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Sample records for porphyromonas gingivalis haemoglobin

  1. Putative respiratory chain of Porphyromonas gingivalis.

    PubMed

    Meuric, Vincent; Rouillon, Astrid; Chandad, Fatiha; Bonnaure-Mallet, Martine

    2010-05-01

    The electron transfer chain in Porphyromonas gingivalis, or periodontopathogens, has not yet been characterized. P. gingivalis, a strict anaerobic bacteria and the second colonizer of the oral cavity, is considered to be a major causal agent involved in periodontal diseases. Primary colonizers create a favorable environment for P. gingivalis growth by decreasing oxygen pressure. Oxygen does not appear to be the final electron acceptor of the respiratory chain. Fumarate and cytochrome b have been implicated as major components of the respiratory activity. However, the P. gingivalis genome shows many other enzymes that could be implicated in aerobic or nitrite respiration. Using bioinformatic tools and literature studies of respiratory pathways, the ATP synthesis mechanism from the sodium cycle and nutrients metabolism, the putative respirasome of P. gingivalis has been proposed.

  2. A combination of both arginine- and lysine-specific gingipain activity of Porphyromonas gingivalis is necessary for the generation of the micro-oxo bishaem-containing pigment from haemoglobin.

    PubMed Central

    Smalley, John W; Thomas, Michael F; Birss, Andrew J; Withnall, Robert; Silver, Jack

    2004-01-01

    The black pigment of Porphyromonas gingivalis is composed of the mu-oxo bishaem complex of Fe(III) protoporphyrin IX (mu-oxo oligomer, dimeric haem), namely [Fe(III)PPIX]2O. P. gingivalis W50 and Rgp (Arg-gingipain)- and Kgp (Lys-gingipain)-deficient mutants K1A, D7, E8 and W501 [Aduse-Opoku, Davies, Gallagher, Hashim, Evans, Rangarajan, Slaney and Curtis (2000) Microbiology 146, 1933-1940] were grown on horse blood/agar for 14 days and examined for the production of mu-oxo bishaem. Mu-oxo Bishaem was detected by UV-visible, Mössbauer and Raman spectroscopies in wild-type W50 and in the black-pigmented RgpA- and RgpB-deficient mutants (W501 and D7 respectively), whereas no haem species were detected in the straw-coloured colonies of Kgp-deficient strain K1A. The dark brown pigment of the double RgpA/RgpB knockout mutant (E8) was not composed of mu-oxo bishaem, but of a high-spin monomeric Fe(III) protoporphyrin IX species (possibly a haem-albumin complex). In vitro incubation of oxyhaemoglobin with cells of the W50 strain and the RgpA- and RgpB-deficient mutants (W501 and D7) resulted in the formation of mu-oxo bishaem via methaemoglobin as an intermediate. Although the Kgp-deficient strain K1A converted oxyhaemoglobin into methaemoglobin, this was not further degraded into mu-oxo bishaem. The double RgpA/RgpB knockout was also not capable of producing mu-oxo bishaem from oxyhaemoglobin, but instead generated a haemoglobin haemichrome. Inhibition of Arg-X protease activity of W50, W501, D7 and K1A with leupeptin, under conditions where Lys-X protease activity was unaffected, prevented the production of mu-oxo bishaem from oxyhaemoglobin, but resulted in the formation of a haemoglobin haemichrome. These results show that one or both of RgpA and RgpB gingipains, in addition to the lysine-specific gingipain, is necessary for the production of mu-oxo bishaem from haemoglobin by whole cells of P. gingivalis. PMID:14741050

  3. Major neutrophil functions subverted by Porphyromonas gingivalis.

    PubMed

    Olsen, Ingar; Hajishengallis, George

    2016-01-01

    Polymorphonuclear leukocytes (neutrophils) constitute an integrated component of the innate host defense in the gingival sulcus/periodontal pocket. However, the keystone periodontal pathogen Porphyromonas gingivalis has in the course of evolution developed a number of capacities to subvert this defense to its own advantage. The present review describes the major mechanisms that P. gingivalis uses to subvert neutrophil homeostasis, such as impaired recruitment and chemotaxis, resistance to granule-derived antimicrobial agents and to the oxidative burst, inhibition of phagocytic killing while promoting a nutritionally favorable inflammatory response, and delay of neutrophil apoptosis. Studies in animal models have shown that at least some of these mechanisms promote the dysbiotic transformation of the periodontal polymicrobial community, thereby leading to inflammation and bone loss. It is apparent that neutrophil-P. gingivalis interactions and subversion of innate immunity are key contributing factors to the pathogenesis of periodontal disease. PMID:26993626

  4. Major neutrophil functions subverted by Porphyromonas gingivalis

    PubMed Central

    Olsen, Ingar; Hajishengallis, George

    2016-01-01

    Polymorphonuclear leukocytes (neutrophils) constitute an integrated component of the innate host defense in the gingival sulcus/periodontal pocket. However, the keystone periodontal pathogen Porphyromonas gingivalis has in the course of evolution developed a number of capacities to subvert this defense to its own advantage. The present review describes the major mechanisms that P. gingivalis uses to subvert neutrophil homeostasis, such as impaired recruitment and chemotaxis, resistance to granule-derived antimicrobial agents and to the oxidative burst, inhibition of phagocytic killing while promoting a nutritionally favorable inflammatory response, and delay of neutrophil apoptosis. Studies in animal models have shown that at least some of these mechanisms promote the dysbiotic transformation of the periodontal polymicrobial community, thereby leading to inflammation and bone loss. It is apparent that neutrophil–P. gingivalis interactions and subversion of innate immunity are key contributing factors to the pathogenesis of periodontal disease. PMID:26993626

  5. Pyocycanin, a Contributory Factor in Haem Acquisition and Virulence Enhancement of Porphyromonas gingivalis in the Lung

    PubMed Central

    Benedyk, Malgorzata; Byrne, Dominic P.; Glowczyk, Izabela; Potempa, Jan; Olczak, Mariusz; Olczak, Teresa; Smalley, John W.

    2015-01-01

    Several recent studies show that the lungs infected with Pseudomonas aeruginosa are often co-colonised by oral bacteria including black-pigmenting anaerobic (BPA) Porphyromonas species. The BPAs have an absolute haem requirement and their presence in the infected lung indicates that sufficient haem, a virulence up-regulator in BPAs, must be present to support growth. Haemoglobin from micro-bleeds occurring during infection is the most likely source of haem in the lung. Porphyromonas gingivalis displays a novel haem acquisition paradigm whereby haemoglobin must be firstly oxidised to methaemoglobin, facilitating haem release, either by gingipain proteolysis or capture via the haem-binding haemophore HmuY. P. aeruginosa produces the blue phenazine redox compound, pyocyanin. Since phenazines can oxidise haemoglobin, it follows that pyocyanin may also facilitate haem acquisition by promoting methaemoglobin production. Here we show that pyocyanin at concentrations found in the CF lung during P. aeruginosa infections rapidly oxidises oxyhaemoglobin in a dose-dependent manner. We demonstrate that methaemoglobin formed by pyocyanin is also susceptible to proteolysis by P. gingivalis Kgp gingipain and neutrophil elastase, thus releasing haem. Importantly, co-incubation of oxyhaemoglobin with pyocyanin facilitates haem pickup from the resulting methemoglobin by the P. gingivalis HmuY haemophore. Mice intra-tracheally challenged with viable P. gingivalis cells plus pyocyanin displayed increased mortality compared to those administered P. gingivalis alone. Pyocyanin significantly elevated both methaemoglobin and total haem levels in homogenates of mouse lungs and increased the level of arginine-specific gingipain activity from mice inoculated with viable P. gingivalis cells plus pyocyanin compared with mice inoculated with P. gingivalis only. These findings indicate that pyocyanin, by promoting haem availability through methaemoglobin formation and stimulating of gingipain

  6. Arginine deiminase inhibits Porphyromonas gingivalis surface attachment

    PubMed Central

    Cugini, Carla; Stephens, Danielle N.; Nguyen, Daniel; Kantarci, Alpdogan

    2013-01-01

    The oral cavity is host to a complex microbial community whose maintenance depends on an array of cell-to-cell interactions and communication networks, with little known regarding the nature of the signals or mechanisms by which they are sensed and transmitted. Determining the signals that control attachment, biofilm development and outgrowth of oral pathogens is fundamental to understanding pathogenic biofilm development. We have previously identified a secreted arginine deiminase (ADI) produced by Streptococcus intermedius that inhibited biofilm development of the commensal pathogen Porphyromonas gingivalis through downregulation of genes encoding the major (fimA) and minor (mfa1) fimbriae, both of which are required for proper biofilm development. Here we report that this inhibitory effect is dependent on enzymic activity. We have successfully cloned, expressed and defined the conditions to ensure that ADI from S. intermedius is enzymically active. Along with the cloning of the wild-type allele, we have created a catalytic mutant (ADIC399S), in which the resulting protein is not able to catalyse the hydrolysis of l-arginine to l-citrulline. P. gingivalis is insensitive to the ADIC399S catalytic mutant, demonstrating that enzymic activity is required for the effects of ADI on biofilm formation. Biofilm formation is absent under l-arginine-deplete conditions, and can be recovered by the addition of the amino acid. Taken together, the results indicate that arginine is an important signal that directs biofilm formation by this anaerobe. Based on our findings, we postulate that ADI functions to reduce arginine levels and, by a yet to be identified mechanism, signals P. gingivalis to alter biofilm development. ADI release from the streptococcal cell and its cross-genera effects are important findings in understanding the nature of inter-bacterial signalling and biofilm-mediated diseases of the oral cavity. PMID:23242802

  7. Porphyromonas gingivalis causing brain abscess in patient with recurrent periodontitis.

    PubMed

    Rae Yoo, Jeong; Taek Heo, Sang; Kim, Miyeon; Lee, Chang Sub; Kim, Young Ree

    2016-06-01

    We report an extremely rare case of Porphyromonas gingivalis causing brain abscess in a patient with recurrent periodontitis. The patient presented with right-sided homonymous hemianopsia and right hemiparesis. Emergent surgical drainage was performed and antibiotics were administered. P. gingivalis was identified from the anaerobic culture of the abscess. The clinical course of the patient improved with full recovery of the neurologic deficit. PMID:27085200

  8. Porphyromonas gingivalis genes isolated by screening for epithelial cell attachment.

    PubMed Central

    Duncan, M J; Emory, S A; Almira, E C

    1996-01-01

    Porphyromonas gingivalis is associated with chronic and severe periodontitis in adults. P. gingivalis and the other periodontal pathogens colonize and interact with gingival epithelial cells, but the genes and molecular mechanisms involved are unknown. To dissect the first steps in these interactions, a P. gingivalis expression library was screened for clones which bound human oral epithelial cells. Insert DNA from the recombinant clones did not contain homology to the P. gingivalis fimA gene, encoding fimbrillin, the subunit protein of fimbriae, but showed various degrees of homology to certain cysteine protease-hemagglutinin genes. The DNA sequence of one insert revealed three putative open reading frames which appeared to be in an operon. The relationship between P. gingivalis attachment to epithelial cells and the activities identified by the screen is discussed. PMID:8751909

  9. Treponema denticola improves adhesive capacities of Porphyromonas gingivalis.

    PubMed

    Meuric, V; Martin, B; Guyodo, H; Rouillon, A; Tamanai-Shacoori, Z; Barloy-Hubler, F; Bonnaure-Mallet, M

    2013-02-01

    Porphyromonas gingivalis, an important etiological agent of periodontal disease, is frequently found associated with Treponema denticola, an anaerobic spirochete, in pathogenic biofilms. However, interactions between these two bacteria are not well understood at the molecular level. In this study, we seek to link the influence of T. denticola on the expression of P. gingivalis proteases with its capacities to adhere and to form biofilms. The P. gingivalis genes encoding Arg-gingipain A (RgpA), Lys-gingipain (Kgp), and hemagglutinin A (HagA) were more strongly expressed after incubation with T. denticola compared with P. gingivalis alone. The amounts of the three resulting proteins, all of which contain hemagglutinin adhesion domains, were increased in culture supernatants. Moreover, incubation of P. gingivalis with T. denticola promoted static and dynamic biofilm formation, primarily via a time-dependent enhancement of P. gingivalis adhesion capacities on bacterial partners such as Streptococcus gordonii. Adhesion of P. gingivalis to human cells was also increased. These results showed that interactions of P. gingivalis with other bacterial species, such as T. denticola, induce increased adhesive capacities on various substrata by hemagglutinin adhesion domain-containing proteins. PMID:23194417

  10. Evidence for the absence of hyaluronidase activity in Porphyromonas gingivalis.

    PubMed Central

    Grenier, D; Michaud, J

    1993-01-01

    The aim of the present study was to evaluate the ability of Porphyromonas gingivalis to degrade hyaluronic acid. No hyaluronidase activity was detected using a turbidimetric method, whereas a standard plate assay showed a positive reaction for P. gingivalis. We postulated that the high proteolytic activity of P. gingivalis may account for this observation. A modified plate assay was designed to avoid false-positive reactions caused by proteolytic bacteria. The new assay, based on the formation of a water-insoluble salt between hyaluronic acid and the polyanion cetylpyridinium chloride, indicated that P. gingivalis does not have hyaluronidase activity. By this modified plate method, it was found that among 24 different oral bacterial species tested, Propionibacterium acnes and Prevotella oris were the only species that possess hyaluronidase activity. Images PMID:8394379

  11. Porphyromonas gingivalis and Treponema denticola Synergistic Polymicrobial Biofilm Development

    PubMed Central

    Zhu, Ying; Dashper, Stuart G.; Chen, Yu-Yen; Crawford, Simon; Slakeski, Nada; Reynolds, Eric C.

    2013-01-01

    Chronic periodontitis has a polymicrobial biofilm aetiology and interactions between key bacterial species are strongly implicated as contributing to disease progression. Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia have all been implicated as playing roles in disease progression. P. gingivalis cell-surface-located protease/adhesins, the gingipains, have been suggested to be involved in its interactions with several other bacterial species. The aims of this study were to determine polymicrobial biofilm formation by P. gingivalis, T. denticola and T. forsythia, as well as the role of P. gingivalis gingipains in biofilm formation by using a gingipain null triple mutant. To determine homotypic and polymicrobial biofilm formation a flow cell system was employed and the biofilms imaged and quantified by fluorescent in situ hybridization using DNA species-specific probes and confocal scanning laser microscopy imaging. Of the three species, only P. gingivalis and T. denticola formed mature, homotypic biofilms, and a strong synergy was observed between P. gingivalis and T. denticola in polymicrobial biofilm formation. This synergy was demonstrated by significant increases in biovolume, average biofilm thickness and maximum biofilm thickness of both species. In addition there was a morphological change of T. denticola in polymicrobial biofilms when compared with homotypic biofilms, suggesting reduced motility in homotypic biofilms. P. gingivalis gingipains were shown to play an essential role in synergistic polymicrobial biofilm formation with T. denticola. PMID:23990979

  12. Presence of Porphyromonas gingivalis in gingival squamous cell carcinoma

    PubMed Central

    Katz, Joseph; Onate, Mairelys D; Pauley, Kaleb M; Bhattacharyya, Indraneel; Cha, Seunghee

    2011-01-01

    Periodontal disease has been recently linked to a variety of systemic conditions such as diabetes, cardiovascular disease, preterm delivery, and oral cancer. The most common bacteria associated with periodontal disease, Porphyromonas gingivalis (P. gingivalis) has not yet been studied in the malignant gingival tissues. The objective of this study was to investigate the presence of P. gingivalis in specimens from squamous cell carcinoma patients. We have performed immunohistochemical staining to investigate the presence of P. gingivalis and Streptococcus gordonii (S. gordonii), a non invasive oral bacteria, in paraffin embedded samples of gingival squamous cell carcinoma (n=10) and normal gingiva (n=5). Staining for P. gingivalis revealed the presence of the bacteria in normal gingival tissues and gingival carcinoma, with higher levels (more than 33%, P<0.05) detected in the carcinoma samples. The staining intensity was also significantly enhanced in the malignant tissue by 2 folds (P<0.023) compared to specimens stained for the non-invasive S. gordonii. P. gingivalis is abundantly present in malignant oral epithelium suggesting a potential association of the bacteria with gingival squamous cell carcinoma. PMID:22010579

  13. Identification of essential genes of the periodontal pathogen Porphyromonas gingivalis

    PubMed Central

    2012-01-01

    Background Porphyromonas gingivalis is a Gram-negative anaerobic bacterium associated with periodontal disease onset and progression. Genetic tools for the manipulation of bacterial genomes allow for in-depth mechanistic studies of metabolism, physiology, interspecies and host-pathogen interactions. Analysis of the essential genes, protein-coding sequences necessary for survival of P. gingivalis by transposon mutagenesis has not previously been attempted due to the limitations of available transposon systems for the organism. We adapted a Mariner transposon system for mutagenesis of P. gingivalis and created an insertion mutant library. By analyzing the location of insertions using massively-parallel sequencing technology we used this mutant library to define genes essential for P. gingivalis survival under in vitro conditions. Results In mutagenesis experiments we identified 463 genes in P. gingivalis strain ATCC 33277 that are putatively essential for viability in vitro. Comparing the 463 P. gingivalis essential genes with previous essential gene studies, 364 of the 463 are homologues to essential genes in other species; 339 are shared with more than one other species. Twenty-five genes are known to be essential in P. gingivalis and B. thetaiotaomicron only. Significant enrichment of essential genes within Cluster of Orthologous Groups ‘D’ (cell division), ‘I’ (lipid transport and metabolism) and ‘J’ (translation/ribosome) were identified. Previously, the P. gingivalis core genome was shown to encode 1,476 proteins out of a possible 1,909; 434 of 463 essential genes are contained within the core genome. Thus, for the species P. gingivalis twenty-two, seventy-seven and twenty-three percent of the genome respectively are devoted to essential, core and accessory functions. Conclusions A Mariner transposon system can be adapted to create mutant libraries in P. gingivalis amenable to analysis by next-generation sequencing technologies. In silico analysis

  14. Porphyromonas gingivalis infection of oral epithelium inhibits neutrophil transepithelial migration.

    PubMed Central

    Madianos, P N; Papapanou, P N; Sandros, J

    1997-01-01

    Periodontal diseases are inflammatory disorders caused by microorganisms of dental plaque that colonize the gingival sulcus and, subsequently, the periodontal pocket. As in other mucosal infections, the host response to plaque bacteria is characterized by an influx of polymorphonuclear leukocytes (PMNs) to the gingival crevice. Neutrophil migration through the epithelial lining of the gingival pocket is thought to be the first line of defense against plaque bacteria. In order to model this phenomenon in vitro, we used the oral epithelial cell line KB and human PMNs in the Transwell system and examined the impact of Porphyromonas gingivalis-epithelial cell interactions on subsequent PMN transepithelial migration. We demonstrate here that P. gingivalis infection of oral epithelial cells failed to trigger transmigration of PMNs. Furthermore, it significantly inhibited neutrophil transmigration actively induced by stimuli such as N-formylmethionyl leucyl phenylalanine, interleukin-8 (IL-8), and the intestinal pathogen enterotoxigenic Escherichia coli. The ability of P. gingivalis to block PMN transmigration was strongly positively correlated with the ability to adhere to and invade epithelial cells. In addition, P. gingivalis attenuated the production of IL-8 and the expression of intercellular adhesion molecule 1 by epithelial cells. The ability of P. gingivalis to block neutrophil migration across an intact epithelial barrier may critically impair the potential of the host to confront the bacterial challenge and thus may play an important role in the pathogenesis of periodontal disease. PMID:9316996

  15. Porphyromonas gingivalis infection-induced tissue and bone transcriptional profiles

    PubMed Central

    Meka, Archana; Bakthavatchalu, Vasudevan; Sathishkumar, Sabapathi; Lopez, M. Cecilia; Verma, Raj K.; Wallet, Shannon M.; Bhattacharyya, Indraneel; Boyce, Brendan F.; Handfield, Martin; Lamont, Richard J.; Baker, Henry V.; Ebersole, Jeffrey L.; Lakshmyya, Kesavalu N.

    2010-01-01

    Introduction Porphyromonas gingivalis has been associated with subgingival biofilms in adult periodontitis. However, the molecular mechanisms of its contribution to chronic gingival inflammation and loss of periodontal structural integrity remain unclear. The objectives of this investigation were to examine changes in the host transcriptional profiles during a P. gingivalis infection using a murine calvarial model of inflammation and bone resorption. Methods P. gingivalis FDC 381 was injected into the subcutaneous soft tissue over the calvaria of BALB/c mice for 3 days, after which the soft tissues and calvarial bones were excised. RNA was isolated from infected soft tissues and calvarial bones and analyzed for transcript profiles using Murine GeneChip® arrays to provide a molecular profile of the events that occur following infection of these tissues. Results After P. gingivalis infection, 5517 and 1900 probe sets in the infected soft tissues and calvarial bone, respectively, were differentially expressed (P ≤ 0.05) and up-regulated. Biological pathways significantly impacted by P. gingivalis infection in tissues and calvarial bone included cell adhesion (immune system) molecules, Toll-like receptors, B cell receptor signaling, TGF-β cytokine family receptor signaling, and MHC class II antigen processing pathways resulting in proinflammatory, chemotactic effects, T cell stimulation, and down regulation of antiviral and T cell chemotactic effects. P. gingivalis-induced inflammation activated osteoclasts, leading to local bone resorption. Conclusion This is the first in vivo evidence that localized P. gingivalis infection differentially induces transcription of a broad array of host genes that differed between inflamed soft tissues and calvarial bone. PMID:20331794

  16. Porphyromonas gingivalis downregulates the immune response of fibroblasts

    PubMed Central

    2013-01-01

    Background Porphyromonas gingivalis is a key pathogen in periodontitis, an inflammatory disease leading to destruction of bone and tooth-supporting tissue. P. gingivalis possesses a number of pathogenic properties to enhance growth and survival, including proteolytic gingipains. Accumulating data shows that gingipains are involved in the regulation of host inflammatory responses. The aim of this study was to determine if P. gingivalis infection modulates the inflammatory response of fibroblasts, including the release of chemokines and cytokines. Human gingival fibroblasts or primary dermal fibroblasts were pre-stimulated with tumor-necrosis factor-α (TNF- α) and cocultured with P. gingivalis. Gingipain inhibitors were used to explore the effect of gingipains. CXCL8 levels were determined with ELISA and the relative levels of various inflammatory mediators were determined by a cytokine assay. Results TNF-α-triggered CXCL8 levels were completely abolished by viable P. gingivalis, whereas heat-killed P. gingivalis did not suppress CXCL8. Accumulation of CXCL8 was partially restored by an arginine-gingipain inhibitor. Furthermore, fibroblasts produced several inflammatory mediators, notably chemokines, all of which were suppressed by viable P. gingivalis. Conclusion These findings provide evidence that fibroblast-derived inflammatory signals are modulated by heat-instable gingipains, whereby the bacteria can escape killing by the host immune system and promote its own growth and establishment. In addition, we show that fibroblasts are important mediators of inflammation in response to infection and thereby play a crucial role in determining the nature and magnitude of the invasion of immune cells. PMID:23841502

  17. Benzamidine derivatives inhibit the virulence of Porphyromonas gingivalis

    PubMed Central

    Fröhlich, Esther; Kantyka, Tomasz; Plaza, Karolina; Schmidt, Karl-Hermann; Pfister, Wolfgang; Potempa, Jan; Eick, Sigrun

    2012-01-01

    SUMMARY We have previously shown that benzamidine-type compoundscan inhibit the activity of arginine-specific cysteine proteinases (gingipainsHRgpA and RgpB); well-known virulence factors of Porphyromonas gingivalis. They also hinder in vitro growth of this important period on to pathogenic bacterium. Apparently growth arrest is not associated with their ability to inhibit these proteases, as pentamidine, which is a 20-fold less efficient inhibitor of gingipainthan 2,6-bis-(4-amidinobenzyl)-cyclohexanone (ACH), blocked P. gingivalis growth far more effectively. To identify targets for benzamidine-derived compounds other than Arg-gingipains, and to explain their bacteriostatic effects, P. gingivalis ATCC 33277 and P. gingivalis M5-1-2 (clinical isolate) cell extracts were subjected to affinity chromatography using a benzamidine-Sepharose column to identify proteins interacting withbenzamidine. In addition to HRgpA and RgpB the analysis revealed heat-shock protein GroELas another ligand forbenzamidine. To better understand the effect ofbenzamidine-derived compounds on P. gingivalis, bacteria were exposed to benzamidine, pentamidine, ACH and heat, and the expression of gingipains and GroEL was determined. Exposure to heat and benzamidine-derived compounds caused significant increasesin GroEL, both at the mRNA and protein levels. Interestingly, despite the fact that gingipains were shown to be the main virulence factors in a fertilized egg model of infection, mortality rates were strongly reduced, not only by ACH, but also bypentamidine, a relatively week gingipain inhibitor. This effect may depend not only ongingipain inhibition but also oninteraction of benzamidine derivatives with GroEL. Therefore these compounds may find use in supportive periodontitis treatment. PMID:23279840

  18. Isolation and characterization of a minor fimbria from Porphyromonas gingivalis.

    PubMed Central

    Hamada, N; Sojar, H T; Cho, M I; Genco, R J

    1996-01-01

    We have discovered two distinctly different fimbriae expressed by the same Porphyromonas gingivalis strain. The construction of a fimA mutant of P. gingivalis ATCC 33277 has previously been reported by N. Hamada et al. (Infect. Immun. 62:1696-1704, 1994). Expression of fimbriae on the surface of the fimA mutant and the wild-type strain, ATCC 33277, were investigated by electron microscopy. The wild-type strain produced long fimbrial structures extending from the cell surface, whereas those structures were not observed on the fimA mutant. However, short fimbrial structures were seen on the surface of the fimA mutant. The short fimbrial protein was purified from the fimA mutant by selective protein precipitation and chromatography on DEAE Sepharose CL-6B. We have found that the second fimbrial structure of P. gingivalis ATCC 33277 is distinct from the 41-kDa (43-kDa) major fimbrial protein (FimA). We provisionally call this protein minor fimbriae. The molecular mass of the minor fimbriae is 67 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions after boiling at 100 degrees C. The component shows a ladder-like pattern at 80 degrees C under nonreducing conditions, suggesting a tendency to aggregate or polymerize. In immunoblotting analysis, anti-minor fimbria serum reacted with both the 100 degrees C- and the 80 degrees C-treated minor fimbriae. The anti-minor fimbria serum also reacts with the same-molecular-size fimbrial preparation from the wild-type strain. Immunogold electron microscopy showed that the anti-minor fimbria serum bound to the minor fimbria on the cell surface of the wild-type strain. This is the first report on the identification of the minor fimbria produced by P. gingivalis. These results suggest that the minor fimbriae appearing on the fimA mutant strain are produced together with numerous long major fimbriae on the wild-type strain. Moreover, the minor fimbriae are different in size and

  19. Selection and phenotypic characterization of nonhemagglutinating mutants of Porphyromonas gingivalis.

    PubMed Central

    Chandad, F; Mayrand, D; Grenier, D; Hinode, D; Mouton, C

    1996-01-01

    To further investigate the relationship between fimbriae and the hemagglutinating adhesin HA-Ag2 of Porphyromonas gingivalis, three spontaneous mutants of the type strain ATCC 33277 were selected by a hemadsorption procedure. They were characterized for hemagglutination, trypsin-like and lectin-binding activities, and hydrophobicity and for the presence of fimbriae. The presence of the 42-kDa (the fimbrilin subunit) and the 43- and 49-kDa (the HA-Ag2 components) polypeptides was investigated by immunoblotting using polyclonal and monoclonal antibodies directed to fimbriae and to the hemagglutinating adhesin HA-Ag2. Cells from two of the three mutants (M1 and M2) exhibited no or little hemagglutination activity and very low trypsin-like activity and did not show the 43- and 49-kDa polypeptides. Abnormal fimbriation in M1 was deduced from the following observations of cells grown for 18 h: absence of the 42-kDa polypeptide and of a 14-kDa polypeptide and no fimbriae visible on electron micrographs. While the cells of mutant M2, irrespective of the age of the culture, were found to lack the 43- and 49-kDa polypeptides and hemagglutination activity, the supernatants of cultures grown for 72 h had high hemagglutination and trypsin-like activities and revealed the presence of the 42-, 43-, and 49-kDa polypeptides. This suggests that M2 may be missing some molecules which anchor the components to the cell surface. Mutant M3 showed levels of activities similar to those of the parental strain but lacked the 43-kDa polypeptide. Other pleiotropic effects observed for the mutants included loss of dark pigmentation and lower hydrophobicity. The data from this study fuel an emerging consensus whereby fimbriation, hemagglutination, and proteolytic activities, as well as other functions in P. gingivalis, are intricate. PMID:8641806

  20. Porphyromonas gingivalis FDC381 multiplies and persists within human oral epithelial cells in vitro.

    PubMed Central

    Madianos, P N; Papapanou, P N; Nannmark, U; Dahlén, G; Sandros, J

    1996-01-01

    Porphyromonas gingivalis FDC381 replication and persistence within KB epithelial cells in vitro were studied by means of an antibiotic protection assay and electron microscopy. Intracellular counts decreased during the first 24 h; showed a threefold increase during the second day, indicating intracellular multiplication; and after 8 days declined to levels approximating 40% of the initial invasion. The ability of P. gingivalis to persist and multiply within epithelial cells may constitute a pathogenic mechanism in periodontal disease. PMID:8550223

  1. Characterisation of the Porphyromonas gingivalis Manganese Transport Regulator Orthologue.

    PubMed

    Zhang, Lianyi; Butler, Catherine A; Khan, Hasnah S G; Dashper, Stuart G; Seers, Christine A; Veith, Paul D; Zhang, Jian-Guo; Reynolds, Eric C

    2016-01-01

    PgMntR is a predicted member of the DtxR family of transcriptional repressors responsive to manganese in the anaerobic periodontal pathogen Porphyromonas gingivalis. Our bioinformatic analyses predicted that PgMntR had divalent metal binding site(s) with elements of both manganous and ferrous ion specificity and that PgMntR has unusual twin C-terminal FeoA domains. We produced recombinant PgMntR and four variants to probe the specificity of metal binding and its impact on protein structure and DNA binding. PgMntR dimerised in the absence of a divalent transition metal cation. PgMntR bound three Mn(II) per monomer with an overall dissociation constant Kd 2.0 x 10(-11) M at pH 7.5. PgMntR also bound two Fe(II) with distinct binding affinities, Kd1 2.5 x 10(-10) M and Kd2 ≤ 6.0 x 10(-8) M at pH 6.8. Two of the metal binding sites may form a binuclear centre with two bound Mn2+ being bridged by Cys108 but this centre provided only one site for Fe2+. Binding of Fe2+ or Mn2+ did not have a marked effect on the PgMntR secondary structure. Apo-PgMntR had a distinct affinity for the promoter region of the gene encoding the only known P. gingivalis manganese transporter, FB2. Mn2+ increased the DNA binding affinity of PgMntR whilst Fe2+ destabilised the protein-DNA complex in vitro. PgMntR did not bind the promoter DNA of the gene encoding the characterised iron transporter FB1. The C-terminal FeoA domain was shown to be essential for PgMntR structure/function, as its removal caused the introduction of an intramolecular disulfide bond and abolished the binding of Mn2+ and DNA. These data indicate that PgMntR is a novel member of the DtxR family that may function as a transcriptional repressor switch to specifically regulate manganese transport and homeostasis in an iron-dependent manner.

  2. Characterisation of the Porphyromonas gingivalis Manganese Transport Regulator Orthologue

    PubMed Central

    Dashper, Stuart G.; Seers, Christine A.; Veith, Paul D.; Zhang, Jian-Guo; Reynolds, Eric C.

