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J Travis

Publications and source records attributed to J Travis.

At least 37 records · Page 2Linked to original sources
PubMed2001

Salivary histatin 5 is an inhibitor of both host and bacterial enzymes implicated in periodontal disease.

One of the salient features of periodontitis and gingivitis is the increase in the levels of bacterial and host-derived proteolytic enzymes in oral inflammatory exudates. This study evaluated the potential of histatin 5, a 24-residue histidine-rich salivary antimicrobial protein, to inhibit these enzymes. Using biotinylated gelatin as a substrate, histatin 5 was found to inhibit the activity of the host matrix metalloproteinases MMP-2 and MMP-9 with 50% inhibitory concentrations (IC50s) of 0.57 and 0.25 microM, respectively. To localize the domain responsible for this inhibition, three peptides containing different regions of histatin 5 were synthesized and tested as inhibitors of MMP-9. Peptides comprising residues 1 to 14 and residues 4 to 15 of histatin 5 showed much lower inhibitory activities (IC50, 21.4 and 20.5 microM, respectively), while a peptide comprising residues 9 to 22 showed identical activity to histatin 5 against MMP-9. These results point to a functional domain localized in the C-terminal part of histatin 5. To evaluate the effect of histatin 5 on bacterial proteases, a detailed characterization of histatin 5 inhibition of gingipains from Porphyromonas gingivalis was carried out using purified Arg- and Lys-specific enzymes. Kinetic analysis of the inhibition of the Arg-gingipain revealed that histatin 5 is a competitive inhibitor, affecting only the Km with a K(i) of 15 microM. In contrast, inhibition of Lys-gingipain affected both the Km and Vmax, suggesting that both competitive and noncompetitive competitive processes underlie this inhibition. The inhibitory activity of histatin 5 against host and bacterial proteases at physiological concentrations points to a new potential biological function of histatin in the oral cavity.

Adhesins, Bacterial↗
PubMed2001

Arginine-specific protease from Porphyromonas gingivalis activates protease-activated receptors on human oral epithelial cells and induces interleukin-6 secretion.

Periodontitis is a chronic inflammatory disease affecting oral tissues. Oral epithelial cells represent the primary barrier against bacteria causing the disease. We examined the responses of such cells to an arginine-specific cysteine proteinase (RgpB) produced by a causative agent of periodontal disease, Porphyromonas gingivalis. This protease caused an intracellular calcium transient in an oral epithelial cell line (KB), which was dependent on its enzymatic activity. Since protease-activated receptors (PARs) might mediate such signaling, reverse transcription-PCR was used to characterize the range of these receptors expressed in the KB cells. The cells were found to express PAR-1, PAR-2, and PAR-3, but not PAR-4. In immunohistochemical studies, human gingival epithelial cells were found to express PAR-1, PAR-2, and PAR-3 on their surface, but not PAR-4, indicating that the cell line was an effective model for the in vivo situation. PAR-1 and PAR-2 expression was confirmed in intracellular calcium mobilization assays by treatment of the cells with the relevant receptor agonist peptides. Desensitization experiments strongly indicated that signaling of the effects of RgpB was occurring through PAR-1 and PAR-2. Studies with cells individually transfected with each of these two receptors confirmed that they were both activated by RgpB. Finally, it was shown that, in the oral epithelial cell line, PAR activation by the bacterial protease-stimulated secretion of interleukin-6. This induction of a powerful proinflammatory cytokine suggests a mechanism whereby cysteine proteases from P. gingivalis might mediate inflammatory events associated with periodontal disease on first contact with a primary barrier of cells.

Adhesins, Bacterial↗
PubMed2001

Novel extracellular x-prolyl dipeptidyl-peptidase (DPP) from Streptococcus gordonii FSS2: an emerging subfamily of viridans Streptococcal x-prolyl DPPs.

