Biomedical subjects
Publications and source records attributed to J Travis.
Evidence is rapidly accumulating that suggests that the growth and proliferation of pathogenic bacteria depend on proteolytic enzymes of the invading organism and of the host. Proteinase inhibitors targeting bacterial proteinases may be useful antimicrobial agents.
Analysis of the available data on the Porphyromonas gingivalis proteinase system suggests that at most three different genes encode arginine-X- and lysine-X-specific cysteine proteinases. These are synthesized as polyproteins containing a prepropeptide, a catalytic domain and a hemagglutinin domain. Post-translational processing of each precursor is responsible for the variety of proteinase forms that are seen.
Infection by Porphyromonas gingivalis is strongly associated with adult periodontitis, with proteinases from this bacterium now considered to be important virulence factors. In order to investigate possible pathological functions of these enzymes, we examined the effect of both free and vesicle-bound forms of the two major cysteine proteinases (gingipains) of P. gingivalis on plasma clot formation by using thrombin time (TT) measurements. Both Lys-gingipain (gingipain-K) and Arg-gingipain (gingipain-R) prolonged plasma TT in a dose- and time-dependent manner, and this was also found with vesicles which are the biological carriers of P. gingivalis proteinases. The increase in plasma TT by vesicles could be completely reversed by treatment with nonspecific cysteine proteinase inhibitors but only partially by compounds selective for either gingipain-K or gingipain-R. Preincubation of vesicles with a gingipain-K-specific inhibitor (z-FK-ck) reduced plasma TT more than a gingipain-R-specific inhibitor (leupeptin), suggesting that under physiological conditions gingipain-K was more effective in fibrinogen destruction. Each purified enzyme also markedly increased fibrinogen TT, gingipain-R being fourfold more potent than gingipain-K. However, in plasma, gingipain-R was ineffective because of the inhibitory effect of albumin. These results imply that cysteine proteinases, especially gingipain-K, abrogate the clotting potential of fibrinogen and, therefore, may contribute to the bleeding tendency and to persistent inflammation in periodontitis sites infected with P. gingivalis.
Porphyromonas gingivalis contains high concentrations of numerous cysteine proteinases with trypsin-like activity which have been implicated as important virulence factors in adult-onset periodontitis. We have analyzed the subfractions of six P. gingivalis strains for the presence of arginine-X- and lysine-X-specific proteinases (Arg-gingipain [RGP] and Lys-gingipain [KGP]) previously purified from P. gingivalis H66. Western blot (immunoblot) analysis using antibodies produced against RGP and the N-terminal peptides of RGP or the catalytic subunit of KGP indicated that these enzymes are synthesized by the strains studied and exist as multiple molecular mass species. The major forms of RGP were identified as 110-, 95-, 70- to 90-, and 50-kDa proteins, the first two being a complex of the 50-kDa catalytic subunit with hemagglutinins, with or without an added membrane anchorage peptide. The other forms are single-chain enzymes. While the 95- and 50-kDa RGP were found predominantly in culture medium, the 110- and 70- to 90-kDa forms associated with membranous fractions of the bacteria. The predominant form of KGP in all strains was a complex of the 60-kDa catalytic domain with hemagglutinins, and vesicle- and membrane-associated KGP was about 15 kDa larger than the 105-kDa enzyme present in culture media. These data explain the apparent complexity of P. gingivalis proteinases and indicate that in all strains tested there are two identical enzymes, one with arginine-X specificity and the other with lysine-X specificity, which, working in concert, are responsible for the trypsin-like activity associated with this bacterium.
