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J Travis

Publications and source records attributed to J Travis.

At least 55 records · Page 3Linked to original sources
PubMed1999

Transposition of the endogenous insertion sequence element IS1126 modulates gingipain expression in Porphyromonas gingivalis.

We have previously reported on a Tn4351-generated mutant of Porphyromonas gingivalis (MSM-3) which expresses enhanced arginine-specific proteinase activity and does not utilize hemin or hemoglobin for growth (C. A. Genco et al., Infect. Immun. 63:2459-2466, 1995). In the process of characterizing the genetic lesion in P. gingivalis MSM-3, we have determined that the endogenous P. gingivalis insertion sequence element IS1126 is capable of transposition within P. gingivalis. We have also determined that IS1126 transposition modulates the transcription of the genes encoding the lysine-specific proteinase, gingipain K (kgp) and the arginine-specific proteinase, gingipain R2 (rgpB). Sequence analysis of P. gingivalis MSM-3 revealed that Tn4351 had inserted 60 bp upstream of the P. gingivalis endogenous IS element IS1126. Furthermore, P. gingivalis MSM-3 exhibited two additional copies of IS1126 compared to the parental strain A7436. Examination of the first additional IS1126 element, IS1126(1), indicated that it has inserted into the putative promoter region of the P. gingivalis kgp gene. Analysis of total RNA extracted from P. gingivalis MSM-3 demonstrated no detectable kgp transcript; likewise, P. gingivalis MSM-3 was devoid of lysine-specific proteinase activity. The increased arginine-specific proteinase activity exhibited by P. gingivalis MSM-3 was demonstrated to correlate with an increase in the rgpA and rgpB transcripts. The second additional IS1126 element, IS1126(2), was found to have inserted upstream of a newly identified gene, hmuR, which exhibits homology to a number of TonB-dependent genes involved in hemin and iron acquisition. Analysis of total RNA from P. gingivalis MSM-3 demonstrated that hmuR is transcribed, indicating that the insertion of IS1126 had not produced a polar effect on hmuR transcription. The hemin-hemoglobin defect in P. gingivalis MSM-3 is proposed to result from the inactivation of Kgp, which has recently been demonstrated to function in hemoglobin binding. Taken together, the results presented here demonstrate that the introduction of Tn4351 into the P. gingivalis chromosome has resulted in two previously undocumented phenomena in P. gingivalis: (i) the transposition of the endogenous insertion sequence element IS1126 and (ii) the modulation of gingipain transcription and translation as a result of IS1126 transposition.

Adhesins, Bacterial↗
PubMed1999

Purification, characterization, and sequence analysis of a potential virulence factor from Porphyromonas gingivalis, peptidylarginine deiminase.

The initiation and progression of adult-onset periodontitis has been associated with infection of the gingival sulcus by Porphyromonas gingivalis. This organism utilizes a multitude of virulence factors to evade host defenses as it establishes itself as one of the predominant pathogens in periodontal pockets. A feature common to many other oral pathogens is the production of ammonia due to its protective effect during acidic cleansing cycles in the mouth. Additionally, ammonia production by P. gingivalis has been proposed as a virulence factor due to its negative effects on neutrophil function. In this study, we describe the first purification of a peptidylarginine deiminase (PAD) from a prokaryote. PAD exhibits biochemical characteristics and properties that suggest that it may be a virulence agent. PAD deiminates the guanidino group of carboxyl-terminal arginine residues on a variety of peptides, including the vasoregulatory peptide-hormone bradykinin, to yield ammonia and a citrulline residue. The soluble protein has an apparent mass of 46 kDa, while the DNA sequence predicts a full-length protein of 61.7 kDa. PAD is optimally active at 55 degrees C, stable at low pH, and shows the greatest activity above pH 9.0. Interestingly, in the presence of stabilizing factors, PAD is resistant to limited proteolysis and retains significant activity after short-term boiling. We propose that PAD, acting in concert with arginine-specific proteinases from P. gingivalis, promotes the growth of the pathogen in the periodontal pocket, initially by enhancing its survivability and then by assisting the organism in its circumvention of host humoral defenses.

