of fibronectin, Arg-Gly-Asp-Ser (250-1,000 micrograms/ml), released 1.94 +/- 0.10 micrograms/ml of elastase from 10(7) neutrophils, in contrast to the lack of release by the control hexapeptide, Arg-Gly-Tyr-Ser-Leu-Gly. Plasmin appeared to be the enzyme responsible for fibronectin cleavage, since neutrophil elastase release in plasma that had been depleted of plasminogen was decreased and reconstitution of plasminogen-deficient plasma with purified plasminogen corrected the abnormal release. Plasmin cleaved fibronectin to multiple degradation products, each less than 200 kD. This fibronectin digest released 1.05 microgram/ml of elastase from 10(7) neutrophils. We suggest that the activation of plasminogen leads to the formation of fibronectin degradation products capable of functioning as agonists for neutrophils.

Antibodies, Monoclonal↗
PubMed1988

Acute cigarette smoke exposure in dogs: the inflammatory response.

Acute cigarette smoke causes polymorphonuclear leukocyte (neutrophil, PMN) recruitment to the lung followed by loss of elastase from the recruited cells. Dogs were exposed to cigarette smoke with different oxidant content, bronchoalveolar lavage (BAL) was performed, and the cell distribution in the recovered alveolar lining fluid was analyzed. Exposures were 1, 3, or 6 cigarettes on one or multiple days with a maximum dose of 42 cigarettes. The mean percent PMN present in control lavage was 2.01%, while the mean percent PMN recovered in BAL after a dose of 42 1R1 cigarettes was 13.05%. Recoverable PMN, after a single exposure to three 1R1 cigarettes, also increased from 1.7 to 10.4% by 15 h after cessation of smoke exposure. The cell response for multiple (2 and 7) day exposures was similar. The elastase content per BAL neutrophil decreased relative to peripheral blood PMN from the same animals. No free elastolytic activity was found in BAL, but PMN elastase antigen was present. Increased frequency of cigarette smoke exposure delayed the return to homeostatic cell conditions. The increased PMN accumulation observed may result in an increased proteolytic load in the pulmonary interstitium and contribute to the pathogenesis of emphysema.

Animals↗