Streptococcus gordonii is generally considered a benign inhabitant of the oral microflora, and yet it is a primary etiological agent in the development of subacute bacterial endocarditis (SBE), an inflammatory state that propagates thrombus formation and tissue damage on the surface of heart valves. Strain FSS2 produced several extracellular aminopeptidase and fibrinogen-degrading activities during growth in culture. In this report we describe the purification, characterization, and cloning of a serine class dipeptidyl-aminopeptidase, an x-prolyl dipeptidyl-peptidase (Sg-xPDPP, for S. gordonii x-prolyl dipeptidyl-peptidase), produced in a pH-controlled batch culture. Purification of this enzyme by anion exchange, gel filtration, and hydrophobic interaction chromatography yielded a protein monomer of approximately 85 kDa, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) under denaturing conditions. However, under native conditions, the protein appeared to be a homodimer on the basis of gel filtration and PAGE. Kinetic studies indicated that purified enzyme had a unique and stringent x-prolyl specificity that is comparable to both the dipeptidyl-peptidase IV/CD26 and lactococcal x-prolyl dipeptidyl-peptidase families. Nested PCR cloning from an S. gordonii library enabled the isolation and sequence analysis of the full-length gene. A 759-amino-acid polypeptide with a theoretical molecular mass of 87,115 Da and a calculated pI of 5.6 was encoded by this open reading frame. Significant homology was found with the PepX gene family from Lactobacillus and Lactococcus spp. and putative x-prolyl dipeptidyl-peptidases from other streptococcal species. Sg-xPDPP may serve as a critical factor for the sustained bacterial growth in vivo and furthermore may aid in the proteolysis of host tissue that is commonly observed during SBE pathology.

Amino Acid Sequence↗
PubMed2001

Degradation of host heme proteins by lysine- and arginine-specific cysteine proteinases (gingipains) of Porphyromonas gingivalis.

Porphyromonas gingivalis can use hemoglobin bound to haptoglobin and heme complexed to hemopexin as heme sources; however, the mechanism by which hemin is released from these proteins has not been defined. In the present study, using a variety of analytical methods, we demonstrate that lysine-specific cysteine proteinase of P. gingivalis (gingipain K, Kgp) can efficiently cleave hemoglobin, hemopexin, haptoglobin, and transferrin. Degradation of hemopexin and transferrin in human serum by Kgp was also detected; however, we did not observe extensive degradation of hemoglobin in serum by Kgp. Likewise the beta-chain of haptoglobin was partially protected from degradation by Kgp in a haptoglobin-hemoglobin complex. Arginine-specific gingipains (gingipains R) were also found to degrade hemopexin and transferrin in serum; however, this was observed only at relatively high concentrations of these enzymes. Growth of P. gingivalis strain A7436 in a minimal media with normal human serum as a source of heme correlated not only with the ability of the organism to degrade hemoglobin, haptoglobin, hemopexin, and transferrin but also with an increase in gingipain K and gingipain R activity. The ability of gingipain K to cleave hemoglobin, haptoglobin, and hemopexin may provide P. gingivalis with a usable source of heme for growth and may contribute to the proliferation of P. gingivalis within periodontal pockets in which erythrocytes are abundant.

Adhesins, Bacterial↗
PubMed2001

pH-regulated secretion of a glyceraldehyde-3-phosphate dehydrogenase from Streptococcus gordonii FSS2: purification, characterization, and cloning of the gene encoding this enzyme.

Streptococcus gordonii and other viridans streptococci (VS) are primary etiologic agents of infective endocarditis, despite being part of the normal oral microflora. Recently, a surface-bound glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been found on the cells of all tested streptococcal species, where it has been implicated as a virulence factor. In contrast, we observed that a soluble extracellular GAPDH was the major secreted protein from S. gordonii FSS2, an endocarditis strain. The biochemical properties and gene sequence of S. gordonii GAPDH are almost identical to those of other streptococcal GAPDHs. Growth at defined pHs showed that secretion of GAPDH is regulated by environmental pH. GAPDH was primarily surface-associated at growth pH 6.5 and shifted to > 90% secreted at growth pH 7.5. Others have identified S. gordonii promoters that are up-regulated by a pH shift similar to that experienced by organisms entering the blood stream (neutral) from the oral cavity (slightly acid). Analysis of our results suggests that secretion of GAPDH may be a similar adaptation by S. gordonii.

Adaptation, Physiological↗
PubMed2001

Isolation and characterization of a highly specific serine endopeptidase from an oral strain of Staphylococcus epidermidis.

