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J Travis

Publications and source records attributed to J Travis.

At least 37 records · Page 2Linked to original sources
PubMed1998

Comparative properties of two cysteine proteinases (gingipains R), the products of two related but individual genes of Porphyromonas gingivalis.

Proteolytic enzymes produced by Porphyromonas gingivalis are important virulence factors of this periodontopathogen. Two of these enzymes, referred to as arginine-specific cysteine proteinases (gingipains R), are the product of two related genes. Here, we describe the purification of an enzyme translated from the rgpB/rgp-2 gene (gingipain R2, RGP-2) and secreted as a single chain protein of 422 residues. The enzyme occurs in several isoforms differing in pI, molecular mass, mobility in gelatin zymography gels, and affinity to arginine-Sepharose. In comparison to the 95-kDa gingipain R1, a complex of catalytic and hemagglutinin/adhesin domains, RGP-2 showed five times lower proteolytic activity, although its activity on various P1-arginine p-nitroanilide substrates was generally higher. Gingipains R amidolytic activity, but not general proteolytic activity, was stimulated by glycyl-glycine. However, in cases of limited proteolysis, such as the inactivation of alpha-1-antichymotrypsin, glycyl-glycine potentiated inhibitor cleavage. In contrast, alpha-1-proteinase inhibitor was not inactivated by gingipains R and only underwent proteolytic degradation during boiling in reducing SDS-polyacrylamide gel electrophoresis treatment buffer. Similarly, native type I collagen was completely resistant to cleavage by gingipains but readily degraded after denaturation. Together, these data explain much of the controversy regarding gingipains structure and substrate specificity and indicate that these enzymes function as P. gingivalis virulence factors by proteolysis of selected target proteins rather than random degradation of host connective tissue components.

Adhesins, Bacterial↗
PubMed1998

The synthesis and biological evaluation of a novel series of indole PDE4 inhibitors I.

This communication describes the synthesis and in vitro evaluation of a novel potent series of phosphodiesterase type (IV) (PDE4) inhibitors. The compounds described contain an indole moiety which replaces the 'rolipram-like' 3-methoxy-4-cyclopentoxy motif. Several of the compounds presented possess low nanomolar IC50's for PDEIV inhibition. In vivo activities determined from measurement of serum TNF-alpha levels in LPS challenged mice (mouse endotoxemia model) are also reported.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1998

Purification and characterization of a novel peptidase (IImes) from mesquite (Prosopis velutina) pollen.

Although the mesquite plant (Prosopis velutina) is not as widely distributed as some other allergenic species, its pollen can induce serious pollinosis in areas where it is localized. We previously isolated and characterized a peptidase from mesquite pollen with trypsin-like specificity (peptidase Imes) (Matheson, N., Schmidt, J., and Travis, J. (1995) Am. J. Respir. Cell Mol. Biol. 12, 441-448). Now we have characterized a second enzyme with specificity for hydrophobic residues (mesquite pollen peptidase IImes). This enzyme has a molecular mass near 92 kDa and activity that was not affected by reducing or chelating agents but was inhibited by specific synthetic serine proteinase inhibitors and the aminopeptidase inhibitor bestatin. However, it was not inhibited by human plasma proteinase inhibitors, nor did it inactivate any of those tested. The enzyme possessed amidolytic activity against p-nitroanilide substrates most effectively after alanine residues and also displayed aminopeptidase activity against non-p-nitroanilide peptides with a preference for phenylalanine. This specificity for hydrophobic amino acid residues was corroborated by inhibition studies with chloromethyl ketone and organophosphonate inhibitors. More interesting from a physiological point of view is that the bioactive peptides, angiotensins I and II and vasoactive intestinal peptide, were also hydrolyzed rapidly, indicating an ability of peptidase IImes to act also as an oligopeptidase. Because these bioactive peptides play a role in the inflammatory responses in allergic asthma, our data suggest that the purified mesquite pollen peptidase IImes may be involved in the degradation of neuro- and vasoactive peptides during pollen-initiated allergic reactions.