    2016-01-01

    PgMntR is a predicted member of the DtxR family of transcriptional repressors responsive to manganese in the anaerobic periodontal pathogen Porphyromonas gingivalis. Our bioinformatic analyses predicted that PgMntR had divalent metal binding site(s) with elements of both manganous and ferrous ion specificity and that PgMntR has unusual twin C-terminal FeoA domains. We produced recombinant PgMntR and four variants to probe the specificity of metal binding and its impact on protein structure and DNA binding. PgMntR dimerised in the absence of a divalent transition metal cation. PgMntR bound three Mn(II) per monomer with an overall dissociation constant Kd 2.0 x 10−11 M at pH 7.5. PgMntR also bound two Fe(II) with distinct binding affinities, Kd1 2.5 x 10−10 M and Kd2 ≤ 6.0 x 10−8 M at pH 6.8. Two of the metal binding sites may form a binuclear centre with two bound Mn2+ being bridged by Cys108 but this centre provided only one site for Fe2+. Binding of Fe2+ or Mn2+ did not have a marked effect on the PgMntR secondary structure. Apo-PgMntR had a distinct affinity for the promoter region of the gene encoding the only known P. gingivalis manganese transporter, FB2. Mn2+ increased the DNA binding affinity of PgMntR whilst Fe2+ destabilised the protein-DNA complex in vitro. PgMntR did not bind the promoter DNA of the gene encoding the characterised iron transporter FB1. The C-terminal FeoA domain was shown to be essential for PgMntR structure/function, as its removal caused the introduction of an intramolecular disulfide bond and abolished the binding of Mn2+ and DNA. These data indicate that PgMntR is a novel member of the DtxR family that may function as a transcriptional repressor switch to specifically regulate manganese transport and homeostasis in an iron-dependent manner. PMID:27007570

  3. Prevalence of fimA genotypes of Porphyromonas gingivalis and other periodontal bacteria in a Spanish population with chronic periodontitis

    PubMed Central

    Puig-Silla, Miriam; Dasí-Fernánde, Francisco; Montiel-Company, José-María

    2012-01-01

    Objectives: The aim of this study was to determine the prevalence of the different fimA genotypes of Porphyromonas gingivalis in adult Spanish patients with chronic periodontitis, patients with gingivitis and periodontally healthy subjects, and the relationship between these genotypes and other periodontopathogenic bacteria. Study design: Samples of subgingival plaque were taken from 86 patients (33 with chronic periodontitis, 16 with gingivitis, and 37 periodontally healthy) in the course of a full periodontal examination. PCR was employed to determine the presence of the 6 fimA genotypes of Porphyromonas gingivalis (I-V and Ib) and of Aggregatibacter actinomycetemcomitans, Tannerella forsythia and Treponema denticola. Results: Porphyromonas gingivalis fimA genotypes II and Ib were present in significantly higher percentages in periodontal patients (39.4% and 12.1% respectively) than in healthy or gingivitis subjects. The prevalence of Tannerella forsythia, Treponema denticola, and Porphyromonas gingivalis fimA genotype IV was significantly higher in the group that presented bleeding greater than 30%. A positive correlation was found between Porphyromonas gingivalis fimA genotype IV and Treponema denticola. Conclusions: A strong association between Porphyromonas gingivalis fimA genotypes II and Ib and chronic periodontitis exists in the Spanish population. The most prevalent genotype in periodontal patients is II. Key words:Periodontitis, Porphyromonas gingivalis, fimA genotype, periodontal bacteria, polymerase chain reaction. PMID:22549664

  4. Role of fimbriae in Porphyromonas gingivalis invasion of gingival epithelial cells.

    PubMed Central

    Weinberg, A; Belton, C M; Park, Y; Lamont, R J

    1997-01-01

    Porphyromonas gingivalis is a periodontal pathogen capable of invading primary cultures of normal human gingival epithelial cells (NHGEC). Involvement of P. gingivalis fimbriae in the invasion process was examined. Purified P. gingivalis 33277 fimbriae blocked invasion of this organism into NHGEC in a dose-dependent manner. DPG3, a P. gingivalis fimbria-deficient mutant, was impaired in its invasion capability approximately eightfold compared to its parent, strain 381. However, adherence of the mutant was only 50% reduced compared to the parent. Biotin labeling of NHGEC surface proteins revealed that two fimbriated strains, but not DPG3, bound a 48-kDa NHGEC protein. Adhesin-receptor interactions, such as fimbriae binding to a 48-kDa NHGEC surface receptor, may trigger activation of eukaryotic proteins involved in signal transduction and/or provoke the generation of surface P. gingivalis molecules required for internalization. PMID:8975930

  5. Transcriptional profiling of human smooth muscle cells infected with gingipain and fimbriae mutants of Porphyromonas gingivalis

    PubMed Central

    Zhang, Boxi; Sirsjö, Allan; Khalaf, Hazem; Bengtsson, Torbjörn

    2016-01-01

    Porphyromonas gingivalis (P. gingivalis) is considered to be involved in the development of atherosclerosis. However, the role of different virulence factors produced by P. gingivalis in this process is still uncertain. The aim of this study was to investigate the transcriptional profiling of human aortic smooth muscle cells (AoSMCs) infected with wild type, gingipain mutants or fimbriae mutants of P. gingivalis. AoSMCs were exposed to wild type (W50 and 381), gingipain mutants (E8 and K1A), or fimbriae mutants (DPG-3 and KRX-178) of P. gingivalis. We observed that wild type P. gingivalis changes the expression of a considerable larger number of genes in AoSMCs compare to gingipain and fimbriae mutants, respectively. The results from pathway analysis revealed that the common differentially expressed genes for AoSMCs infected by 3 different wild type P. gingivalis strains were enriched in pathways of cancer, cytokine-cytokine receptor interaction, regulation of the actin cytoskeleton, focal adhesion, and MAPK signaling pathway. Disease ontology analysis showed that various strains of P. gingivalis were associated with different disease profilings. Our results suggest that gingipains and fimbriae, especially arginine-specific gingipain, produced by P. gingivalis play important roles in the association between periodontitis and other inflammatory diseases, including atherosclerosis. PMID:26907358

  6. Comparative genomics and proteomics of 13 Porphyromonas gingivalis strains.

    PubMed

    Chen, Tsute; Siddiqui, Huma; Olsen, Ingar

    2015-01-01

    At the current time, genome sequences of a total of 13 Porphyromonas gingivalis strains are available, including five completed genomes (strains ATCC 33277, HG66, TDC60, JCVISC001, and W83) and eight high-coverage draft sequences (F0185, F0566, F0568, F0569, F0570, SJD2, W4087, and W50) that are assembled into fewer than 300 contigs. This study compared these genomes at both nucleotide and protein sequence levels in order to understand their phylogenetic and functional relatedness. There are four copies of 16S rRNA gene sequences in each of the strains of ATCC 33277, HG66, TDC60, and W83 and one copy in the other nine genomes. These 25 16S rRNA sequences represent only 13 unique sequences. The five copies in W83 and W50 are identical and the three copies in HG66 are identical to the four copies in ATCC 33277, suggesting close evolutionary lineage between W83 and W50, as well as HG66 and ATCC 33277. Genome-wide comparison based on "Rapid Annotation using Subsystem Technology" (RAST) also showed that for the overall biological functions of the genomes, W83 is closer to W50, and HG66 to ATCC33277, than to other genomes. The comparison of the RAST subsystems identified biological functions that are unique to individual, shared by some, or by all genomes. Functions unique to individual genomes include: a tetracycline resistance protein TetQ, DNA metabolism gene YcfH, and DNA repair gene exonuclease SbcC (only in SJD2); very-short-patch mismatch repair endonuclease and a phage packaging terminase similar to Bacteroides phage B124-14 (in W4087); an internalin similar to a Listeria surface virulence protein (W83); a Type I restriction-modification system (F0569); an iron acquisition/heme transport protein (F0566); colicin I receptor and carbamoylputrescine amidase (W50); L-serine dehydratase (TDC60); and spermidine synthase and ribokinase (JCVISC001). The results also identified biological functions that are missing in individual or several genomes. For example, JCVISC001

  7. The peptidylarginine deiminase gene is a conserved feature of Porphyromonas gingivalis

    PubMed Central

    Gabarrini, Giorgio; de Smit, Menke; Westra, Johanna; Brouwer, Elisabeth; Vissink, Arjan; Zhou, Kai; A. Rossen, John W.; Stobernack, Tim; van Dijl, Jan Maarten; Jan van Winkelhoff, Arie

    2015-01-01

    Periodontitis is an infective process that ultimately leads to destruction of the soft and hard tissues that support the teeth (the periodontium). Periodontitis has been proposed as a candidate risk factor for development of the autoimmune disease rheumatoid arthritis (RA). Porphyromonas gingivalis, a major periodontal pathogen, is the only known prokaryote expressing a peptidyl arginine deiminase (PAD) enzyme necessary for protein citrullination. Antibodies to citrullinated proteins (anti-citrullinated protein antibodies, ACPA) are highly specific for RA and precede disease onset. Objective of this study was to assess P. gingivalis PAD (PPAD) gene expression and citrullination patterns in representative samples of P. gingivalis clinical isolates derived from periodontitis patients with and without RA and in related microbes of the Porphyromonas genus. Our findings indicate that PPAD is omnipresent in P. gingivalis, but absent in related species. No significant differences were found in the composition and expression of the PPAD gene of P. gingivalis regardless of the presence of RA or periodontal disease phenotypes. From this study it can be concluded that if P. gingivalis plays a role in RA, it is unlikely to originate from a variation in PPAD gene expression. PMID:26403779

  8. Humoral immune response to an antigen from Porphyromonas gingivalis 381 in periodontal disease.

    PubMed Central

    Kurihara, H; Nishimura, F; Nakamura, T; Nakagawa, M; Tanimoto, I; Nomura, Y; Kokeguchi, S; Kato, K; Murayama, Y

    1991-01-01

    The humoral immune responses of patients with periodontitis were evaluated to characterize the host response to Porphyromonas gingivalis. A sonic extract of P. gingivalis 381 from whole cells was fractionated by gel chromatography and ion-exchange chromatography. The fractionated extracts were evaluated by Western blot (immunoblot) analyses with patient sera. A dominant antigen was identified from the sonic extract with an apparent molecular mass of 53 kDa. The 53-kDa protein antigen (Ag53) was purified by affinity chromatography by using a monoclonal antibody. Ag53 was detected on the vesicle surface of P. gingivalis 381 by immunoelectron microscopy by using the monoclonal antibody and was detected as a major protein in the outer membrane and in vesicles by Western blot analysis. Monoclonal antibody cross-reactivity to Ag53 in the sonic extracts of P. gingivalis ATCC 33277, P. gingivalis 1021, and Porphyromonas endodontalis ATCC 35406 was revealed. Seventy-seven patients with periodontitis were examined for their responses to Ag53. Serum immunoglobulin G (IgG) from 54 patients reacted strongly to Ag53; however, serum IgG from the remaining 23 patients did not exhibit detectable reactivity at all to Ag53, even though the patients had high serum IgG titers to the sonic extract. Ag53 is a new marker that represents an interesting aspect of the humoral immune response to P. gingivalis in patients with periodontitis. Images PMID:1855992

  9. Inhibitory Effect of Enterococcus faecium WB2000 on Volatile Sulfur Compound Production by Porphyromonas gingivalis

    PubMed Central

    Higuchi, Takuya; Nakajima, Masato; Fujimoto, Akie; Hanioka, Takashi; Hirofuji, Takao

    2016-01-01

    Volatile sulfur compounds (VSCs) produced by oral anaerobes are the major compounds responsible for oral malodor. Enterococcus faecium WB2000 is recognized as an antiplaque probiotic bacterium. In this study, the effect of E. faecium WB2000 on VSC production by Porphyromonas gingivalis was evaluated, and the mechanism of inhibition of oral malodor was investigated. P. gingivalis ATCC 33277 was cultured in the presence of four lactic acid bacteria, including E. faecium WB2000. Subsequently, P. gingivalis ATCC 33277, W50, W83, and two clinical isolates were cultured in the presence or absence of E. faecium WB2000, and the emission of VSCs from spent culture medium was measured by gas chromatography. The number of P. gingivalis ATCC 33277 in mixed culture with E. faecium WB2000 decreased at 6 h, and the rate of decrease was higher than that in mixed cultures with the other lactic acid bacteria. The numbers of five P. gingivalis strains decreased at similar rates in mixed culture with E. faecium WB2000. The concentration of methyl mercaptan was lower in spent culture medium from P. gingivalis and E. faecium WB2000 cultures compared with that from P. gingivalis alone. Therefore, E. faecium WB2000 may reduce oral malodor by inhibiting the growth of P. gingivalis and neutralizing methyl mercaptan. PMID:27799940

  10. Lipid raft-dependent uptake, signaling, and intracellular fate of Porphyromonas gingivalis in mouse macrophages

    PubMed Central

    Wang, Min; Hajishengallis, George

    2009-01-01

    Summary Lipid rafts are cholesterol-enriched microdomains involved in cellular trafficking and implicated as portals for certain pathogens. We sought to determine whether the oral pathogen Porphyromonas gingivalis enters macrophages via lipid rafts, and if so, to examine the impact of raft entry on its intracellular fate. Using J774A.1 mouse macrophages, we found that P. gingivalis colocalizes with lipid rafts in a cholesterol-dependent way. Depletion of cellular cholesterol using methyl-β-cyclodextrin resulted in about 50% inhibition of P. gingivalis uptake, although this effect was reversed by cholesterol reconstitution. The intracellular survival of P. gingivalis was dramatically inhibited in cholesterol-depleted cells relative to untreated or cholesterol-reconstituted cells, even when infections were adjusted to allow equilibration of the initial intracellular bacterial load. P. gingivalis thus appeared to exploit raft-mediated uptake for promoting its survival. Consistent with this, lipid raft disruption enhanced the colocalization of internalized P. gingivalis with lysosomes. In contrast, raft disruption did not affect the expression of host receptors interacting with P. gingivalis, although it significantly inhibited signal transduction. In summary, P. gingivalis uses macrophage lipid rafts as signaling and entry platforms, which determine its intracellular fate to the pathogen’s own advantage. PMID:18547335

  11. Porphyromonas gingivalis Gingipain-Dependently Enhances IL-33 Production in Human Gingival Epithelial Cells

    PubMed Central

    Tada, Hiroyuki; Matsuyama, Takashi; Nishioka, Takashi; Hagiwara, Makoto; Kiyoura, Yusuke; Shimauchi, Hidetoshi; Matsushita, Kenji

    2016-01-01

    The cytokine IL-33 is constitutively expressed in epithelial cells and it augments Th2 cytokine-mediated inflammatory responses by regulating innate immune cells. We aimed to determine the role of the periodontal pathogen, Porphyromonas gingivalis, in the enhanced expression of IL-33 in human gingival epithelial cells. We detected IL-33 in inflamed gingival epithelium from patients with chronic periodontitis, and found that P. gingivalis increased IL-33 expression in the cytoplasm of human gingival epithelial cells in vitro. In contrast, lipopolysaccharide, lipopeptide, and fimbriae derived from P. gingivalis did not increase IL-33 expression. Specific inhibitors of P. gingivalis proteases (gingipains) suppressed IL-33 mRNA induction by P. gingivalis and the P. gingivalis gingipain-null mutant KDP136 did not induce IL-33 expression. A small interfering RNA for protease-activated receptor-2 (PAR-2) as well as inhibitors of phospholipase C, p38 and NF-κB inhibited the expression of IL-33 induced by P. gingivalis. These results indicate that the PAR-2/IL-33 axis is promoted by P. gingivalis infection in human gingival epithelial cells through a gingipain-dependent mechanism. PMID:27058037

  12. Porphyromonas gingivalis Lipopolysaccharide Induced Proliferation and Activation of Natural Killer Cells in Vivo.

    PubMed

    Wang, Yuhua; Zhang, Wei; Xu, Li; Jin, Jun-O

    2016-01-01

    Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) promoted different innate immune activation than that promoted by Escherichia coli (E. coli) LPS. In this study, we examined the effect of P. gingivalis LPS on the proliferation and activation of natural killer (NK) cells in vivo and compared that function with that of E. coli LPS. Administration of P. gingivalis LPS to C57BL/6 mice induced stronger proliferation of NK cells in the spleen and submandibular lymph nodes (sLNs) and increased the number of circulating NK cells in blood compared to those treated with E. coli LPS. However, P. gingivalis LPS did not induce interferon-gamma (IFN-γ) production and CD69 expression in the spleen and sLN NK cells in vivo, and this was attributed to the minimal activation of the spleen and sLN dendritic cells (DCs), including low levels of co-stimulatory molecule expression and pro-inflammatory cytokine production. Furthermore, P. gingivalis LPS-treated NK cells showed less cytotoxic activity against Yac-1 target cells than E. coli LPS-treated NK cells. Hence, these data demonstrated that P. gingivalis LPS promoted limited activation of spleen and sLN NK cells in vivo, and this may play a role in the chronic inflammatory state observed in periodontal disease. PMID:27548133

  13. Green tea epigallocatechin-3-gallate attenuates Porphyromonas gingivalis-induced atherosclerosis.

    PubMed

    Cai, Yu; Kurita-Ochiai, Tomoko; Hashizume, Tomomi; Yamamoto, Masafumi

    2013-02-01

    The purpose of this study was to determine whether epigallocatechin-3-gallate (EGCG) ameliorates Porphyromonas gingivalis-induced atherosclerosis. EGCG is a polyphenol extract from green tea with health benefits and P. gingivalis is shown here to accelerate atheroma formation in a murine model. Apolipoprotein E knockout mice were administered EGCG or vehicle in drinking water; they were then fed high-fat diets and injected with P. gingivalis three times a week for 3 weeks. Mice were then killed at 15 weeks. Atherosclerotic plaques in the proximal aorta were determined by Oil Red O staining. Atherosclerosis risk factors in serum, liver or aorta were analysed using cytokine antibody arrays, enzyme-linked immunosorbent assay and real-time PCR. Atherosclerotic lesion areas of the aortic sinus caused by P. gingivalis infection decreased in EGCG-treated groups, wherein EGCG reduced the production of C-reactive protein, monocyte chemoattractant protein-1, and oxidized low-density lipoprotein (LDL), and slightly lowered LDL/very LDL cholesterol in P. gingivalis-challenged mice serum. Furthermore, the increase in CCL2, MMP-9, ICAM-1, HSP60, CD44, LOX-1, NOX-4, p22phox and iNOS gene expression levels in the aorta of P. gingivalis-challenged mice were reduced in EGCG-treated mice. However, HO-1 mRNA levels were elevated by EGCG treatment, suggesting that EGCG, as a natural substance, inhibits P. gingivalis-induced atherosclerosis through anti-inflammatory and antioxidative effects. PMID:23620122

  14. Lactobacillus rhamnosus could inhibit Porphyromonas gingivalis derived CXCL8 attenuation

    PubMed Central

    Mendi, Ayşegül; Köse, Sevil; Uçkan, Duygu; Akca, Gülçin; Yilmaz, Derviş; Aral, Levent; Gültekin, Sibel Elif; Eroğlu, Tamer; Kiliç, Emine; Uçkan, Sina

    2016-01-01

    ABSTRACT An increasing body of evidence suggests that the use of probiotic bacteria is a promising intervention approach for the treatment of inflammatory diseases with a polymicrobial etiology. P. gingivalis has been noted to have a different way of interacting with the innate immune response of the host compared to other pathogenic bacteria, which is a recognized feature that inhibits CXCL8 expression. Objective The aim of the study was to determine if P. gingivalis infection modulates the inflammatory response of gingival stromal stem cells (G-MSSCs), including the release of CXCL8, and the expression of TLRs and if immunomodulatory L. rhamnosus ATCC9595 could prevent CXCL8 inhibition in experimental inflammation. Material and Methods G-MSSCs were pretreated with L. rhamnosus ATCC9595 and then stimulated with P. gingivalis ATCC33277. CXCL8 and IL-10 levels were investigated with ELISA and the TLR-4 and 2 were determined through flow cytometer analysis. Results CXCL8 was suppressed by P. gingivalis and L. rhamnosus ATCC9595, whereas incubation with both strains did not abolish CXCL8. L. rhamnosus ATCC9595 scaled down the expression of TLR4 and induced TLR2 expression when exposed to P. gingivalis stimulation (p<0.01). Conclusions These findings provide evidence that L. rhamnosus ATCC9595 can modulate the inflammatory signals and could introduce P. gingivalis to immune systems by inducing CXCL8 secretion. PMID:27008259

  15. Draft Genome Sequence of Low-Passage Clinical Isolate Porphyromonas gingivalis MP4-504.

    PubMed

    To, Thao T; Liu, Quanhui; Watling, Michael; Bumgarner, Roger E; Darveau, Richard P; McLean, Jeffrey S

    2016-04-07

    We present the draft genome ofPorphyromonas gingivalisMP4-504, a low-passage clinical isolate obtained from a periodontitis patient. The genome is composed of 92 contigs for a length of 2,373,453 bp and a G+C of 48.3%. ThetraA-Qconjugative transfer locus is genetically distinct from W83 but highly similar to ATCC 33277.

  16. Draft Genome Sequence of Low-Passage Clinical Isolate Porphyromonas gingivalis MP4-504

    PubMed Central

    Liu, Quanhui; Watling, Michael; Bumgarner, Roger E.; Darveau, Richard P.

    2016-01-01

    We present the draft genome of Porphyromonas gingivalis MP4-504, a low-passage clinical isolate obtained from a periodontitis patient. The genome is composed of 92 contigs for a length of 2,373,453 bp and a G+C of 48.3%. The traA-Q conjugative transfer locus is genetically distinct from W83 but highly similar to ATCC 33277. PMID:27056232

  17. Phagocytosis of virulent Porphyromonas gingivalis by human polymorphonuclear leukocytes requires specific immunoglobulin G.

    PubMed Central

    Cutler, C W; Kalmar, J R; Arnold, R R

    1991-01-01

    No studies to date clearly define the interactions between Porphyromonas gingivalis and human peripheral blood polymorphonuclear leukocytes (PMN), nor has a protective role for antibody to P. gingivalis been defined. Using a fluorochrome phagocytosis microassay, we investigated PMN phagocytosis and killing of P. gingivalis as a function of P. gingivalis-specific antibody. Sera from a nonimmune rabbit and a healthy human subject were not opsonic for virulent P. gingivalis A7436, W83, and HG405; phagocytosis of these strains (but not 33277) required opsonization with hyperimmune antiserum (RaPg). Diluting RaPg with a constant complement source decreased proportionally the number of P. gingivalis A7436 cells phagocytosed per phagocytic PMN. Enriching for the immunoglobulin G fraction of RAPg A7436 enriched for opsonic activity toward A7436. An opsonic evaluation of 18 serum samples from adult periodontitis patients revealed that only 3 adult periodontitis sera of 17 with elevated immunoglobulin G to P. gingivalis A7436 were opsonic for A7436 and, moreover, that the serum sample with the highest enzyme-linked immunosorbent assay titer was most opsonic (patient 1). However, the opsonic activity of serum from patient 1 was qualitatively and not just quantitatively different from that of the nonopsonic human sera (but was less effective opsonin than RaPg). Strain variability was observed in resistance of P. gingivalis to phagocytosis, and opsonization was strain specific for some, but not all, strains tested. An evaluation of killing of A7436 revealed that serum killing and extracellular killing of P. gingivalis were less effective alone when compared with intracellular PMN killing alone. PMID:2037370

  18. Evidence that Porphyromonas (Bacteroides) gingivalis fimbriae function in adhesion to Actinomyces viscosus.

    PubMed Central

    Goulbourne, P A; Ellen, R P

    1991-01-01

    Porphyromonas (Bacteroides) gingivalis adheres to gram-positive bacteria, such as Actinomyces viscosus, when colonizing the tooth surface. However, little is known of the adhesins responsible for this interaction. A series of experiments were performed to determine whether P. gingivalis fimbriae function in its coadhesion with A. viscosus. Fimbriae typical of P. gingivalis were isolated from strain 2561 (ATCC 33277) by the method of Yoshimura et al. (F. Yoshimura, K. Takahashi, Y. Nodasaka, and T. Suzuki, J. Bacteriol. 160:949-957, 1984) in fractions enriched with a 40-kDa subunit, the fimbrillin monomer, P. gingivalis-A. viscosus coaggregation was inhibited by purified rabbit antifimbrial immunoglobulin G (IgG) at dilutions eightfold higher than those of preimmune IgG, providing indirect evidence implicating P. gingivalis fimbriae in coadhesion. Three types of direct binding assays further supported this observation. (i) Mixtures of isolated P. gingivalis fimbriae and A. viscosus WVU627 cells were incubated for 1 h, washed vigorously with phosphate-buffered saline (pH 7.2), and subjected to electrophoresis. Transblots onto nitrocellulose were probed with antifimbrial antiserum. Fimbrillin labeled positively on these blots. No reaction occurred with the control protein, porcine serum albumin, when blots were exposed to anti-porcine serum albumin, (ii) A. viscosus cells incubated with P. gingivalis fimbriae were agglutinated only after the addition of antifimbrial antibodies. (iii) Binding curves generated from an enzyme immunoassay demonstrated concentration-dependent binding of P. gingivalis fimbriae to A. viscosus cells. From these lines of evidence, P. gingivalis fimbriae appear to be capable of binding to A. viscosus and mediating the coadhesion of these species. Images PMID:1679428

  19. The periodontal pathogen Porphyromonas gingivalis sensitises human blood platelets to epinephrine.

    PubMed

    Nylander, M; Lindahl, T L; Bengtsson, T; Grenegård, M

    2008-08-01

    Recent studies indicate connections between periodontitis and atherothrombosis, and the periodontal pathogen Porphyromonas gingivalis has been found within atherosclerotic lesions. P. gingivalis-derived proteases, designated gingipains activate human platelets, probably through a "thrombin-like" activity on protease-activated receptors (PARs). However, the potential interplay between P. gingivalis and other physiological platelet activators has not been investigated. The aim of this study was to elucidate consequences and mechanisms in the interaction between P. gingivalis and the stress hormone epinephrine. By measuring changes in light transmission through platelet suspensions, we found that P. gingivalis provoked aggregation, whereas epinephrine alone never had any effect. Intriguingly, pre-treatment of platelets with a low, sub-threshold number of P. gingivalis (i.e. a density that did not directly provoke platelet aggregation) resulted in a marked aggregation response when epinephrine was added. This synergistic action was not inhibited by the cyclooxygenas inhibitor aspirin. Furthermore, fura-2-measurements revealed that epinephrine caused an intracellular Ca(2+) mobilization in P. gingivalis pre-treated platelets, whereas epinephrine alone had no effect. Inhibition of the arg-specific gingipains, but not the lys-specific gingipains, abolished the aggregation and the Ca(2+) response provoked by epinephrine. Similar results were achieved by separate blockage of platelet alpha(2)-adrenergic receptors and PARs. In conclusion, the present study shows that a sub-threshold number of P. gingivalis sensitizes platelets to epinephrine. We suggest that P. gingivalis-derived arg-specific gingipains activates a small number of PARs on the surface of the platelets. This leads to an unexpected Ca(2+) mobilization and a marked aggregation response when epinephrine subsequently binds to the alpha(2)-adrenergic receptor. The present results are consistent with a direct

  20. Altered antigenic profiling and infectivity of Porphyromonas gingivalis in smokers and non-smokers with periodontitis

    PubMed Central

    Zeller, Iris; Hutcherson, Justin A.; Lamont, Richard J.; Demuth, Donald R.; Gumus, Pinar; Nizam, Nejat; Buduneli, Nurcan; Scott, David A.

    2014-01-01

    Background Cigarette smokers are more susceptible to periodontal diseases and are more likely to be infected with Porphyromonas gingivalis than non-smokers. Furthermore, smoking is known to alter the expression of P. gingivalis surface components and to compromise IgG generation. The aim of this study was to evaluate whether IgG response to P. gingivalis is suppressed in smokers in vivo and whether previously established in vitro tobacco-induced phenotypic P. gingivalis changes would be reflected in vivo. Methods We examined the humoral response to several P. gingivalis strains as well as specific tobacco-regulated outer membrane proteins (FimA and RagB) by ELISA in biochemically-validated (salivary cotinine) smokers and non-smokers with chronic (CP, n = 13) or aggressive (AP, n = 20) periodontitis. We also monitored the local and systemic presence of P. gingivalis DNA by PCR. Results Smoking was associated with decreased total IgG responses against clinical (10512, 5607, and 10208C; all p < 0.05) but not laboratory (ATCC 33277, W83) P. gingivalis strains. Smoking did not influence IgG produced against specific cell surface proteins, although a non-significant pattern towards increased total FimA-specific IgG in CP subjects, but not AP subjects, was observed. Seropositive smokers were more likely to be infected orally and systemically with P. gingivalis (p < 0.001), as determined by 16S RNA analysis. Conclusions Smoking alters the humoral response against P. gingivalis, strengthening the evidence that mechanisms of periodontal disease progression in smokers may differ from non-smokers with the same disease classification. PMID:24147843

  1. Abrogation of neuraminidase reduces biofilm formation, capsule biosynthesis, and virulence of Porphyromonas gingivalis.

    PubMed

    Li, Chen; Kurniyati; Hu, Bo; Bian, Jiang; Sun, Jianlan; Zhang, Weiyan; Liu, Jun; Pan, Yaping; Li, Chunhao

    2012-01-01

    The oral bacterium Porphyromonas gingivalis is a key etiological agent of human periodontitis, a prevalent chronic disease that affects up to 80% of the adult population worldwide. P. gingivalis exhibits neuraminidase activity. However, the enzyme responsible for this activity, its biochemical features, and its role in the physiology and virulence of P. gingivalis remain elusive. In this report, we found that P. gingivalis encodes a neuraminidase, PG0352 (SiaPg). Transcriptional analysis showed that PG0352 is monocistronic and is regulated by a sigma70-like promoter. Biochemical analyses demonstrated that SiaPg is an exo-α-neuraminidase that cleaves glycosidic-linked sialic acids. Cryoelectron microscopy and tomography analyses revealed that the PG0352 deletion mutant (ΔPG352) failed to produce an intact capsule layer. Compared to the wild type, in vitro studies showed that ΔPG352 formed less biofilm and was less resistant to killing by the host complement. In vivo studies showed that while the wild type caused a spreading type of infection that affected multiple organs and all infected mice were killed, ΔPG352 only caused localized infection and all animals survived. Taken together, these results demonstrate that SiaPg is an important virulence factor that contributes to the biofilm formation, capsule biosynthesis, and pathogenicity of P. gingivalis, and it can potentially serve as a new target for developing therapeutic agents against P. gingivalis infection.

  2. Gingipains: Critical Factors in the Development of Aspiration Pneumonia Caused by Porphyromonas gingivalis.

    PubMed

    Benedyk, Małgorzata; Mydel, Piotr Mateusz; Delaleu, Nicolas; Płaza, Karolina; Gawron, Katarzyna; Milewska, Aleksandra; Maresz, Katarzyna; Koziel, Joanna; Pyrc, Krzysztof; Potempa, Jan

    2016-01-01

    Aspiration pneumonia is a life-threatening infectious disease often caused by oral anaerobic and periodontal pathogens such as Porphyromonas gingivalis. This organism produces proteolytic enzymes, known as gingipains, which manipulate innate immune responses and promote chronic inflammation. Here, we challenged mice with P. gingivalis W83 and examined the role of gingipains in bronchopneumonia, lung abscess formation, and inflammatory responses. Although gingipains were not required for P. gingivalis colonization and survival in the lungs, they were essential for manifestation of clinical symptoms and infection-related mortality. Pathologies caused by wild-type (WT) P. gingivalis W83, including hemorrhage, necrosis, and neutrophil infiltration, were absent from lungs infected with gingipain-null isogenic strains or WT bacteria preincubated with gingipain-specific inhibitors. Damage to lung tissue correlated with systemic inflammatory responses, as manifested by elevated levels of TNF, IL-6, IL-17, and C-reactive protein. These effects were unequivocally dependent on gingipain activity. Gingipain activity was also implicated in the observed increase in IL-17 in lung tissues. Furthermore, gingipains increased platelet counts in the blood and activated platelets in the lungs. Arginine-specific gingipains made a greater contribution to P. gingivalis-related morbidity and mortality than lysine-specific gingipains. Thus, inhibition of gingipain may be a useful adjunct treatment for P. gingivalis-mediated aspiration pneumonia. PMID:26613585

  3. Antimicrobial Efficacy of Various Essential Oils at Varying Concentrations against Periopathogen Porphyromonas gingivalis

    PubMed Central

    Grover, Harpreet Singh; Deswal, Himanshu; Agarwal, Preeti

    2016-01-01

    Introduction Porphyromonas gingivalis (P.gingivalis) is a notorious perio-pathogen with the ability to evade host defense mechanism and invade into the periodontal tissues. Many antimicrobial agents have been tested that curb its growth, although these agents tend to produce side effects such as antibiotic resistance and opportunistic infections. Therefore search for naturally occurring anti-microbials with lesser side effects is the need of the hour. Aim The aim of this study was to substantiate the antimicrobial activity of various essential oils; eucalyptus oil, chamomile oil, tea tree oil and turmeric oil against P. gingivalis. Materials and Methods Pure cultures of P. gingivalis were grown on selective blood agar. Antimicrobial efficacy of various concentrations of essential oils (0%, 25%, 50% and 100%) was assessed via disc diffusion test. Zone of inhibition were measured around disc after 48 hours in millimeters. Results Zones of inhibition were directly proportional to the concentration of essential oils tested. At 100% concentration all the tested oils possess antimicrobial activity against P.gingivalis with eucalyptus oil being most effective followed by tea tree oil, chamomile oil and turmeric oil. Conclusion All essential oils tested were effective against P.gingivalis. After testing for their clinical safety they could be developed into local agents to prevent and treat periodontitis. PMID:27790572

  4. Abrogation of Neuraminidase Reduces Biofilm Formation, Capsule Biosynthesis, and Virulence of Porphyromonas gingivalis

    PubMed Central

    Li, Chen; Kurniyati; Hu, Bo; Bian, Jiang; Sun, Jianlan; Zhang, Weiyan; Liu, Jun; Pan, Yaping

    2012-01-01

    The oral bacterium Porphyromonas gingivalis is a key etiological agent of human periodontitis, a prevalent chronic disease that affects up to 80% of the adult population worldwide. P. gingivalis exhibits neuraminidase activity. However, the enzyme responsible for this activity, its biochemical features, and its role in the physiology and virulence of P. gingivalis remain elusive. In this report, we found that P. gingivalis encodes a neuraminidase, PG0352 (SiaPg). Transcriptional analysis showed that PG0352 is monocistronic and is regulated by a sigma70-like promoter. Biochemical analyses demonstrated that SiaPg is an exo-α-neuraminidase that cleaves glycosidic-linked sialic acids. Cryoelectron microscopy and tomography analyses revealed that the PG0352 deletion mutant (ΔPG352) failed to produce an intact capsule layer. Compared to the wild type, in vitro studies showed that ΔPG352 formed less biofilm and was less resistant to killing by the host complement. In vivo studies showed that while the wild type caused a spreading type of infection that affected multiple organs and all infected mice were killed, ΔPG352 only caused localized infection and all animals survived. Taken together, these results demonstrate that SiaPg is an important virulence factor that contributes to the biofilm formation, capsule biosynthesis, and pathogenicity of P. gingivalis, and it can potentially serve as a new target for developing therapeutic agents against P. gingivalis infection. PMID:22025518

  5. Proteomic and transcriptional analysis of interaction between oral microbiota Porphyromonas gingivalis and Streptococcus oralis.

    PubMed

    Maeda, Kazuhiko; Nagata, Hideki; Ojima, Miki; Amano, Atsuo

    2015-01-01

    Porphyromonas gingivalis, a major periodontal pathogen, forms biofilm with other oral bacteria such as streptococci. Here, by using shotgun proteomics, we examined the molecular basis of mixed-biofilm formation by P. gingivalis with Streptococcus oralis. We identified a total of 593 bacterial proteins in the biofilm. Compared to the expression profile in the P. gingivalis monobiofilm, the expression of three proteins was induced and that of 31 proteins was suppressed in the mixed biofilm. Additionally, the expression of two S. oralis proteins was increased, while that of two proteins was decreased in the mixed biofilm, as compared to its monotypic profile. mRNA expression analysis of selected genes using a quantitative reverse transcription polymerase chain reaction confirmed the proteomics data, which included overexpression of P. gingivalis FimA and S. oralis glyceraldehyde-3-phosphate dehydrogenase in association with the biofilm. The results also indicated that S. oralis regulates the transcriptional activity of P. gingivalis luxS to influence autoinducer-2-dependent signaling. These findings suggest that several functional molecules are involved in biofilm formation between P. gingivalis and S. oralis.

  6. Transglutaminase 2 is essential for adherence of Porphyromonas gingivalis to host cells

    PubMed Central

    Boisvert, Heike; Lorand, Laszlo; Duncan, Margaret J.

    2014-01-01

    Porphyromonas gingivalis is the major causative agent of periodontitis, and it may also be involved in the development of systemic diseases (atherosclerosis, rheumatoid arthritis). P. gingivalis is found on and within oral and gingival epithelial cells following binding to surface components of host cells, which serve as receptors for the bacterium. Evidence is presented in this study that shows that transglutaminase 2 (TG2) plays a critical role in the adherence of P. gingivalis to host cells. Studies of confocal microscopy indicate colocalization of P. gingivalis with TG2 on the surface of HEp-2 epithelial cells, with clusters of TG2 seen at bacterial attachment sites. By silencing the expression of TG2 with siRNA in HEp-2 cells, P. gingivalis association was greatly diminished. The bacterium does not bind well to a mouse fibroblast cell line that produces low amounts of surface TG2, but binding can be restored by introduction of TG2 expressed on a plasmid. TG2 can form very tight complexes with fibronectin (FN), and the complementary binding sites of the two proteins are known. A synthetic peptide that mimics the main FN-binding sequence of TG2 blocks the formation of TG2–FN complexes and is highly effective in inhibiting adherence of P. gingivalis to host cells. These findings provide evidence of a role for cell-surface TG2 in bacterial attachment and subsequent internalization. PMID:24706840

  7. Evaluation of the antimicrobial effect of lactoferrin on Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens.

    PubMed

    Aguilera, O; Andrés, M T; Heath, J; Fierro, J F; Douglas, C W

    1998-05-01

    The antimicrobial effect of lactoferrin (apoLf) on the oral, black-pigmented anaerobes Porphyromonas gingivalis, Prevotella intermedia and P. nitrescens has been studied. ApoLf did not kill any of these species but it did inhibit the growth of P. gingivalis, while iron-saturated Lf (FeLf) had no effect. The other two species were unaffected by apoLf. This growth inhibitory effect of apoLf could not be explained on the basis of chelation of inorganic iron, since growth of P. gingivalis occurred in the presence of ethylenediamine di-o-hydroxyphenylacetic acid provided haemin was added. Both apoLf and FeLf reduced haemin uptake by all three species and caused the release of cell-bound haemin in a dose-dependent manner. In addition, haemin reduced the binding of both apoLf and FeLf to P. intermedia and P. nigrescens but stimulated the binding of Lf by P. gingivalis. These data suggest that Lf forms complexes with haemin in solution and competes for the binding of haemin to certain cell receptors, possibly lipopolysaccharides, but this is not sufficient to inhibit growth of the bacteria. P. gingivalis appears to bind Lf-haemin complexes, as well as haemin alone, which may facilitate access of the Lf to the outer and cytoplasmic membranes of P. gingivalis, so disrupting function.