Infection by Staphylococcus epidermidis, an opportunistic pathogen, has become a major problem due to the increased use of implanted medical devices and the growing number of patients who are therapeutically or infectiously immunosuppressed. These infections appear to proceed via modulation of the coagulation and complement systems. In this communication we describe the purification and characterization of a novel extracellular proteinase from an oral strain of S. epidermidis that can degrade fibrinogen, complement protein C5, and several other proteins. This proteinase has a strong preference for cleavage after glutamic acid residues, but not after aspartic acid. The S. epidermidis enzyme may be a multifunctional protein which not only provides this organism with both the ability to evade the complement defense system and to dysregulate the coagulation cascade, but also supplies nutrients for its growth through the degradation of Glu-rich proteins.

Amino Acid Sequence↗
PubMed2001

Arginine-specific cysteine proteinase from porphyromonas gingivalis as a convenient tool in protein chemistry.

RgpB, a cysteine proteinase produced by Porphyromonas gingivalis, exhibits proteolytic activity selectively directed against peptide bonds containing an arginine residue in the P1 position. Here we show that this enzyme can be used for very efficient and specific protein cleavage. RgpB is highly active even at high concentrations of denaturing agents, including urea (up to 6 M) and SDS (0.1%), both of them being commonly used for solubilization of insoluble proteins and peptides. Moreover, RgpB is able to digest polypeptide chains in buffers supplemented with 1% Triton X-100, 1% octyl or decylpyranoside, detergents employed for the enzymatic digestion of proteins transferred onto nitrocellulose membranes. These features render RgpB a suitable tool for use in protein chemistry.

Amino Acid Sequence↗
PubMed2000

Porphyromonas gingivalis DPP-7 represents a novel type of dipeptidylpeptidase.

A novel dipeptidylpeptidase (DPP-7) was purified from the membrane fraction of Porphyromonas gingivalis. This enzyme, with an apparent molecular mass of 76 kDa, has the specificity for both aliphatic and aromatic residues in the P1 position. Although it belongs to the serine class of peptidases, it does not resemble other known dipeptidylpeptidases. Interestingly, the amino acid sequence around the putative active site serine residue shows significant similarity to the C-terminal region of the Staphylococcus aureus V-8 endopeptidase. The genes encoding homologues of DPP-7 were found in genomes of Xylella fastidiosa, Shewanella putrefaciens, and P. gingivalis. It is likely that at least in P. gingivalis, DPP-7 and its homologue, in concert with other di- and tripeptidases, serve nutritional functions by providing dipeptides to this asaccharolytic bacterium.

Amino Acid Sequence↗
PubMed2000

Mechanism of interleukin-1- and tumor necrosis factor alpha-dependent regulation of the alpha 1-antichymotrypsin gene in human astrocytes.

The expression of alpha(1)-antichymotrypsin (ACT) is significantly enhanced in affected brain regions in Alzheimer's disease. This serine proteinase inhibitor specifically colocalizes with filamentous beta-amyloid deposits and recently has been shown to influence both formation and destabilization of beta-amyloid fibrils. In the brain, ACT is expressed in astrocytes, and interleukin-1 (IL-1), tumor necrosis factor alpha (TNF), oncostatin M (OSM), and IL-6/soluble IL-6 receptor complexes control synthesis of this inhibitor. Here, we characterize a molecular mechanism responsible for both IL-1 and TNF-induced expression of ACT gene in astrocytes. We identify the 5' distal IL-1/TNF-responsive enhancer of the ACT gene located 13 kb upstream of the transcription start site. This 413-bp-long enhancer contains three elements, two of which bind nuclear factor kB (NF-kB) and one that binds activating protein 1 (AP-1). All of these elements contribute to the full responsiveness of the ACT gene to both cytokines, as determined by deletion and mutational analysis. The 5' NF-kB high-affinity binding site and AP-1 element contribute most to the enhancement of gene transcription in response to TNF and IL-1. In addition, we demonstrate that the 5' untranslated region of the ACT mRNA does not contribute to cytokine-mediated activation. Finally, we find that overexpression of the NF-kB inhibitor (IkB) totally inhibits any activation mediated by the newly identified IL-1/TNF enhancer of the ACT gene.

5' Untranslated Regions↗
PubMed2000

Bacterial proteinases as targets for the development of second-generation antibiotics.