Infection with Porphyromonas gingivalis is strongly associated with adult periodontitis, and proteinases are considered to be important virulent factors of the bacterium. In order to investigate the function of proteinases in disease development we examined vesicles, a biological carrier of these enzymes, for the generation of vascular permeability enhancement (VPE) activity, believed to correlate with the exudation of gingival crevicular fluid. The vesicles generated VPE activity from human plasma in a dose-dependent manner which could be inhibited 90% by antipain, a specific inhibitor of the Arg-specific cysteine proteinases (Arg-gingipains [RGPs] from P. gingivalis. Incubation of vesicles with high-molecular-weight-kininogen (HMWK)-deficient plasma did not result in VPE activity. On this basis, RGPs associated with vesicles were assumed to be responsible for most of the VPE activity generation via plasma prekallikrein activation and subsequent bradykinin production. The secondary pathway for VPE activity production was dependent on the direct release of bradykinin from HMWK by the concerted action of RGP and a Lys-specific cysteine proteinase (Lys-gingipain [KGP]), also associated with vesicles. These results indicate that RGP and KGP are biologically important VPE factors acting either via prekallikrein activation (RGP) and/or HMWK cleavage (RGP and KGP) to release BK and, thereby, contributing to the production of gingival crevicular fluid at periodontal sites infected with P. gingivalis.
Biochemical studies of pollen proteins have been focused, primarily, in investigating their roles as allergens. These molecules, some of which have enzymatic activity, act as antigens and initiate the production of IgE antibodies, leading to allergic and/or asthmatic responses. Included in this mixture of proteins are proteinases which, although they may or may not be allergenic, could still be involved in airway dysfunction. We have isolated an arginine-specific endopeptidase to homogeneity from mesquite (Prosopis velutina) pollen, a known wind-borne allergen, which has a molecular mass near 84 kDa by NaDodSO4-gel electrophoresis, a pH optimum in the neutral to alkaline range, and a requirement for Ca2+ for stabilization. The enzyme is inhibited by diisopropyl fluorophosphate (DFP) and N-p-tosyl-L-lysine chloromethylketone but not by N-p-tosyl-L-phenylalanine chloromethylketone, EDTA, or iodoacetamide. It was also not inhibited by human plasma proteinase inhibitors nor several other naturally occurring plant and animal inhibitors. Cleavage by the endopeptidase was primarily on the carboxy-terminal side of arginine residues in peptides, whereas proteins such as kallikrein and prothrombin were only activated and/or degraded extremely slowly. Several bioactive peptides that may be involved in maintaining normal lung function were readily fragmented, including angiotensin II, a vasoconstrictor, and atrial natriuretic peptide, a modulator of vascular permeability, both of which were rapidly cleaved at low enzyme:substrate molar ratios. Thus, the pollen endopeptidase could be involved in exacerbating the development of asthma by inactivating bioactive peptides that have ameliorating effects in maintaining lung airway homeostasis.
Although it is a well known fact that hepatocytes are the primary source of plasma proteinase inhibitors, including alpha 1-antichymotrypsin, this protein has also been detected in lung epithelial cells, which may suggest its local production. We have demonstrated that lung-derived epithelial cells are capable of synthesizing high levels of alpha 1-antichymotrypsin. In normal bronchial epithelial cells, as well as in the HTB55 human adenocarcinoma cell line, alpha 1-antichymotrypsin synthesis was under the control of inflammatory cytokines, of which oncostatin M was the most potent stimulator. This finding is consistent with a role for this inhibitor in protecting the lung epithelium from damage by chymotrypsin-like enzymes released from phagocytes such as neutrophils following pathogen invasion.
Paraformaldehyde (PFA) fixation was optimized to facilitate the immobilization and labeling of multiple granule antigens, using short fixation regimens and cryoultramicrotomy of unembedded neutrophils (PMNs). In the optimal protocol, extraction of azurophil granule antigens (especially of the abundant elastase) was obviated by manipulating the polymeric state of PFA, and hence its rate of cross-linking, by altering its concentration and pH in a multistep process. Primary fixation conditions used (4% PFA, pH 8.0, 5 min) favor fixative penetration and rapid cross-linking. Stable cross-linking of the antigen was achieved in a secondary fixation step using conditions that favor larger, more cross-linking polymeric forms of PFA (8% PFA, pH 7.2, 15 min). Immobilization of granule antigens was enhanced by flotation of cut sections on fixative (8% PFA, pH 8.0) before labeling and by using post-labeling fixation with 1% glutaraldehyde. The optimized protocol facilitated immobilization and immunolabeling of elastase, myeloperoxidase, lactoferrin, and cathepsin D in highly hydrated, unembedded PMNs.