Allergens↗
PubMed1998

Inactivation of alpha1-proteinase inhibitor as a broad screen for detecting proteolytic activities in unknown samples.

The need for a quick, simple screening method for the detection of general proteolytic activity prompted us to determine whether cleavage within the reactive site loop region (RSL) of alpha1-proteinase inhibitor (alpha1-PI), a well-characterized member of the serpin family known to be susceptible to proteolytic inactivation, can be utilized for this purpose. Inactivation of alpha1-PI in the RSL region can be measured by loss of residual inhibitory capacity of alpha1-PI against its target proteinase. While we originally utilized this assay to detect a new proteinase from culture supernatants of Porphyromonas gingivalis, the feasibility of extending this assay to scan for proteolytic activity from other systems was also assessed. As an example, we found that the serine proteinase from Staphylococcus aureus (SSP) had virtually the same catalytic efficiency in inactivating alpha1-PI in our assay as it did in the hydrolysis of the synthetic substrate Z-Phe-Leu-Glu-pNA (kcat/Km value of 2 x 10(4) M-1 s-1 vs 2.6 x 10(4) M-1 s-1, respectively). Additionally, in both assays activity could be readily detected in less than a 1 h incubation at SSP concentrations in the picomolar range. This assay is unique in that proteinases which hydrolyze peptide bonds within the RSL of alpha1-PI can readily be detected as measured by loss of alpha1-PI inhibitory activity.

Amino Acid Sequence↗
PubMed1998

Spontaneous inactivation of human lung tryptase as probed by size-exclusion chromatography and chemical cross-linking: dissociation of active tetrameric enzyme into inactive monomers is the primary event of the entire process.

A unique property of human mast cell tryptase is its spontaneous inactivation, which may be relevant to the regulation of the activity of this enzyme in vivo. We have found, using size-exclusion chromatography, that the dissociation of the tetrameric active enzyme into the inactive monomer occurred immediately from the beginning of the inactivation process and at a rate significantly faster than that of the appearance of the inactive, tetrameric form. Eventually, a relatively long-lived state of apparent equilibrium between all three forms (active tetramer, inactive monomer, inactive tetramer) was reached. When tryptase was extensively cross-linked with several heterobifunctional photoactivatable reagents, this modified enzyme exhibited a long-term stability in low-ionic-strength buffer and at elevated temperature, unlike that of the native enzyme. Its is suggested that cross-linking prevents the spontaneous inactivation and dissociation of tryptase by 'freezing' the normal association state of the enzyme and supports the hypothesis that the dissociation of native tetrameric tryptase into inactive monomer is the primary event for the entire process of spontaneous inactivation of this enzyme.

Biopolymers↗
PubMed1998

Proteolytic enzymes from larvae of the fire ant, Solenopsis invicta. Isolation and characterization of four serine endopeptidases.

The imported red fire ant (Solenopsis invicta) is a problematic pest in the Southern United States. The stages of development for these ants are as follows: egg, 1st, 2nd, 3rd, and 4th instar larvae, prepupae, pupae, and adult. The 4th instar larvae plays an important role in the survival of the colony in that it is totally responsible for the digestion of solid foods and the source of nutrients for the queen and adult workers. In our studies we have been successful in purifying and characterizing four proteinases from the 4th instar larvae. Based on substrate specificity, they appear to represent two chymotrypsin-like and two elastase-like proteinases. These are referred to as Soli C1, Soli C2, Soli E1, and Soli E2, with molecular masses of 25, 28, 23, and 24 kDa, respectively, based on SDS-PAGE. All enzymes were inhibited by diisopropyl fluorophosphate, a general serine class inhibitor. Various synthetic substrates with either Phe or Val in the P1 position, were readily cleaved by Soli C1/C2 or E1/E2, respectively. Each enzyme has been characterized as to pH optimum, pH stability, isoelectrofocusing and susceptibility to inhibition by a broad range of natural and synthetic proteinase inhibitors. Such compounds may prove useful for the development of insecticides to control fire ant infestation.