Amino Acid Sequence↗
PubMed1999

Activation of protein C by arginine-specific cysteine proteinases (gingipains-R) from Porphyromonas gingivalis.

In order to determine the effect of bacterial proteinases on activation of the protein C system, a negative regulator of blood coagulation, two arginine-specific cysteine proteinases (gingipains R) from Porphyromonas gingivalis, a causative bacterium of adult periodontitis, were examined. Each enzyme activated human protein C in a dose- and incubation time-dependent manner. Interestingly, the form of enzyme being composed of a non-covalent complex containing both catalytic and adhesion domains (RgpA) produced activated protein C 14-fold more efficiently than RgpB which contained the catalytic domain alone. The kcat/Km value of RgpA was 18-fold higher than that of RgpB and comparable to that of the thrombin-thrombomodulin complex, the physiological activator of protein C. RgpA catalyzed protein C activation was augmented 1.4-fold by phospholipids, ubiquitous cell membrane components. Furthermore, RgpA, but not RgpB, could activate protein C in plasma and this resulted in a decrease of the protein C concentration in plasma, which is often observed in patients with sepsis during the development of disseminated intravascular coagulation (DIC). These data indicate that RgpA is a more potent activator of protein C than RgpB and suggest that only the former enzyme can cause protein C activation in vivo. The present study further suggests that bacterial proteinases may possibly contribute to the consumption of plasma protein C which predisposes to DIC and/or promotes a thrombotic tendency towards DIC in sepsis.

Adhesins, Bacterial↗
PubMed1999

Comparative cleavage sites within the reactive-site loop of native and oxidized alpha1-proteinase inhibitor by selected bacterial proteinases.

Human alpha1-proteinase inhibitor (alpha1-PI) is responsible for the tight control of neutrophil elastase activity which, if down regulated, may cause local excessive tissue degradation. Many bacterial proteinases can inactivate alpha1-PI by hydrolytic cleavage within its reactive site, resulting in the down regulation of elastase, and this mechanism is likely to contribute to the connective tissue damage often associated with bacterial infections. Another pathway of the inactivation of alpha1-PI is reversible and involves oxidation of a critical active-site methionine residue that may influence inhibitor susceptibility to proteolytic inactivation. Hence, the aim of this work was to determine whether this oxidation event might affectthe rate and pattern of the cleavage of the alpha1-PI reactive-site loop by selected bacterial proteinases, including thermolysin, aureolysin, serralysin, pseudolysin, Staphylococcus aureus serine proteinase, streptopain, and periodontain. A shift of cleavage specificity was observed after alpha1-PI oxidation, with a preference for the Glu354-Ala355 bond by most of the proteinases tested. Only aureolysin and serralysin cleave the oxidized form of alpha1-PI faster than the native inhibitor, suggesting that bacteria which secrete these metalloproteinases may specifically take advantage of the host defense oxidative mechanism to accelerate elimination of alpha1-PI and, consequently, tissue degradation by neutrophil elastase.

Amino Acid Sequence↗
PubMed1998

A novel mechanism for bradykinin production at inflammatory sites. Diverse effects of a mixture of neutrophil elastase and mast cell tryptase versus tissue and plasma kallikreins on native and oxidized kininogens.

Coprocessing of kininogens by a mixture of human mast cell tryptase and neutrophil elastase was explored as a potential substitute for the kallikrein-dependent pathway for kinin generation during inflammation. Tryptase easily excised bradykinin from the synthetic heptadecapeptide, ISLMKRPPGFSPFRSSR, but was unable to produce significant amounts of kinin by proteolysis of kininogens. However, a mixture of tryptase and elastase released bradykinin from each protein with a yield comparable to that of human plasma kallikrein. Significantly, neither plasma nor tissue kallikrein was able to effectively process N-chlorosuccinimide-oxidized high molecular weight kininogen, an effect attributed to the oxidation of a methionine residue upstream from the N terminus of the kinin domain. In support of these results the model heptadecapetide, ISL(MO)KRPPGFSPFRSSR, was also resistant to hydrolysis by either kallikrein. In contrast, the release of bradykinin from oxidized peptide or protein substrates by the tryptase/elastase mixture was not altered. Because kininogen modification may occur at inflammatory sites, as a result of the oxidative burst of recruited neutrophils and macrophages, these results suggest an alternative pathway for kinin production and the necessity for the novel utilization of two specific proteinases known to be released from these cells during inflammatory episodes.