  8. Susceptibility of Porphyromonas gingivalis and Streptococcus mutans to Antibacterial Effect from Mammea americana.

    PubMed

    Herrera Herrera, Alejandra; Franco Ospina, Luis; Fang, Luis; Díaz Caballero, Antonio

    2014-01-01

    The development of periodontal disease and dental caries is influenced by several factors, such as microorganisms of bacterial biofilm or commensal bacteria in the mouth. These microorganisms trigger inflammatory and immune responses in the host. Currently, medicinal plants are treatment options for these oral diseases. Mammea americana extracts have reported antimicrobial effects against several microorganisms. Nevertheless, this effect is unknown against oral bacteria. Therefore, the aim of this study was to evaluate the antibacterial effect of M. americana extract against Porphyromonas gingivalis and Streptococcus mutans. For this, an experimental study was conducted. Ethanolic extract was obtained from seeds of M. americana (one oil phase and one ethanolic phase). The strains of Porphyromonas gingivalis ATCC 33277 and Streptococcus mutans ATCC 25175 were exposed to this extract to evaluate its antibacterial effect. Antibacterial activity was observed with the two phases of M. americana extract on P. gingivalis and S. mutans with lower MICs (minimum inhibitory concentration). Also, bactericidal and bacteriostatic activity was detected against S. mutans, depending on the concentration of the extract, while on M. americana extract presented only bacteriostatic activity against P. gingivalis. These findings provide important and promising information allowing for further exploration in the future.

  9. Susceptibility of Porphyromonas gingivalis and Streptococcus mutans to Antibacterial Effect from Mammea americana

    PubMed Central

    Herrera Herrera, Alejandra; Franco Ospina, Luis; Fang, Luis; Díaz Caballero, Antonio

    2014-01-01

    The development of periodontal disease and dental caries is influenced by several factors, such as microorganisms of bacterial biofilm or commensal bacteria in the mouth. These microorganisms trigger inflammatory and immune responses in the host. Currently, medicinal plants are treatment options for these oral diseases. Mammea americana extracts have reported antimicrobial effects against several microorganisms. Nevertheless, this effect is unknown against oral bacteria. Therefore, the aim of this study was to evaluate the antibacterial effect of M. americana extract against Porphyromonas gingivalis and Streptococcus mutans. For this, an experimental study was conducted. Ethanolic extract was obtained from seeds of M. americana (one oil phase and one ethanolic phase). The strains of Porphyromonas gingivalis ATCC 33277 and Streptococcus mutans ATCC 25175 were exposed to this extract to evaluate its antibacterial effect. Antibacterial activity was observed with the two phases of M. americana extract on P. gingivalis and S. mutans with lower MICs (minimum inhibitory concentration). Also, bactericidal and bacteriostatic activity was detected against S. mutans, depending on the concentration of the extract, while on M. americana extract presented only bacteriostatic activity against P. gingivalis. These findings provide important and promising information allowing for further exploration in the future. PMID:24864137

  10. Green tea epigallocatechin-3-gallate alleviates Porphyromonas gingivalis-induced periodontitis in mice.

    PubMed

    Cai, Yu; Chen, ZhiBin; Liu, Hao; Xuan, Yan; Wang, XiaoXuan; Luan, QingXian

    2015-12-01

    Porphyromonas gingivalis causes inflammation, and leads to the periodontitis in gingival tissue damage and bone resorption. Epigallocatechin-3-gallate (EGCG) is a major polyphenol extract from green tea with plenty of pharmacological functions. The aim of this study was to determine whether continuous oral intake of EGCG would alleviate P. gingivalis-induced periodontitis. Eight-week BALB/c mice were administered with EGCG (0.02%) or vehicle in drinking water. They were fed normal food and orally infected with P. gingivalis every 2days, up to a total of 20 times, and then sacrificed at 15weeks of age. The P. gingivalis-challenged group markedly increased alveolar bone resorption of the maxillae in BALB/c mice by Micro-CT detection, and administration of EGCG resulted in a significant reduction in bone loss. Inflammation cytokine antibody array and enzyme linked immunosorbent assay revealed that some inflammatory mediators in serum were increased by P. gingivalis infection, but were lowered after EGCG treatment. High positive areas of IL-17 and IL-1β in the gingival tissue were observed in the P. gingivalis-challenged mice, and were reduced by EGCG treatment. Real-time polymerase chain reaction (PCR) analyses also showed the expressions of IL-1β, IL-6, IL-17, IL-23, TNF-α and other mediators in gingival tissue were higher in P. gingivalis-challenged mice, and were down-regulated with EGCG treatment, except IL-23. Our results suggest that EGCG, as a natural healthy substance, probably alleviates P. gingivalis-induced periodontitis by anti-inflammatory effect.

  11. A Major Fimbrilin Variant of Mfa1 Fimbriae in Porphyromonas gingivalis.

    PubMed

    Nagano, K; Hasegawa, Y; Yoshida, Y; Yoshimura, F

    2015-08-01

    The periodontal pathogen Porphyromonas gingivalis is known to express 2 distinct types of fimbriae: FimA and Mfa1 fimbriae. However, we previously reported that fimbria-like structures were found in a P. gingivalis strain in which neither FimA nor Mfa1 fimbriae were detected. In this study, we identified a major protein in the bacterial lysates of the strain, which has been reported as the 53-kDa major outer membrane protein of P. gingivalis (53K protein) and subsequently reported as a major fimbrilin of a novel-type fimbria. Sequencing of the chromosomal DNA of the strain showed that the 53k gene (encoding the 53K protein) was located at a locus corresponding to the mfa1 gene (encoding the Mfa1 protein, which is a major fimbrilin of Mfa1 fimbriae) of the ATCC 33277 type strain. However, the 53K and Mfa1 proteins showed a low amino acid sequence homology and different antigenicity. The 53K protein was detected in 34 of 84 (41%) P. gingivalis strains, while the Mfa1 protein was detected in 44% of the strains. No strain expressed both 53K and Mfa1 proteins. Additionally, fimbriae were normally expressed in mutants in which the 53k and mfa1 genes were interchanged. These results indicate that the 53K protein is another major fimbrilin of Mfa1 fimbriae in P. gingivalis. PMID:26001707

  12. Porphyromonas gingivalis Accelerates Inflammatory Atherosclerosis in the Innominate Artery of ApoE Deficient Mice

    PubMed Central

    Hayashi, Chie; Viereck, Jason; Hua, Ning; Phinikaridou, Alkystis; Madrigal, Andres G.; Gibson, Frank C.; Hamilton, James A.; Genco, Caroline A.

    2011-01-01

    Objective Studies in humans support a role for the oral pathogen Porphyromonas gingivalis in the development of inflammatory atherosclerosis. The goal of this study was to determine if P. gingivalis infection accelerates inflammation and atherosclerosis in the innominate artery of mice, an artery which has been reported to exhibit many features of human atherosclerotic disease, including plaque rupture. Methods and Results Apolipoprotein E-deficient (ApoE−/−) mice were orally infected with P. gingivalis, and Magnetic Resonance Imaging (MRI) was used to monitor the progression of atherosclerosis in live mice. P. gingivalis infected mice exhibited a statistically significant increase in atherosclerotic plaque in the innominate artery as compared to uninfected mice. Polarized light microscopy and immunohistochemistry revealed that the innominate arteries of infected mice had increased lipids, macrophages and T cells as compared to uninfected mice. Increases in plaque, total cholesterol esters and cholesterol monohydrate crystals, macrophages, and T cells were prevented by immunization with heat-killed P. gingivalis prior to pathogen exposure. Conclusions These are the first studies to demonstrate progression of inflammatory plaque accumulation in the innominate arteries by in-vivo MRI analysis following pathogen exposure, and to document protection from plaque progression in the innominate artery via immunization. PMID:21251656

  13. Multilocus Sequence Typing of Porphyromonas gingivalis Strains from Different Geographic Origins

    PubMed Central

    Enersen, Morten; Olsen, Ingar; van Winkelhoff, Arie J.; Caugant, Dominique A.

    2006-01-01

    Porphyromonas gingivalis is an important periodontal pathogen that can be isolated from both active and inactive periodontal lesions. Apparently, differences in virulence between P. gingivalis strains exist, but the mechanisms underlying these differences are not yet fully understood. To obtain more information about pathogenicity and virulence of P. gingivalis, it is relevant to assess the genetic population structure of the species and to examine the occurrence of putative virulence factors against the genetic background. Presently, multilocus sequence typing (MLST) is the best method for analyzing bacterial population structures. Forty P. gingivalis strains from worldwide sources were analyzed by MLST. Internal 310- to 420-bp DNA fragments of the eight ubiquitous chromosomal genes, ftsQ, hagB, gdpxJ, pepO, mcmA, recA, pga, and nah, were amplified by PCR and then sequenced. The number of alleles at individual loci ranged from 2 to 19, and a total of 33 allelic profiles, or sequence types (STs), were identified. Nucleotide variation between alleles was located at one or a few sites. Identical or similar STs were found in isolates from different geographic regions. Our results showed signs of a clonal population structure with a level of recombination not as high as that previously suggested for the species. We also found that P. gingivalis isolates from individual patients were genetically heterogeneous. PMID:16390944

  14. Development of a novel plasmid vector pTIO-1 adapted for electrotransformation of Porphyromonas gingivalis.

    PubMed

    Tagawa, Junpei; Inoue, Tetsuyoshi; Naito, Mariko; Sato, Keiko; Kuwahara, Tomomi; Nakayama, Masaaki; Nakayama, Koji; Yamashiro, Takashi; Ohara, Naoya

    2014-10-01

    We report here the construction of a plasmid vector designed for the efficient electrotransformation of the periodontal pathogen Porphyromonas gingivalis. The novel Escherichia coli-Bacteroides/P. gingivalis shuttle vector, designated pTIO-1, is based on the 11.0-kb E. coli-Bacteroides conjugative shuttle vector, pVAL-1 (a pB8-51 derivative). To construct pTIO-1, the pB8-51 origin of replication and erythromycin resistance determinant of pVAL-1 were cloned into the E. coli cloning vector pBluescript II SK(-) and non-functional regions were deleted. pTIO-1 has an almost complete multiple cloning site from pBluescript II SK(-). The size of pTIO-1 is 4.5kb, which is convenient for routine gene manipulation. pTIO-1 was introduced into P. gingivalis via electroporation, and erythromycin-resistant transformants carrying pTIO-1 were obtained. We characterized the transformation efficiency, copy number, host range, stability, and insert size capacity of pTIO-1. An efficient plasmid electrotransformation of P. gingivalis will facilitate functional analysis and expression of P. gingivalis genes, including the virulence factors of this bacterium.

  15. Unprimed, M1 and M2 Macrophages Differentially Interact with Porphyromonas gingivalis.

    PubMed

    Lam, Roselind S; O'Brien-Simpson, Neil M; Holden, James A; Lenzo, Jason C; Fong, Shao B; Reynolds, Eric C

    2016-01-01

    Porphyromonas gingivalis is a keystone pathogen in the development of chronic periodontitis. Tissue macrophages are amongst the first immune cells to respond to bacteria and depending on the cytokine profile at the infection site, macrophages are primed to react to infection in different ways. Priming of naive macrophages with IFN-γ produces a classical pro-inflammatory, antibacterial M1 macrophage after TLR ligation, whereas priming with IL-4 induces an anti-inflammatory tissue-repair M2 phenotype. Previous work has shown that M1 are preferentially generated in gingival tissue following infection with P. gingivalis. However, few studies have investigated the interactions of macrophage subsets with P. gingivalis cells. The aim of this study was to determine the ability of naive, M1 and M2 macrophages to phagocytose P. gingivalis and investigate how this interaction affects both the bacterial cell and the macrophage. M1 and M2 macrophages were both found to have enhanced phagocytic capacity compared with that of naive macrophages, however only the naive and M1 macrophages were able to produce a respiratory burst in order to clear the bacteria from the phagosome. P. gingivalis was found to persist in naive and M2, but not M1 macrophages for 24 hours. Phagocytosis of P. gingivalis also induced high levels of TNF-α, IL-12 and iNOS in M1 macrophages, but not in naive or M2 macrophages. Furthermore, infection of macrophages with P. gingivalis at high bacteria to macrophage ratios, while inducing an inflammatory response, was also found to be deleterious to macrophage longevity, with high levels of apoptotic cell death found in macrophages after infection. The activation of M1 macrophages observed in this study may contribute to the initiation and maintenance of a pro-inflammatory state during chronic periodontitis. PMID:27383471

  16. Unprimed, M1 and M2 Macrophages Differentially Interact with Porphyromonas gingivalis

    PubMed Central

    Lenzo, Jason C.; Fong, Shao B.; Reynolds, Eric C.

    2016-01-01

    Porphyromonas gingivalis is a keystone pathogen in the development of chronic periodontitis. Tissue macrophages are amongst the first immune cells to respond to bacteria and depending on the cytokine profile at the infection site, macrophages are primed to react to infection in different ways. Priming of naive macrophages with IFN-γ produces a classical pro-inflammatory, antibacterial M1 macrophage after TLR ligation, whereas priming with IL-4 induces an anti-inflammatory tissue-repair M2 phenotype. Previous work has shown that M1 are preferentially generated in gingival tissue following infection with P. gingivalis. However, few studies have investigated the interactions of macrophage subsets with P. gingivalis cells. The aim of this study was to determine the ability of naive, M1 and M2 macrophages to phagocytose P. gingivalis and investigate how this interaction affects both the bacterial cell and the macrophage. M1 and M2 macrophages were both found to have enhanced phagocytic capacity compared with that of naive macrophages, however only the naive and M1 macrophages were able to produce a respiratory burst in order to clear the bacteria from the phagosome. P. gingivalis was found to persist in naive and M2, but not M1 macrophages for 24 hours. Phagocytosis of P. gingivalis also induced high levels of TNF-α, IL-12 and iNOS in M1 macrophages, but not in naive or M2 macrophages. Furthermore, infection of macrophages with P. gingivalis at high bacteria to macrophage ratios, while inducing an inflammatory response, was also found to be deleterious to macrophage longevity, with high levels of apoptotic cell death found in macrophages after infection. The activation of M1 macrophages observed in this study may contribute to the initiation and maintenance of a pro-inflammatory state during chronic periodontitis. PMID:27383471

  17. Characterization of the α- and β-Mannosidases of Porphyromonas gingivalis

    PubMed Central

    Aduse-Opoku, Joseph; Hashim, Ahmed; Paramonov, Nikolay; Curtis, Michael A.

    2013-01-01

    Mannose is an important sugar in the biology of the Gram-negative bacterium Porphyromonas gingivalis. It is a major component of the oligosaccharides attached to the Arg-gingipain cysteine proteases, the repeating units of an acidic lipopolysaccharide (A-LPS), and the core regions of both types of LPS produced by the organism (O-LPS and A-LPS) and a reported extracellular polysaccharide (EPS) isolated from spent culture medium. The organism occurs at inflamed sites in periodontal tissues, where it is exposed to host glycoproteins rich in mannose, which may be substrates for the acquisition of mannose by P. gingivalis. Five potential mannosidases were identified in the P. gingivalis W83 genome that may play a role in mannose acquisition. Four mannosidases were characterized in this study: PG0032 was a β-mannosidase, whereas PG0902 and PG1712 were capable of hydrolyzing p-nitrophenyl α-d-mannopyranoside. PG1711 and PG1712 were α-1→3 and α-1→2 mannosidases, respectively. No enzyme function could be assigned to PG0973. α-1→6 mannobiose was not hydrolyzed by P. gingivalis W50. EPS present in the culture supernatant was shown to be identical to yeast mannan and a component of the medium used for culturing P. gingivalis and was resistant to hydrolysis by mannosidases. Synthesis of O-LPS and A-LPS and glycosylation of the gingipains appeared to be unaffected in all mutants. Thus, α- and β-mannosidases of P. gingivalis are not involved in the harnessing of mannan/mannose from the growth medium for these biosynthetic processes. P. gingivalis grown in chemically defined medium devoid of carbohydrate showed reduced α-mannosidase activity (25%), suggesting these enzymes are environmentally regulated. PMID:24056103

  18. Adhesion of Actinomyces viscosus to Porphyromonas (Bacteroides) gingivalis-coated hexadecane droplets.

    PubMed Central

    Rosenberg, M; Buivids, I A; Ellen, R P

    1991-01-01

    Interbacterial adhesion (coadhesion) is considered a major determinant of dental plaque ecology. In this report, we studied several aspects of the adhesion of Porphyromonas (Bacteroides) gingivalis to hexadecane in order to use the liquid hydrocarbon as a convenient substratum for coadhesion assays. Washed suspensions of hydrophobic P. gingivalis 2561 cells were vortexed with hexadecane to yield highly stable cell-coated droplets. Kinetics of coadhesion between Actinomyces viscosus cells and P. gingivalis-coated hexadecane droplets (PCHD) was subsequently studied. Aliquots of PCHD were added to A. viscosus suspensions, and the mixtures were gently rotated. Avid adhesion of A. viscosus cells to the immobilized P. gingivalis layer could be readily measured by the decrease in turbidity in the aqueous phase, following phase separation. Despite the ability of A. viscosus cells to adsorb to hexadecane following vigorous mixing, gentle mixing did not appreciably promote adhesion to bare hexadecane. Moreover, extensive microscopic examinations revealed that A. viscosus cells adhered exclusively to the bound P. gingivalis cells rather than to exposed areas of hexadecane. Coadhesion of A. viscosus to the PCHD appeared to follow first-order kinetics, attaining 80% levels within 30 min. Electron micrographs revealed A. viscosus cells adhering to the P. gingivalis cell layer adsorbed at the hexadecane-water interface. Interestingly, P. gingivalis cells did not appear to penetrate the hexadecane. A viscosus mutants lacking type 1 or type 2 fimbriae or both were still able to bind to the PCHD. No obvious correlation was observed between relative hydrophobicity of A. viscosus strains and their binding to PCHD. However, defatted bovine serum albumin, an inhibitor of hydrophobic interactions, was the most potent inhibitor among those tested. The data suggest that this approach provides a simple, quantitative technique for studying kinetics of bacterial coadhesion which is amenable

  19. Crystallization and preliminary X-ray characterization of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis

    SciTech Connect

    Nakajima, Yoshitaka; Ito, Kiyoshi Xu, Yue; Yamada, Nozomi; Onohara, Yuko; Ito, Takashi; Yoshimoto, Tadashi

    2005-12-01

    P. gingivalis prolyl tripeptidyl aminopeptidase has been crystallized by the vapour-diffusion method. Diffraction data have been collected and processed to 2.1 Å resolution. A recombinant form of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis has been crystallized by the hanging-drop vapour-diffusion method using potassium sodium tartrate as a precipitating agent. The crystals belong to the hexagonal space group P6{sub 3}22, with unit-cell parameters a = b = 149.4, c = 159.7 Å. The crystals are most likely to contain one subunit of a dimer in the asymmetric unit, with a V{sub M} value of 3.14 Å{sup 3} Da{sup −1}. Diffraction data were collected to 2.1 Å resolution using synchrotron radiation at the BL5 station of the Photon Factory.

  20. Porphyromonas gingivalis: An Overview of Periodontopathic Pathogen below the Gum Line

    PubMed Central

    How, Kah Yan; Song, Keang Peng; Chan, Kok Gan

    2016-01-01

    Periodontal disease represents a group of oral inflammatory infections initiated by oral pathogens which exist as a complex biofilms on the tooth surface and cause destruction to tooth supporting tissues. The severity of this disease ranges from mild and reversible inflammation of the gingiva (gingivitis) to chronic destruction of connective tissues, the formation of periodontal pocket and ultimately result in loss of teeth. While human subgingival plaque harbors more than 500 bacterial species, considerable research has shown that Porphyromonas gingivalis, a Gram-negative anaerobic bacterium, is the major etiologic agent which contributes to chronic periodontitis. This black-pigmented bacterium produces a myriad of virulence factors that cause destruction to periodontal tissues either directly or indirectly by modulating the host inflammatory response. Here, this review provides an overview of P. gingivalis and how its virulence factors contribute to the pathogenesis with other microbiome consortium in oral cavity. PMID:26903954

  1. Porphyromonas gingivalis: An Overview of Periodontopathic Pathogen below the Gum Line.

    PubMed

    How, Kah Yan; Song, Keang Peng; Chan, Kok Gan

    2016-01-01

    Periodontal disease represents a group of oral inflammatory infections initiated by oral pathogens which exist as a complex biofilms on the tooth surface and cause destruction to tooth supporting tissues. The severity of this disease ranges from mild and reversible inflammation of the gingiva (gingivitis) to chronic destruction of connective tissues, the formation of periodontal pocket and ultimately result in loss of teeth. While human subgingival plaque harbors more than 500 bacterial species, considerable research has shown that Porphyromonas gingivalis, a Gram-negative anaerobic bacterium, is the major etiologic agent which contributes to chronic periodontitis. This black-pigmented bacterium produces a myriad of virulence factors that cause destruction to periodontal tissues either directly or indirectly by modulating the host inflammatory response. Here, this review provides an overview of P. gingivalis and how its virulence factors contribute to the pathogenesis with other microbiome consortium in oral cavity. PMID:26903954

  2. The core genome of the anaerobic oral pathogenic bacterium Porphyromonas gingivalis

    PubMed Central

    2010-01-01

    Background The Gram negative anaerobic bacterium Porphyromonas gingivalis has long been recognized as a causative agent of periodontitis. Periodontitis is a chronic infectious disease of the tooth supporting tissues eventually leading to tooth-loss. Capsular polysaccharide (CPS) of P. gingivalis has been shown to be an important virulence determinant. Seven capsular serotypes have been described. Here, we used micro-array based comparative genomic hybridization analysis (CGH) to analyze a representative of each of the capsular serotypes and a non-encapsulated strain against the highly virulent and sequenced W83 strain. We defined absent calls using Arabidopsis thaliana negative control probes, with the aim to distinguish between aberrations due to mutations and gene gain/loss. Results Our analyses allowed us to call aberrant genes, absent genes and divergent regions in each of the test strains. A conserved core P. gingivalis genome was described, which consists of 80% of the analyzed genes from the sequenced W83 strain. The percentage of aberrant genes between the test strains and control strain W83 was 8.2% to 13.7%. Among the aberrant genes many CPS biosynthesis genes were found. Most other virulence related genes could be found in the conserved core genome. Comparing highly virulent strains with less virulent strains indicates that hmuS, a putative CobN/Mg chelatase involved in heme uptake, may be a more relevant virulence determinant than previously expected. Furthermore, the description of the 39 W83-specific genes could give more insight in why this strain is more virulent than others. Conclusion Analyses of the genetic content of the P. gingivalis capsular serotypes allowed the description of a P. gingivalis core genome. The high resolution data from three types of analysis of triplicate hybridization experiments may explain the higher divergence between P. gingivalis strains than previously recognized. PMID:20920246

  3. Dipeptidyl peptidase with strict substrate specificity of an anaerobic periodontopathogen Porphyromonas gingivalis.

    PubMed

    Fujimura, Setsuo; Hirai, Kaname; Shibata, Yukinaga

    2002-03-19

    A dipeptidyl peptidase which hydrolyzed Xaa-Ala-p-nitroanilide was purified to homogeneity by sequential procedures including ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic interaction chromatography, gel filtration and isoelectric focusing from the cell extract of Porphyromonas gingivalis. The purified enzyme hydrolyzed p-nitroanilide derivatives of Lys-Ala, Ala-Ala, and Val-Ala, but not Xaa-Pro. Enzyme activity was maximum at neutral pHs. Its molecular mass was 64 kDa with an isoelectric point of 5.7. The enzyme belonged to the family of serine peptidases. PMID:12007665

  4. Synthesis of sulfonamides with effective inhibitory action against Porphyromonas gingivalis γ-carbonic anhydrase.

    PubMed

    Ceruso, Mariangela; Del Prete, Sonia; AlOthman, Zeid; Osman, Sameh M; Scozzafava, Andrea; Capasso, Clemente; Supuran, Claudiu T

    2014-08-15

    New benzenesulfonamides incorporating GABA or N-α-acetyl-L-lysine scaffolds as well as guanidine functionalities as water solubilizing moieties were obtained, using 4-aminoethyl/methyl-benzenesulfonamide and metanilamide/sulfanilamide as zinc-binding motives. The new compounds were medium potency inhibitors of the widespread cytosolic human carbonic anhydrase (CA, EC 4.2.1.1) isoforms I and II and more effective inhibitors (KIs low nanomolar range) of the bacterial γ-CA from the oral pathogen Porphyromonas gingivalis. These sulfonamides may be useful tools for understanding the physiological role of bacterial CAs in pathogenesis of some infectious disease. PMID:25011913

  5. Photodynamic destruction of Porphyromonas gingivalis induced by delta-aminolaevulinic acid

    NASA Astrophysics Data System (ADS)

    Sieron, Aleksander; Wiczkowski, Andrzej; Adamek, Mariusz; Dyla, Lucja; Mazur, Sebastian; Wierucka-Mlynarczyk, Beata

    2004-09-01

    Photodynamic therapy (PDT) is one of a novel modalities which has recently been exploited to eradicate various microorganisms. In our study we have evaluated bactericidal efficacy of PDT in the presence of 5-δ aminolaevulinic acid (ALA). Porphyromonas gingivalis were incubated with increasing concentration of ALA and subsequently irradiated by progressive light doses. Complete killing effect was obtained for bacteria irradiated with 25J/cm2 in ALA solution final concentration of 1mM, 5mM, 10mM. Statistical analysis has revealed ALA concentration to be a major factor responsible for eradication of bacteria. The latter may be attributable to the known ALA dark toxicity.

  6. Isolation and characterization of a cloned Porphyromonas gingivalis hemagglutinin from an avirulent strain of Salmonella typhimurium.

    PubMed Central

    Dusek, D M; Progulske-Fox, A; Whitlock, J; Brown, T A

    1993-01-01

    Identification of surface macromolecules of Porphyromonas gingivalis that act as virulence factors in periodontal disease has important implications for studying host-parasite interactions as well as for potential vaccine development. The objective of this study was to determine whether a cloned, P. gingivalis hemagglutinin gene could be expressed in an intact form in an avirulent Salmonella typhimurium vaccine construct and to characterize the recombinant protein. The recombinant protein was purified from the vaccine strain, characterized, and tested for biological activity as a competitive inhibitor of hemagglutination. Cells of S. typhimurium SL3261/pST7 grown in Luria broth were broken by sonic disruption and fractionated. The purified recombinant protein was found to inhibit hemagglutination of erythrocytes by whole P. gingivalis cells. The same purified protein was analyzed for its N-terminal amino acid sequence and amino acid composition and found to match that predicted from the nucleotide sequence of the cloned gene. These results indicate that a surface macromolecule of P. gingivalis can be expressed in an intact and biologically active form in a Salmonella carrier strain. Images PMID:8381773

  7. In Situ Anabolic Activity of Periodontal Pathogens Porphyromonas gingivalis and Filifactor alocis in Chronic Periodontitis.

    PubMed

    Spooner, Ralee; Weigel, Kris M; Harrison, Peter L; Lee, KyuLim; Cangelosi, Gerard A; Yilmaz, Özlem

    2016-01-01

    Porphyromonas gingivalis and Filifactor alocis are fastidious anaerobic bacteria strongly associated with chronic forms of periodontitis. Our understanding of the growth activities of these microorganisms in situ is very limited. Previous studies have shown that copy numbers of ribosomal-RNA precursor (pre-rRNA) of specific pathogen species relative to genomic-DNA (gDNA) of the same species (P:G ratios) are greater in actively growing bacterial cells than in resting cells. The method, so-called steady-state pre-rRNA-analysis, represents a novel culture-independent approach to study bacteria. This study employed this technique to examine the in situ growth activities of oral bacteria in periodontitis before and after non-surgical periodontal therapy. Sub-gingival paper-point samples were taken at initial and re-evaluation appointments. Pre-rRNA and gDNA levels of P. gingivalis and F. alocis were quantified and compared using reverse-transcriptase qPCR. The results indicate significantly reduced growth activity of P. gingivalis, but not F. alocis, after therapy. The P:G ratios of P. gingivalis and F. alocis were compared and a low-strength, but statistically significant inter-species correlation was detected. Our study demonstrates that steady-state pre-rRNA-analysis can be a valuable culture-independent approach to studying opportunistic bacteria in periodontitis. PMID:27642101

  8. Comparison of inherently essential genes of Porphyromonas gingivalis identified in two transposon-sequencing libraries.

    PubMed

    Hutcherson, J A; Gogeneni, H; Yoder-Himes, D; Hendrickson, E L; Hackett, M; Whiteley, M; Lamont, R J; Scott, D A

    2016-08-01

    Porphyromonas gingivalis is a Gram-negative anaerobe and keystone periodontal pathogen. A mariner transposon insertion mutant library has recently been used to define 463 genes as putatively essential for the in vitro growth of P. gingivalis ATCC 33277 in planktonic culture (Library 1). We have independently generated a transposon insertion mutant library (Library 2) for the same P. gingivalis strain and herein compare genes that are putatively essential for in vitro growth in complex media, as defined by both libraries. In all, 281 genes (61%) identified by Library 1 were common to Library 2. Many of these common genes are involved in fundamentally important metabolic pathways, notably pyrimidine cycling as well as lipopolysaccharide, peptidoglycan, pantothenate and coenzyme A biosynthesis, and nicotinate and nicotinamide metabolism. Also in common are genes encoding heat-shock protein homologues, sigma factors, enzymes with proteolytic activity, and the majority of sec-related protein export genes. In addition to facilitating a better understanding of critical physiological processes, transposon-sequencing technology has the potential to identify novel strategies for the control of P. gingivalis infections. Those genes defined as essential by two independently generated TnSeq mutant libraries are likely to represent particularly attractive therapeutic targets.

  9. Mechanisms of Resistance of Porphyromonas gingivalis to Killing by Serum Complement

    PubMed Central

    Slaney, Jennifer M.; Gallagher, Alexandra; Aduse-Opoku, Joseph; Pell, Keith; Curtis, Michael A.

    2006-01-01

    The complement system plays an important role in the host defense against infection, and the formation of the terminal complement complex on the bacterial surface has been shown to be particularly important in killing of gram-negative bacteria. The gram-negative periodontal pathogen Porphyromonas gingivalis is resistant to complement killing, and possible mechanisms suggested for this resistance include protease production and capsule formation. In this study, P. gingivalis Arg- and Lys-gingipain deletion mutants and polysaccharide synthesis deletion mutants have been used to investigate these hypotheses. When Arg- and Lys-gingipain protease mutants were incubated in 20% normal human serum, deposition of complement components on the cell surface was significantly increased compared to that for the wild-type organism. However, despite the increased deposition, the protease mutants maintained resistance to killing and their viability was equal to that seen with heat-inactivated serum. Similar data were obtained when the wild-type organism was treated with gingipain protease inhibitors. K-antigen expression mutants were also resistant to killing. However, mutants which no longer synthesized a surface anionic polysaccharide (APS) (a phosphorylated branched mannan) were extremely sensitive to serum killing. These mutants lack the organized dense glycan surface layer present on the parent strain on the basis of electron microscopy. We conclude that the production of APS at the surface of P. gingivalis rather than Arg- and Lys-gingipain synthesis is the principal mechanism of serum resistance in P. gingivalis. PMID:16926430

  10. In Situ Anabolic Activity of Periodontal Pathogens Porphyromonas gingivalis and Filifactor alocis in Chronic Periodontitis

    PubMed Central

    Spooner, Ralee; Weigel, Kris M.; Harrison, Peter L.; Lee, KyuLim; Cangelosi, Gerard A.; Yilmaz, Özlem

    2016-01-01

    Porphyromonas gingivalis and Filifactor alocis are fastidious anaerobic bacteria strongly associated with chronic forms of periodontitis. Our understanding of the growth activities of these microorganisms in situ is very limited. Previous studies have shown that copy numbers of ribosomal-RNA precursor (pre-rRNA) of specific pathogen species relative to genomic-DNA (gDNA) of the same species (P:G ratios) are greater in actively growing bacterial cells than in resting cells. The method, so-called steady-state pre-rRNA-analysis, represents a novel culture-independent approach to study bacteria. This study employed this technique to examine the in situ growth activities of oral bacteria in periodontitis before and after non-surgical periodontal therapy. Sub-gingival paper-point samples were taken at initial and re-evaluation appointments. Pre-rRNA and gDNA levels of P. gingivalis and F. alocis were quantified and compared using reverse-transcriptase qPCR. The results indicate significantly reduced growth activity of P. gingivalis, but not F. alocis, after therapy. The P:G ratios of P. gingivalis and F. alocis were compared and a low-strength, but statistically significant inter-species correlation was detected. Our study demonstrates that steady-state pre-rRNA-analysis can be a valuable culture-independent approach to studying opportunistic bacteria in periodontitis. PMID:27642101

  11. Effect of Porphyromonas gingivalis on epithelial cell MMP-9 type IV collagenase production.

    PubMed Central

    Fravalo, P; Ménard, C; Bonnaure-Mallet, M

    1996-01-01

    Porphyromonas gingivalis is reportedly capable of stimulating the expression of host cell matrix metalloproteinases (MMP), contributing to tissue destruction. However, the impact of this bacterium on specific molecules remains to be determined. In this study, we evaluate the effect of P. gingivalis on regulation of MMP-9 expression in human gingival epithelial cells (HGEC). Various inocula of P. gingivalis were added to cultures of HGEC. The effects of live bacteria, heat-killed bacteria, and outer membrane extract were analyzed. MMP-9 secretion by HGEC was evaluated by enzyme-linked immunosorbent assay. For inocula smaller than one bacterium per cell, the quantity of MMP-9 secreted by HGEC was increased in comparison to control conditions. For inocula from 2.5 to 250 bacteria per cell, an inhibition of MMP-9 secretion in a dose-response fashion was observed, with a maximum reduction (ranging from 80 to 95% in five experiments) at 50 bacteria per cell. Gelatin zymograms confirmed the decrease in MMP-9 secretion. A band of 83 kDa, corresponding to activated enzyme, was present for inocula of 0.5 to 50 bacteria. Inhibition took place without any alteration of epithelial cell viability. Heat-killed bacteria and outer membrane extract also provoked proenzyme activation but did not inhibit MMP-9 secretion. These results demonstrate a direct effect of P. gingivalis on HGEC, suggesting a specific action on the collagen renewal process at the interface between the epithelium and connective tissue. PMID:8945530

  12. Structures of the Porphyromonas gingivalis OxyR regulatory domain explain differences in expression of the OxyR regulon in Escherichia coli and P. gingivalis

    SciTech Connect

    Svintradze, David V.; Peterson, Darrell L.; Collazo-Santiago, Evys A.; Lewis, Janina P.; Wright, H. Tonie

    2013-10-01

    Differences in OxyR regulated expression of oxidative stress genes between Escherichia coli and Porphyromonas gingivalis are explained by very minor differences in structure and amino-acid sequence of the respective oxidized and reduced OxyR regulatory domains. These differences affect OxyR quaternary structures and are predicted from model building of full length OxyR–DNA complexes to confer distinct modes of DNA binding on this transcriptional regulator. OxyR transcriptionally regulates Escherichia coli oxidative stress response genes through a reversibly reducible cysteine disulfide biosensor of cellular redox status. Structural changes induced by redox changes in these cysteines are conformationally transmitted to the dimer subunit interfaces, which alters dimer and tetramer interactions with DNA. In contrast to E. coli OxyR regulatory-domain structures, crystal structures of Porphyromonas gingivalis OxyR regulatory domains show minimal differences in dimer configuration on changes in cysteine disulfide redox status. This locked configuration of the P. gingivalis OxyR regulatory-domain dimer closely resembles the oxidized (activating) form of the E. coli OxyR regulatory-domain dimer. It correlates with the observed constitutive activation of some oxidative stress genes in P. gingivalis and is attributable to a single amino-acid insertion in P. gingivalis OxyR relative to E. coli OxyR. Modelling of full-length P. gingivalis, E. coli and Neisseria meningitidis OxyR–DNA complexes predicts different modes of DNA binding for the reduced and oxidized forms of each.