The emergence of bacterial pathogen resistance to common antibiotics strongly supports the necessity to develop alternative mechanisms for combating drug-resistant forms of these infective organisms. Currently, few pharmaceutical companies have attempted to investigate the possibility of interrupting metabolic pathways other than those that are known to be involved in cell wall biosynthesis. In this review, we describe multiple, novel roles for bacterial proteinases during infection using, as a specific example, the enzymes from the organism Porphyromonas gingivalis, a periodontopathogen, which is known to be involved in the development and progression of periodontal disease. In this manner, we are able to justify the concept of developing synthetic inhibitors against members of this class of enzymes as potential second-generation antibiotics. Such compounds could not only prove valuable in retarding the growth and proliferation of bacterial pathogens but also lead to the use of this class of inhibitors against invasion by other infective organisms.

Amino Acid Sequence↗
PubMed2000

Comparison of pathogenic properties between two types of arginine-specific cysteine proteinases (gingipains-R) from Porphyromonas gingivalis.

Two major arginine-specific cysteine proteinases (gingipains R) from Porphyromonas gingivalis have been compared with regard to their potential participation in the pathology of periodontal disease. Both the high and low molecular mass forms, HRgpA and RgpB, cleaved oligopeptide fluorogenic substrates at the P1-arginine residue with essentially identical specificity but different efficiencies, with HRgpA being about 1.5 to seven-fold less potent than RgpB. In contrast HRgpA, which occurs as a non-covalent complex of catalytic and hemagglutinin/adhesion domains, was about two-fold more active than RgpB in degrading fibrinogen and fibrin, while both enzymes activated prekallikrein with similar efficiency. These data indicate the likelihood that both activities could be involved in both the bleeding tendency and production of gingival crevicular fluid, which occur at infected periodontitis sites. Significantly, however, is the fact that HRgpA, but not RgpB, was able to bind phospholipids in the presence of calcium ions, the effect dramatically enhancing the activation of clotting factors by this proteinase. This suggests that HRgpA may play a more important role in the virulence of Porphyromonas gingivalis, relative to RgpB, almost certainly because of the presence of the hemagglutinin/adhesion domain which can bind phospholipid and apparently modulate enzyme activity.

Adhesins, Bacterial↗
PubMed2000

The role of bacterial and host proteinases in periodontal disease.

It is abundantly obvious that the uncontrolled degradation and/or activation of host defense pathways is the major pathway by which the periodontal pathogen P. gingivalis promotes its growth and proliferation. By being able to shed host receptors, degrade cytokines, and activate coagulation, complement, and kallikrein/kinin pathways it is clear that this organism has found a mechanism(s) to evade host defense and at the same time develop a system for cannibalizing host proteins for its own nutritional usage (Fig 2). Thus, it seems only logical that the development of inhibitors against these bacterial proteinases would be a useful method for negating their activities and making such pathogens more susceptible to attack by host phagocyte cells. In this respect, the structure of the truncated form of RGP has just been elucidated. Thus, it should only be a question of time before inhibitors to this enzyme will be developed and, hopefully, be used to reduce the pathologies associated with the development of periodontitis and/or eliminate the disease altogether.

Adhesins, Bacterial↗
PubMed2000

Experimental evolution in Heterandria formosa, a livebearing fish: group selection on population size.

Group selection has historically been an important and controversial subject in evolutionary biology. There is now a compelling body of evidence, both theoretical and experimental, that group selection not only can be effective, but can be effective in situations when individual selection is not. However, experiments in which true population-level traits have been shown to evolve in response to group selection are currently limited to two species of flour beetle in the genus Tribolium and RNA viruses. Here we report the results of an experiment wherein we imposed group selection via differential extinction for increased and decreased population size at 6-week intervals, a true population-level trait, in the poeciliid fish Heterandria formosa. In contrast to most other group selection experiments, we observed no evolutionary response after six rounds of group selection in either the up- or down-selected lines. Populational heritability for population size was low, if not actually negative. Our results suggest that group selection via differential extinction may be effective only if population sizes are very small and/or migration rates are low.

Animals↗
PubMed2000

Unaltered expression of the major protease genes in a non-virulent recA-defective mutant of Porphyromonas gingivalis W83.