Amino Acid Sequence↗
PubMed1998

Modulation of interleukin-8 activity by gingipains from Porphyromonas gingivalis: implications for pathogenicity of periodontal disease.

Gingipains are the major cysteine proteinases synthesized by Porphyromonas gingivalis which, in soluble form, are able to initially convert IL-8 (77 amino acid residues) to a more potent species truncated at the amino terminus, followed by slow degradation and destruction of chemokine biological activity. In contrast, the same enzymes when associated with bacterial outer-membrane blebs (vesicles), instantly degrade this chemokine. This division of enhancing and inactivating activity between soluble and membrane-bound gingipains can cause the compartmentalization of pro- and anti-inflammatory reactions to distal and proximal positions from bacterial plaque, respectively, which may explain why, despite the massive neutrophil accumulation at periodontitis sites, there is no elimination of infection.

Adhesins, Bacterial↗
PubMed1998

Orally active indole N-oxide PDE4 inhibitors.

This communication describes the synthesis and in vitro and in vivo evaluation of a novel potent series of phosphodiesterase type (IV) (PDE4) inhibitors. Several of the compounds presented possess low nanomolar IC50's for PDE4 inhibition and excellent in vivo activity for inhibition of TNF-alpha levels in LPS challenged mice (mouse endotoxemia model). Emesis studies (dog) and efficacy in a SCW arthritis model for the most potent PDE4 inhibitors are presented.

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

Amino-acid sequence and three-dimensional structure of the Staphylococcus aureus metalloproteinase at 1.72 A resolution.

BACKGROUND: Aureolysin is an extracellular zinc-dependent metalloproteinase from the pathogenic bacterium Staphylococcus aureus. This enzyme exhibits in vitro activity against several molecules of biological significance for the host, indicating that it is involved in the pathology of staphylococcal diseases. RESULTS: Here we report the amino-acid sequence and inhibitor-free X-ray crystal structure of aureolysin, a member of the thermolysin family of zinc-dependent metalloproteinases. This enzyme, which binds one zinc and three calcium ions, comprises a single chain of 301 amino acids that consists of a beta-strand-rich upper domain and an alpha-helix-rich lower domain. CONCLUSIONS: The overall structure of aureolysin is very similar to that of the other three members of this family whose structures are known - thermolysin (TLN) from Bacillus thermoproteolyticus, neutral protease (NP) from Bacillus cereus and elastase (PAE) from Pseudomonas aeruginosa. But an important difference has been encountered: in contrast to what has been observed in the other three members of this family (TLN, NP and PAE), inhibitor-free aureolysin displays a 'closed' active site cleft conformation. This new structure therefore raises questions about the universality of the hinge-bending motion model for the neutral metalloproteinases.

Amino Acid Sequence↗
PubMed1998

Cleavage and activation of proteinase-activated receptor-2 on human neutrophils by gingipain-R from Porphyromonas gingivalis.

Gingipain-R, the major arginine-specific proteinase from Porphyromonas gingivalis, a causative agent of adult periodontal disease, was found to cleave a model peptide representing the cleavage site of proteinase-activated receptor-2 (PAR-2), a G-protein-coupled receptor found on the surface of neutrophils. The bacterial proteinase was also shown to induce an increase in the intracellular calcium concentration of enzyme-treated neutrophils, most probably due to PAR-2 activation. This response by neutrophils to gingipain-R may be a mechanism for the development of inflammation associated with periodontal disease.

Adhesins, Bacterial↗
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↗