  13. Effects of Porphyromonas gingivalis LipopolysaccharideTolerized Monocytes on Inflammatory Responses in Neutrophils

    PubMed Central

    Chen, Yang; Cheng, Xiao-fan; Qiu, Jia-ying; Xu, Yan; Sun, Ying

    2016-01-01

    Periodontitis is a chronic inflammatory disease induced by bacteria. Exposure of the host to periodontal pathogens and their virulence factors induces a state of hyporesponsiveness to subsequent stimulations, which is termed endotoxin tolerance. The role and mechanism of lipopolysaccharide (LPS)–tolerized monocytes in inflammatory responses in neutrophils are currently unclear. Here, conditioned supernatants were collected from THP-1 cells treated with or without repeated 1 μg/ml Porphyromonas gingivalis (P.gingivalis) LPS. The chemotactic response of freshly isolated neutrophils recruited by supernatants was determined by a transwell migration assay, which demonstrated a reduced migration of neutrophils stimulated with supernatants from tolerized THP-1 cells in comparison to non-tolerized THP-1 cells. In addition, there was a marked increase in reactive oxygen species (ROS) generation and a significant decrease in Caspase 3 activities in neutrophils treated with supernatants from THP-1 cells that were treated repeatedly with P.gingivalis LPS in comparison to single treatment. A cytokine antibody array was then used to assess cytokine expression patterns in THP-1 cells. In tolerized THP-1 cells, 43 cytokine (43/170) expression levels were decreased, including chemokine ligand 23 (CCL23) and IFN-γ, while 11 cytokine (11/170) expression levels were increased, such as death receptor 6 (DR6). Furthermore, there was decreased production of IFN-γ and epithelial neutrophil activating peptide-78 (ENA-78) in THP-1 cells after stimulation with repeated P. gingivalis LPS in comparison to single challenge, which was confirmed by ELISA. Therefore, P.gingivalis LPS- tolerized THP-1 cells were able to depress neutrophil chemotaxis and apoptosis, and contribute to respiratory burst, which might be related to the changes in cytokine expression patterns in THP-1 cells. PMID:27536946

  14. Peptidyl arginine deiminase from Porphyromonas gingivalis abolishes anaphylatoxin C5a activity.

    PubMed

    Bielecka, Ewa; Scavenius, Carsten; Kantyka, Tomasz; Jusko, Monika; Mizgalska, Danuta; Szmigielski, Borys; Potempa, Barbara; Enghild, Jan J; Prossnitz, Eric R; Blom, Anna M; Potempa, Jan

    2014-11-21

    Evasion of killing by the complement system, a crucial part of innate immunity, is a key evolutionary strategy of many human pathogens. A major etiological agent of chronic periodontitis, the Gram-negative bacterium Porphyromonas gingivalis, produces a vast arsenal of virulence factors that compromise human defense mechanisms. One of these is peptidylarginine deiminase (PPAD), an enzyme unique to P. gingivalis among bacteria, which converts Arg residues in polypeptide chains into citrulline. Here, we report that PPAD citrullination of a critical C-terminal arginine of the anaphylatoxin C5a disabled the protein function. Treatment of C5a with PPAD in vitro resulted in decreased chemotaxis of human neutrophils and diminished calcium signaling in monocytic cell line U937 transfected with the C5a receptor (C5aR) and loaded with a fluorescent intracellular calcium probe: Fura-2 AM. Moreover, a low degree of citrullination of internal arginine residues by PPAD was also detected using mass spectrometry. Further, after treatment of C5 with outer membrane vesicles naturally shed by P. gingivalis, we observed generation of C5a totally citrullinated at the C-terminal Arg-74 residue (Arg74Cit). In stark contrast, only native C5a was detected after treatment with PPAD-null outer membrane vesicles. Our study suggests reduced antibacterial and proinflammatory capacity of citrullinated C5a, achieved via lower level of chemotactic potential of the modified molecule, and weaker cell activation. In the context of previous studies, which showed crosstalk between C5aR and Toll-like receptors, as well as enhanced arthritis development in mice infected with PPAD-expressing P. gingivalis, our findings support a crucial role of PPAD in the virulence of P. gingivalis. PMID:25324545

  15. Effects of Porphyromonas gingivalis LipopolysaccharideTolerized Monocytes on Inflammatory Responses in Neutrophils.

    PubMed

    Zhu, Xiang-Qing; Lu, Wei; Chen, Yang; Cheng, Xiao-Fan; Qiu, Jia-Ying; Xu, Yan; Sun, Ying

    2016-01-01

    Periodontitis is a chronic inflammatory disease induced by bacteria. Exposure of the host to periodontal pathogens and their virulence factors induces a state of hyporesponsiveness to subsequent stimulations, which is termed endotoxin tolerance. The role and mechanism of lipopolysaccharide (LPS)-tolerized monocytes in inflammatory responses in neutrophils are currently unclear. Here, conditioned supernatants were collected from THP-1 cells treated with or without repeated 1 μg/ml Porphyromonas gingivalis (P.gingivalis) LPS. The chemotactic response of freshly isolated neutrophils recruited by supernatants was determined by a transwell migration assay, which demonstrated a reduced migration of neutrophils stimulated with supernatants from tolerized THP-1 cells in comparison to non-tolerized THP-1 cells. In addition, there was a marked increase in reactive oxygen species (ROS) generation and a significant decrease in Caspase 3 activities in neutrophils treated with supernatants from THP-1 cells that were treated repeatedly with P.gingivalis LPS in comparison to single treatment. A cytokine antibody array was then used to assess cytokine expression patterns in THP-1 cells. In tolerized THP-1 cells, 43 cytokine (43/170) expression levels were decreased, including chemokine ligand 23 (CCL23) and IFN-γ, while 11 cytokine (11/170) expression levels were increased, such as death receptor 6 (DR6). Furthermore, there was decreased production of IFN-γ and epithelial neutrophil activating peptide-78 (ENA-78) in THP-1 cells after stimulation with repeated P. gingivalis LPS in comparison to single challenge, which was confirmed by ELISA. Therefore, P.gingivalis LPS- tolerized THP-1 cells were able to depress neutrophil chemotaxis and apoptosis, and contribute to respiratory burst, which might be related to the changes in cytokine expression patterns in THP-1 cells. PMID:27536946

  16. Functional Analysis of Porphyromonas gingivalis W83 CRISPR-Cas Systems

    PubMed Central

    Burmistrz, Michał; Dudek, Bartosz; Staniec, Dominika; Rodriguez Martinez, Jose Ignacio; Bochtler, Matthias; Potempa, Jan

    2015-01-01

    ABSTRACT The CRISPR-Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) system provides prokaryotic cells with an adaptive and heritable immune response to foreign genetic elements, such as viruses, plasmids, and transposons. It is present in the majority of Archaea and almost half of species of Bacteria. Porphyromonas gingivalis is an important human pathogen that has been proven to be an etiological agent of periodontitis and has been linked to systemic conditions, such as rheumatoid arthritis and cardiovascular disease. At least 95% of clinical strains of P. gingivalis carry CRISPR arrays, suggesting that these arrays play an important function in vivo. Here we show that all four CRISPR arrays present in the P. gingivalis W83 genome are transcribed. For one of the arrays, we demonstrate in vivo activity against double-stranded DNA constructs containing protospacer sequences accompanied at the 3′ end by an NGG protospacer-adjacent motif (PAM). Most of the 44 spacers present in the genome of P. gingivalis W83 share no significant similarity with any known sequences, although 4 spacers are similar to sequences from bacteria found in the oral cavity and the gastrointestinal tract. Four spacers match genomic sequences of the host; however, none of these is flanked at its 3′ terminus by the appropriate PAM element. IMPORTANCE The CRISPR-Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) system is a unique system that provides prokaryotic cells with an adaptive and heritable immunity. In this report, we show that the CRISPR-Cas system of P. gingivalis, an important human pathogen associated with periodontitis and possibly also other conditions, such as rheumatoid arthritis and cardiovascular disease, is active and provides protection from foreign genetic elements. Importantly, the data presented here may be useful for better understanding the communication between cells in larger bacterial

  17. Structure and mechanism of a bacterial host-protein citrullinating virulence factor, Porphyromonas gingivalis peptidylarginine deiminase

    PubMed Central

    Goulas, Theodoros; Mizgalska, Danuta; Garcia-Ferrer, Irene; Kantyka, Tomasz; Guevara, Tibisay; Szmigielski, Borys; Sroka, Aneta; Millán, Claudia; Usón, Isabel; Veillard, Florian; Potempa, Barbara; Mydel, Piotr; Solà, Maria; Potempa, Jan; Gomis-Rüth, F. Xavier

    2015-01-01

    Citrullination is a post-translational modification of higher organisms that deiminates arginines in proteins and peptides. It occurs in physiological processes but also pathologies such as multiple sclerosis, fibrosis, Alzheimer’s disease and rheumatoid arthritis (RA). The reaction is catalyzed by peptidylarginine deiminases (PADs), which are found in vertebrates but not in lower organisms. RA has been epidemiologically associated with periodontal disease, whose main infective agent is Porphyromonas gingivalis. Uniquely among microbes, P. gingivalis secretes a PAD, termed PPAD (Porphyromonas peptidylarginine deiminase), which is genetically unrelated to eukaryotic PADs. Here, we studied function of PPAD and its substrate-free, substrate-complex, and substrate-mimic-complex structures. It comprises a flat cylindrical catalytic domain with five-fold α/β-propeller architecture and a C-terminal immunoglobulin-like domain. The PPAD active site is a funnel located on one of the cylinder bases. It accommodates arginines from peptide substrates after major rearrangement of a “Michaelis loop” that closes the cleft. The guanidinium and carboxylate groups of substrates are tightly bound, which explains activity of PPAD against arginines at C-termini but not within peptides. Catalysis is based on a cysteine-histidine-asparagine triad, which is shared with human PAD1-PAD4 and other guanidino-group modifying enzymes. We provide a working mechanism hypothesis based on 18 structure-derived point mutants. PMID:26132828

  18. PERIODONTAL DISEASE AND BONE PATHOGENESIS: THE CROSSTALK BETWEEN CYTOKINES AND PORPHYROMONAS GINGIVALIS.

    PubMed

    Ballini, A; Cantore, S; Farronato, D; Cirulli, N; Inchingolo, F; Papa, F; Malcangi, G; Inchingolo, A D; Dipalma, G; Sardaro, N; Lippolis, R; Santacroce, L; Coscia, M F; Pettini, F; De Vito, D; Scacco, S

    2015-01-01

    Periodontal disease is the most frequent cause of tooth loss among adults. It is defined as a plaque-induced inflammation of the periodontal tissues that results in a loss of support of the affected teeth. This process is characterized by destruction of the periodontal attachment apparatus, increased bone resorption with loss of crestal alveolar bone, apical migration of the epithelial attachment, and formation of periodontal pockets. Although the presence of periodontal pathogens such as Porphyromonas gingivalis is a prerequisite, the progression of periodontal disease is dependent on the host response to pathogenic bacteria that colonize the tooth surface. Nowadays, a growing body of literature has accumulated to investigate the association between bone diseases, periodontal pathogens and periodontal diseases. The integration of pathogen-associated molecular patterns from microorganisms with their surface receptors in the immune cells, induces the production of several cytokines and chemokines that present either a pro- and/or anti-inflammatory role and the activation of mechanisms of controlling this and the related disease, such as osteoporosis and rheumatoid arthritis. This review focuses on the evidence and significance of bone host cell invasion by Porphyromonas gingivalis in the pathogenesis of bone disorders, as well as the different lines of evidence supporting the role of cytokines in bone diseases. PMID:26122214

  19. Loop-mediated isothermal amplification combined with PCR and immunohistochemistry for detecting Porphyromonas gingivalis in periapical periodontitis.

    PubMed

    Kitano, Taiichi; Mikami, Yoshikazu; Iwase, Takashi; Asano, Masatake; Komiyama, Kazuo

    2016-01-01

    Porphyromonas gingivalis is important in the development of marginal periodontitis. However, the precise role and localization of P. gingivalis in chronic periapical periodontitis remain unclear. Thus, methods that can detect P. gingivalis in formalin-fixed and paraffin-embedded (FFPE) tissue samples are needed. We assessed a technique combining loop-mediated isothermal amplification (LAMP) with PCR (PCR-LAMP) for detection of P. gingivalis, using 110 FFPE tissue samples of chronic apical periodontitis. PCR-LAMP specifically detected P. gingivalis with high sensitivity in FFPE tissue samples, and the sensitivity of the technique was higher than that of PCR or LAMP alone. The results of immunohistochemistry (IHC) confirmed the specificity of PCR-LAMP. IHC showed that P. gingivalis was localized in a granular layer of chronic apical periodontitis, a region that correlated with the localization of macrophages. This is the first study to describe the localization of P. gingivalis in human periapical periodontitis. In conclusion, PCR-LAMP was an effective tool for detecting P. gingivalis in periapical periodontitis. In addition, IHC results improve our understanding of the role of P. gingivalis in the progression of periapical periodontitis. (J Oral Sci 58, 163-169, 2016).

  20. Structure determination and analysis of a haemolytic gingipain adhesin domain from Porphyromonas gingivalis

    SciTech Connect

    Li, N.; Yun, P.; Nadkarni, M.A.; Ghadikolaee, N.B.; Nguyen, K.A.; Lee, M.; Hunter, N.; Collyer, C.A.

    2010-08-27

    Porphyromonas gingivalis is an obligately anaerobic bacterium recognized as an aetiological agent of adult periodontitis. P. gingivalis produces cysteine proteinases, the gingipains. The crystal structure of a domain within the haemagglutinin region of the lysine gingipain (Kgp) is reported here. The domain was named K2 as it is the second of three homologous structural modules in Kgp. The K2 domain structure is a 'jelly-roll' fold with two anti-parallel {beta}-sheets. This fold topology is shared with adhesive domains from functionally diverse receptors such as MAM domains, ephrin receptor ligand binding domains and a number of carbohydrate binding modules. Possible functions of K2 were investigated. K2 induced haemolysis of erythrocytes in a dose-dependent manner that was augmented by the blocking of anion transport. Further, cysteine-activated arginine gingipain RgpB, which degrades glycophorin A, sensitized erythrocytes to the haemolytic effect of K2. Cleaved K2, similar to that found in extracted Kgp, lacks the haemolytic activity indicating that autolysis of Kgp may be a staged process which is artificially enhanced by extraction of the protein. The data indicate a functional role for K2 in the integrated capacity conferred by Kgp to enable the porphyrin auxotroph P. gingivalis to capture essential haem from erythrocytes.

  1. Genetic structure of populations of Porphyromonas gingivalis associated with periodontitis and other oral infections.

    PubMed Central

    Loos, B G; Dyer, D W; Whittam, T S; Selander, R K

    1993-01-01

    One hundred isolates of the oral pathogenic bacterium Porphyromonas gingivalis were genetically characterized by determining the electrophoretic mobilities of 16 metabolic enzymes and the presence or absence of catalase activity. A total of 78 distinct electrophoretic types (ETs), representing multilocus genotypes, were identified, and cluster analysis placed them in three major phylogenetic divisions. Division I (71 ETs) included all 88 human isolates examined, most of which had been recovered from patients with periodontitis, together with 4 monkey isolates. The strains in division II (four ETs) and division III (three ETs) are strongly differentiated from those in division I and apparently represent two previously unclassified (cryptic) species. The mean genetic diversity per enzyme locus among the 92 isolates of division I (P. gingivalis, strict sense) was 0.321, and the strains were distributed among 14 phylogenetic clusters and single-ET lineages. The population structure is basically clonal, with some clonal genotypes being widespread, and even global, in distribution. There was no evidence of association between specific genetic lineages or clusters of ETs and the type of disease (periodontitis or root canal infections), invasive potential, serogroup, or fimbrial restriction fragment length polymorphism group. The finding that dental patients are infected by strains of a wide variety of chromosomal genotypes suggests that interstrain variation in pathogenicity is small. On the basis of the observed genetic structure of natural populations of P. gingivalis, we hypothesize that the role of this microorganism in the pathogenesis of periodontitis and other dental infections is largely opportunistic. PMID:8380281

  2. Effects of temperature stress on expression of fimbriae and superoxide dismutase by Porphyromonas gingivalis.

    PubMed Central

    Amano, A; Sharma, A; Sojar, H T; Kuramitsu, H K; Genco, R J

    1994-01-01

    We examined the biosynthesis of fimbriae and superoxide dismutase (SOD) produced by the periodontopathic bacterium Porphyromonas gingivalis in response to elevated temperature. P. gingivalis 2561, grown at 37 degrees C to mid-logarithmic phase, was subsequently incubated at 39, 41, and 43 degrees C, respectively, to stationary phase. There was no difference in the growth of cells at 37 and 39 degrees C. However, at 39 degrees C there was a 54% reduction in the amount of fimbrillin (fimbriae) as well as decreased expression of mRNA for fimA. On the other hand, under the same conditions, a more than twofold increase in the amount of SOD activity, as well as in the levels of SOD mRNA, was observed. Moreover, cells cultured for 20 h at 39 degrees C showed an 86% decrease of fimbrillin protein and a threefold increase in SOD activity. These observations suggest that P. gingivalis may undergo alterations in its virulence and susceptibility to host immune responses as a result of the elevated temperatures found in inflamed periodontal pockets. Images PMID:7927742

  3. Role of Porphyromonas gingivalis HmuY in Immunopathogenesis of Chronic Periodontitis

    PubMed Central

    Gomes-Filho, I. S.; Meyer, R.; Olczak, T.; Xavier, M. T.; Trindade, S. C.

    2016-01-01

    Periodontitis is a multifactorial disease, with participation of bacterial, environmental, and host factors. It results from synergistic and dysbiotic multispecies microorganisms, critical “keystone pathogens,” affecting the whole bacterial community. The purpose of this study was to review the role of Porphyromonas gingivalis in the immunopathogenesis of chronic periodontitis, with special attention paid to HmuY. The host response during periodontitis involves the innate and adaptive immune system, leading to chronic inflammation and progressive destruction of tooth-supporting tissues. In this proinflammatory process, the ability of P. gingivalis to evade the host immune response and access nutrients in the microenvironment is directly related to its survival, proliferation, and infection. Furthermore, heme is an essential nutrient for development of these bacteria, and HmuY is responsible for its capture from host heme-binding proteins. The inflammatory potential of P. gingivalis HmuY has been shown, including induction of high levels of proinflammatory cytokines and CCL2, decreased levels of IL-8, and increased levels of anti-HmuY IgG and IgG1 antibodies in individuals with chronic periodontitis. Therefore, the HmuY protein might be a promising target for therapeutic strategies and for development of diagnostic methods in chronic periodontitis, especially in the case of patients with chronic periodontitis not responding to treatment, monitoring, and maintenance therapy. PMID:27403039

  4. Identification of an O-antigen chain length regulator, WzzP, in Porphyromonas gingivalis

    PubMed Central

    Shoji, Mikio; Yukitake, Hideharu; Sato, Keiko; Shibata, Yasuko; Naito, Mariko; Aduse-Opoku, Joseph; Abiko, Yoshimitsu; Curtis, Michael A; Nakayama, Koji

    2013-01-01

    The periodontal pathogen Porphyromonas gingivalis has two different lipopolysaccharides (LPSs) designated O-LPS and A-LPS, which are a conventional O-antigen polysaccharide and an anionic polysaccharide that are both linked to lipid A-cores, respectively. However, the precise mechanisms of LPS biosynthesis remain to be determined. In this study, we isolated a pigment-less mutant by transposon mutagenesis and identified that the transposon was inserted into the coding sequence PGN_2005, which encodes a hypothetical protein of P. gingivalis ATCC 33277. We found that (i) LPSs purified from the PGN_2005 mutant were shorter than those of the wild type; (ii) the PGN_2005 protein was located in the inner membrane fraction; and (iii) the PGN_2005 gene conferred Wzz activity upon an Escherichia coli wzz mutant. These results indicate that the PGN_2005 protein, which was designated WzzP, is a functional homolog of the Wzz protein in P. gingivalis. Comparison of amino acid sequences among WzzP and conventional Wzz proteins indicated that WzzP had an additional fragment at the C-terminal region. In addition, we determined that the PGN_1896 and PGN_1233 proteins and the PGN_1033 protein appear to be WbaP homolog proteins and a Wzx homolog protein involved in LPS biosynthesis, respectively. PMID:23509024

  5. Role of Porphyromonas gingivalis HmuY in Immunopathogenesis of Chronic Periodontitis.

    PubMed

    Carvalho-Filho, P C; Gomes-Filho, I S; Meyer, R; Olczak, T; Xavier, M T; Trindade, S C

    2016-01-01

    Periodontitis is a multifactorial disease, with participation of bacterial, environmental, and host factors. It results from synergistic and dysbiotic multispecies microorganisms, critical "keystone pathogens," affecting the whole bacterial community. The purpose of this study was to review the role of Porphyromonas gingivalis in the immunopathogenesis of chronic periodontitis, with special attention paid to HmuY. The host response during periodontitis involves the innate and adaptive immune system, leading to chronic inflammation and progressive destruction of tooth-supporting tissues. In this proinflammatory process, the ability of P. gingivalis to evade the host immune response and access nutrients in the microenvironment is directly related to its survival, proliferation, and infection. Furthermore, heme is an essential nutrient for development of these bacteria, and HmuY is responsible for its capture from host heme-binding proteins. The inflammatory potential of P. gingivalis HmuY has been shown, including induction of high levels of proinflammatory cytokines and CCL2, decreased levels of IL-8, and increased levels of anti-HmuY IgG and IgG1 antibodies in individuals with chronic periodontitis. Therefore, the HmuY protein might be a promising target for therapeutic strategies and for development of diagnostic methods in chronic periodontitis, especially in the case of patients with chronic periodontitis not responding to treatment, monitoring, and maintenance therapy. PMID:27403039

  6. Bactericidal effect of a 405-nm diode laser on Porphyromonas gingivalis

    NASA Astrophysics Data System (ADS)

    Kotoku, Y.; Kato, J.; Akashi, G.; Hirai, Y.; Ishihara, K.

    2009-05-01

    The study was conducted to determine the effect of 405-nm diode laser irradiation on periodontopathic bacteria such as Porphyromonas gingivalis in vitro. A diluted suspension of P. gingivalis was irradiated directly with a 405-nm diode laser under conditions of 100 mW-10 sec, 100 mW-20 sec, 200 mW-5 sec, 200 mW-10 sec, 200 mW-20 sec, 400 mW-5 sec, 400 mW-10 sec, and 400 mW-20 sec. The energy density ranged from 2.0 to 16.0 J/cm2. The irradiated bacterial suspension was spread on a blood agar plate and growth of the colonies was examined after an anaerobic culture for 7 days. Bacterial growth was inhibited under all irradiation conditions, but the bactericidal effect of the 405-nm diode laser depended on the energy density. More than 97% of bacterial growth was inhibited with irradiation at an energy density > 4.0 J/cm2. The mechanism of the bactericidal effect is photochemical, rather than photothermal. These findings suggest that a 405-nm diode laser has a high bactericidal effect on P. gingivalis.

  7. Phototoxic effect of visible light on Porphyromonas gingivalis and Fusobacterium nucleatum: an in vitro study.

    PubMed

    Feuerstein, Osnat; Persman, Nir; Weiss, Ervin I

    2004-01-01

    The antibacterial effect of visible light irradiation combined with photosensitizers has been reported. The objective of this was to test the effect of visible light irradiation without photosensitizers on the viability of oral microorganisms. Strains of Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus mutans and Streptococcus faecalis in suspension or grown on agar were exposed to visible light at wavelengths of 400-500 nm. These wavelengths are used to photopolymerize composite resins widely used for dental restoration. Three photocuring light sources, quartz-tungsten-halogen lamp, light-emitting diode and plasma-arc, at power densities between 260 and 1300 mW/cm2 were used for up to 3 min. Bacterial samples were also exposed to a near-infrared diode laser (wavelength, 830 nm), using identical irradiation parameters for comparison. The results show that blue light sources exert a phototoxic effect on P. gingivalis and F. nucleatum. The minimal inhibitory dose for P. gingivalis and F. nucleatum was 16-62 J/cm2, a value significantly lower than that for S. mutans and S. faecalis (159-212 J/cm2). Near-infrared diode laser irradiation did not affect any of the bacteria tested. Our results suggest that visible light sources without exogenous photosensitizers have a phototoxic effect mainly on Gram-negative periodontal pathogens. PMID:15623322

  8. Antibacterial activity against Porphyromonas gingivalis and biological characteristics of antibacterial stainless steel.

    PubMed

    Zhang, Dan; Ren, Ling; Zhang, Yang; Xue, Nan; Yang, Ke; Zhong, Ming

    2013-05-01

    To evaluate the possibility of an alternative to the traditional orthodontic stainless steel implants, the antibacterial activity against Porphyromonas gingivalis (P. gingivalis) and the related cytotoxicity of a type 304 Cu bearing antibacterial stainless steel were studied. The results indicated that the antibacterial stainless steel showed excellent antibacterial property against P. gingivalis, compared with the control steel (a purchased medical grade 304 stainless steel). Compared to the control steel, there were fewer bacteria on the surface of the antibacterial stainless steel, with significant difference in morphology. The cytotoxicities of the antibacterial stainless steel to both MG-63 and KB cells were all grade 1, the same as those of the control steel. There were no significant differences in the apoptosis rates on MG-63 and KB cells between the antibacterial stainless steel and the control steel. This study demonstrates that the antibacterial stainless steel is possible to reduce the incidence of implant-related infections and can be a more suitable material for the micro-implant than the conventional stainless steel in orthodontic treatment.

  9. Porphyromonas gingivalis manipulates complement and TLR signaling to uncouple bacterial clearance from inflammation and promote dysbiosis

    PubMed Central

    Maekawa, Tomoki; Krauss, Jennifer L.; Abe, Toshiharu; Jotwani, Ravi; Triantafilou, Martha; Triantafilou, Kathy; Hashim, Ahmed; Hoch, Shifra; Curtis, Michael A.; Nussbaum, Gabriel; Lambris, John D.; Hajishengallis, George

    2014-01-01

    SUMMARY Certain low-abundance bacterial species, such as the periodontitis-associated oral bacterium Porphyromonas gingivalis can subvert host immunity to remodel a normally symbiotic microbiota into a dysbiotic, disease-provoking state. However, such pathogens also exploit inflammation to thrive in dysbiotic conditions. How these bacteria evade immunity while maintaining inflammation is unclear. As previously reported, P. gingivalis remodels the oral microbiota into a dysbiotic state by exploiting complement. Now we show that in neutrophils P. gingivalis disarms a host-protective TLR2-MyD88 pathway via proteasomal degradation of MyD88, whereas it activates an alternate TLR2-Mal-PI3K pathway. This alternate TLR2-Mal-PI3K pathway blocks phagocytosis, provides ‘bystander’ protection to otherwise susceptible bacteria, and promotes dysbiotic inflammation in vivo. This mechanism to disengage bacterial clearance from inflammation required an intimate crosstalk between TLR2 and the complement receptor C5aR, and can contribute to the persistence of microbial communities that drive dysbiotic diseases. PMID:24922578

  10. Arg-Gingipain A DNA Vaccine Induces Protective Immunity against Infection by Porphyromonas gingivalis in a Murine Model

    PubMed Central

    Yonezawa, Hideo; Ishihara, Kazuyuki; Okuda, Katsuji

    2001-01-01

    Arginine-specific cysteine proteinases (RgpA and RgpB) produced by the periodontal pathogen Porphyromonas gingivalis are suspected virulence factors and are involved in interrupting host defense mechanisms as well as in penetrating and destroying periodontal connective tissues. To induce a protective immune response against P. gingivalis, we constructed an rgpA DNA vaccine. BALB/c mice were immunized intradermally by Gene Gun with plasmid DNA carrying rgpA. Antibody responses against P. gingivalis were determined by an enzyme-linked immunosorbent assay. The rgpA DNA vaccine induced high levels of serum antibodies against P. gingivalis. Sera from the rgpA DNA vaccine-immunized mice diminished the proteolytic activity of RgpA and RgpB and inhibited the binding of P. gingivalis to a type I collagen sponge. Moreover, the sera effectively reduced the hemagglutination of P. gingivalis, indicating that the hemagglutinin activity of the organism is associated with RgpA. We found with a murine abscess model that mice immunized with the rgpA DNA vaccine were resistant to an invasive P. gingivalis W50 challenge. These results suggest that the rgpA DNA vaccine induced specific antibodies against the enzyme and that this vaccine could confer protective immunity against P. gingivalis infection. PMID:11292699

  11. Gingipains from Porphyromonas gingivalis W83 induce cell adhesion molecule cleavage and apoptosis in endothelial cells.

    PubMed

    Sheets, Shaun M; Potempa, Jan; Travis, James; Casiano, Carlos A; Fletcher, Hansel M

    2005-03-01

    The presence of Porphyromonas gingivalis in the periodontal pocket and the high levels of gingipain activity detected in gingival crevicular fluid could implicate a role for gingipains in the destruction of the highly vascular periodontal tissue. To explore the effects of these proteases on endothelial cells, we exposed bovine coronary artery endothelial cells and human microvascular endothelial cells to gingipain-active extracellular protein preparations and/or purified gingipains from P. gingivalis. Treated cells exhibited a rapid loss of cell adhesion properties that was followed by apoptotic cell death. Cleavage of N- and VE-cadherin and integrin beta1 was observed in immunoblots of cell lysates. There was a direct correlation between the kinetics of cleavage of N- and VE-cadherin and loss of cell adhesion properties. Loss of cell adhesion, as well as N- and VE-cadherin and integrin beta1 cleavage, could be inhibited or significantly delayed by preincubation of P. gingivalis W83 gingipain-active extracellular extracts with the cysteine protease inhibitor Nalpha-p-tosyl-l-lysine chloromethylketone. Furthermore, purified gingipains also induced endothelial cell detachment and apoptosis. Apoptosis-associated events, including annexin V positivity, caspase-3 activation, and cleavage of the caspase substrates poly(ADP-ribose) polymerase and topoisomerase I (Topo I), were observed in endothelial cells after detachment. All of the effects observed were correlated with the different levels of cysteine-dependent proteolytic activity of the extracts tested. Taken together, these results indicate that gingipains from P. gingivalis can alter cell adhesion molecules and induce endothelial cell death, which could have implications for the pathogenicity of this organism. PMID:15731052

  12. Erythritol alters microstructure and metabolomic profiles of biofilm composed of Streptococcus gordonii and Porphyromonas gingivalis.

    PubMed

    Hashino, E; Kuboniwa, M; Alghamdi, S A; Yamaguchi, M; Yamamoto, R; Cho, H; Amano, A

    2013-12-01

    The effects of sugar alcohols such as erythritol, xylitol, and sorbitol on periodontopathic biofilm are poorly understood, though they have often been reported to be non-cariogenic sweeteners. In the present study, we evaluated the efficacy of sugar alcohols for inhibiting periodontopathic biofilm formation using a heterotypic biofilm model composed of an oral inhabitant Streptococcus gordonii and a periodontal pathogen Porphyromonas gingivalis. Confocal microscopic observations showed that the most effective reagent to reduce P. gingivalis accumulation onto an S. gordonii substratum was erythritol, as compared with xylitol and sorbitol. In addition, erythritol moderately suppressed S. gordonii monotypic biofilm formation. To examine the inhibitory effects of erythritol, we analyzed the metabolomic profiles of erythritol-treated P. gingivalis and S. gordonii cells. Metabolome analyses using capillary electrophoresis time-of-flight mass spectrometry revealed that a number of nucleic intermediates and constituents of the extracellular matrix, such as nucleotide sugars, were decreased by erythritol in a dose-dependent manner. Next, comparative analyses of metabolites of erythritol- and sorbitol-treated cells were performed using both organisms to determine the erythritol-specific effects. In P. gingivalis, all detected dipeptides, including Glu-Glu, Ser-Glu, Tyr-Glu, Ala-Ala and Thr-Asp, were significantly decreased by erythritol, whereas they tended to be increased by sorbitol. Meanwhile, sorbitol promoted trehalose 6-phosphate accumulation in S. gordonii cells. These results suggest that erythritol has inhibitory effects on dual species biofilm development via several pathways, including suppression of growth resulting from DNA and RNA depletion, attenuated extracellular matrix production, and alterations of dipeptide acquisition and amino acid metabolism.

  13. Por Secretion System-Dependent Secretion and Glycosylation of Porphyromonas gingivalis Hemin-Binding Protein 35

    PubMed Central

    Shoji, Mikio; Sato, Keiko; Yukitake, Hideharu; Kondo, Yoshio; Narita, Yuka; Kadowaki, Tomoko; Naito, Mariko; Nakayama, Koji

    2011-01-01

    The anaerobic Gram-negative bacterium Porphyromonas gingivalis is a major pathogen in severe forms of periodontal disease and refractory periapical perodontitis. We have recently found that P. gingivalis has a novel secretion system named the Por secretion system (PorSS), which is responsible for secretion of major extracellular proteinases, Arg-gingipains (Rgps) and Lys-gingipain. These proteinases contain conserved C-terminal domains (CTDs) in their C-termini. Hemin-binding protein 35 (HBP35), which is one of the outer membrane proteins of P. gingivalis and contributes to its haem utilization, also contains a CTD, suggesting that HBP35 is translocated to the cell surface via the PorSS. In this study, immunoblot analysis of P. gingivalis mutants deficient in the PorSS or in the biosynthesis of anionic polysaccharide-lipopolysaccharide (A-LPS) revealed that HBP35 is translocated to the cell surface via the PorSS and is glycosylated with A-LPS. From deletion analysis with a GFP-CTD[HBP35] green fluorescent protein fusion, the C-terminal 22 amino acid residues of CTD[HBP35] were found to be required for cell surface translocation and glycosylation. The GFP-CTD fusion study also revealed that the CTDs of CPG70, peptidylarginine deiminase, P27 and RgpB play roles in PorSS-dependent translocation and glycosylation. However, CTD-region peptides were not found in samples of glycosylated HBP35 protein by peptide map fingerprinting analysis, and antibodies against CTD-regions peptides did not react with glycosylated HBP35 protein. These results suggest both that the CTD region functions as a recognition signal for the PorSS and that glycosylation of CTD proteins occurs after removal of the CTD region. Rabbits were used for making antisera against bacterial proteins in this study. PMID:21731719

  14. Identification and Characterization of Prokaryotic Dipeptidyl-peptidase 5 from Porphyromonas gingivalis *

    PubMed Central

    Ohara-Nemoto, Yuko; Rouf, Shakh M. A.; Naito, Mariko; Yanase, Amie; Tetsuo, Fumi; Ono, Toshio; Kobayakawa, Takeshi; Shimoyama, Yu; Kimura, Shigenobu; Nakayama, Koji; Saiki, Keitarou; Konishi, Kiyoshi; Nemoto, Takayuki K.