Amino Acid Sequence↗
PubMed1998

Inactivation of tumor necrosis factor-alpha by proteinases (gingipains) from the periodontal pathogen, Porphyromonas gingivalis. Implications of immune evasion.

Porphyromonas gingivalis is one of the major pathogens associated with adult periodontitis, a major chronic inflammatory disease. Potent proteinases elaborated by these bacteria aid directly and indirectly in both the development of the pathophysiology of the disease and in host defense evasion. For these reasons they are considered key virulence factors. To investigate whether possible immune evasion mechanisms involve the dysregulation of the host cytokine network, we examined the ability of P. gingivalis cysteine proteinases, including Arg-specific gingipains HRGP and RGP2 and Lys-specific KGP, to degrade the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). All three gingipains rapidly degraded TNF-alpha as exhibited by immunoblot analysis. Moreover, all biological activity was significantly reduced over extended incubation periods with the proteinases tested, whereas the host neutrophil proteinases were ineffective. These results indicate that the gingipain proteinases elaborated by P. gingivalis are capable of disrupting the cytokine network at the site of infection through the degradation of the proinflammatory cytokine TNF-alpha, suggesting the removal of one of several mediators important to the function of polymorphonuclear leukocytes. Such a mechanism is likely to be utilized by other infective organisms not only for survival but also for growth and proliferation.

Adhesins, Bacterial↗
PubMed1998

Oncostatin M and the interleukin-6 and soluble interleukin-6 receptor complex regulate alpha1-antichymotrypsin expression in human cortical astrocytes.

alpha1-Antichymotrypsin (ACT) is an acute phase protein expressed in the brain which specifically colocalizes with amyloid-beta during Alzheimer's disease. We analyzed ACT synthesis in cultured human cortical astrocytes in response to various cytokines and growth factors. Oncostatin M (OSM) and interleukin (IL)-1beta were potent stimulators of ACT mRNA expression, whereas tumor necrosis factor-alpha had modest activity, and IL-6 and leukemia inhibitory factor (LIF) were ineffective. The finding that OSM, but not LIF or IL-6, stimulated ACT expression suggests that human astrocytes express a "specific" OSM receptor, but not IL-6 or LIF receptors. However, cotreatment of human astrocytes with soluble IL-6 receptor (sIL-6R).IL-6 complex did result in potent stimulation of ACT expression. When the human ACT gene was cloned, two elements binding STAT1 and STAT3 (signal transducer and activator of transcription) in response to OSM or IL-6.sIL-6R complexes could be identified and characterized. Taken together, these findings indicate that OSM or IL-6.sIL-6 complexes may regulate ACT expression in human astrocytes and thus directly or indirectly contribute to the pathogenesis of Alzheimer's disease.

Acute-Phase Proteins↗
PubMed1998

Oncostatin M, leukaemia-inhibitory factor and interleukin 6 trigger different effects on alpha1-proteinase inhibitor synthesis in human lung-derived epithelial cells.

Interleukin 6 (IL-6), oncostatin M (OSM) and leukaemia-inhibitory factor (LIF) share a common signal-transducing subunit in each of their receptors and thus mediate an overlapping spectrum of biological activities. Although all of these cytokines stimulate the production of alpha1-proteinase inhibitor (alpha1-PI) in hepatocyte-derived cells, only OSM is able to up-regulate levels of this inhibitor in epithelial cells originating from the lung. In this study we characterized human lung-derived epithelial-like HTB58 cells for their ability to synthesize alpha1-PI after treatment with IL-6, OSM and LIF. The results demonstrate that the resistance of HTB58 cells to the effects of IL-6 and LIF was not because of a lack of their individual functional receptors and suggest that OSM utilizes two different receptors, gp130/LIF receptor and gp130/OSM receptor, in lung-derived epithelial cells.

Blotting, Northern↗
PubMed1998

Crystallization and preliminary X-ray diffraction analysis of gingipain R2 from Porphyromonas gingivalis in complex with H-D-Phe-Phe-Arg-chloromethylketone.