Nutritional deficits have long been recognized as contributing factors to morbidity and mortality of hospitalized patients. A multi-disciplinary team approach to nutritional support has been shown to be superior to non-team approaches. A formal consultive nutritional support team has been active at St. Dominic Hospital since 1991. The benefits of our nutritional support team at St. Dominic Hospital are described. Acquisition of an Indirect calorimeter has improved measurements of caloric and substrate requirements. Individualized nutritional prescriptions more closely meet assessed patient needs. Utilization of less expensive, more physiologic enteral feeding routes have increased. Also, the number of total parenteral nutrition (TPN) bags admixed and wasted have decreased, despite an increase in the percent of patients with moderate to severe nutritional risk. The nutritional support service at St. Dominic Hospital has improved patient care while reducing cost to both the institutional and patient.
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In order better to understand the pathophysiology of the equine form of emphysema, two elastinolytic enzymes from horse neutrophils, referred to as proteinases 2A and 2B, have been extensively characterized and compared with the human neutrophil proteinases, proteinase-3 and elastase. Specificity studies using both the oxidized insulin B-chain and synthetic peptides revealed that cleavage of peptide bonds with P1 alanine or valine residues was preferred. Further characterization of the two horse elastases by N-terminal sequence and reactive-site analyses indicated that proteinases 2A and 2B have considerable sequence similarity to each other, to proteinase-3 from human neutrophils (proteinase 2A), to human neutrophil elastase (proteinase 2B) and to a lesser extent to pig pancreatic elastase. Horse and human elastases differed somewhat in their interaction with some natural protein proteinase inhibitors. For example, in contrast with its action on human neutrophil elastase, aprotinin did not inhibit either of the horse proteinases. However, the Val15, alpha-aminobutyric acid-15 (Abu15), alpha-aminovaleric acid-15 (Nva15) and Ala15 reactive-site variants of aprotinin were good inhibitors of proteinase 2B (Ki < 10(-9) M) but only weak inhibitors of proteinase 2A (Ki > 10(-7) M). In summary, despite these differences, the horse neutrophil elastases were found to resemble closely their human counterparts, thus implicating them in the pathological degradation of connective tissue in chronic lung diseases in the equine species.
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BACKGROUND: The pathogenesis of osteofibrous dysplasia, a rare fibroosseous lesion occurring almost exclusively in the tibia of children younger than 10 years of age, is not known. One theory is that osteofibrous dysplasia results from excessive resorption of bone with fibrous repair of the defect. Alternatively, osteofibrous dysplasia has been considered a congenital lesion or a variant of fibrous dysplasia. It has been hypothesized that osteofibrous dysplasia is a secondary reactive process to adamantinoma. Cytogenetic analysis is one form of investigation that has been instrumental in determining the origin of many disorders. METHODS: Short-term cultures of two separate osteofibrous dysplasia specimens (approximately 1 year apart) from the tibia of an 11-year-old boy and 2 separate specimens (approximately 2 years apart) from the tibia of a 16-year-old boy were cytogenetically examined using standard procedures. Additionally, fluorescence in situ hybridization (FISH) studies were performed on uncultured cells of both specimens of the first patient using an alpha-satellite probe for chromosome 12. RESULTS: Cytogenetic and FISH analysis revealed trisomy 12 in both specimens of the first patient. Trisomy for chromosomes 7, 8, and 22 was seen in both specimens of the second patient. CONCLUSIONS: Osteofibrous dysplasia has not previously been subjected to cytogenetic analysis. Trisomy 7 and 12, however, have been reported in a clonally aberrant adamantinoma, potentially providing further support for a relationship between these two lesions. Most importantly, these findings demonstrate a clonal and possibly neoplastic origin for osteofibrous dysplasia of long bone.