    2014-01-01

    Porphyromonas gingivalis, a Gram-negative asaccharolytic anaerobe, is a major causative organism of chronic periodontitis. Because the bacterium utilizes amino acids as energy and carbon sources and incorporates them mainly as dipeptides, a wide variety of dipeptide production processes mediated by dipeptidyl-peptidases (DPPs) should be beneficial for the organism. In the present study, we identified the fourth P. gingivalis enzyme, DPP5. In a dpp4-7-11-disrupted P. gingivalis ATCC 33277, a DPP7-like activity still remained. PGN_0756 possessed an activity indistinguishable from that of the mutant, and was identified as a bacterial orthologue of fungal DPP5, because of its substrate specificity and 28.5% amino acid sequence identity with an Aspergillus fumigatus entity. P. gingivalis DPP5 was composed of 684 amino acids with a molecular mass of 77,453, and existed as a dimer while migrating at 66 kDa on SDS-PAGE. It preferred Ala and hydrophobic residues, had no activity toward Pro at the P1 position, and no preference for hydrophobic P2 residues, showed an optimal pH of 6.7 in the presence of NaCl, demonstrated Km and kcat/Km values for Lys-Ala-MCA of 688 μm and 11.02 μm−1 s−1, respectively, and was localized in the periplasm. DPP5 elaborately complemented DPP7 in liberation of dipeptides with hydrophobic P1 residues. Examinations of DPP- and gingipain gene-disrupted mutants indicated that DPP4, DPP5, DPP7, and DPP11 together with Arg- and Lys-gingipains cooperatively liberate most dipeptides from nutrient oligopeptides. This is the first study to report that DPP5 is expressed not only in eukaryotes, but also widely distributed in bacteria and archaea. PMID:24398682

  15. Activation of serum complement by polysaccharide-containing antigens of Porphyromonas gingivalis.

    PubMed

    Schifferle, R E; Wilson, M E; Levine, M J; Genco, R J

    1993-07-01

    We previously reported that hot aqueous phenol extraction of Porphyromonas gingivalis yields a preparation containing both lipopolysaccharide (LPS) and an antigenically distinct capsular polysaccharide (PS). In the present study, we examined the capacity of phenol-water extracts from a number of strains of P. gingivalis to activate human serum complement. Anticomplementary activity of extracts from two invasive and two noninvasive strains of P. gingivalis was assessed in a sheep erythrocyte hemolytic assay and in an alternative pathway-selective rabbit erythrocyte hemolytic assay. In the sheep erythrocyte assay, extracts from noninvasive strains were found to exhibit greater anticomplementary activity than extracts derived from invasive strains. A phenol-water extract from invasive strain ATCC 53977 was further resolved into its LPS and PS fractions. Whereas isolated LPS from this strain exhibited strong anticomplementary activity, the PS fraction was only weakly active. Phenol-water extracts from three of four strains were found to be potent activators of the alternative pathway, with extracts from the two noninvasive strains being most active. The extract from the remaining strain (ATCC 53977) was a poor activator of the alternative pathway. Further analysis of this extract revealed, however, that the LPS fraction was a potent activator of the alternative pathway, although the PS fraction exhibited negligible activity. The results of this study indicate that phenol-water extracts of invasive and noninvasive strains of P. gingivalis differ in their respective anticomplementary activities, with invasive strains being less active. Although extracts from both invasive and noninvasive strains activated the alternative pathway, this activity appears to be attributable to the LPS, rather than the PS, component.

  16. Porphyromonas (Bacteroides) gingivalis fimbrillin: size, amino-terminal sequence, and antigenic heterogeneity.

    PubMed Central

    Lee, J Y; Sojar, H T; Bedi, G S; Genco, R J

    1991-01-01

    Bacterial fimbriae mediate cell adhesion and are important in colonization. Fimbrial proteins from strains of Porphyromonas (Bacteroides) gingivalis isolated from different individuals were compared for their size, amino-terminal sequence, and antigenic diversity. Two major protein components of the crude fimbrial preparations differed in apparent molecular mass, ranging from 41 to 49 kDa for the fimbrillin monomer and from 61 to 78 kDa for the other major protein. The amino-terminal sequence of the antigenically related group of proteins of the fimbrillin monomer in the 41- to 49-kDa range showed significant homology; however, minor sequence heterogeneity was observed, mainly in residues 4 to 6. One of the observed amino-terminal sequences, AFGVGDDESKVAKLTVMVYNG, resembled the deduced sequence of P. gingivalis 381 (D.P. Dickinson, M. K. Kubiniec, F. Yoshimura, and R.J. Genco, J. Bacteriol. 170:1658-1665, 1988). Fimbriae from all the strains of P. gingivalis showing this sequence contained a fimbrillin monomer of 43 kDa and showed a strong reaction with both polyclonal and monoclonal antibodies directed to the fimbriae from P. gingivalis 2561 (381). Fimbriae from strains showing amino-terminal sequence variations in residues 4 to 6 (i.e., substitution of VGD with either E or NAG) were more diverse in their molecular sizes. Most of these variant fimbriae showed weak reactions with the polyclonal antibodies and no reaction with the monoclonal antibodies induced to the fimbriae of strain 2561. No correlation could be established between the molecular size and immunological reactivity of the fimbrillin monomer of P. gingivalis strains. Strains 9-14K-1 and HG 564 not only showed markedly different sequences from the other three amino-terminal sequences but also did not react with either polyclonal or monoclonal antibodies to the fimbriae of strain 2561. Strains W50, W83, and AJW 5 failed to show any immunological reactivity with the antibodies to fimbrillin or fimbriae

  17. Honey – a potential agent against Porphyromonas gingivalis: an in vitro study

    PubMed Central

    2014-01-01

    Background Honey has been discussed as a therapeutic option in wound healing since ancient time. It might be also an alternative to the commonly used antimicrobials in periodontitis treatment. The in-vitro study was aimed to determine the antimicrobial efficacy against Porphyromonas gingivalis as a major periodontopathogen. Methods One Manuka and one domestic beekeeper honey have been selected for the study. As a screening, MICs of the honeys against 20 P. gingivalis strains were determined. Contents of methylglyoxal and hydrogen peroxide as the potential antimicrobial compounds were determined. These components (up to 100 mg/l), propolis (up to 200 mg/l) as well as the two honeys (up to 10% w/v) were tested against four P. gingivalis strains in planktonic growth and in a single-species biofilm. Results 2% of Manuka honey inhibited the growth of 50% of the planktonic P. gingivalis, the respective MIC50 of the German beekeeper honey was 5%. Manuka honey contained 1.87 mg/kg hydrogen peroxide and the domestic honey 3.74 mg/kg. The amount of methylglyoxal was found to be 2 mg/kg in the domestic honey and 982 mg/kg in the Manuka honey. MICs for hydrogen peroxide were 10 mg/l - 100 mg/l, for methylglyoxal 5 – 20 mg/l, and for propolis 20 mg/l – 200 mg/l. 10% of both types of honey inhibited the formation of P. gingivalis biofilms and reduced the numbers of viable bacteria within 42 h-old biofilms. Neither a total prevention of biofilm formation nor a complete eradication of a 42 h-old biofilm by any of the tested compounds and the honeys were found. Conclusions Honey acts antibacterial against P. gingivalis. The observed pronounced effects of Manuka honey against planktonic bacteria but not within biofilm can be attributed to methylglyoxal as the characteristic antimicrobial component. PMID:24666777

  18. Porphyromonas gingivalis HmuY stimulates expression of Bcl-2 and Fas by human CD3+ T cells

    PubMed Central

    2013-01-01

    Background Apoptosis is a highly controlled process of cell death that can be induced by periodontopathogens. The present study aimed to investigate the expression of Fas and Bcl-2 proteins by CD3+ T cells in vitro under stimulation by total Porphyromonas gingivalis antigens and purified recombinant P. gingivalis HmuY protein. Results CD3+ T cells derived from CP patients and stimulated with HmuY expressed higher levels of Bcl-2 compared to identical cells stimulated with P. gingivalis crude extract or cells derived from NP control subjects (p = 0.043). Conclusion The authors hypothesize that P. gingivalis HmuY plays a role in the pathogenesis of chronic periodontitis, possibly by reducing or delaying apoptosis in T cells through a pathway involving the Bcl-2 protein. PMID:24025186

  19. A Two-Component System Regulates Hemin Acquisition in Porphyromonas gingivalis

    PubMed Central

    Scott, Jodie C.; Klein, Brian A.; Duran-Pinedo, Ana; Hu, Linden; Duncan, Margaret J.

    2013-01-01

    Porphyromonas gingivalis is a Gram-negative oral anaerobe associated with infection of the periodontia. The organism has a small number of two-component signal transduction systems, and after comparing genome sequences of strains W83 and ATCC 33277 we discovered that the latter was mutant in histidine kinase (PGN_0752), while the cognate response regulator (PGN_0753) remained intact. Microarray-based transcriptional profiling and ChIP-seq assays were carried out with an ATCC 33277 transconjugant containing the functional histidine kinase from strain W83 (PG0719). The data showed that the regulon of this signal transduction system contained genes that were involved in hemin acquisition, including gingipains, at least three transport systems, as well as being self-regulated. Direct regulation by the response regulator was confirmed by electrophoretic mobility shift assays. In addition, the system appears to be activated by hemin and the regulator acts as both an activator and repressor. PMID:24039921

  20. Intraspecies Variability Affects Heterotypic Biofilms of Porphyromonas gingivalis and Prevotella intermedia: Evidences of Strain-Dependence Biofilm Modulation by Physical Contact and by Released Soluble Factors.

    PubMed

    Barbosa, Graziela Murta; Colombo, Andrea Vieira; Rodrigues, Paulo Henrique; Simionato, Maria Regina Lorenzetti

    2015-01-01

    It is well known that strain and virulence diversity exist within the population structure of Porphyromonas gingivalis. In the present study we investigate intra- and inter-species variability in biofilm formation of Porphyromonas gingivalis and partners Prevotella intermedia and Prevotella nigrescens. All strains tested showed similar hydrophobicity, except for P. gingivalis W83 which has roughly half of the hydrophobicity of P. gingivalis ATCC33277. An intraspecies variability in coaggregation of P. gingivalis with P. intermedia was also found. The association P. gingivalis W83/P. intermedia 17 produced the thickest biofilm and strain 17 was prevalent. In a two-compartment system P. gingivalis W83 stimulates an increase in biomass of strain 17 and the latter did not stimulate the growth of P. gingivalis W83. In addition, P. gingivalis W83 also stimulates the growth of P. intermedia ATCC25611 although strain W83 was prevalent in the association with P. intermedia ATCC25611. P. gingivalis ATCC33277 was prevalent in both associations with P. intermedia and both strains of P. intermedia stimulate the growth of P. gingivalis ATCC33277. FISH images also showed variability in biofilm structure. Thus, the outcome of the association P. gingivalis/P. intermedia seems to be strain-dependent, and both soluble factors and physical contact are relevant. The association P. gingivalis-P. nigrescens ATCC33563 produced larger biomass than each monotypic biofilm, and P. gingivalis was favored in consortia, while no differences were found in the two-compartment system. Therefore, in consortia P. gingivalis-P. nigrescens physical contact seems to favor P. gingivalis growth. The intraspecies variability found in our study suggests strain-dependence in ability of microorganisms to recognize molecules in other bacteria which may further elucidate the dysbiosis event during periodontitis development giving additional explanation for periodontal bacteria, such as P. gingivalis and P

  1. Porphyromonas gingivalis attenuates ATP-mediated inflammasome activation and HMGB1 release through expression of a nucleoside-diphosphate kinase

    PubMed Central

    Johnson, Larry; Atanasova, Kalina R.; Bui, Phuong Q.; Lee, Jungnam; Hung, Shu-Chen; Yilmaz, Özlem; Ojcius, David M.

    2015-01-01

    Many intracellular pathogens evade the innate immune response in order to survive and proliferate within infected cells. We show that Porphyromonas gingivalis, an intracellular opportunistic pathogen, uses a nucleoside-diphosphate kinase (NDK) homolog to inhibit innate immune responses due to stimulation by extracellular ATP, which acts as a danger signal that binds to P2X7 receptors and induces activation of an inflammasome and caspase-1. Thus, infection of gingival epithelial cells (GECs) with wild-type P. gingivalis results in inhibition of ATP-induced caspase-1 activation. However, ndk-deficient P. gingivalis is less effective than wild-type P. gingivalis in reducing ATP-mediated caspase-1 activation and secretion of the proinflammatory cytokine, IL-1β, from infected GECs. Furthermore, P. gingivalis NDK modulates release of high-mobility group protein B1 (HMGB1), a pro-inflammatory danger signal, which remains associated with chromatin in healthy cells. Unexpectedly, infection with either wild-type or ndk-deficient P. gingivalis causes release of HMGB1 from the nucleus to the cytosol. But HMGB1 is released to the extracellular space when uninfected GECs are further stimulated with ATP, and there is more HMGB1 released from the cells when ATP-treated cells are infected with ndk-deficient mutant than wild-type P. gingivalis. Our results reveal that NDK plays a significant role in inhibiting P2X7-dependent inflammasome activation and HMGB1 release from infected GECs. PMID:25828169

  2. Diagnostic evaluation of a nanobody with picomolar affinity towards the protease RgpB from Porphyromonas gingivalis

    PubMed Central

    Skottrup, Peter Durand; Leonard, Paul; Kaczmarek, Jakub Zbigniew; Veillard, Florian; Enghild, Jan J.; O’Kennedy, Richard; Sroka, Aneta; Clausen, Rasmus Prætorius; Potempa, Jan; Riise, Erik

    2011-01-01

    Porphyromonas gingivalis is one of the major periodontitis-causing pathogens. P. gingivalis secretes a group of proteases, termed gingipains, and in this study we have utilized the RgpB gingipain as a biomarker for P. gingivalis. We constructed a naïve camel nanobody library and used phage display to select one nanobody towards RgpB with picomolar affinity. The nanobody was used in an inhibition assay for detection of RgpB in buffer as well as in saliva. The nanobody was highly specific for RgpB as it did not bind to the homologous gingipain HRgpA. This indicated a presence of a binding epitope within the immunoglobulin-like domain of RgpB. A subtractive inhibition assay was used to demonstrate that the nanobody could bind native RgpB in the context of intact cells. The nanobody bound exclusively to the P. gingivalis membrane-bound RgpB isoform (mt-RgpB) and to secreted, soluble RgpB. Further cross-reactivity studies with P. gingivalis gingipain deletion mutants showed that the nanobody could discriminate between native RgpB and native Kgp and RgpA in complex bacterial samples. This study demonstrates that RgpB can be used as a specific biomarker for P. gingivalis detection and that the presented nanobody-based assay could supplement existing methods for P. gingivalis detection. PMID:21569755

  3. Protein Analysis of Sapienic Acid-Treated Porphyromonas gingivalis Suggests Differential Regulation of Multiple Metabolic Pathways

    PubMed Central

    Dawson, Deborah V.; Blanchette, Derek R.; Drake, David R.; Wertz, Philip W.; Brogden, Kim A.

    2015-01-01

    ABSTRACT Lipids endogenous to skin and mucosal surfaces exhibit potent antimicrobial activity against Porphyromonas gingivalis, an important colonizer of the oral cavity implicated in periodontitis. Our previous work demonstrated the antimicrobial activity of the fatty acid sapienic acid (C16:1Δ6) against P. gingivalis and found that sapienic acid treatment alters both protein and lipid composition from those in controls. In this study, we further examined whole-cell protein differences between sapienic acid-treated bacteria and untreated controls, and we utilized open-source functional association and annotation programs to explore potential mechanisms for the antimicrobial activity of sapienic acid. Our analyses indicated that sapienic acid treatment induces a unique stress response in P. gingivalis resulting in differential expression of proteins involved in a variety of metabolic pathways. This network of differentially regulated proteins was enriched in protein-protein interactions (P = 2.98 × 10−8), including six KEGG pathways (P value ranges, 2.30 × 10−5 to 0.05) and four Gene Ontology (GO) molecular functions (P value ranges, 0.02 to 0.04), with multiple suggestive enriched relationships in KEGG pathways and GO molecular functions. Upregulated metabolic pathways suggest increases in energy production, lipid metabolism, iron acquisition and processing, and respiration. Combined with a suggested preferential metabolism of serine, which is necessary for fatty acid biosynthesis, these data support our previous findings that the site of sapienic acid antimicrobial activity is likely at the bacterial membrane. IMPORTANCE P. gingivalis is an important opportunistic pathogen implicated in periodontitis. Affecting nearly 50% of the population, periodontitis is treatable, but the resulting damage is irreversible and eventually progresses to tooth loss. There is a great need for natural products that can be used to treat and/or prevent the overgrowth of

  4. Serine dipeptide lipids of Porphyromonas gingivalis inhibit osteoblast differentiation: Relationship to Toll-like receptor 2.

    PubMed

    Wang, Yu-Hsiung; Nemati, Reza; Anstadt, Emily; Liu, Yaling; Son, Young; Zhu, Qiang; Yao, Xudong; Clark, Robert B; Rowe, David W; Nichols, Frank C

    2015-12-01

    Porphyromonas gingivalis is a periodontal pathogen strongly associated with loss of attachment and supporting bone for teeth. We have previously shown that the total lipid extract of P. gingivalis inhibits osteoblast differentiation through engagement of Toll-like receptor 2 (TLR2) and that serine dipeptide lipids of P. gingivalis engage both mouse and human TLR2. The purpose of the present investigation was to determine whether these serine lipids inhibit osteoblast differentiation in vitro and in vivo and whether TLR2 engagement is involved. Osteoblasts were obtained from calvaria of wild type or TLR2 knockout mouse pups that also express the Col2.3GFP transgene. Two classes of serine dipeptide lipids, termed Lipid 654 and Lipid 430, were tested. Osteoblast differentiation was monitored by cell GFP fluorescence and osteoblast gene expression and osteoblast function was monitored as von Kossa stained mineral deposits. Osteoblast differentiation and function were evaluated in calvarial cell cultures maintained for 21 days. Lipid 654 significantly inhibited GFP expression, osteoblast gene expression and mineral nodule formation and this inhibition was dependent on TLR2 engagement. Lipid 430 also significantly inhibited GFP expression, osteoblast gene expression and mineral nodule formation but these effects were only partially attributed to engagement of TLR2. More importantly, Lipid 430 stimulated TNF-α and RANKL gene expression in wild type cells but not in TLR2 knockout cells. Finally, osteoblast cultures were observed to hydrolyze Lipid 654 to Lipid 430 and this likely occurs through elevated PLA2 activity in the cultured cells. In conclusion, our results show that serine dipeptide lipids of P. gingivalis inhibit osteoblast differentiation and function at least in part through engagement of TLR2. The Lipid 430 serine class also increased the expression of genes that could increase osteoclast activity. We conclude that Lipid 654 and Lipid 430 have the potential

  5. Identification of Small-Molecule Inhibitors against Meso-2, 6-Diaminopimelate Dehydrogenase from Porphyromonas gingivalis

    PubMed Central

    Stone, Victoria N.; Parikh, Hardik I.; El-rami, Fadi; Ge, Xiuchun; Chen, Weihau; Zhang, Yan; Kellogg, Glen E.; Xu, Ping

    2015-01-01

    Species-specific antimicrobial therapy has the potential to combat the increasing threat of antibiotic resistance and alteration of the human microbiome. We therefore set out to demonstrate the beginning of a pathogen-selective drug discovery method using the periodontal pathogen Porphyromonas gingivalis as a model. Through our knowledge of metabolic networks and essential genes we identified a “druggable” essential target, meso-diaminopimelate dehydrogenase, which is found in a limited number of species. We adopted a high-throughput virtual screen method on the ZINC chemical library to select a group of potential small-molecule inhibitors. Meso-diaminopimelate dehydrogenase from P. gingivalis was first expressed and purified in Escherichia coli then characterized for enzymatic inhibitor screening studies. Several inhibitors with similar structural scaffolds containing a sulfonamide core and aromatic substituents showed dose-dependent inhibition. These compounds were further assayed showing reasonable whole-cell activity and the inhibition mechanism was determined. We conclude that the establishment of this target and screening strategy provides a model for the future development of new antimicrobials. PMID:26544875

  6. Role of Porphyromonas gingivalis protease activity in colonization of oral surfaces.

    PubMed Central

    Tokuda, M; Duncan, M; Cho, M I; Kuramitsu, H K

    1996-01-01

    Cysteine proteases, including Arg-gingipain of Porphyromonas gingivalis, have been implicated as important virulence factors in periodontal diseases. These enzymes are also involved in the hemagglutinating activity of the organisms. In order to determine the role of proteases in the colonization of the gingival margin, we have compared the attachment properties of P. gingivalis 381 with those of its Arg-gingipain-defective mutant, G-102. Interactions with gram-positive bacteria, human oral epithelial cells, extracellular matrix proteins, and type I collagen were evaluated. In all cases, mutant G-102 was deficient in attachment relative to the parental strain. The mutant's defects could be explained, in part, by the weak autoaggregation displayed by the mutant, which appeared to result from altered fimbrial expression. Both Western blot (immunoblot) and Northern (RNA) blot analyses indicated reduced expression of the major 43-kDa fimbrillin subunit in the mutant. These results suggest that Arg-gingipain may play both direct and indirect roles in the colonization of the gingival margin. In addition, fimbriae may play a direct role in interacting with some host surfaces. PMID:8926070

  7. Role of Arg-Gingipain A in Virulence of Porphyromonas gingivalis

    PubMed Central

    Tokuda, Masayuki; Karunakaran, Thonthi; Duncan, Margaret; Hamada, Nobushiro; Kuramitsu, Howard

    1998-01-01

    In order to access the role of the Porphyromonas gingivalis Arg-gingipain proteases in the virulence of this organism, a mutant defective in the rgpA gene was constructed in strain 381. This mutant, MT10, displayed only 40% of the Arg-specific cysteine protease activity of the wild-type strain. In addition, MT10, as well as the recently characterized protease mutant G-102, which is defective in the rgpB gene, displayed reduced self-aggregation, hemagglutination, and the ability to bind to immobilized type I collagen compared to levels of the wild-type parent. However, unlike mutant G-102, the rgpA mutant displayed increased binding to epithelial cells relative to that of the parental organism. Mutant MT10 also did not express detectable levels of the FimA protein as assessed by both Western and Northern blotting or fimbriae visible by electron microscopy of the cells. Furthermore, the ability of MT10 to degrade rat tail collagen fibers when it was cultured at 37°C was markedly attenuated compared to that of strain 381. These results suggest that Arg-gingipain A may play a significant role in the pathogenicity of P. gingivalis by altering the colonization and toxic properties of the organism. PMID:9488409

  8. The chronicles of Porphyromonas gingivalis: the microbium, the human oral epithelium and their interplay

    PubMed Central

    Yilmaz, Özlem

    2009-01-01

    The microbiota of the human oral mucosa consists of a myriad of bacterial species that normally exist in commensal harmony with the host. Porphyromonas gingivalis, an aetiological agent in severe forms of periodontitis (a chronic inflammatory disease), is a prominent component of the oral microbiome and a successful colonizer of the oral epithelium. This Gram-negative anaerobe can also exist within the host epithelium without the existence of overt disease. Gingival epithelial cells, the outer lining of the gingival mucosa, which function as an important part of the innate immune system, are among the first host cells colonized by P. gingivalis. This review describes recent studies implicating the co-existence and intracellular adaptation of the organism in these target host cells. Specifically, recent findings on the putative mechanisms of persistence, intercellular dissemination and opportunism are highlighted. These new findings may also represent an original and valuable model for mechanistic characterization of other successful host-adapted, self-limiting, persistent intracellular bacteria in human epithelial tissues. PMID:18832296

  9. Acute Toxicity and the Effect of Andrographolide on Porphyromonas gingivalis-Induced Hyperlipidemia in Rats

    PubMed Central

    Al-Bayaty, Fouad; Al-Obaidi, Mazen M. Jamil; Abdulla, Mahmood A.

    2013-01-01

    The aim of the current study is to evaluate the effect of andrographolide on hyperlipidemia induced by Porphyromonas gingivalis in rats. Thirty male Sprague Dawley (SD) rats were divided into five groups as follows: group 1 (vehicle) and four experimental groups (groups 2, 3, 4, and 5) were challenged orally with P. gingivalis ATCC 33277 (0.2 mL of 1.5 ×1012 bacterial cells/mL in 2% carboxymethylcellulose (CMC) with phosphate-buffered saline (PBS)) five times a week for one month to induce hyperlipidemia. Then, group 3 received a standard oral treatment with simvastatin 100 mg/kg, and groups 4 and 5 received oral treatment with andrographolide 20 mg/kg and 10 mg/kg, respectively, for another month. The results showed that total cholesterol (TC), low-density lipoprotein (LDL-C), and triglycerides (TG) were reduced significantly in groups treated with andrographolide. The malondialdehyde (MDA) level was low in treated groups, while antioxidant enzymes, superoxide dismutase (SOD), and glutathione peroxidase (GPx) were significantly increased in these groups (P < 0.05). Liver tissues of the groups treated with andrographolide reduce the accumulation of lipid droplets in hepatic tissue cells. An acute toxicity test did not show any toxicological symptoms in rats. PMID:23844365

  10. Tetratricopeptide repeat protein-associated proteins contribute to the virulence of Porphyromonas gingivalis.

    PubMed

    Kondo, Yoshio; Ohara, Naoya; Sato, Keiko; Yoshimura, Mamiko; Yukitake, Hideharu; Naito, Mariko; Fujiwara, Taku; Nakayama, Koji

    2010-06-01

    Porphyromonas gingivalis is one of the most etiologically important microorganisms in periodontal disease. We found in a previous study that PG1385 (TprA) protein, a tetratricopeptide repeat (TPR) protein, was upregulated in P. gingivalis wild-type cells placed in a mouse subcutaneous chamber and that a tprA mutant was clearly less virulent in the mouse subcutaneous abscess model (M. Yoshimura et al., Oral Microbiol. Immunol. 23:413-418, 2008). In the present study, we investigated the gene expression profile of tprA mutant cells placed in a mouse subcutaneous chamber and found that 9 genes, including PG2102 (tapA), PG2101 (tapB), and PG2100 (tapC) genes, were downregulated in the tprA mutant compared with those in the wild type. Expression of a cluster of tapA, tapB, and tapC genes of the mutant was also downregulated in an in vitro culture with enriched brain heart infusion medium. The TprA protein has three TPR motifs known as a protein-protein interaction module. Yeast two-hybrid system analysis and in vitro protein binding assays with immunoprecipitation and surface plasmon resonance detection revealed that the TprA protein could bind to TapA and TapB proteins. TprA and TapB proteins were located in the periplasmic space, whereas TapA, which appeared to be one of the C-terminal domain family proteins, was located at the outer membrane. We constructed tapA, tapB, and tapC single mutants and a tapA-tapB-tapC deletion mutant. In the mouse subcutaneous infection experiment, all of the mutants were less virulent than the wild type. These results suggest that TprA, TapA, TapB, and TapC are cooperatively involved in P. gingivalis virulence. PMID:20351137

  11. Expression, purification and characterization of enoyl-ACP reductase II, FabK, from Porphyromonas gingivalis

    SciTech Connect

    Hevener, Kirk E.; Mehboob, Shahila; Boci, Teuta; Truong, Kent; Santarsiero, Bernard D.; Johnson, Michael E.

    2012-10-25

    The rapid rise in bacterial drug resistance coupled with the low number of novel antimicrobial compounds in the discovery pipeline has led to a critical situation requiring the expedient discovery and characterization of new antimicrobial drug targets. Enzymes in the bacterial fatty acid synthesis pathway, FAS-II, are distinct from their mammalian counterparts, FAS-I, in terms of both structure and mechanism. As such, they represent attractive targets for the design of novel antimicrobial compounds. Enoyl-acyl carrier protein reductase II, FabK, is a key, rate-limiting enzyme in the FAS-II pathway for several bacterial pathogens. The organism, Porphyromonas gingivalis, is a causative agent of chronic periodontitis that affects up to 25% of the US population and incurs a high national burden in terms of cost of treatment. P. gingivalis expresses FabK as the sole enoyl reductase enzyme in its FAS-II cycle, which makes this a particularly appealing target with potential for selective antimicrobial therapy. Herein we report the molecular cloning, expression, purification and characterization of the FabK enzyme from P. gingivalis, only the second organism from which this enzyme has been isolated. Characterization studies have shown that the enzyme is a flavoprotein, the reaction dependent upon FMN and NADPH and proceeding via a Ping-Pong Bi-Bi mechanism to reduce the enoyl substrate. A sensitive assay measuring the fluorescence decrease of NADPH as it is converted to NADP{sup +} during the reaction has been optimized for high-throughput screening. Finally, protein crystallization conditions have been identified which led to protein crystals that diffract x-rays to high resolution.

  12. Insights into the antiatherogenic molecular mechanisms of andrographolide against Porphyromonas gingivalis-induced atherosclerosis in rabbits.

    PubMed

    Al Batran, Rami; Al-Bayaty, Fouad; Al-Obaidi, Mazen M Jamil; Ashrafi, Amer

    2014-12-01

    Atherosclerosis is the commonest and most important vascular disease. Andrographolide (AND) is the main bioactive component of the medicinal plant Andrographis paniculata and is used in traditional medicine. This study was aimed to evaluate the antiatherogenic effect of AND against atherosclerosis induced by Porphyromonas gingivalis in White New Zealand rabbits. Thirty rabbits were divided into five groups as follows: G1, normal group; G2-5, were orally challenged with P. gingivalis five times a week over 12 weeks; G2, atherogenic control group; G3, standard group treated with atorvastatin (AV) 5 mg/kg; and G4 and G5, treatment groups treated with AND 10 and 20 mg/kg, respectively over 12 weeks. Serums were subjected to antioxidant enzymatic and anti-inflammatory activities, and the aorta was subjected to histological analyses. Groups treated with AND showed a significant reversal of liver and renal biochemical changes, compared with the atherogenic control group. In the same groups, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), total glutathione (GSH) levels in serum were significantly increased (P < 0.05), and lipid peroxidation (malondialdehyde (MDA)) levels were significantly decreased (P < 0.05), respectively. Furthermore, treated groups with AV and AND showed significant decrease in the level of VCAM-1 and ICAM-1 compared with the atherogenic control group. In aortic homogenate, the level of nitrotyrosine was significantly increased, while the level of MCP1 was significantly decreased in AV and AND groups compared with the atherogenic control group. In addition, staining the aorta with Sudan IV showed a reduction in intimal thickening plaque in AV and AND groups compared with the atherogenic control group. AND has showed an antiatherogenic property as well as the capability to reduce lipid, liver, and kidney biomarkers in atherogenic serum that prevents atherosclerosis complications caused by P. gingivalis.

  13. Expression, Purification and Characterization of Enoyl-ACP Reductase II, FabK, from Porphyromonas gingivalis

    PubMed Central

    Hevener, Kirk E.; Mehboob, Shahila; Boci, Teuta; Truong, Kent; Santarsiero, Bernard D.; Johnson, Michael E.

    2012-01-01

    The rapid rise in bacterial drug resistance coupled with the low number of novel antimicrobial compounds in the discovery pipeline has led to a critical situation requiring the expedient discovery and characterization of new antimicrobial drug targets. Enzymes in the bacterial fatty acid synthesis pathway, FAS-II, are distinct from their mammalian counterparts, FAS-I, in terms of both structure and mechanism. As such, they represent attractive targets for the design of novel antimicrobial compounds. Enoyl-acyl carrier protein reductase II, FabK, is a key, rate-limiting enzyme in the FAS-II pathway for several bacterial pathogens. The organism, Porphyromonas gingivalis, is a causative agent of chronic periodontitis that affects up to 25% of the U.S. population and incurs a high national burden in terms of cost of treatment. P. gingivalis expresses FabK as the sole enoyl reductase enzyme in its FAS-II cycle, which makes this a particularly appealing target with potential for selective antimicrobial therapy. Herein we report the molecular cloning, expression, purification and characterization of the FabK enzyme from P. gingivalis, only the second organism from which this enzyme has been isolated. Characterization studies have shown that the enzyme is a flavoprotein, the reaction dependent upon FMN and NADPH and proceeding via a Ping-Pong Bi-Bi mechanism to reduce the enoyl substrate. A sensitive assay measuring the fluorescence decrease of NADPH as it is converted to NADP+ during the reaction has been optimized for high-throughput screening. Finally, protein crystallization conditions have been identified which led to protein crystals that diffract x-rays to high resolution. PMID:22820244

  14. Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells.

    PubMed

    Wunsch, Christopher M; Lewis, Janina P

    2015-12-17

    Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth. The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2',7'-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal

  15. Genetic Exchange of Fimbrial Alleles Exemplifies the Adaptive Virulence Strategy of Porphyromonas gingivalis

    PubMed Central

    Kerr, Jennifer E.; Abramian, Jared R.; Dao, Doan-Hieu V.; Rigney, Todd W.; Fritz, Jamie; Pham, Tan; Gay, Isabel; Parthasarathy, Kavitha; Wang, Bing-yan; Zhang, Wenjian; Tribble, Gena D.

    2014-01-01

    Porphyromonas gingivalis is a gram–negative anaerobic bacterium, a member of the human oral microbiome, and a proposed “keystone” pathogen in the development of chronic periodontitis, an inflammatory disease of the gingiva. P. gingivalis is a genetically diverse species, and is able to exchange chromosomal DNA between strains by natural competence and conjugation. In this study, we investigate the role of horizontal DNA transfer as an adaptive process to modify behavior, using the major fimbriae as our model system, due to their critical role in mediating interactions with the host environment. We show that P. gingivalis is able to exchange fimbrial allele types I and IV into four distinct strain backgrounds via natural competence. In all recombinants, we detected a complete exchange of the entire fimA allele, and the rate of exchange varies between the different strain backgrounds. In addition, gene exchange within other regions of the fimbrial genetic locus was identified. To measure the biological implications of these allele swaps we compared three genotypes of fimA in an isogenic background, strain ATCC 33277. We demonstrate that exchange of fimbrial allele type results in profound phenotypic changes, including the quantity of fimbriae elaborated, membrane blebbing, auto-aggregation and other virulence-associated phenotypes. Replacement of the type I allele with either the type III or IV allele resulted in increased invasion of gingival fibroblast cells relative to the isogenic parent strain. While genetic variability is known to impact host-microbiome interactions, this is the first study to quantitatively assess the adaptive effect of exchanging genes within the pan genome cloud. This is significant as it presents a potential mechanism by which opportunistic pathogens may acquire the traits necessary to modify host-microbial interactions. PMID:24626479

  16. High In Vitro Antibacterial Activity of Pac-525 against Porphyromonas gingivalis Biofilms Cultured on Titanium

    PubMed Central

    Li, Ji-yin; Wang, Xue-jin; Wang, Li-na; Ying, Xiao-xia; Ren, Xiang; Liu, Hui-ying; Xu, Li; Ma, Guo-wu

    2015-01-01

    In order to investigate the potential of short antimicrobial peptides (AMPs) as alternative antibacterial agents during the treatment of peri-implantitis, the cytotoxic activity of three short AMPs, that is, Pac-525, KSL-W, and KSL, was determined using the MTT assay. The antimicrobial activity of these AMPs, ranging in concentration from 0.0039 mg/mL to 0.5 mg/mL, against the predominant planktonic pathogens, including Streptococcus sanguis, Fusobacterium nucleatum, and Porphyromonas gingivalis, involved in peri-implantitis was investigated. Furthermore, 2-day-old P. gingivalis biofilms cultured on titanium surfaces were treated with Pac-525 and subsequently observed and analysed using confocal laser scanning microscopy (CLSM). The average cell proliferation curve indicated that there was no cytotoxicity due to the three short AMPs. The minimum inhibitory concentration and minimum bactericidal concentration values of Pac-525 were 0.0625 mg/mL and 0.125 mg/mL, respectively, for P. gingivalis and 0.0078 mg/mL and 0.0156 mg/mL, respectively, for F. nucleatum. Using CLSM, we confirmed that compared to 0.1% chlorhexidine, 0.5 mg/mL of Pac-525 caused a significant decrease in biofilm thickness and a decline in the percentage of live bacteria. These data indicate that Pac-525 has unique properties that might make it suitable for the inhibition the growth of pathogenic bacteria around dental implants. PMID:25710035

  17. Identification of signaling pathways in macrophage exposed to Porphyromonas gingivalis or to its purified cell wall components.

    PubMed

    Zhou, Qingde; Amar, Salomon

    2007-12-01

    Porphyromonas gingivalis (P. gingivalis) can trigger an inflammatory condition leading to the destruction of periodontal tissues. However P. gingivalis LPS and its fimbriae (FimA) play different roles compared with the live bacteria in the context of intracellular molecule induction and cytokine secretion. To elucidate whether this difference results from different signaling pathways in host immune response to P. gingivalis, its LPS, or its FimA, we examined gene expression profile of human macrophages exposed to P. gingivalis, its LPS, or its FimA. A comparison of gene expression resulted in the identification of three distinct groups of expressed genes. Furthermore, computer-assisted promoter analysis of a subset of each group of differentially regulated genes revealed four putative transcriptional regulation models that associate with transcription factors NFkappaB, IRF7, and KLF4. Using gene knockout mice and siRNA to silence mouse genes, we showed that both TLR2 and TLR7 are essential for the induction of NFkappaB-containing genes and NFkappaB-IFN-sensitive response element (ISRE) cocontaining genes by either P. gingivalis or its purified components. The gene induction via either TLR2 or TLR7 is dependent on both MyD88 and p38 MAPK. However, the unique induction of IFN-beta by P. gingivalis LPS requires TLR7 and IFNalphabetaR cosignaling, and the induction of ISRE-containing gene is dependent on the activation of IFN-beta autocrine loop. Taken together, these data demonstrate that P. gingivalis and its components induce NFkappaB-containing genes through either TLR2- or TLR7-MyD88-p38 MAPK pathway, while P. gingivalis LPS uniquely induces ISRE-containing genes, which requires IFNalphabetaR signaling involving IRF7, KLF4, and pY701 STAT1. PMID:18025224

  18. Porphyromonas gingivalis Lipopolysaccharide Weakly Activates M1 and M2 Polarized Mouse Macrophages but Induces Inflammatory Cytokines

    PubMed Central

    Holden, James A.; Attard, Troy J.; Laughton, Katrina M.; Mansell, Ashley; O'Brien-Simpson, Neil M.