Gingipain R2 is a 50 kDa proteinase from the oral pathogenic bacterium Porphyromonas gingivalis. This proteinase, which displays no significant sequence homology to any protein previously analyzed by X-ray crystallography, has been crystallized using the vapor diffusion method. Two different crystal forms were obtained from a solution containing polyethylene glycol (MW 8,000) (space group P2(1)2(1)2(1)) or magnesium sulfate (space group R3) as precipitating agent. Complete diffraction data sets have been collected up to 2.0 and 2.9 A resolution, respectively. Cell dimensions are a = 51.9 A, b = 79.9 A, and c = 99.6 A (P2(1)2(1)2(1)), and a = b = 176.6 A, and c = 143.4 A (R3). Considerations of the possible values of Vm accounts for the presence of one monomer per asymmetric unit in the case of the orthorhombic crystal form, whereas the rhombohedral crystal form, together with the analysis of the self-rotation function, could accommodate a tetramer in the asymmetric unit.

Adhesins, Bacterial↗
PubMed1998

Ragweed pollen proteolytic enzymes: possible roles in allergies and asthma.

Extracts from ragweed pollen grains contain novel trypsin and chymotrypsin-like serine peptidases which are described in this report. The molecular mass of the chymotrypsin-like enzyme was 82 kDa, had a pH optimum near 9.0, and its activity was unaffected by chelating or reducing agents. It was inhibited by diisopropyl fluorophosphate (DFP), a general serine class inhibitor, and more specifically N-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK), a chymotrypsin-like proteinase inhibitor. In addition to various synthetic substrates, the neuropeptides, vasoactive intestinal peptide (VIP) and substance P, which are required for normalized lung functions, were also rapidly hydrolysed. Activity toward protein substrates was not detected with the exception of the inactivation of alpha-1-proteinase inhibitor (alpha-1-PI) which occurred through cleavage within the reactive site loop. The 'trypsin-like' enzyme has a molecular mass near 80 kDa, a blocked N-terminus, a pH optimum near 9.0, and requires Ca++ for stability and activity, but not reducing agents. It is inhibited by DFP, and more specifically the trypsin-like proteinase inhibitor, N-p-tosyl-L-lysine chloromethyl ketone (TLCK). Again, activity toward protein substrates was not detected, but various synthetic substrates and biologically active peptides were efficiently cleaved. Significantly, atrial natriuretic peptide (ANP) and angiotensin 2 (ATII), whose degradation would amplify kinin activity and influence inflammatory diseases of the respiratory tract and nasal passages, were also rapidly hydrolyzed.

Allergens↗
PubMed1998

Effect of oxidation of beta-amyloid precursor protein on its beta-secretase cleavage. A model study with synthetic peptides and candidate beta-secretases.

As the amyloidogenic processing of beta-amyloid precursor protein (betaAPP) proceeds under conditions of oxidative stress, the methionine-596 residue at the beta-secretase cleavage point is likely in an oxidized state. In the present work, possible consequences of the oxidation of Met-596 for the generation of the N-terminus of amyloid beta protein were modeled using synthetic peptide substrates, matching 587-606 sequence fragment of betaAPP and containing either intact methionine or methionine sulfoxide. Patterns and rates for the cleavage of these substrates by purified mast cell chymase, cathepsin G, cathepsin D, matrix metalloproteinase-3 and neutrophil elastase, were compared. Only the three first proteases, all previously suggested as candidate beta-secretases, preferentially cleaved the "intact" substrate after Met-596. For chymase and cathepsin G, the specificity of this cleavage increased upon a shift from optimal alkaline pH to acidic pH, which is also more compatible with the plausible intracellular localization of amyloidogenic betaAPP processing. The substitution of methionine sulfoxide for methionine in the substrate slowed down the cleavage rate for all the enzymes tested, by a factor of 6-15. This was associated with shifts of cleavage preferences to points of minor importance for the "intact" peptide, suggesting a specific resistance of the peptide bond after MetSO-596 against proteolysis.