Porphyromonas gingivalis FLL32, a recA mutant, was isolated during construction of a recA defective mutant of P. gingivalis W83 by allelic exchange mutagenesis. In contrast to W83 and FLL33, the typical recA- mutant previously reported, FLL32 was non-pigmented, lacked beta-hemolytic activity on blood agar and produced significantly less proteolytic activity. The proteolytic activity in FLL32 was mostly soluble. Expression of the rgpA, rgpB and kgp protease genes was unaltered in FLL32 when compared to FLL33 and the wild-type strain. FLL32 exhibited reduced virulence in a murine model and partially protected the animals immunized with that strain against a subsequent lethal challenge by the wild-type strain. These results indicate that the reduced level of proteolytic activity in FLL32 may be due to a defect in the processing of the proteases. Further, immunization with a non-virulent recA defective mutant of P. gingivalis can partially protect against a lethal wild-type challenge. The results from this study suggest that the recA locus may be involved in expression and regulation of proteolytic activity in P. gingivalis.

Animals↗
PubMed2000

Role of bacterial proteinases in matrix destruction and modulation of host responses.

Recently accumulated large bodies of evidence have strongly implicated proteolytic enzymes released by subgingival plaque bacteria in the pathogenicity of periodontal disease. With regard to proteolytic power, however, the contribution from different microbial species considered as periodontal pathogens is not equal. Two of these bacteria, P. gingivalis and T. denticola, have developed an elaborate proteolytic systems composed of several surface-located or secreted enzymes, which apparently serve a role to provide bacteria with nutrients in the form of small peptides and amino acids. Of these two species, proteinases of P. gingivalis are the most intensively studied, and during the last decade an impressive array of information has been accumulated with respect to the biochemical characterization of purified proteinases and structure of the genes encoding them, the regulation of expression and the effects of these enzymes on host systems. In addition, studies on proteinase-deficient isogenic mutants has shed light on both their housekeeping functions and potential role(s) in the pathogenicity of periodontitis. Among several proteinases produced by P. gingivalis, the cysteine proteinases, referred to as gingipains, are clearly in the spotlight. They are the subject of several recent reviews and generally considered as the major virulence factors of this periodontal pathogen (59, 105, 139, 182, 183, 186, 281, 284, 286, 289). Gingipains seem to be key players in subverting host defense systems with, significantly, the complement and neutrophils being the main target. In addition, through uncontrolled activation of kallikrein/kinin pathway and coagulation cascade they contribute to local generation of bradykinin and thrombin, two synergistically working pro-inflammatory reagents with a strongly, although indirectly, stimulatory effect on bone resorption. Furthermore, the ability to interact with the cytokine networking systems has the potential to dysregulate the local inflammatory reaction. Finally, gingipains have a strong effect on mechanisms controlling host matrix metalloproteinase activity at the level of gene expression and zymogen activation (Fig. 10). Collectively, at the periodontal lesion site, the non-restrained action of gingipains, supported by other proteinases locally produced by subgingival plaque bacteria, would dysregulate most mechanisms controlling inflammatory reaction. Although successful in limiting infection to the periodontium, the ultimate effect of uncontrolled inflammatory processes would be the destruction of periodontal connective tissue, certainly the hallmark of periodontitis.

Adhesins, Bacterial↗
PubMed2000

Direct and correlated responses to artificial selection on acute thermal stress tolerance in a livebearing fish.

Tradeoffs in performance or fitness across environments have important implications regarding the nature of evolutionary constraints. It remains controversial whether tradeoffs such as these reflect genetic correlations that are genuine evolutionary constraints. However, if such long-term genetic constraints do exist, they must be due to underlying pleiotropy such that alleles that confer high performance in one environment invariably confer low performance in another. The distribution of genetic correlations within and among populations can provide insight about the existence of such pleiotropic tradeoffs. The long-term association of certain teleost fish taxa with particular abiotic environments suggests that tradeoffs in performance across environments have constrained the geographic distribution of those taxa. Here we report the results of an experiment in which we artificially selected on acute heat- and cold-stress tolerance in two stocks of the poeciliid fish Heterandria formosa from source populations with different thermal histories. Unexpectedly, we observed no direct responses to selection. Under certain conditions, fish from the different source populations differed significantly in cold tolerance, but not in heat tolerance. The results suggest there are no strong pleiotropic tradeoffs between heat- and cold-stress tolerance in these populations.

Animals↗