    2014-01-01

    Porphyromonas gingivalis is associated with chronic periodontitis, an inflammatory disease of the tooth's supporting tissues. Macrophages are important in chronic inflammatory conditions, infiltrating tissue and becoming polarized to an M1 or M2 phenotype. As responses to stimuli differ between these phenotypes, we investigated the effect of P. gingivalis lipopolysaccharide (LPS) on M1 and M2 macrophages. M1 and M2 polarized macrophages were produced from murine bone marrow macrophages (BMMϕ) primed with gamma interferon (IFN-γ) or interleukin-4 (IL-4), respectively, and incubated with a low or high dose of P. gingivalis LPS or control TLR2 and TLR4 ligands. In M1-Mϕ, the high dose of P. gingivalis LPS (10 μg/ml) significantly increased the expression of CD40, CD86, inducible nitric oxide synthase, and nitric oxide secretion. The low dose of P. gingivalis LPS (10 ng/ml) did not induce costimulatory or antibacterial molecules but did increase the secretion of IL-1α, IL-6, IL-12p40, IL-12p70, and tumor necrosis factor alpha (TNF-α). P. gingivalis LPS marginally increased the expression of CD206 and YM-1, but it did enhance arginase expression by M2-Mϕ. Furthermore, the secretion of the chemokines KC, RANTES, eotaxin, and MCP-1 from M1, M2, and nonpolarized Mϕ was enhanced by P. gingivalis LPS. TLR2/4 knockout macrophages combined with the TLR activation assays indicated that TLR2 is the main activating receptor for P. gingivalis LPS and whole cells. In conclusion, although P. gingivalis LPS weakly activated M1-Mϕ or M2-Mϕ compared to control TLR ligands, it induced the secretion of inflammatory cytokines, particularly TNF-α from M1-Mϕ and IL-10 from M2-Mϕ, as well as chemotactic chemokines from polarized macrophages. PMID:25047849

  19. Detection of antimicrobial activity of banana peel (Musa paradisiaca L.) on Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans: An in vitro study

    PubMed Central

    Kapadia, Suraj Premal; Pudakalkatti, Pushpa S.; Shivanaikar, Sachin

    2015-01-01

    Introduction and Aim: Banana is used widely because of its nutritional values. In past, there are studies that show banana plant parts, and their fruits can be used to treat the human diseases. Banana peel is a part of banana fruit that also has the antibacterial activity against microorganisms but has not been studied extensively. Since, there are no studies that relate the antibacterial activity of banana peel against periodontal pathogens. Hence, the aim of this study is to determine the antimicrobial activity of banana peel extract on Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans). Material and Methods: Standard strains of P. gingivalis and A. actinomycetemcomitans were used in this study which was obtained from the in-house bacterial bank of Department of Molecular Biology and Immunology at Maratha Mandal's Nathajirao G. Halgekar Institute of Dental Sciences and Research Centre. The banana peel extract was prepared, and the antibacterial activity was assessed using well agar diffusion method and minimum inhibitory concentration was assessed using serial broth dilution method. Results: In the current study, both the tested microorganisms showed antibacterial activity. In well diffusion method, P. gingivalis and A. actinomycetemcomitans showed 15 mm and 12 mm inhibition zone against an alcoholic extract of banana peel, respectively. In serial broth dilution method P. gingivalis and A. actinomycetemcomitans were sensitive until 31.25 μg/ml dilutions. Conclusion: From results of the study, it is suggested that an alcoholic extract of banana peel has antimicrobial activity against P. gingivalis and A. actinomycetemcomitans. PMID:26681854

  20. Porphyromonas gingivalis Type IX Secretion Substrates Are Cleaved and Modified by a Sortase-Like Mechanism

    PubMed Central

    Chen, Dina; Seers, Christine A.; Mitchell, Helen A.; Chen, Yu-Yen; Glew, Michelle D.; Dashper, Stuart G.; Reynolds, Eric C.

    2015-01-01

    The type IX secretion system (T9SS) of Porphyromonas gingivalis secretes proteins possessing a conserved C-terminal domain (CTD) to the cell surface. The C-terminal signal is essential for these proteins to translocate across the outer membrane via the T9SS. On the surface the CTD of these proteins is cleaved prior to extensive glycosylation. It is believed that the modification on these CTD proteins is anionic lipopolysaccharide (A-LPS), which enables the attachment of CTD proteins to the cell surface. However, the exact site of modification and the mechanism of attachment of CTD proteins to the cell surface are unknown. In this study we characterized two wbaP (PG1964) mutants that did not synthesise A-LPS and accumulated CTD proteins in the clarified culture fluid (CCF). The CTDs of the CTD proteins in the CCF were cleaved suggesting normal secretion, however, the CTD proteins were not glycosylated. Mass spectrometric analysis of CTD proteins purified from the CCF of the wbaP mutants revealed the presence of various peptide/amino acid modifications from the growth medium at the C-terminus of the mature CTD proteins. This suggested that modification occurs at the C-terminus of T9SS substrates in the wild type P. gingivalis. This was confirmed by analysis of CTD proteins from wild type, where a 648 Da linker was identified to be attached at the C-terminus of mature CTD proteins. Importantly, treatment with proteinase K released the 648 Da linker from the CTD proteins demonstrating a peptide bond between the C-terminus and the modification. Together, this is suggestive of a mechanism similar to sortase A for the cleavage and modification/attachment of CTD proteins in P. gingivalis. PG0026 has been recognized as the CTD signal peptidase and is now proposed to be the sortase-like protein in P. gingivalis. To our knowledge, this is the first biochemical evidence suggesting a sortase-like mechanism in Gram-negative bacteria. PMID:26340749

  1. A Dual Role for P2X7 Receptor during Porphyromonas gingivalis Infection

    PubMed Central

    Ramos-Junior, E.S.; Morandini, A.C.; Almeida-da-Silva, C.L.C.; Franco, E.J.; Potempa, J.; Nguyen, K.A.; Oliveira, A.C.; Zamboni, D.S.; Ojcius, D.M.; Scharfstein, J.

    2015-01-01

    Emerging evidence suggests a role for purinergic signaling in the activation of multiprotein intracellular complexes called inflammasomes, which control the release of potent inflammatory cytokines, such as interleukin (IL) -1β and -18. Porphyromonas gingivalis is intimately associated with periodontitis and is currently considered one of the pathogens that can subvert the immune system by limiting the activation of the NLRP3 inflammasome. We recently showed that P. gingivalis can dampen eATP-induced IL-1β secretion by means of its fimbriae in a purinergic P2X7 receptor–dependent manner. Here, we further explore the role of this purinergic receptor during eATP-induced IL-1β processing and secretion by P. gingivalis–infected macrophages. We found that NLRP3 was necessary for eATP-induced IL-1β secretion as well as for caspase 1 activation irrespective of P. gingivalis fimbriae. Additionally, although the secretion of IL-1β from P. gingivalis–infected macrophages was dependent on NLRP3, its adaptor protein ASC, or caspase 1, the cleavage of intracellular pro-IL-1β to the mature form was found to occur independently of NLRP3, its adaptor protein ASC, or caspase 1. Our in vitro findings revealed that P2X7 receptor has a dual role, being critical not only for eATP-induced IL-1β secretion but also for intracellular pro-IL-1β processing. These results were relevant in vivo since P2X7 receptor expression was upregulated in a P. gingivalis oral infection model, and reduced IFN-γ and IL-17 were detected in draining lymph node cells from P2rx7-/- mice. Furthermore, we demonstrated that P2X7 receptor and NLRP3 transcription were modulated in human chronic periodontitis. Overall, we conclude that the P2X7 receptor has a role in periodontal immunopathogenesis and suggest that targeting of the P2X7/NLRP3 pathway should be considered in future therapeutic interventions in periodontitis. PMID:26152185

  2. Assessment of outer membrane vesicles of periodontopathic bacterium Porphyromonas gingivalis as possible mucosal immunogen.

    PubMed

    Nakao, Ryoma; Hasegawa, Hideki; Dongying, Bai; Ohnishi, Makoto; Senpuku, Hidenobu

    2016-08-31

    Periodontitis is the most prevalent infectious disease and related to oral and systemic health, therefore novel prophylaxis to prevent the disease is highly desirable. Here, we assessed the outer membrane vesicles (OMVs) of a keystone periodontal pathogen, Porphyromonas gingivalis, as a candidate mucosal immunogen and adjuvant for a periodontitis vaccine. The structural and functional stability of OMVs, demonstrated by proteinase K resistance and ability to withstand long-term storage, are considered advantageous for carrying the OMV components into the host immune system. Intranasal vaccination of OMVs in mice elicited production of P. gingivalis-specific antibodies in blood and saliva by OMVs in a dose-dependent manner, which was dramatically enhanced by addition of a TLR3 agonist, Poly(I:C). Serum samples from mice immunized with OMVs plus Poly(I:C) adjuvant [OMV+Poly(I:C)] showed significant inhibition of gingipain proteolytic activity of not only the vaccine strain, but also heterologous strains. The viability of P. gingivalis was also decreased by preincubation with OMV+Poly(I:C)-immunized sera, while the killing effect was partially blocked by heat-inactivation of the sera. Saliva samples from mice immunized with OMV+Poly(I:C) enhanced bacterial agglutination of both the vaccine and heterologous strains. In an oral infection mouse model, the numbers of P. gingivalis in the oral cavity were significantly decreased in mice intranasally immunized with OMV+Poly(I:C) as compared to mock (only Poly[I:C])-immunized mice. The high levels of serum IgG (including IgG1 and IgG2a) and salivary S-IgA were elicited in mice intranasally immunized with OMV+Poly(I:C), which were maintained for at least 28 and 18weeks, respectively, after immunization. An experiment examining the accumulation of OMVs after intranasal immunization in proximal organs and an intracerebral injection experiment confirmed the safety of OMVs. Based on our results, we propose that intranasal

  3. Porphyromonas gingivalis Type IX Secretion Substrates Are Cleaved and Modified by a Sortase-Like Mechanism.

    PubMed

    Gorasia, Dhana G; Veith, Paul D; Chen, Dina; Seers, Christine A; Mitchell, Helen A; Chen, Yu-Yen; Glew, Michelle D; Dashper, Stuart G; Reynolds, Eric C

    2015-09-01

    The type IX secretion system (T9SS) of Porphyromonas gingivalis secretes proteins possessing a conserved C-terminal domain (CTD) to the cell surface. The C-terminal signal is essential for these proteins to translocate across the outer membrane via the T9SS. On the surface the CTD of these proteins is cleaved prior to extensive glycosylation. It is believed that the modification on these CTD proteins is anionic lipopolysaccharide (A-LPS), which enables the attachment of CTD proteins to the cell surface. However, the exact site of modification and the mechanism of attachment of CTD proteins to the cell surface are unknown. In this study we characterized two wbaP (PG1964) mutants that did not synthesise A-LPS and accumulated CTD proteins in the clarified culture fluid (CCF). The CTDs of the CTD proteins in the CCF were cleaved suggesting normal secretion, however, the CTD proteins were not glycosylated. Mass spectrometric analysis of CTD proteins purified from the CCF of the wbaP mutants revealed the presence of various peptide/amino acid modifications from the growth medium at the C-terminus of the mature CTD proteins. This suggested that modification occurs at the C-terminus of T9SS substrates in the wild type P. gingivalis. This was confirmed by analysis of CTD proteins from wild type, where a 648 Da linker was identified to be attached at the C-terminus of mature CTD proteins. Importantly, treatment with proteinase K released the 648 Da linker from the CTD proteins demonstrating a peptide bond between the C-terminus and the modification. Together, this is suggestive of a mechanism similar to sortase A for the cleavage and modification/attachment of CTD proteins in P. gingivalis. PG0026 has been recognized as the CTD signal peptidase and is now proposed to be the sortase-like protein in P. gingivalis. To our knowledge, this is the first biochemical evidence suggesting a sortase-like mechanism in Gram-negative bacteria.

  4. Porphyromonas gingivalis Peptidylarginine Deiminase, a Key Contributor in the Pathogenesis of Experimental Periodontal Disease and Experimental Arthritis

    PubMed Central

    Gully, Neville; Bright, Richard; Marino, Victor; Marchant, Ceilidh; Cantley, Melissa; Haynes, David; Butler, Catherine; Dashper, Stuart; Reynolds, Eric; Bartold, Mark

    2014-01-01

    Objectives To investigate the suggested role of Porphyromonas gingivalis peptidylarginine deiminase (PAD) in the relationship between the aetiology of periodontal disease and experimentally induced arthritis and the possible association between these two conditions. Methods A genetically modified PAD-deficient strain of P. gingivalis W50 was produced. The effect of this strain, compared to the wild type, in an established murine model for experimental periodontitis and experimental arthritis was assessed. Experimental periodontitis was induced following oral inoculation with the PAD-deficient and wild type strains of P. gingivalis. Experimental arthritis was induced via the collagen antibody induction process and was monitored by assessment of paw swelling and micro-CT analysis of the radio-carpal joints. Experimental periodontitis was monitored by micro CT scans of the mandible and histological assessment of the periodontal tissues around the mandibular molars. Serum levels of anti-citrullinated protein antibodies (ACPA) and P. gingivalis were assessed by ELISA. Results The development of experimental periodontitis was significantly reduced in the presence of the PAD-deficient P. gingivalis strain. When experimental arthritis was induced in the presence of the PAD-deficient strain there was less paw swelling, less erosive bone damage to the joints and reduced serum ACPA levels when compared to the wild type P. gingivalis inoculated group. Conclusion This study has demonstrated that a PAD-deficient strain of P. gingivalis was associated with significantly reduced periodontal inflammation. In addition the extent of experimental arthritis was significantly reduced in animals exposed to prior induction of periodontal disease through oral inoculation of the PAD-deficient strain versus the wild type. This adds further evidence to the potential role for P. gingivalis and its PAD in the pathogenesis of periodontitis and exacerbation of arthritis. Further studies are now

  5. NOX1/2 activation in human gingival fibroblasts by Fusobacterium nucleatum facilitates attachment of Porphyromonas gingivalis.

    PubMed

    Ahn, Sun Hee; Song, Ji-Eun; Kim, Suhee; Cho, Sung-Hyun; Lim, Yun Kyong; Kook, Joong-Ki; Kook, Min-Suk; Lee, Tae-Hoon

    2016-08-01

    Periodontal diseases are infectious polymicrobial inflammatory diseases that lead to destruction of the periodontal ligament, gingiva, and alveolar bone. Sequential colonization of a broad range of bacteria, including Fusobacterium nucleatum and Porphyromonas gingivalis, is an important phenomenon in this disease model. F. nucleatum is a facultative anaerobic species thought to be a key mediator of dental plaque maturation due to its extensive coaggregation with other oral bacteria, while P. gingivalis is an obligate anaerobic species that induces gingival inflammation by secreting various virulence factors. The formation of a bacterial complex by these two species is central to the pathogenesis of periodontal disease. Reactive oxygen species (ROS) are produced during bacterial infections and are involved in intracellular signaling. However, the impact of oral bacteria-induced ROS on the ecology of F. nucleatum and P. gingivalis has yet to be clarified. In the present study, we investigated ROS production induced in primary human oral cells by F. nucleatum and P. gingivalis and its effect on the formation of their bacterial complexes and further host cell apoptosis. We found that in primary human gingival fibroblasts (GFs), two NADPH oxidase isoforms, NOX1 and NOX2, were activated in response to F. nucleatum infection but not P. gingivalis infection. Accordingly, increased NADPH oxidase activity and production of superoxide anion were observed in GFs after F. nucleatum infection, but not after P. gingivalis infection. Interestingly, in NOX1, NOX2, or NOX1/NOX2 knockdown cells, the number of P. gingivalis decreased when the cells were coinfected with F. nucleatum. A similar pattern of host cell apoptosis was observed. This implies that F. nucleatum contributes to attachment of P. gingivalis by triggering activation of NADPH oxidase in host cells, which may provide an environment more favorable to strict anaerobic bacteria and have a subsequent effect on apoptosis of

  6. Development and evaluation of a saliva-based chair-side diagnostic for the detection of Porphyromonas gingivalis.

    PubMed

    O'Brien-Simpson, Neil M; Burgess, Kate; Brammar, Gail C; Darby, Ivan B; Reynolds, Eric C

    2015-01-01

    Porphyromonas gingivalis is a key pathogen in the polymicrobial biofilm that is associated with the oral disease chronic periodontitis. A number of studies have shown that in humans the level of P. gingivalis in the polymicrobial biofilm is positively correlated with disease progression. The aim of this study was to develop a P. gingivalis diagnostic that has high specificity and sensitivity for P. gingivalis using a range of laboratory and clinical isolates and then compare the efficacy of the diagnostic with RTPCR using samples from chronic periodontitis patients and age- and sex-matched healthy controls. Key parameters for the kit were to use saliva as the biological fluid as this is a most convenient medium for chair-side sampling and to give a positive reading for the reported threshold for detection of 5×10(5) P. gingivalis cells/mL that indicates disease progression. We initially screened a range of monoclonal antibodies for recognition of the P. gingivalis conserved virulence factor RgpA-Kgp complex and identified two mAbs that could be used in a capture and detection ELISA system. These mAbs were used to formulate and manufacture the GC P. gingivalis saliva diagnostic kit used in the study. To validate the saliva kit, saliva (P. gingivalis free) was spiked with known concentrations of viable P. gingivalis whole cells of W50, 381, A7A1-28, and ATCC 33277; P. gingivalis clinical isolates; P. gingivalis vesicles; and the secreted form of the RgpA-Kgp complex. Laboratory findings indicated that the kit was able to detect all laboratory and clinical isolate strains of P. gingivalis at 5×10(4)/mL to 5×10(5)/mL. It was also able to detect the RgpA-Kgp complex and vesicles at 5×10(4) and 5×10(5) cell equivalent doses, respectively. Saliva and plaque were then collected from 50 subjects with moderate-severe chronic periodontitis and 50 age- and sex-matched subjects with healthy periodontium. Real-time PCR was utilised to analyse levels of P. gingivalis in both

  7. Lysine acetylation is a common post-translational modification of key metabolic pathway enzymes of the anaerobe Porphyromonas gingivalis.

    PubMed

    Butler, Catherine A; Veith, Paul D; Nieto, Matthew F; Dashper, Stuart G; Reynolds, Eric C

    2015-10-14

    Porphyromonas gingivalis is a Gram-negative anaerobe considered to be a keystone pathogen in the development of the bacterial-associated inflammatory oral disease chronic periodontitis. Although post-translational modifications (PTMs) of proteins are commonly found to modify protein function in eukaryotes and prokaryotes, PTMs such as lysine acetylation have not been examined in P. gingivalis. Lysine acetylation is the addition of an acetyl group to a lysine which removes this amino acid's positive charge and can induce changes in a protein's secondary structure and reactivity. A proteomics based approach combining immune-affinity enrichment with high sensitivity Orbitrap mass spectrometry identified 130 lysine acetylated peptides from 92 P. gingivalis proteins. The majority of these peptides (71) were attributed to 45 proteins with predicted metabolic activity; these proteins could be mapped to several P. gingivalis metabolic pathways where enzymes catalysing sequential reactions within the same pathway were often found acetylated. In particular, the catabolic pathways of complex anaerobic fermentation of amino acids to produce energy had 12 enzymes lysine acetylated. The results suggest that lysine acetylation may be an important mechanism in metabolic regulation in P. gingivalis, which is vital for P. gingivalis survival and adaptation of its metabolism throughout infection. Statement of significance. Porphyromonas gingivalis is a keystone pathogen in the development of chronic periodontitis, an inflammatory disease of the supporting tissues of the teeth. The ability of the pathogen to induce dysbiosis and disease is related to an array of specific virulence factors and metabolic regulation that enables the bacterium to proliferate in an inflamed periodontal pocket. The mechanisms P. gingivalis uses to adapt to a changing and hostile environment are poorly understood and here we show, for the first time, that enzymes of critical metabolic pathways for energy

  8. Identification of Porphyromonas gingivalis proteins secreted by the Por secretion system.

    PubMed

    Sato, Keiko; Yukitake, Hideharu; Narita, Yuka; Shoji, Mikio; Naito, Mariko; Nakayama, Koji

    2013-01-01

    The Gram-negative bacterium Porphyromonas gingivalis possesses a number of potential virulence factors for periodontopathogenicity. In particular, cysteine proteinases named gingipains are of interest given their abilities to degrade host proteins and process other virulence factors such as fimbriae. Gingipains are translocated on the cell surface or into the extracellular milieu by the Por secretion system (PorSS), which consists of a number of membrane or periplasmic proteins including PorK, PorL, PorM, PorN, PorO, PorP, PorQ, PorT, PorU, PorV (PG27, LptO), PorW and Sov. To identify proteins other than gingipains secreted by the PorSS, we compared the proteomes of P. gingivalis strains kgp rgpA rgpB (PorSS-proficient strain) and kgp rgpA rgpB porK (PorSS-deficient strain) using two-dimensional gel electrophoresis and peptide-mass fingerprinting. Sixteen spots representing 10 different proteins were present in the particle-free culture supernatant of the PorSS-proficient strain but were absent or faint in that of the PorSS-deficient strain. These identified proteins possessed the C-terminal domains (CTDs), which had been suggested to form the CTD protein family. These results indicate that the PorSS is used for secretion of a number of proteins other than gingipains and that the CTDs of the proteins are associated with the PorSS-dependent secretion. PMID:23075153

  9. Identification of Porphyromonas gingivalis proteins secreted by the Por secretion system.

    PubMed

    Sato, Keiko; Yukitake, Hideharu; Narita, Yuka; Shoji, Mikio; Naito, Mariko; Nakayama, Koji

    2013-01-01

    The Gram-negative bacterium Porphyromonas gingivalis possesses a number of potential virulence factors for periodontopathogenicity. In particular, cysteine proteinases named gingipains are of interest given their abilities to degrade host proteins and process other virulence factors such as fimbriae. Gingipains are translocated on the cell surface or into the extracellular milieu by the Por secretion system (PorSS), which consists of a number of membrane or periplasmic proteins including PorK, PorL, PorM, PorN, PorO, PorP, PorQ, PorT, PorU, PorV (PG27, LptO), PorW and Sov. To identify proteins other than gingipains secreted by the PorSS, we compared the proteomes of P. gingivalis strains kgp rgpA rgpB (PorSS-proficient strain) and kgp rgpA rgpB porK (PorSS-deficient strain) using two-dimensional gel electrophoresis and peptide-mass fingerprinting. Sixteen spots representing 10 different proteins were present in the particle-free culture supernatant of the PorSS-proficient strain but were absent or faint in that of the PorSS-deficient strain. These identified proteins possessed the C-terminal domains (CTDs), which had been suggested to form the CTD protein family. These results indicate that the PorSS is used for secretion of a number of proteins other than gingipains and that the CTDs of the proteins are associated with the PorSS-dependent secretion.

  10. Characterization of the binding activities of proteinase-adhesin complexes from Porphyromonas gingivalis.

    PubMed Central

    Pike, R N; Potempa, J; McGraw, W; Coetzer, T H; Travis, J

    1996-01-01

    Adhesins from oral bacteria perform an important function in colonizing target tissues within the dentogingival cavity. In Porphyromonas gingivalis certain of these adhesion proteins exist as a complex with either of two major proteinases referred to as gingipain R (arginine-specific gingipain) and gingipain K (lysine-specific gingipain) (R. N. Pike, W. T. McGraw, J. Potempa, and J. Travis, J. Biol. Chem. 269:406-411, 1994). With specific proteinase inhibitors, it was shown that hemagglutination by either proteinase-adhesin complex could occur independently of proteinase activity. Significantly, low concentrations of fibrinogen, fibronectin, and laminin inhibited hemagglutination, indicating that adherence to these proteins and not the hemagglutination activity was a primary property of the adhesin activity component of complexes. Binding studies with gingipain K and gingipain R suggest that interaction with fibrinogen is a major function of the adhesin domain, with dissociation constants for binding to fibrinogen being 4 and 8.5 nM, respectively. Specific association with fibronectin and laminin was also found. All bound proteins were degraded by the functional proteinase domain, with gingipain R being more active on laminin and fibronectin and gingipain K being more effective in the digestion of fibrinogen. Cumulatively, these data suggest that gingipain R and gingipain K, acting as proteinase-adhesin complexes, progressively attach to, degrade, and detach from target proteins. Since such complexes appear to be present on the surfaces of both vesicles and membranes of P. gingivalis, they may play an important role in the attachment of this bacterium to host cell surfaces. PMID:8631676

  11. Purification and characterization of three types of proteases from culture supernatants of Porphyromonas gingivalis.

    PubMed Central

    Hinode, D; Hayashi, H; Nakamura, R

    1991-01-01

    Three types of caseinolytic proteases (Pase-A, Pase-B, and Pase-C) were isolated and purified from culture supernatants of Porphyromonas gingivalis 381 by the combined procedures of acetone precipitation, gel filtration, solubilization with octylthioglucoside followed by affinity chromatography on arginine-Sepharose 4B, high-performance liquid chromatography (HPLC) on Biofine IEC-DEAE, and HPLC on TSK-G4000SW. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Pase-A and -B showed diffuse protein bands of 105 to 110 and 72 to 80 kDa, respectively, and Pase-C showed a clear band of about 44 kDa. Pase-B and -C hydrolyzed some synthetic substrates for trypsin, but Pase-B did not act on the carboxyl side of lysine in insulin chain B or on a synthetic substrate which trypsin and Pase-C acted on. Pase-A did not act on the synthetic substrates but cleaved the peptide bonds Glu-Ala and Ala-Leu of insulin. Leupeptin inhibition of the caseinolytic activity of both Pase-A and -B was similar to its inhibition of Pase-C. Phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate strongly inhibited Pase-A, but no significant effect on the other enzymes was observed, suggesting that only Pase-A is a serine protease. The inhibitory characteristics of Pase-B and -C were very similar. Pase-A was not thiol dependent for enzyme activity, but Pase-B was strongly dependent, i.e., even more so than Pase-C. Pase-A inactivated the inhibitory activity of plasma alpha-1-antitrypsin, but the other two did not. These results show that P. gingivalis produces different types of proteases other than the trypsinlike protease generally reported. Images PMID:1879930

  12. Isolation and characterization of fimbriae from a sparsely fimbriated strain of Porphyromonas gingivalis.

    PubMed Central

    Sojar, H T; Hamada, N; Genco, R J

    1997-01-01

    Porphyromonas gingivalis W50 (ATCC 53978) possesses the gene for fimbriae; however, the surface-expressed fimbriae are sparse and have not been previously isolated and characterized. We purified fimbriae from strain W50 to homogeneity by ammonium sulfate precipitation and reverse-phase high-performance liquid chromatography [H. T. Sojar, N. Hamada, and R. J. Genco, Protein Expr. Purif. 9(1):49-52, 1997]. Negative staining of purified fimbriae viewed by electron microscopy revealed that the fimbriae were identical in diameter to fimbriae of other P. gingivalis strains, such as 2561, but were shorter in length. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, the apparent molecular weight of isolated fimbrillin from strain W50 was found to be identical to that of the fimbrillin molecule of strain 2561. Unlike 2561 fimbriae, W50 fimbriae, under reducing condition, exhibited a monomeric structure on SDS-PAGE at room temperature. However, under nonreduced conditions, even at 100 degrees C, no monomer was observed. In immunoblot analysis as well as immunogold labeling of isolated fimbriae, polyclonal antibodies against 2561 fimbriae, as well as antibodies against peptide I (V-V-M-A-N-T-G-A-M-E-V-G-K-T-L-A-E-V-K-Cys) and peptide J (A-L-T-T-E-L-T-A-E-N-Q-E-A-A-G-L-I-M-T-A-E-P-Cys), reacted. However, antifimbrial antibodies against strain 2561 reacted very weakly compared to anti-peptide I and anti-peptide J. Negative staining of whole W50 cells, as well as immunogold electron microscopy with anti-peptide I and anti-peptide J, showed fimbriae shorter in length and very few in number compared to those of strain 2561. Purified fimbriae showed no hemagglutinating activity. Amino acid composition was very similar to that of previously reported fimbriae of the 2561 strain. PMID:9172351

  13. Expression Profiles of TGF-β and TLR Pathways in Porphyromonas gingivalis and Prevotella intermedia Challenged Osteoblasts

    PubMed Central

    Aydin, Kubra; Ekinci, Fatma Yesim; Korachi, May

    2015-01-01

    Background: The presence of certain oral pathogens at implant sites can hinder the osseointegration process. However, it is unclear how and by what microorganisms it happens. Objectives: This study investigated whether the presence of oral pathogens of Porphyromonas gingivalis and Prevotella intermedia individually, play a role in the failure of bone formation by determining the expression profiles of Transforming Growth Factor Beta (TGF-β/Bone Morphogenic Protein (BMP) and Toll-Like Receptor (TLR) pathways in challenged osteoblasts. Materials and Methods: Cell viability of P. gingivalis and P. intermedia challenged osteoblasts were determined by WST assay. Changes in osteoblast morphology and inhibition of mineralization were observed by Scanning Electron Microscopy (SEM) and Von Kossa staining, respectively. Expression of TGF-β and TLR pathway genes on challenged cells were identified by RT profiler array. Both P. gingivalis and P. intermedia challenges resulted in reduced viability and mineralization of osteoblasts. Results: Viability was reduced to 56.8% (P. gingivalis) and 52.75% (P. intermedia) at 1000 multiplicity. Amongst 48 genes examined, expressions of BMPER, SMAD1, IL8 and NFRKB were found to be highly upregulated by both bacterial challenges (Fold Change > 4). Conclusions: P. gingivalis and P. intermedia could play a role in implant failure by changing the expression profiles of genes related to bone formation and resorption. PMID:26034550

  14. Subcutaneous vaccination with Porphyromonas gingivalis ameliorates periodontitis by modulating Th17/Treg imbalance in a murine model.

    PubMed

    Wang, Linyuan; Guan, Ning; Jin, Ying; Lin, Xiaoping; Gao, Hong

    2015-03-01

    To date, Porphyromonas gingivalis (P. gingivalis) vaccination has been studied only in animals, and no effective prophylactic human periodontal vaccine has been developed, with the reason for the failure of prophylactic human periodontal vaccines unknown. T helper 17 cell (Th17)/regulatory T (Treg) cell responses play an important role in the development of periodontitis, and a Th17/Treg imbalance causes the pathogenesis of periodontitis. However, whether vaccination with P. gingivalis can prevent periodontitis through modulation of the Th17/Treg imbalance remains unknown. In this study, mice were subcutaneously vaccinated with formalin-killed P. gingivalis and then orally challenged with P. gingivalis. The vaccination protected the mice from alveolar bone resorption and inflammation. These protective effects might be ascribed to downregulation of Th17 cells and interleukin (IL)-17A production, upregulation of Treg and receptor activator of nuclear factor-kappa B ligand (RANKL)(+)CD4(+)T cells, and IL-10 and transforming growth factor-β1 production, and inhibition of lymphocyte proliferation. Our findings may provide a direction for the development of a vaccine or therapy against periodontitis by alteration of the Th17/Treg imbalance.

  15. Evaluation of the effect of andrographolide on atherosclerotic rabbits induced by Porphyromonas gingivalis.

    PubMed

    Al Batran, Rami; Al-Bayaty, Fouad; Al-Obaidi, Mazen M Jamil; Hussain, Saba F; Mulok, Tengku Z

    2014-01-01

    Epidemiologic evidence has demonstrated significant associations between atherosclerosis and Porphyromonas gingivalis (Pg). We had investigated the effect of andrographolide (AND) on atherosclerosis induced by Pg in rabbits. For experimental purpose, we separated thirty male white New Zealand rabbits into 5 groups. Group 1 received standard food pellets; Groups 2-5 were orally challenged with Pg; Group 3 received atorvastatin (AV, 5 mg/kg), and Groups 4-5 received 10 and 20 mg/kg of AND, respectively, over 12 weeks. Groups treated with AND showed significant decrease in TC, TG, and LDL levels (P<0.05) and significant increase in HDL level in the serum of rabbits. Furthermore, the treated groups (G3-G5) exhibited reductions in interleukins (IL-1β and IL-6) and C-reactive protein (CRP) as compared to atherogenicgroup (G2). The histological results showed that the thickening of atherosclerotic plaques were less significant in treated groups (G3-G5) compared with atherogenicgroup (G2). Also, alpha-smooth muscle actin (α-SMA) staining decreased within the plaques of atherogenicgroup (G2), while it was increased in treated groups (G3-G5). Lastly, groups treated with AV and AND (G3-G5) showed significant reduction of CD36 expression (P<0.05) compared to atherogenicgroup (G2). These results substantially proved that AND contain antiatherogenic activity.

  16. Gender-Specific Associations of Serum Antibody to Porphyromonas gingivalis and Inflammatory Markers

    PubMed Central

    Furuta, Michiko; Shimazaki, Yoshihiro; Tanaka, Shunichi; Takeuchi, Kenji; Shibata, Yukie; Takeshita, Toru; Nishimura, Fusanori; Yamashita, Yoshihisa

    2015-01-01

    It remains unclear whether serum antibody titer against Porphyromonas gingivalis (Pg) and inflammatory components lead to periodontal deterioration in each gender, as periodontal and systemic status is influenced by gender. The present study investigates the gender-specific probable effects of titer against Pg and inflammatory markers on periodontal health status in a longitudinal study. A retrospective study design was used. At two time points over an 8-year period (in 2003 and 2011), 411 individuals (295 males with a mean age of 57.6 ± 11.2 years and 116 females with a mean age of 59.2 ± 10.3 years) were surveyed. Periodontal status, serum antibody titer against Pg, and high-sensitive C-reactive protein (hsCRP) were evaluated. Poisson regression analyses revealed that the elevated titer against Pg and hsCRP significantly predicted the persistence of periodontal disease 8 years later in females with periodontal disease in 2003. Elevated hsCRP was significantly associated with the incidence of periodontal disease 8 years later in females who were periodontally healthy in 2003. Males had a weaker association among titer against Pg, inflammatory markers, and periodontal disease. These findings suggest that immune response to Pg infection in addition to inflammatory components affects periodontal deterioration in females. PMID:25756052

  17. Streptococcus salivarius promotes mucin putrefaction and malodor production by Porphyromonas gingivalis.

    PubMed

    Sterer, N; Rosenberg, M

    2006-10-01

    Although the contribution of the oral microbiota to oral malodor is well-documented, the potential role of Gram-positive micro-organisms is unclear. In the current study, we tested the hypothesis that Gram-positive micro-organisms contribute to malodor production by deglycosylating oral glycoproteins, rendering them susceptible to subsequent proteolysis. To this end, we examined the effect of Streptococcus salivarius on Porphyromonas gingivalis-mediated putrefaction of a model glycoprotein (pig gastric mucin). Malodor was scored by two odor judges, and volatile sulfides were determined with the use of a sulfide monitor. Mucin degradation was followed by electrophoresis on SDS-PAGE. Results showed that the addition of S. salivarius or beta-galactosidase promoted mucin degradation and concomitant malodor production. Addition of glycosidic inhibitors (p-APTG and glucose) inhibited this process. These results suggest that Gram-positive micro-organisms such as S. salivarius contribute to oral malodor production by deglycosylating salivary glycoproteins, thus exposing their protein core to further degradation by Gram-negative micro-organisms. PMID:16998130

  18. Evaluation of the Effect of Andrographolide on Atherosclerotic Rabbits Induced by Porphyromonas gingivalis

    PubMed Central

    Al-Bayaty, Fouad; Al-Obaidi, Mazen M. Jamil; Hussain, Saba F.; Mulok, Tengku Z.