Amyloid Precursor Protein Secretases↗
PubMed1998

A relationship between proteinase activity and clinical parameters in the treatment of periodontal disease.

The objective of this research was to determine the effectiveness of a biochemical assay which measures proteolytic enzyme activity in gingival crevicular fluid (GCF) and to relate this enzyme activity to clinical parameters traditionally utilized for periodontitis detection. A clinical trial was conducted on 8 periodontitis subjects with > or =4 sites exhibiting a loss of attachment of > or =5 mm and probing depths of > or =5 mm with bleeding on probing. On each subject, a plaque index was performed, followed by GCF sampling at those sites which exhibited a loss of attachment and probing depths. GCF was analyzed for activity against benzoyl-L-arginine-p-nitroanilide in the presence (BAPNA w/gly-gly) and the absence (BAPNA w/o gly-gly) of glycyl-glycine and against MeOSuc-Ala-Ala-Pro-Val-pNA and Suc-Ala-Ala-Pro-Phe-pNA for neutrophil serine proteinases activity (elastase and cathepsin G, respectively). Subsequently, a gingival index was performed, attachment levels and probing depths were recorded using a constant force probe with bleeding on probing being noted. A split-mouth design was employed and half mouths were randomly assigned to the following treatment groups: group A, half of the mouth received scaling/root planing and polishing: group B, half of the mouth received no treatment (control). Subjects were treated, then instructed on toothbrushing and interdental cleaning. After 4 weeks, subjects returned to receive a plaque index; GCF sampling, gingival index, attachment levels, probing depths and bleeding on probing as described above. Using a paired Student t-test, the findings suggest that BAPNA w/gly-gly was significantly less in treatment sites than in non-treated control sites (p=0.05). No such correlation was found for other activities, including neutrophil serine proteinases which were shown to occur in GCF in free, proteolytically active forms. In addition, significant treatment effects were detected for probing depths (p= 0.03) which reduced by 1.3 mm and attachment levels (p=0.02) which gained 0.7 mm. The reduction of P. gingivalis from treated periodontitis sites as detected by a significant decrease in BAPNA w/ gly-gly may prove to be a valuable marker for periodontal disease activity.

Benzoylarginine Nitroanilide↗
PubMed1998

A peptide domain on gingipain R which confers immunity against Porphyromonas gingivalis infection in mice.

The cysteine proteinases referred to as gingipains R (gingipain R1 and gingipain R2) and gingipain K produced by Porphyromonas gingivalis are virulence factors of this periodontal pathogen which likely act by interrupting host defense mechanisms and by participating in the penetration and destruction of host connective tissue. To examine the effect of immunization with gingipains R on the ability of P. gingivalis to colonize and invade in the mouse chamber model, BALB/c mice were immunized intraperitoneally with the 95-kDa gingipain R1, the 50-kDa gingipain R2, or multiple antigenic peptide (MAP)-conjugated gingipain R-derived peptides and then challenged with P. gingivalis. Immunization of mice with the 95-kDa gingipain R1, the 50-kDa gingipain R2, or a peptide derived from the N-terminal sequence of the catalytic domain of gingipains R (peptide A) followed by challenge with P. gingivalis A7436 resulted in protection from P. gingivalis invasion. In contrast, immunization with peptides corresponding to either a sequence encompassing the catalytic cysteine residue of gingipains R (peptide B) or an identical sequence within the catalytic domains of gingipain R1 and gingipain K (peptide C), followed by challenge with P. gingivalis, did not protect animals, nor did immunization with a peptide corresponding to sequences within the adhesion/hemagglutinin domain of gingipain R1 (peptide D) which have been shown to be directly involved in the hemagglutinin activity of gingipain R1. However, the immunoglobulin G (IgG) titer obtained following immunization with peptide D was comparable to that obtained following immunization with the N-terminal peptide (peptide A). Competitive enzyme-linked immunosorbent assays, using either the 95-kDa gingipain R1 or gingipain K as the competing soluble antigen, indicated that 42 and 53% of the antibodies induced by immunization with heat-killed bacteria recognize gingipain R1 and gingipain K, respectively; however, even at very high concentrations, the 50-kDa gingipain R2 did not hinder IgG binding to P. gingivalis. These results indicate that antibodies directed to the amino-terminal region of the catalytic domain of gingipains R are capable of inducing a protective immune response against P. gingivalis infection in the mouse chamber model.