    2014-01-01

    Epidemiologic evidence has demonstrated significant associations between atherosclerosis and Porphyromonas gingivalis (Pg). We had investigated the effect of andrographolide (AND) on atherosclerosis induced by Pg in rabbits. For experimental purpose, we separated thirty male white New Zealand rabbits into 5 groups. Group 1 received standard food pellets; Groups 2–5 were orally challenged with Pg; Group 3 received atorvastatin (AV, 5 mg/kg), and Groups 4-5 received 10 and 20 mg/kg of AND, respectively, over 12 weeks. Groups treated with AND showed significant decrease in TC, TG, and LDL levels (P < 0.05) and significant increase in HDL level in the serum of rabbits. Furthermore, the treated groups (G3–G5) exhibited reductions in interleukins (IL-1β and IL-6) and C-reactive protein (CRP) as compared to atherogenicgroup (G2). The histological results showed that the thickening of atherosclerotic plaques were less significant in treated groups (G3–G5) compared with atherogenicgroup (G2). Also, alpha-smooth muscle actin (α-SMA) staining decreased within the plaques of atherogenicgroup (G2), while it was increased in treated groups (G3–G5). Lastly, groups treated with AV and AND (G3–G5) showed significant reduction of CD36 expression (P < 0.05) compared to atherogenicgroup (G2). These results substantially proved that AND contain antiatherogenic activity. PMID:25215291

  19. Bortezomib Inhibits Osteoclastogenesis and Porphyromonas gingivalis Lipopolysaccharide-induced Alveolar Bone Resorption.

    PubMed

    Kim, Y-G; Kang, J H; Kim, H J; Kim, H J; Kim, H-H; Kim, J-Y; Lee, Y

    2015-09-01

    Healthy bone is maintained by the coordinated activities of osteoblast-mediated bone formation and osteoclast-dependent bone resorption. Pathologic conditions such as hormonal imbalance and inflammation cause increased osteoclastogenesis resulting in osteoporosis, rheumatoid arthritis, and periodontitis. Bortezomib is novel antimyeloma agent that has a direct beneficial effect on bone formation. However, the role of bortezomib in osteoclastogenesis and underlying mechanisms remains to be fully comprehended. In the present study, we show that bortezomib directly inhibited the receptor activator of nuclear factor κB ligand (RANKL)- and lipopolysaccharide-dependent osteoclast differentiation. Interestingly, the bortezomib-mediated inhibition of osteoclastogenesis was transient, since the removal of bortezomib from culture completely restored osteoclast differentiation. Bortezomib impeded the induction and nuclear localization of nuclear factor of activated T cells, cytoplasmic 1 and reduced both macrophage colony-stimulating factor- and RANKL-induced extracellular-signal-regulated kinase (ERK) phosphorylation. In a mouse model of periodontitis, bortezomib prevented alveolar bone erosion induced by Porphyromonas gingivalis lipopolysaccharide. These data not only suggest a previously unappreciated mechanism by which bortezomib regulates bone resorption but also propose novel applications of bortezomib beyond its use as an antimyeloma agent.

  20. Rosiglitazone impedes Porphyromonas gingivalis-accelerated atherosclerosis by downregulating the TLR/NF-κB signaling pathway in atherosclerotic mice.

    PubMed

    Pan, Shengbo; Lei, Lang; Chen, Shuai; Li, Houxuan; Yan, Fuhua

    2014-12-01

    Porphyromonas gingivalis,a predominant periodontal pathogen, is known to accelerate atherosclerosis in hyperlipidemic animals via aberrant inflammatory responses. Peroxisome proliferator-activated receptor gamma (PPARγ) agonists have been reported to exert anti-inflammatory effects in vitro. The purpose of the present study was to investigate the potential protective role of the PPARγ agonist rosiglitazone in pathogen accelerated atherosclerosis in an apolipoprotein E-deficient (ApoE-/-) mouse model. ApoE-/- mice were inoculated intravenously with live P. gingivalis (strain 33277) or the buffer vehicle and treated with rosiglitazone or saline over a 10-week period. Their atherosclerotic status in aortic artery was assessed through histomorphometric analysis, inflammatory agent and lipid profiles in blood was determined by ELISA, and levels of relevant cytokines and Toll-like receptors (TLRs) in aortic tissues were evaluated using immunohistochemistry and quantitative PCR. P. gingivalis inoculation was associated with increased atherosclerotic plaque formation in the aorta and higher levels of serum pro-inflammatory cytokines (tumor necrosis factor-α, monocyte chemotactic protein-1 and interleukin-1β), but the serum lipid profile was not affected by P. gingivalis infection. Levels of tumor necrosis factor-α, monocyte chemotactic protein-1 intercellular cell adhesion molecule-1 and TLRs were higher in the aortic tissues of mice exposed to P. gingivalis, and activation of nuclear factor-κB was also observed. In both P. gingivalis-treated and -untreated ApoE-/- mice, rosiglitazone treatment was associated with less atherosclerotic plaque formation; lower serum inflammatory cytokines, total cholesterol, and low density lipoprotein cholesterol; higher levels of PPARγ, lower amounts of TLR2/4 and downregulated nuclear factor-κB activity in aortic tissues. These findings suggest that rosiglitazone mitigates or prevents P. gingivalis-accelerated atherosclerosis by

  1. Xylitol, an Anticaries Agent, Exhibits Potent Inhibition of Inflammatory Responses in Human THP-1-Derived Macrophages Infected With Porphyromonas gingivalis

    PubMed Central

    Park, Eunjoo; Na, Hee Sam; Kim, Sheon Min; Wallet, Shannon; Cha, Seunghee; Chung, Jin

    2016-01-01

    Background Xylitol is a well-known anticaries agent and has been used for the prevention and treatment of dental caries. In this study, the anti-inflammatory effects of xylitol are evaluated for possible use in the prevention and treatment of periodontal infections. Methods Cytokine expression was stimulated in THP-1 (human monocyte cell line)-derived macrophages by live Porphyromonas gingivalis, and enzyme-linked immunosorbent assay and a commercial multiplex assay kit were used to determine the effects of xylitol on live P. gingivalis–induced production of cytokine. The effects of xylitol on phagocytosis and the production of nitric oxide were determined using phagocytosis assay, viable cell count, and Griess reagent. The effects of xylitol on P. gingivalis adhesion were determined by immunostaining, and costimulatory molecule expression was examined by flow cytometry. Results Live P. gingivalis infection increased the production of representative proinflammatory cytokines, such as tumor necrosis factor-α and interleukin (IL)-1β, in a multiplicity of infection– and time-dependent manner. Live P. gingivalis also enhanced the release of cytokines and chemokines, such as IL-12 p40, eotaxin, interferon γ–induced protein 10, monocyte chemotactic protein-1, and macrophage inflammatory protein-1. The pretreatment of xylitol significantly inhibited the P. gingivalis– induced cytokines production and nitric oxide production. In addition, xylitol inhibited the attachment of live P. gingivalis on THP-1-derived macrophages. Furthermore, xylitol exerted anti-phagocytic activity against both Escherichia coli and P. gingivalis. Conclusion These findings suggest that xylitol acts as an antiinflammatory agent in THP-1-derived macrophages infected with live P. gingivalis, which supports its use in periodontitis. PMID:24592909

  2. Aging and contribution of MyD88 and TRIF in expression of TLR pathway associated genes to Porphyromonas gingivalis

    PubMed Central

    Shaik-Dasthagirisaheb, Yazdani B.; Huang, Nasi; Weinberg, Ellen O.; Shen, Steve S.; Genco, Caroline A.; Gibson, Frank C.

    2014-01-01

    BACKGROUND AND OBJECTIVE Periodontal disease is a highly complex chronic inflammatory disease of the oral cavity. Multiple factors influence periodontal disease including socioeconomic status, genetics, age, however, inflammation elicited by the presence of specific bacteria in the subgingival space is thought to drive the majority of soft and hard tissue destruction. Porphyromonas gingivalis is closely associated with periodontal disease. Toll-like receptors (TLRs) and their intracellular signaling pathways play roles in host responses to P. gingivalis. The focus of current study was to use microarray analysis to define the contributions that TLR adaptor molecules MyD88 and TRIF, and aging have on TLR pathway associated mRNA expression in response to P. gingivalis. MATERIALS AND METHODS Bone marrow derived macrophages (BMØ) from wild type (Wt), MyD88-KO and TrifLps2 mice at 2-months and 12-months of age were cultured with P. gingivalis. Expression of genes in BMØ cultured with P. gingivalis was determined in comparison to medium alone control. RESULTS Using a two-fold cut-off in mRNA expression criteria, differential expression of 32 genes was observed when Wt BMØ from 2-month old mice were cultured with P. gingivalis compared with medium alone control. When compared with 2-month old Wt, 21 and 12 genes were differentially expressed (P<0.05) as a result of MyD88 or TRIF mutations respectively. The expression of 5 genes was significantly (P<0.05) reduced in the 12-month group compared to the 2-month group in Wt BMØ following culture with P. gingivalis. Age also influenced expression of genes in MyD88-KO and TrifLps2 mice challenged with P. gingivalis. CONCLUSION Our results indicate that P. gingivalis induces differential expression of TLR pathway associated genes, and both MyD88, and TRIF play roles in the expression of these genes. Age also played a role in the expression of TLR-associated genes following stimulation of BMØ with P. gingivalis. PMID:24862405

  3. Assessing the Antimicrobial Effect of the Essential Oil of Myrtus communis on the Clinical Isolates of Porphyromonas gingivalis: An in vitro Study

    PubMed Central

    Hedayati, Azita; Khosropanah, Hengameh; Bazargani, Abdollah; Abed, Molud; Emami, Amir

    2013-01-01

    Background One of the major diseases affecting the oral health is periodontal disease. Various therapeutic methods have been introduced to eliminate the periodonto-pathic subgingival microflora. Among these, Porphyromonas gingivalis (P. gingivalis) has a major role in the pathogenesis of different forms of periodontal diseases. Objectives The present study investigated the antimicrobial effect of the essential oil of Myrtus communis on Porphyromonas gingivalis (P. gingivalis) as the most destructive periodontal pathogens. Materials and Methods The subjects included 27 male and 3 female patients with advanced chronic periodontitis. The mean age of the patients was 47.6 ± 2.0 years old. P. gingivalis was isolated from the samples and identified by various diagnostic tests, including Gram staining, Indol test, and fluorescent test. Minimum inhibitory concentration (MIC) of the essential oil against isolated P. gingivalis was determined by broth micro-dilution method. Results In this study, 0.12 - 64 μL/mL Myrtus communis essence were used for 30 P. gingivalis isolates and the MIC50 and MIC90 concentration of Myrtus communis essence against the isolates was equal to 1 and 8 μL/mL respectively. Conclusions The results showed that Myrtus communis has antimicrobial effects against P. gingivalis. Further studies are suggested to include this essence in therapeutic protocols of periodontal disease. PMID:24624208

  4. Quantification of Porphyromonas gingivalis in chronic periodontitis patients associated with diabetes mellitus using real-time polymerase chain reaction

    PubMed Central

    Padmalatha, GV; Bavle, Radhika M; Satyakiran, Gadavalli Vera Venkata; Paremala, K; Sudhakara, M; Makarla, Soumya

    2016-01-01

    Introduction: Periodontal diseases, if left untreated, can lead to tooth loss and affect at least one tooth in 80% of adults worldwide, with the main cause being a bacterial plaque. Among subgingival plaque bacterial species, Porphyromonas gingivalis has been implicated as a major etiological agent causing tooth loss. Diabetics and smokers are two patient groups at high risk for periodontal disease. The increase in the number of this organism with the coexistence of other pathogenic microbes leads to rapid destruction of the periodontium, premature loss of teeth and also because of its virulence has implications in systemic pathology. Our aim was to observe the involvement of P. gingivalis in diabetes mellitus (DM) patients associated with periodontitis with and without tobacco-associated habits and to compare them with periodontitis patients having no other systemic pathologies. Materials and Methods: Subgingival plaque samples from a total of seventy subjects were included in the study. DNA was isolated from the collected sample and was quantified using spectrophotometer for standardizing the polymerase chain reaction. The quantity of the isolated DNA was checked in a ultraviolet-visible spectrophotomer. Statistics: One-way ANOVA and Tukey's multiple post hoc procedures were carried out. Results: The maximum score of P. gingivalis was seen in periodontitis patients having DM, whereas the least score was seen in periodontitis patients having DM with tobacco smoking habit compared to the other groups. Conclusion: P. gingivalis count is significantly reduced in periodontitis patients having DM with smoking habit; it is concluded that P. gingivalis might not be a key causative organism responsible for the periodontal destruction in case of smokers despite the DM condition. The decrease in counts may be attributed to change in the local environment like chemical (tobacco nitrosamines) and physical changes preventing the growth of P. gingivalis. PMID:27721606

  5. Porphyromonas gingivalis and related bacteria: from colonial pigmentation to the type IX secretion system and gliding motility

    PubMed Central

    Nakayama, K

    2015-01-01

    Porphyromonas gingivalis is a gram-negative, non-motile, anaerobic bacterium implicated as a major pathogen in periodontal disease. P. gingivalis grows as black-pigmented colonies on blood agar, and many bacteriologists have shown interest in this property. Studies of colonial pigmentation have revealed a number of important findings, including an association with the highly active extracellular and surface proteinases called gingipains that are found in P. gingivalis. The Por secretion system, a novel type IX secretion system (T9SS), has been implicated in gingipain secretion in studies using non-pigmented mutants. In addition, many potent virulence proteins, including the metallocarboxypeptidase CPG70, 35 kDa hemin-binding protein HBP35, peptidylarginine deiminase PAD and Lys-specific serine endopeptidase PepK, are secreted through the T9SS. These findings have not been limited to P. gingivalis but have been extended to other bacteria belonging to the phylum Bacteroidetes. Many Bacteroidetes species possess the T9SS, which is associated with gliding motility for some of these bacteria. PMID:25546073

  6. Oral mucosal lipids are antibacterial against Porphyromonas gingivalis, induce ultrastructural damage, and alter bacterial lipid and protein compositions

    PubMed Central

    Fischer, Carol L; Walters, Katherine S; Drake, David R; Dawson, Deborah V; Blanchette, Derek R; Brogden, Kim A; Wertz, Philip W

    2013-01-01

    Oral mucosal and salivary lipids exhibit potent antimicrobial activity for a variety of Gram-positive and Gram-negative bacteria; however, little is known about their spectrum of antimicrobial activity or mechanisms of action against oral bacteria. In this study, we examine the activity of two fatty acids and three sphingoid bases against Porphyromonas gingivalis, an important colonizer of the oral cavity implicated in periodontitis. Minimal inhibitory concentrations, minimal bactericidal concentrations, and kill kinetics revealed variable, but potent, activity of oral mucosal and salivary lipids against P. gingivalis, indicating that lipid structure may be an important determinant in lipid mechanisms of activity against bacteria, although specific components of bacterial membranes are also likely important. Electron micrographs showed ultrastructural damage induced by sapienic acid and phytosphingosine and confirmed disruption of the bacterial plasma membrane. This information, coupled with the association of treatment lipids with P. gingivalis lipids revealed via thin layer chromatography, suggests that the plasma membrane is a likely target of lipid antibacterial activity. Utilizing a combination of two-dimensional in-gel electrophoresis and Western blot followed by mass spectroscopy and N-terminus degradation sequencing we also show that treatment with sapienic acid induces upregulation of a set of proteins comprising a unique P. gingivalis stress response, including proteins important in fatty acid biosynthesis, metabolism and energy production, protein processing, cell adhesion and virulence. Prophylactic or therapeutic lipid treatments may be beneficial for intervention of infection by supplementing the natural immune function of endogenous lipids on mucosal surfaces. PMID:23867843

  7. Punica granatum L. (Pomegranate) Extract: In Vivo Study of Antimicrobial Activity against Porphyromonas gingivalis in Galleria mellonella Model

    PubMed Central

    Aparecida Procópio Gomes, Livia; Alves Figueiredo, Lívia Mara; Corrêa Geraldo, Barbara Maria; Isler Castro, Kelly Cristine; Ruano de Oliveira Fugisaki, Luciana; Olavo Cardoso Jorge, Antônio; Dias de Oliveira, Luciane; Campos Junqueira, Juliana

    2016-01-01

    Due to the increase of bacterial resistance, medicinal alternatives are being explored. Punica granatum L. is an effective herbal extract with broad spectrum of action and bactericidal, antifungal, anthelmintic potential and being able to modulate the immune response. The aim was to evaluate the antimicrobial activity of pomegranate glycolic extract (PGE) against the periodontal pathogen Porphyromonas gingivalis by using Galleria mellonella as in vivo model. Fifteen larvae were used per group. Injection of high concentration (200, 100, and 25 mg/mL) of PGE showed a toxic effect, leading them to death. A suspension of P. gingivalis (106 cells/mL) was inoculated in the left last proleg and PGE (12.5, 6.25, 3.1, and 2.5 mg/mL) were injected into the right proleg. The larvae were then kept at 37°C under the dark. Injection of PGE at any dose statistically improved larvae survival rates. The data were analysed (log-rank test, Mantel-Cox, P < 0.05) and showed that all concentrations of PGE (12.5, 6.25, 3.1, and 2.5 mg/mL) presented higher larval survival rates, with significant statistical difference in relation to control group (P. gingivalis). In conclusion, the PGE had antimicrobial action against P. gingivalis in vivo model using G. mellonella. PMID:27668280

  8. Punica granatum L. (Pomegranate) Extract: In Vivo Study of Antimicrobial Activity against Porphyromonas gingivalis in Galleria mellonella Model

    PubMed Central

    Aparecida Procópio Gomes, Livia; Alves Figueiredo, Lívia Mara; Corrêa Geraldo, Barbara Maria; Isler Castro, Kelly Cristine; Ruano de Oliveira Fugisaki, Luciana; Olavo Cardoso Jorge, Antônio; Dias de Oliveira, Luciane; Campos Junqueira, Juliana

    2016-01-01

    Due to the increase of bacterial resistance, medicinal alternatives are being explored. Punica granatum L. is an effective herbal extract with broad spectrum of action and bactericidal, antifungal, anthelmintic potential and being able to modulate the immune response. The aim was to evaluate the antimicrobial activity of pomegranate glycolic extract (PGE) against the periodontal pathogen Porphyromonas gingivalis by using Galleria mellonella as in vivo model. Fifteen larvae were used per group. Injection of high concentration (200, 100, and 25 mg/mL) of PGE showed a toxic effect, leading them to death. A suspension of P. gingivalis (106 cells/mL) was inoculated in the left last proleg and PGE (12.5, 6.25, 3.1, and 2.5 mg/mL) were injected into the right proleg. The larvae were then kept at 37°C under the dark. Injection of PGE at any dose statistically improved larvae survival rates. The data were analysed (log-rank test, Mantel-Cox, P < 0.05) and showed that all concentrations of PGE (12.5, 6.25, 3.1, and 2.5 mg/mL) presented higher larval survival rates, with significant statistical difference in relation to control group (P. gingivalis). In conclusion, the PGE had antimicrobial action against P. gingivalis in vivo model using G. mellonella.

  9. Porphyromonas gingivalis and Disease-Related Autoantibodies in Individuals at Increased Risk of Rheumatoid Arthritis

    PubMed Central

    Mikuls, Ted R.; Thiele, Geoffrey M.; Deane, Kevin D.; Payne, Jeffrey B.; O'Dell, James R.; Yu, Fang; Sayles, Harlan; Weisman, Michael H.; Gregersen, Peter K.; Buckner, Jane H.; Keating, Richard M.; Derber, Lezlie A.; Robinson, William H.; Holers, V. Michael; Norris, Jill M.

    2012-01-01

    Purpose To examine the relationship of Porphyromonas gingivalis (Pg) with the presence of autoantibodies in individuals at risk for rheumatoid arthritis (RA). Methods Participants included: 1) a cohort enriched with HLA-DR4 and 2) those at risk for RA by virtue of having a first-degree relative with RA. None satisfied 1987 ACR RA classification criteria. Autoantibodies measured included anti-citrullinated protein antibody (ACPA) and rheumatoid factor (RF; nephelometry, IgA, IgM, IgG). Individuals were considered autoantibody positive (n = 113) with ≥ 1 positive autoantibody with individuals further categorized as `high-risk' (n = 38; positive ACPA or ≥ 2 RF assays). Autoantibody negative individuals served as comparators (n = 171). Antibody to Pg, P. intermedia (Pi), and F. nucleatum (Fn) were measured. Associations of bacterial antibodies with group status were examined using logistic regression. Results Anti-Pg concentrations were higher in high-risk (p = 0.011) and autoantibody positive group (p = 0.010) than in the autoantibody negative group. There were no group differences in anti-Pi or anti-Fn concentrations. After multivariable adjustment, anti-Pg concentrations (but not anti-Pi or anti-Fn) were significantly associated with autoantibody positive and high-risk status (p < 0.05). Conclusion Immunity to Pg, but not Pi or Fn, is significantly associated with the presence of RA-related autoantibodies in individuals at risk for RA. These results support the hypothesis that infection with Pg may play a central role in the early loss of tolerance to self-antigens in RA pathogenesis. PMID:22736291

  10. Porphyromonas gingivalis within Placental Villous Mesenchyme and Umbilical Cord Stroma Is Associated with Adverse Pregnancy Outcome

    PubMed Central

    Vanterpool, Sizzle F.; Been, Jasper V.; Houben, Michiel L.; Nikkels, Peter G. J.; De Krijger, Ronald R.; Zimmermann, Luc J. I.; Kramer, Boris W.; Progulske-Fox, Ann; Reyes, Leticia

    2016-01-01

    Intrauterine presence of Porphyromonas gingivalis (Pg), a common oral pathobiont, is implicated in preterm birth. Our aim was to determine if the location of Pg within placental and/or umbilical cord sections was associated with a specific delivery diagnosis at preterm delivery (histologic chorioamnionitis, chorioamnionitis with funisitis, preeclampsia, and preeclampsia with HELLP-syndrome, small for gestational age). The prevalence and location of Pg within archived placental and umbilical cord specimens from preterm (25 to 32 weeks gestation) and term control cohorts were evaluated by immunofluorescent histology. Detection of Pg was performed blinded to pregnancy characteristics. Multivariate analyses were performed to evaluate independent effects of gestational age, being small for gestational age, specific preterm delivery diagnosis, antenatal steroids, and delivery mode, on the odds of having Pg in the preterm tissue. Within the preterm cohort, 49 of 97 (51%) placentas and 40 of 97 (41%) umbilical cord specimens were positive for Pg. Pg within the placenta was significantly associated with shorter gestation lengths (OR 0.63 (95%CI: 0.48–0.85; p = 0.002) per week) and delivery via caesarean section (OR 4.02 (95%CI: 1.15–14.04; p = 0.03), but not with histological chorioamnionitis or preeclampsia. However, the presence of Pg in the umbilical cord was significantly associated with preeclampsia: OR 6.73 (95%CI: 1.31–36.67; p = 0.02). In the term cohort, 2 of 35 (6%) placentas and no umbilical cord term specimens were positive for Pg. The location of Pg within the placenta was different between preterm and term groups in that Pg within the villous mesenchyme was only detected in the preterm cohort, whereas Pg associated with syncytiotrophoblasts was found in both preterm and term placentas. Taken together, our results suggest that the presence of Pg within the villous stroma or umbilical cord may be an important determinant in Pg-associated adverse pregnancy

  11. Leptomeningeal Cells Transduce Peripheral Macrophages Inflammatory Signal to Microglia in Reponse to Porphyromonas gingivalis LPS

    PubMed Central

    Zhang, Xinwen; Ni, Junjun; Yu, Weixian; Nakanishi, Hiroshi

    2013-01-01

    We report here that the leptomeningeal cells transduce inflammatory signals from peripheral macrophages to brain-resident microglia in response to Porphyromonas gingivalis (P.g.) LPS. The expression of Toll-like receptor 2 (TLR2), TLR4, TNF-α, and inducible NO synthase was mainly detected in the gingival macrophages of chronic periodontitis patients. In in vitro studies, P.g. LPS induced the secretion of TNF-α and IL-1β from THP-1 human monocyte-like cell line and RAW264.7 mouse macrophages. Surprisingly, the mean mRNA levels of TNF-α and IL-1β in leptomeningeal cells after treatment with the conditioned medium from P.g. LPS-stimulated RAW264.7 macrophages were significantly higher than those after treatment with P.g. LPS alone. Furthermore, the mean mRNA levels of TNF-α and IL-1β in microglia after treatment with the conditioned medium from P.g. LPS-stimulated leptomeningeal cells were significantly higher than those after P.g. LPS alone. These observations suggest that leptomeninges serve as an important route for transducing inflammatory signals from macrophages to microglia by secretion of proinflammatory mediators during chronic periodontitis. Moreover, propolis significantly reduced the P.g. LPS-induced TNF-α and IL-1 β production by leptomeningeal cells through inhibiting the nuclear factor-κB signaling pathway. Together with the inhibitory effect on microglial activation, propolis may be beneficial in preventing neuroinflammation during chronic periodontitis. PMID:24363500

  12. Serpine1 Mediates Porphyromonas gingivalis Induced Insulin Secretion in the Pancreatic Beta Cell Line MIN6

    PubMed Central

    Bhat, Uppoor G.; Watanabe, Keiko

    2015-01-01

    Periodontitis is an inflammatory disease resulting in destruction of gingiva and alveolar bone caused by an exuberant host immunological response to periodontal pathogens. Results from a number of epidemiological studies indicate a close association between diabetes and periodontitis. Results from cross-sectional studies indicate that subjects with periodontitis have a higher odds ratio of developing insulin resistance (IR). However, the mechanisms by which periodontitis influences the development of diabetes are not known. Results from our previous studies using an animal model of periodontitis suggest that periodontitis accelerates the onset of hyperinsulinemia and IR. In addition, LPS from a periodontal pathogen, Porphyromonas gingivalis (Pg), stimulates Serpine1 expression in the pancreatic beta cell line MIN6. Based on these observations, we hypothesized that a periodontal pathogen induces hyperinsulinemia and Serpine1 may be involved in this process. To test this hypothesis, we co-incubated Pg with the pancreatic beta cell line MIN6 and measured the effect on insulin secretion by MIN6 cells. We further determined the involvement of Serpine1 in insulin secretion by downregulating Serpine1 expression. Our results indicated that Pg stimulated insulin secretion by approximately 3.0 fold under normoglycemic conditions. In a hyperglycemic state, Pg increased insulin secretion by 1.5 fold. Pg significantly upregulated expression of the Serpine1 gene and this was associated with increased secretion of insulin by MIN6 cells. However, cells with downregulated Serpine1 expression were resistant to Pg stimulated insulin secretion under normoglycemic conditions. We conclude that the periodontal pathogen, Pg, induced insulin secretion by MIN6 cells and this induction was, in part, Serpine1 dependent. Thus, Serpine1 may play a pivotal role in insulin secretion during the accelerated development of hyperinsulinemia and the resulting IR in the setting of periodontitis. PMID

  13. Antibacterial Effect of an Herbal Product Persica on Porphyromonas Gingivalis and Aggregatibacter Actinomycetemcomitans: An In-Vitro Study

    PubMed Central

    Jelvehgaran Esfahani, Zahra; Kadkhoda, Zeinab; Eshraghi, Seyed Saeed; Salehi Surmaghi, Mohammad Hossein

    2014-01-01

    Objective: The plant Salvadora persica is used for oral hygiene in many parts of the world. It has been suggested that it has antibacterial properties, in addition to its ability to mechanically remove plaques. The aim of this study was to assess the antimicrobial activity of the herbal product Persica containing Salvadora persica against periodontopathogens Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans in vitro. Materials and Methods: Fifty patients with moderate and severe periodontitis were recruited. Using paper points, subgingival plaque samples were taken from pockets with attachment loss ≥ 3mm. The samples were subjected to microbial culture to yield P. gingivalis and A. actinomycetemcomitans. The ditch plate method was used for antimicrobial susceptibility testing of the bacteria to Persica compared to chlorhexidine and distilled water. The growth inhibition zones of microorganisms around the ditches were measured in millimeters. The data were analyzed using SPSS 16. Freidman test and Wilcoxon signed ranks test with Bonferroni adjustment were used for analysis of variance with 5% significance level. P<0.05 for main comparisons and P< 0.017 for multiple comparisons were considered statistically significant. Results: P. gingivalis was sensitive to chlorhexidine and persica. There was a significant difference (P=0.001) between antimicrobial activity of chlorhexidine (mean 28.733mm, SD 5.216) and Persica (mean 16.333mm, SD 5.259) compared to water against P. gingivalis. There was a significant difference (P< 0.001) between the antimicrobial activity of chlorhexidine (24.045mm, SD 3.897) and Persica (0.545mm, SD 2.558) with respect to A. actinomycetemcomitans. There was no significant difference (P=0.317) between the antimicrobial activity of Persica and water against A. actinomycetemcomitans. Conclusion: The herbal product Persica had significant antimicrobial activity against P. gingivalis and negligible antimicrobial activity against A

  14. Involvement of PG2212 Zinc Finger Protein in the Regulation of Oxidative Stress Resistance in Porphyromonas gingivalis W83

    PubMed Central

    Dou, Yuetan; Aruni, Wilson; Luo, Tianlong; Roy, Francis; Wang, Charles

    2014-01-01

    The adaptation of Porphyromonas gingivalis to H2O2-induced stress while inducible is modulated by an unknown OxyR-independent mechanism. Previously, we reported that the PG_2212 gene was highly upregulated in P. gingivalis under conditions of prolonged oxidative stress. Because this gene may have regulatory properties, its function in response to H2O2 was further characterized. PG2212, annotated as a hypothetical protein of unknown function, is a 10.3-kDa protein with a cysteine 2-histidine 2 (Cys2His2) zinc finger domain. The isogenic mutant P. gingivalis FLL366 (ΔPG_2212) showed increased sensitivity to H2O2 and decreased gingipain activity compared to the parent strain. Transcriptome analysis of P. gingivalis FLL366 revealed that approximately 11% of the genome displayed altered expression (130 downregulated genes and 120 upregulated genes) in response to prolonged H2O2-induced stress. The majority of the modulated genes were hypothetical or of unknown function, although some are known to participate in oxidative stress resistance. The promoter region of several of the most highly modulated genes contained conserved motifs. In electrophoretic mobility shift assays, the purified rPG2212 protein did not bind its own promoter region but bound a similar region in several of the genes modulated in the PG_2212-deficient mutant. A metabolome analysis revealed that PG2212 can regulate a number of genes coding for proteins involved in metabolic pathways critical for its survival under the conditions of oxidative stress. Collectively, our data suggest that PG2212 is a transcriptional regulator that plays an important role in oxidative stress resistance and virulence regulation in P. gingivalis. PMID:25225267

  15. The hemagglutinin gene A (hagA) of Porphyromonas gingivalis 381 contains four large, contiguous, direct repeats.

    PubMed Central

    Han, N; Whitlock, J; Progulske-Fox, A

    1996-01-01

    Porphyromonas gingivalis is a gram-negative anaerobic bacterial species strongly associated with adult periodontitis. One of its distinguishing characteristics and putative virulence properties is the ability to agglutinate erythrocytes. We have previously reported the cloning of multiple hemagglutinin genes from P. gingivalis 381. Subsequent sequencing of clone ST 2 revealed that the cloned fragment contained only an internal portion of the gene which lacked both start and stop codons. We here report the cloning and sequencing of the entire gene, designated hagA, as well as its relationship to other genes of this species. By use of inverse PCR technology and the construction of several additional genomic libraries, the complete open reading frame of hagA was found to be 7,887 bp in length, encoding a protein of 2,628 amino acids with a molecular mass of 283.3 kDa, which is among the largest genes ever cloned from a prokaryote to date. Within its open reading frame, four large, contiguous, direct repeats (varying from 1,318 to 1,368 bp) were identified. The repeat unit (HArep), which is assumed to contain the hemagglutinin domain, is also present in other recently reported protease and hemagglutinin genes in P. gingivalis. Thus, we propose that hagA and the other genes which share the HArep sequence form a multigene family with hagA as a central member. PMID:8926061

  16. Inhibitory effect of gels loaded with a low concentration of antibiotics against biofilm formation by Enterococcus faecalis and Porphyromonas gingivalis.

    PubMed

    A Algarni, Amnah; H Yassen, Ghaeth; L Gregory, Richard

    2015-09-01

    We explored longitudinally the inhibitory effect of gels loaded with 1 mg/mL modified triple antibiotic paste (MTAP) or double antibiotic paste (DAP) against biofilm formation by Enterococcus faecalis and Porphyromonas gingivalis. Methylcellulose-based antibiotic gels of MTAP (ciprofloxacin, metronidazole and clindamycin) and DAP (ciprofloxacin and metronidazole) were prepared at a concentration of 1 mg/mL. Individually cultured E. faecalis and P. gingivalis bacterial suspensions were treated with MTAP, DAP, or placebo (vehicle only) gels at different dilutions and allowed to grow in 96-well microtiter plates. Untreated bacterial suspensions served as a negative control. Crystal violet assays were used to evaluate biofilm formation after 48 h. The ability of the gels to inhibit biofilm formation was determined immediately, and at 1 month and 3 months after the gels had been prepared. Data were analyzed using a mixed-model ANOVA. The MTAP and DAP gels significantly reduced biofilm formation by both bacterial species at all time points, regardless of the tested dilution. No-significant differences in biofilm-inhibitory effects between MTAP and DAP gels were observed at the majority of the tested dilutions through various time points. Gels loaded with 1 mg/mL MTAP and DAP demonstrated a significant antibiofilm effect against E.faecalis and P. gingivalis. PMID:26369485

  17. Inhibitory Effect of Dodonaea viscosa var. angustifolia on the Virulence Properties of the Oral Pathogens Streptococcus mutans and Porphyromonas gingivalis

    PubMed Central

    Owotade, Foluso John

    2013-01-01

    Aim. This study investigated the effect of Dodonaea viscosa var. angustifolia (DVA) on the virulence properties of cariogenic Streptococcus mutans and Porphyromonas gingivalis implicated in periodontal diseases. Methods. S. mutans was cultured in tryptone broth containing a crude leaf extract of DVA for 16 hours, and the pH was measured after 10, 12, 14, and 16 h. Biofilms of S. mutans were grown on glass slides for 48 hours and exposed to plant extract for 30 minutes; the adherent cells were reincubated and the pH was measured at various time intervals. Minimum bactericidal concentration of the extracts against the four periodontal pathogens was determined. The effect of the subinhibitory concentration of plant extract on the production of proteinases by P. gingivalis was also evaluated. Results. DVA had no effect on acid production by S. mutans biofilms; however, it significantly inhibited acid production in planktonic cells. Periodontal pathogens were completely eliminated at low concentrations ranging from 0.09 to 0.02 mg/mL of crude plant extracts. At subinhibitory concentrations, DVA significantly reduced Arg-gingipain (24%) and Lys-gingipain (53%) production by P. gingivalis (P ≤ 0.01). Conclusions. These results suggest that DVA has the potential to be used to control oral infections including dental caries and periodontal diseases. PMID:24223061

  18. The survival rate of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Bacteroides forsythus following 4 randomized treatment modalities.