Adhesins, Bacterial↗
PubMed1998

Purification and characterization of an arginine-specific peptidase from ragweed (Ambrosia artemisiifolia) pollen.

Ragweed (Ambrosia artemisiifolia) is clinically the most important source of seasonal aeroallergens, as it is responsible for the majority and most severe cases of hay fever (allergic rhinitis). Extracts from pollen grains have been shown to contain numerous proteins with various functions, including a novel serine proteolytic enzyme with chymotrypsin-like specificity that has been previously described (J. Biol. Chem. 1996; 271:26227-26232). We now report the isolation and properties of a second, trypsin-like enzyme with a molecular mass near 80 kD, from ragweed pollen extracts. This enzyme has a blocked N-terminus, a pH optimum near 9.0, and requires Ca2+ for stability and activity, but not reducing agents. The enzyme is inhibited by diisopropyl fluorophosphate, a general serine class proteinase inhibitor, and more specifically by N-p-tosyl-L-lysine chloromethyl ketone. Activity toward protein substrates was not detected, but various synthetic substrates and small biologically active peptides were efficiently cleaved, with a strong preference for Arg in the P1 position and either Arg or Gly in the P2 position. This specificity was confirmed through inhibition studies with both peptidyl chloromethyl ketone and organophosphate inhibitors. Significantly, atrial natriuretic peptide and angiotensin 2, whose degradation would amplify kinin activity and influence inflammatory diseases of the respiratory tract and nasal passages, were also rapidly hydrolyzed. Thus, the ragweed pollen endopeptidase may be involved in the inactivation of regulatory neuropeptides during pollen-initiated allergic reactions.

Amino Acid Sequence↗
PubMed1998

Genetic variation of Porphyromonas gingivalis genes encoding gingipains, cysteine proteinases with arginine or lysine specificity.

Porphyromonas gingivalis hemagglutinating cysteine proteinases (gingipains) are considered as important virulence factors in the development of adult periodontitis. Using Southern blot analysis it was determined that genes of three distinct but related gingipains (gingipain-R1, gingipain-R2 and gingipain-K) were present in all tested strains including HG66, ATCC 33277, W50, E-20-1, EM-3, 381, A7436, and IKG5, with a region encoding a part of the hemagglutinin domain of gingipain-R1 and gingipain-K showing considerable variability. In contrast, the loci encoding gingipain-R1 (rgp1) and gingipain-R2 (rgp2) were strongly conserved excluding the concurrent occurrence of other gingipain-R-like genes such as cpgR and agp. Significantly, no evidence could be found to support the expression of a gene coding for the putative proteinase porphypain, an enzyme suggested to have both gingipain-R and gingipain-K activity.

Adhesins, Bacterial↗
PubMed1997

Oncostatin M stimulates the expression and release of the IL-6 receptor in human hepatoma HepG2 cells.

Soluble IL-6R (sIL-6R), which lacks the transmembrane domain, has been suggested to be a potent immunomodulator of IL-6 biologic activity. In this study, the ability of cells of hepatic origin to generate the sIL-6R was investigated. It was found that oncostatin M alone or in combination with the glucocorticoid analogue dexamethasone significantly up-regulated IL-6R release. Oncostatin M appeared to generate the sIL-6R primarily through an alternative splicing mechanism. Since sIL-6R is able to form biologically active complexes with IL-6, the release of the sIL-6R from hepatocytes may be important to sensitize cells lacking the membrane-bound receptor, particularly during an acute phase reaction.

Alternative Splicing↗