    PubMed

    Shiloah, J; Patters, M R; Dean, J W; Bland, P; Toledo, G

    1997-08-01

    The overall goal of this clinical study was to determine the short-term anti-infective effects of four randomized treatment modalities on Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), and Bacteroides forsythus (Bf) and determine the effects of bacterial survival on treatment outcomes in patients with adult periodontitis. Twelve adult patients requiring therapy for moderate periodontitis were selected for this study. All patients had at least one tooth in each quadrant that had an inflamed pocket of probing depth > or =5 mm with probing attachment loss that harbored at least one of the following three periodontal pathogens: Aa, Pg, or Bf. The number of target organisms per site was determined pre-operatively, at 1 week, and 1 month and 3 months postoperatively utilizing DNA probes. One quadrant in each patient was randomly assigned to each one of the following four treatment groups: 1) scaling and root planing (SRP group); 2) pocket reduction through osseous surgery and apically-positioned flap (OS group); 3) modified Widman flap (MWF group); and 4) modified Widman flap and topical application of saturated citric acid at pH 1 for 3 minutes (CA group). The 4 treatment modalities were performed in one appointment. No postoperative antibiotics were used. Patients were instructed to supplement their daily oral hygiene with chlorohexidine oral rinse during the study. The results of this investigation indicated that: 1) none of the treatment modalities was effective in eliminating the target species; 2) the incidence of infected sites for all groups was 100% preoperatively; 62.5%, 33.3%, and 31.3% at 1 week, and 1 and 3 months postoperatively, respectively; 3) these infected sites lost 1.1 +/- 0.4 mm of probing attachment compared to gain of 0.0 +/- 0.3 mm for uninfected sites; 4) the infected sites had higher plaque and bleeding on probing 0.9 +/- 0.3, 73 +/- 12%, respectively, compared to 0.3 +/- 0.1 and 30 +/- 8% for the uninfected sites

  19. Porphyromonas gingivalis Lipopolysaccharide Upregulates Insulin Secretion From Pancreatic β Cell Line MIN6

    PubMed Central

    Bhat, Uppoor G.; Ilievski, Vladimir; Unterman, Terry G.; Watanabe, Keiko

    2015-01-01

    Background A close association between periodontitis and diabetes has been demonstrated in human cross-sectional studies, but an exact relationship between periodontitis and prediabetes has not been established. Previous studies using animal model systems consistently have shown that hyperinsulinemia occurs in animals with periodontitis compared to animals with healthy periodontium (while maintaining normoglycemia). Because bacterial lipopolysaccharide (LPS) plays an important role in the pathogenesis of periodontitis, we hypothesized that LPS may stimulate insulin secretion through a direct effect on β cell function. To test this hypothesis, pancreatic β cell line MIN6 cells were used to determine the effect of Porphyromonas gingivalis (Pg) LPS on insulin secretion. Furthermore, expression of genes altered by Pg LPS in innate immunity and insulin-signaling pathways was determined. Methods MIN6 cells were grown in medium with glucose concentration of normoglycemia (5.5 mM). Pg LPS was added to each well at final concentrations of 50, 200, and 500 ng/mL. Insulin secretion was measured using enzyme-linked immunosorbent assay. Gene expression levels altered by Pg LPS were determined by polymerase chain reaction (PCR) array for mouse innate and adaptive immunity response and mouse insulin-signaling pathways, and results were confirmed for specific genes of interest by quantitative PCR. Results Pg LPS stimulated insulin secretion in the normoglycemic condition by ≈1.5- to 3.0-fold depending on the concentration of LPS. Pg LPS treatment altered the expression of several genes involved in innate and adaptive immune response and insulin-signaling pathway. Pg LPS upregulated the expression of the immune response–related genes cluster of differentiation 8a (Cd8a), Cd14, and intercellular adhesion molecule-1 (Icam1) by about two-fold. LPS also increased the expression of two insulin signaling–related genes, glucose-6-phosphatase catalytic subunit (G6pc) and insulin

  20. Increased levels of Porphyromonas gingivalis are associated with ischemic and hemorrhagic cerebrovascular disease in humans: an in vivo study

    PubMed Central

    GHIZONI, Janaina Salomon; TAVEIRA, Luís Antônio de Assis; GARLET, Gustavo Pompermaier; GHIZONI, Marcos Flávio; PEREIRA, Jefferson Ricardo; DIONÍSIO, Thiago José; BROZOSKI, Daniel Thomas; SANTOS, Carlos Ferreira; SANT'ANA, Adriana Campos Passanezi

    2012-01-01

    Objective: This study investigated the role of periodontal disease in the development of stroke or cerebral infarction in patients by evaluating the clinical periodontal conditions and the subgingival levels of periodontopathogens. Material and Methods: Twenty patients with ischemic (I-CVA) or hemorrhagic (H-CVA) cerebrovascular episodes (test group) and 60 systemically healthy patients (control group) were evaluated for: probing depth, clinical attachment level, bleeding on probing and plaque index. Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans were both identified and quantified in subgingival plaque samples by conventional and real-time PCR, respectively. Results: The test group showed a significant increase in each of the following parameters: pocket depth, clinical attachment loss, bleeding on probing, plaque index and number of missing teeth when compared to control values (p<0.05, unpaired t-test). Likewise, the test group had increased numbers of sites that were contaminated with P. gingivalis (60%x10%; p<0.001; chi-squared test) and displayed greater prevalence of periodontal disease, with an odds ratio of 48.06 (95% CI: 5.96-387.72; p<0.001). Notably, a positive correlation between probing depth and the levels of P. gingivalis in ischemic stroke was found (r=0.60; p=0.03; Spearman's rank correlation coefficient test). A. actinomycetemcomitans DNA was not detected in any of the groups by conventional or real-time PCR. Conclusions: Stroke patients had deeper pockets, more severe attachment loss, increased bleeding on probing, increased plaque indexes, and in their pockets harbored increased levels of P. gingivalis. These findings suggest that periodontal disease is a risk factor for the development of cerebral hemorrhage or infarction. Early treatment of periodontitis may counteract the development of cerebrovascular episodes. PMID:22437687

  1. Initial serum antibody titer to Porphyromonas gingivalis influences development of antibody avidity and success of therapy for chronic periodontitis.

    PubMed Central

    Mooney, J; Adonogianaki, E; Riggio, M P; Takahashi, K; Haerian, A; Kinane, D F

    1995-01-01

    This study assessed the effect of periodontal therapy on specific serum antibody concentration, expressed as titer, and antibody binding strength, expressed as relative avidity. The immune responses to Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans were investigated. Antibody titer was assayed by enzyme-linked immunosorbent assay (ELISA) and relative avidity was measured by thiocyanate elution in 17 adult periodontitis patients before and after therapy. Immunoglobulin G (IgG) avidities (expressed as thiocyanate molarity) to P. gingivalis increased from 1.01 to 1.38 M (P = 0.05) and IgA titers (expressed as ELISA units [EU]) increased from 89 to 237 EU (P = 0.012). There were no significant changes in avidity to A. actinomycetemcomitans, but the titer of all three immunoglobulin classes increased significantly (P < 0.03). More specifically, when patients were divided into subgroups which had originally been either IgG seropositive (i.e., having an IgG titer to this organism > 2 times the control median) or seronegative for P. gingivalis, only patients who were initially seropositive showed a significant increase in antibody avidity (P = 0.026; mean difference, 0.69 M). Patients who were originally seropositive in terms of IgG and IgA titer to P. gingivalis had demonstrably better treatment outcomes in terms of a reduced number of deep pockets and sites which bled on probing (P < 0.05). These findings suggest that periodontal therapy affects the magnitude and quality of the humoral immune response to suspected periodontopathogens, that this effect is dependent on initial serostatus, and that initial serostatus may have a bearing on treatment outcome. PMID:7642270

  2. Anchoring and length regulation of Porphyromonas gingivalis Mfa1 fimbriae by the downstream gene product Mfa2

    PubMed Central

    Hasegawa, Yoshiaki; Iwami, Jun; Sato, Keiko; Park, Yoonsuk; Nishikawa, Kiyoshi; Atsumi, Tatsuo; Moriguchi, Keiichi; Murakami, Yukitaka; Lamont, Richard J.; Nakamura, Hiroshi; Ohno, Norikazu; Yoshimura, Fuminobu

    2009-01-01

    Porphyromonas gingivalis, a causative agent of periodontitis, has at least two types of thin, single-stranded fimbriae, termed FimA and Mfa1 (according to the names of major subunits), which can be discriminated by filament length and by the size of their major fimbrilin subunits. FimA fimbriae are long filaments that are easily detached from cells, whereas Mfa1 fimbriae are short filaments that are tightly bound to cells. However, a P. gingivalis ATCC 33277-derived mutant deficient in mfa2, a gene downstream of mfa1, produced long filaments (10 times longer than those of the parent), easily detached from the cell surface, similar to FimA fimbriae. Longer Mfa1 fimbriae contributed to stronger autoaggregation of bacterial cells. Complementation of the mutant with the wild-type mfa2 allele in trans restored the parental phenotype. Mfa2 is present in the outer membrane of P. gingivalis, but does not co-purify with the Mfa1 fimbriae. However, co-immunoprecipitation demonstrated that Mfa2 and Mfa1 are associated with each other in whole P. gingivalis cells. Furthermore, immunogold microscopy, including double labelling, confirmed that Mfa2 was located on the cell surface and likely associated with Mfa1 fimbriae. Mfa2 may therefore play a role as an anchor for the Mfa1 fimbriae and also as a regulator of Mfa1 filament length. Two additional downstream genes (pgn0289 and pgn0290) are co-transcribed with mfa1 (pgn0287) and mfa2 (pgn0288), and proteins derived from pgn0289, pgn0290 and pgn0291 appear to be accessory fimbrial components. PMID:19589838

  3. Antibacterial effect of copper-bearing titanium alloy (Ti-Cu) against Streptococcus mutans and Porphyromonas gingivalis

    NASA Astrophysics Data System (ADS)

    Liu, Rui; Memarzadeh, Kaveh; Chang, Bei; Zhang, Yumei; Ma, Zheng; Allaker, Robert P.; Ren, Ling; Yang, Ke

    2016-07-01

    Formation of bacterial biofilms on dental implant material surfaces (titanium) may lead to the development of peri-implant diseases influencing the long term success of dental implants. In this study, a novel Cu-bearing titanium alloy (Ti-Cu) was designed and fabricated in order to efficiently kill bacteria and discourage formation of biofilms, and then inhibit bacterial infection and prevent implant failure, in comparison with pure Ti. Results from biofilm based gene expression studies, biofilm growth observation, bacterial viability measurements and morphological examination of bacteria, revealed antimicrobial/antibiofilm activities of Ti-Cu alloy against the oral specific bacterial species, Streptococcus mutans and Porphyromonas gingivalis. Proliferation and adhesion assays with mesenchymal stem cells, and measurement of the mean daily amount of Cu ion release demonstrated Ti-Cu alloy to be biocompatible. In conclusion, Ti-Cu alloy is a promising dental implant material with antimicrobial/antibiofilm activities and acceptable biocompatibility.

  4. Antibacterial effect of copper-bearing titanium alloy (Ti-Cu) against Streptococcus mutans and Porphyromonas gingivalis

    PubMed Central

    Liu, Rui; Memarzadeh, Kaveh; Chang, Bei; Zhang, Yumei; Ma, Zheng; Allaker, Robert P.; Ren, Ling; Yang, Ke

    2016-01-01

    Formation of bacterial biofilms on dental implant material surfaces (titanium) may lead to the development of peri-implant diseases influencing the long term success of dental implants. In this study, a novel Cu-bearing titanium alloy (Ti-Cu) was designed and fabricated in order to efficiently kill bacteria and discourage formation of biofilms, and then inhibit bacterial infection and prevent implant failure, in comparison with pure Ti. Results from biofilm based gene expression studies, biofilm growth observation, bacterial viability measurements and morphological examination of bacteria, revealed antimicrobial/antibiofilm activities of Ti-Cu alloy against the oral specific bacterial species, Streptococcus mutans and Porphyromonas gingivalis. Proliferation and adhesion assays with mesenchymal stem cells, and measurement of the mean daily amount of Cu ion release demonstrated Ti-Cu alloy to be biocompatible. In conclusion, Ti-Cu alloy is a promising dental implant material with antimicrobial/antibiofilm activities and acceptable biocompatibility. PMID:27457788

  5. Antibacterial effect of copper-bearing titanium alloy (Ti-Cu) against Streptococcus mutans and Porphyromonas gingivalis.

    PubMed

    Liu, Rui; Memarzadeh, Kaveh; Chang, Bei; Zhang, Yumei; Ma, Zheng; Allaker, Robert P; Ren, Ling; Yang, Ke

    2016-01-01

    Formation of bacterial biofilms on dental implant material surfaces (titanium) may lead to the development of peri-implant diseases influencing the long term success of dental implants. In this study, a novel Cu-bearing titanium alloy (Ti-Cu) was designed and fabricated in order to efficiently kill bacteria and discourage formation of biofilms, and then inhibit bacterial infection and prevent implant failure, in comparison with pure Ti. Results from biofilm based gene expression studies, biofilm growth observation, bacterial viability measurements and morphological examination of bacteria, revealed antimicrobial/antibiofilm activities of Ti-Cu alloy against the oral specific bacterial species, Streptococcus mutans and Porphyromonas gingivalis. Proliferation and adhesion assays with mesenchymal stem cells, and measurement of the mean daily amount of Cu ion release demonstrated Ti-Cu alloy to be biocompatible. In conclusion, Ti-Cu alloy is a promising dental implant material with antimicrobial/antibiofilm activities and acceptable biocompatibility. PMID:27457788

  6. An Overview of the Carbonic Anhydrases from Two Pathogens of the Oral Cavity: Streptococcus mutans and Porphyromonas gingivalis.

    PubMed

    Capasso, Clemente; Supuran, Claudiu T

    2016-01-01

    Among the crowd of bacteria provoking disease of the oral cavity during the weakened of immune system, Streptococcus mutans and Porphyromonas gingivalis are the main microorganisms implicated in caries formation and periodontitis, respectively. The life cycle of the pathogens, such as protozoa, fungi and bacteria, is influenced by a superfamily of enzymes, called carbonic anhydrases (CAs, EC 4.2.1.1). These metalloenzymes, being crucial for the survival of the pathogen, have been considered as novel anti-infective targets. In fact, bicarbonate and protons, produced by the CA catalyzed carbon dioxide as substrate, are two fundamental ions implicated in the pH regulation, biosynthetic reactions, and adaptation of the pathogen to the host or in the possibility of the pathogen to avoid the host immune system. Bacteria genome encodes for the α-, β- and γ-CAs. Recently, our groups using the recombinant DNA technology prepared and characterized the CAs belonging to the β- and γ-classes encoded by the genome of the two oral cavity pathogens S. mutans and P. gingivalis. An extensive inhibition study was carried out using typical anion/sulfonamide inhibitors of these classes of CAs. We discovered numerous inhibitors, which had in vitro an effective inhibitory activity against the bacterial CAs considered, here, as alternative anti-infective targets.

  7. Bifidobacteria inhibit the growth of Porphyromonas gingivalis but not of Streptococcus mutans in an in vitro biofilm model.

    PubMed

    Jäsberg, Heli; Söderling, Eva; Endo, Akihito; Beighton, David; Haukioja, Anna

    2016-06-01

    There is growing interest in the use of probiotic bifidobacteria for enhancement of the therapy, and in the prevention, of oral microbial diseases. However, the results of clinical studies assessing the effects of bifidobacteria on the oral microbiota are controversial, and the mechanisms of actions of probiotics in the oral cavity remain largely unknown. In addition, very little is known about the role of commensal bifidobacteria in oral health. Our aim was to study the integration of the probiotic Bifidobacterium animalis subsp. lactis Bb12 and of oral Bifidobacterium dentium and Bifidobacterium longum isolates in supragingival and subgingival biofilm models and their effects on other bacteria in biofilms in vitro using two different in vitro biofilms and agar-overlay assays. All bifidobacteria integrated well into the subgingival biofilms composed of Porphyromonas gingivalis, Actinomyces naeslundii, and Fusobacterium nucleatum and decreased significantly only the number of P. gingivalis in the biofilms. The integration of bifidobacteria into the supragingival biofilms containing Streptococcus mutans and A. naeslundii was less efficient, and bifidobacteria did not affect the number of S. mutans in biofilms. Therefore, our results suggest that bifidobacteria may have a positive effect on subgingival biofilm and thereby potential in enhancing gingival health; however, their effect on supragingival biofilm may be limited. PMID:27061393

  8. Heterogeneous Porphyromonas gingivalis LPS modulates immuno-inflammatory response, antioxidant defense and cytoskeletal dynamics in human gingival fibroblasts

    PubMed Central

    Herath, Thanuja D. K.; Darveau, Richard P.; Seneviratne, Chaminda J.; Wang, Cun-Yu; Wang, Yu; Jin, Lijian

    2016-01-01

    Periodontal (gum) disease is a highly prevalent infection and inflammation accounting for the majority of tooth loss in adult population worldwide. Porphyromonas gingivalis is a keystone periodontal pathogen and its lipopolysaccharide (PgLPS) acts as a major virulence attribute to the disease. Herein, we deciphered the overall host response of human gingival fibroblasts (HGFs) to two featured isoforms of tetra-acylated PgLPS1435/1449 and penta-acylated PgLPS1690 with reference to E. coli LPS through quantitative proteomics. This study unraveled differentially expressed novel biomarkers of immuno-inflammatory response, antioxidant defense and cytoskeletal dynamics in HGFs. PgLPS1690 greatly upregulated inflammatory proteins (e.g. cyclophilin, inducible nitric oxide synthase, annexins, galectin, cathepsins and heat shock proteins), whereas the anti-inflammatory proteins (e.g. Annexin A2 and Annexin A6) were significantly upregulated by PgLPS1435/1449. Interestingly, the antioxidants proteins such as mitochondrial manganese-containing superoxide dismutase and peroxiredoxin 5 were only upregulated by PgLPS1690. The cytoskeletal rearrangement-related proteins like myosin were differentially regulated by these PgLPS isoforms. The present study gives new insight into the biological properties of P. gingivalis LPS lipid A moiety that could critically modulate immuno-inflammatory response, antioxidant defense and cytoskeletal dynamics in HGFs, and thereby enhances our understanding of periodontal pathogenesis. PMID:27538450

  9. Heightened immune response to autocitrullinated Porphyromonas gingivalis peptidylarginine deiminase: a potential mechanism for breaching immunologic tolerance in rheumatoid arthritis

    PubMed Central

    Quirke, Anne-Marie; Lugli, Elena Birgitta; Wegner, Natalia; Hamilton, Bart C; Charles, Peter; Chowdhury, Muslima; Ytterberg, A Jimmy; Zubarev, Roman A; Potempa, Jan; Culshaw, Shauna; Guo, Yonghua; Fisher, Benjamin A; Thiele, Geoffrey; Mikuls, Ted R; Venables, Patrick JW

    2014-01-01

    Background Rheumatoid arthritis (RA) is characterised by autoimmunity to citrullinated proteins, and there is increasing epidemiologic evidence linking Porphyromonas gingivalis to RA. P gingivalis is apparently unique among periodontal pathogens in possessing a citrullinating enzyme, peptidylarginine deiminase (PPAD) with the potential to generate antigens driving the autoimmune response. Objectives To examine the immune response to PPAD in patients with RA, individuals with periodontitis (PD) and controls (without arthritis), confirm PPAD autocitrullination and identify the modified arginine residues. Methods PPAD and an inactivated mutant (C351A) were cloned and expressed and autocitrullination of both examined by immunoblotting and mass spectrometry. ELISAs using PPAD, C351A and another P gingivalis protein arginine gingipain (RgpB) were developed and antibody reactivities examined in patients with RA (n=80), individuals with PD (n=44) and controls (n=82). Results Recombinant PPAD was a potent citrullinating enzyme. Antibodies to PPAD, but not to Rgp, were elevated in the RA sera (median 122 U/ml) compared with controls (median 70 U/ml; p<0.05) and PD (median 60 U/ml; p<0.01). Specificity of the anti-peptidyl citrullinated PPAD response was confirmed by the reaction of RA sera with multiple epitopes tested with synthetic citrullinated peptides spanning the PPAD molecule. The elevated antibody response to PPAD was abolished in RA sera if the C351A mutant was used on ELISA. Conclusions The peptidyl citrulline-specific immune response to PPAD supports the hypothesis that, as a bacterial protein, it might break tolerance in RA, and could be a target for therapy. PMID:23463691

  10. Fur homolog regulates Porphyromonas gingivalis virulence under low-iron/heme conditions through a complex regulatory network.

    PubMed

    Ciuraszkiewicz, J; Smiga, M; Mackiewicz, P; Gmiterek, A; Bielecki, M; Olczak, M; Olczak, T

    2014-12-01

    Porphyromonas gingivalis is a key pathogen responsible for initiation and progression of chronic periodontitis. Little is known about the regulatory mechanisms of iron and heme uptake that allow P. gingivalis to express virulence factors and survive in the hostile environment of the oral cavity, so we initiated characterization of a P. gingivalis Fur homolog (PgFur). Many Fur paralogs found in microbial genomes, including Bacteroidetes, confirm that Fur proteins have a tendency to be subjected to a sub- or even neofunctionalization process. PgFur revealed extremely high sequence divergence, which could be associated with its functional dissimilarity in comparison with other Fur homologs. A fur mutant strain constructed by insertional inactivation exhibited retarded growth during the early growth phase and a significantly lower tendency to form a homotypic biofilm on abiotic surfaces. The mutant also showed significantly weaker adherence and invasion to epithelial cells and macrophages. Transcripts of many differentially regulated genes identified in the fur mutant strain were annotated as hypothetical proteins, suggesting that PgFur can play a novel role in the regulation of gene expression. Inactivation of the fur gene resulted in decreased hmuY gene expression, increased expression of other hmu components and changes in the expression of genes encoding hemagglutinins and proteases (mainly gingipains), HtrA, some extracytoplasmic sigma factors and two-component systems. Our data suggest that PgFur can influence in vivo growth and virulence, at least in part by affecting iron/heme acquisition, allowing efficient infection through a complex regulatory network.

  11. Blocking Pro-Inflammatory Cytokine Release Modulates Peripheral Blood Mononuclear Cell Response to Porphyromonas Gingivalis

    PubMed Central

    Berker, Ezel; Kantarci, Alpdogan; Hasturk, Hatice; Van Dyke, Thomas E.

    2013-01-01

    Background Chronic periodontitis is an inflammatory disease in which cytokines play a major role in the progression of disease. Anti-inflammatory cytokines (IL-4 and IL-10) were reported to be absent or reduced in diseased periodontal tissues, suggesting an imbalance between the pro- and anti-inflammatory mediators. We have tested the hypothesis that there is cellular cross-talk mediated by pro- and anti-inflammatory cytokines and that blocking pro-inflammatory cytokine (TNF-α and IL-1) production will enhance anti-inflammatory cytokine (IL-4 and IL-10) production from peripheral blood mononuclear cells (PBMC) in response to P. gingivalis. Methods PBMC were isolated from individuals diagnosed with chronic periodontitis or healthy individuals and cultured for 24 hours. Concanavalin-A (ConA) was used as an activator of lymphocyte function. Live and heat-killed P .gingivalis or lipopolysaccharide from P. gingivalis was used as the bacterial stimulants. TNF-α and IL-1 production was neutralized by specific antibodies against TNF-α and IL-1α or β. Culture supernatants were evaluated by ELISA for TNF-α, IL-1β, IL-4, and IL-10 production. Results Live P. gingivalis did not result in any significant IL-10 or IL-4 release while heat-killed P. gingivalis led to a significant increase in IL-10 levels compared to unstimulated or live P. gingivalis-stimulated cells from both healthy and periodontitis individuals. Overall, PBMC from patients with chronic periodontitis produced significantly lower IL-10 in response to ConA and P. gingivalis suggesting chronic suppression of the anti-inflammatory cytokine production. Blocking the pro-inflammatory cytokine response did not result in any substantial change in IL-10 or IL-4 response to live P. gingivalis. Blocking the pro-inflammatory cytokine response restored IL-10 production by cells from chronic periodontitis in response to P. gingivalis LPS. Conclusion These findings suggest that PBMC from patients with chronic

  12. Coinvasion of dentinal tubules by Porphyromonas gingivalis and Streptococcus gordonii depends upon binding specificity of streptococcal antigen I/II adhesin.

    PubMed

    Love, R M; McMillan, M D; Park, Y; Jenkinson, H F

    2000-03-01

    Cell wall-anchored polypeptides of the antigen I/II family are produced by many species of oral streptococci. These proteins mediate adhesion of streptococci to salivary glycoproteins and to other oral microorganisms and promote binding of cells to collagen type I and invasion of dentinal tubules. Since infections of the root canal system have a mixed anaerobic bacterial etiology, we investigated the hypothesis that coadhesion of anaerobic bacteria with streptococci may facilitate invasive endodontic disease. Porphyromonas gingivalis ATCC 33277 cells were able to invade dentinal tubules when cocultured with Streptococcus gordonii DL1 (Challis) but not when cocultured with Streptococcus mutans NG8. An isogenic noninvasive mutant of S. gordonii, with production of SspA and SspB (antigen I/II family) polypeptides abrogated, was deficient in binding to collagen and had a 40% reduced ability to support adhesion of P. gingivalis. Heterologous expression of the S. mutans SpaP (antigen I/II) protein in this mutant restored collagen binding and tubule invasion but not adhesion to P. gingivalis or the ability to promote P. gingivalis coinvasion of dentin. An isogenic afimbrial mutant of P. gingivalis had 50% reduced binding to S. gordonii cells but was unaffected in the ability to coinvade dentinal tubules with S. gordonii wild-type cells. Expression of the S. gordonii SspA or SspB polypeptide on the surface of Lactococcus lactis cells endowed these bacteria with the abilities to bind P. gingivalis, penetrate dentinal tubules, and promote P. gingivalis coinvasion of dentin. The results demonstrate that collagen-binding and P. gingivalis-binding properties of antigen I/II polypeptides are discrete functions. Specificity of antigen I/II polypeptide recognition accounts for the ability of P. gingivalis to coinvade dentinal tubules with S. gordonii but not with S. mutans. This provides evidence that the specificity of interbacterial coadhesion may influence directly the etiology

  13. Effects of the antimicrobial peptide cathelicidin (LL-37) on immortalized gingival fibroblasts infected with Porphyromonas gingivalis and irradiated with 625-nm LED light.

    PubMed

    Kim, JiSun; Kim, SangWoo; Lim, WonBong; Choi, HongRan; Kim, OkJoon

    2015-11-01

    Porphyromonas gingivalis causes chronic inflammatory diseases (periodontal diseases) that destroy the periodontal ligament and alveolar bone. Antimicrobial peptides are crucial components of the host defense response required to maintain cellular homeostasis during microbial invasion. Because light-emitting diode (LED) irradiation influences the host defense response against bacterial infections, we investigated its effect on immortalized gingival fibroblasts (IGFs) infected with P. gingivalis. IGFs were incubated with P. gingivalis following LED irradiation at 425, 525, and 625 nm. The dark 1 group comprised noninfected, nonirradiated IGFs, and the dark 2 group comprised nonirradiated IGFs infected with P. gingivalis. These groups served as controls. Infected cells and controls were assayed for reactive oxygen species (ROS) and were subjected to RT-PCR and Western blotting analyses to determine the levels of expression of antimicrobial peptides. LED irradiation enhanced the bactericidal effects of the antimicrobial peptide LL-37 in cells infected with P. gingivalis. Irradiation at 625 nm decreased inflammatory responses involving the release of prostaglandin E2 induced by ROS in P. gingivalis-infected IGFs. LED irradiation at 625 nm induces an anti-inflammatory response that elicits the production of antimicrobial peptides, providing an efficacious method of treatment for periodontal diseases.

  14. Detection of Porphyromonas gingivalis fimA Type I Genotype in Gingivitis by Real-Time PCR–A Pilot Study

    PubMed Central

    Krishnan, Mahalakshmi; Chandrasekaran, S.C.

    2016-01-01

    Introduction Published literature till date reveals a high prevalence of Porphyromonas gingivalis fimA type I genotype among healthy subjects. Quite a few studies have reported its prevalence also in periodontitis patients. Nevertheless incidence of this genotype in gingivitis is lacking in adult population. Aim The present study was chosen to detect P. gingivalis fimA type I genotype among chronic gingivitis patients. Materials and Methods A total of 46 subgingival plaque samples collected from chronic marginal gingivitis (n=23) and chronic periodontitis subjects (control group) (n=23) were subjected to Real-Time Polymerase Chain Reaction to detect the P. gingivalis fimA type I gene. Statistical analysis was performed using chi-square test. Results Prevalence of P. gingivalis fimA type I gene among chronic periodontitis and chronic gingivitis patients were 8.7% and 30.4% respectively. P. gingivalis fimA type I genotype prevalence was found to be statistically insignificant between the two study groups (p=0.135). Conclusion The avirulent P. gingivalis fimA type I genotype, occurred in high prevalence among chronic gingivitis patients, while its presence was low in chronic periodontitis patients. Presence of this avirulent genotype in chronic marginal gingivitis signifies its reversible condition. PMID:27504406

  15. Dual Action of Myricetin on Porphyromonas gingivalis and the Inflammatory Response of Host Cells: A Promising Therapeutic Molecule for Periodontal Diseases

    PubMed Central

    Grenier, Daniel; Chen, Huangqin; Ben Lagha, Amel; Fournier-Larente, Jade; Morin, Marie-Pierre

    2015-01-01

    Periodontitis that affects the underlying structures of the periodontium, including the alveolar bone, is a multifactorial disease, whose etiology involves interactions between specific bacterial species of the subgingival biofilm and the host immune components. In the present study, we investigated the effects of myricetin, a flavonol largely distributed in fruits and vegetables, on growth and virulence properties of Porphyromonas gingivalis as well as on the P. gingivalis-induced inflammatory response in host cells. Minimal inhibitory concentration values of myricetin against P. gingivalis were in the range of 62.5 to 125 μg/ml. The iron-chelating activity of myricetin may contribute to the antibacterial activity of this flavonol. Myricetin was found to attenuate the virulence of P. gingivalis by reducing the expression of genes coding for important virulence factors, including proteinases (rgpA, rgpB, and kgp) and adhesins (fimA, hagA, and hagB). Myricetin dose-dependently prevented NF-κB activation in a monocyte model. Moreover, it inhibited the secretion of IL-6, IL-8 and MMP-3 by P. gingivalis-stimulated gingival fibroblasts. In conclusion, our study brought clear evidence that the flavonol myricetin exhibits a dual action on the periodontopathogenic bacterium P. gingivalis and the inflammatory response of host cells. Therefore, myricetin holds promise as a therapeutic agent for the treatment/prevention of periodontitis. PMID:26121135

  16. A study of the uptake of toluidine blue O by Porphyromonas gingivalis and the mechanism of lethal photosensitization.

    PubMed

    Bhatti, M; MacRobert, A; Meghji, S; Henderson, B; Wilson, M

    1998-09-01

    The purpose of the study was to determine the distribution of the photosensitizer toluidine blue O (TBO) within Porphyromonas gingivalis and the possible mechanism(s) involved in the lethal photosensitization of this organism. The distribution of TBO was determined by incubating P. gingivalis with tritiated TBO (3H-TBO) and fractionating the cells into outer membrane (OM), plasma membrane (PM), cytoplasmic proteins, other cytoplasmic constituents and DNA. The percentage of TBO in each of the fractions was found to be, 86.7, 5.4, 1.9, 5.7 and 0.3%, respectively. The involvement of cytotoxic species in the lethal photosensitization induced by light from a heliumneon (HeNe) laser and TBO was investigated by using deuterium oxide (D2O), which prolongs the lifetime of singlet oxygen, and the free radical and signlet oxygen scavenger L-tryptophan. There were 9.0 log10 and 2 log10 reductions in the presence of D2O and H2O (saline solutions), respectively, at a light dose of 0.44 J (energy density = 0.22 J/cm2), suggesting the involvement of singlet oxygen. Decreased kills were attained in the presence of increasing concentrations of L-tryptophan. The effect of lethal photosensitization on whole cell proteins was determined by measuring tryptophan fluorescence, which decreased by 30% using 4.3 J (energy density = 4.3 J/cm2) of light. Effects on the OM and PM proteins were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. There was evidence of change in the molecular masses of several PM proteins and OM proteins compared to controls. There was evidence of damage to the DNA obtained from irradiated cells. Scanning electron microscopic studies showed that there was coaggregation of P. gingivalis cells when sensitized and then exposed to laser light. These results suggest that lethal photosensitization of P. gingivalis may involve changes in OM and/or PM proteins and DNA damage mediated by singlet oxygen.

  17. Porphyromonas gingivalis Stimulates Bone Resorption by Enhancing RANKL (Receptor Activator of NF-κB Ligand) through Activation of Toll-like Receptor 2 in Osteoblasts*

    PubMed Central

    Kassem, Ali; Henning, Petra; Lundberg, Pernilla; Souza, Pedro P. C.; Lindholm, Catharina; Lerner, Ulf H.

    2015-01-01

    Periodontitis has been associated with rheumatoid arthritis. In experimental arthritis, concomitant periodontitis caused by oral infection with Porphyromonas gingivalis enhances articular bone loss. The aim of this study was to investigate how lipopolysaccharide (LPS) from P. gingivalis stimulates bone resorption. The effects by LPS P. gingivalis and four other TLR2 ligands on bone resorption, osteoclast formation, and gene expression in wild type and Tlr2-deficient mice were assessed in ex vivo cultures of mouse parietal bones and in an in vivo model in which TLR2 agonists were injected subcutaneously over the skull bones. LPS P. gingivalis stimulated mineral release and matrix degradation in the parietal bone organ cultures by increasing differentiation and formation of mature osteoclasts, a response dependent on increased RANKL (receptor activator of NF-κB ligand). LPS P. gingivalis stimulated RANKL in parietal osteoblasts dependent on the presence of TLR2 and through a MyD88 and NF-κB-mediated mechanism. Similarly, the TLR2 agonists HKLM, FSL1, Pam2, and Pam3 stimulated RANKL in osteoblasts and parietal bone resorption. LPS P. gingivalis and Pam2 robustly enhanced osteoclast formation in periosteal/endosteal cell cultures by increasing RANKL. LPS P. gingivalis and Pam2 also up-regulated RANKL and osteoclastic genes in vivo, resulting in an increased number of periosteal osteoclasts and immense bone loss in wild type mice but not in Tlr2-deficient mice. These data demonstrate that LPS P. gingivalis stimulates periosteal osteoclast formation and bone resorption by stimulating RANKL in osteoblasts via TLR2. This effect might be important for periodontal bone loss and for the enhanced bone loss seen in rheumatoid arthritis patients with concomitant periodontal disease. PMID:26085099

  18. Unique Structure and Stability of HmuY, a Novel Heme-Binding Protein of Porphyromonas gingivalis

    PubMed Central

    Wójtowicz, Halina; Guevara, Tibisay; Tallant, Cynthia; Olczak, Mariusz; Sroka, Aneta; Potempa, Jan; Solà, Maria; Olczak, Teresa; Gomis-Rüth, F. Xavier

    2009-01-01

    Infection, survival, and proliferation of pathogenic bacteria in humans depend on their capacity to impair host responses and acquire nutrients in a hostile environment. Among such nutrients is heme, a co-factor for oxygen storage, electron transport, photosynthesis, and redox biochemistry, which is indispensable for life. Porphyromonas gingivalis is the major human bacterial pathogen responsible for severe periodontitis. It recruits heme through HmuY, which sequesters heme from host carriers and delivers it to its cognate outer-membrane transporter, the TonB-dependent receptor HmuR. Here we report that heme binding does not significantly affect the secondary structure of HmuY. The crystal structure of heme-bound HmuY reveals a new all-β fold mimicking a right hand. The thumb and fingers pinch heme iron through two apical histidine residues, giving rise to highly symmetric octahedral iron co-ordination. The tetrameric quaternary arrangement of the protein found in the crystal structure is consistent with experiments in solution. It shows that thumbs and fingertips, and, by extension, the bound heme groups, are shielded from competing heme-binding proteins from the host. This may also facilitate heme transport to HmuR for internalization. HmuY, both in its apo- and in its heme-bound forms, is resistant to proteolytic digestion by trypsin and the major secreted proteases of P. gingivalis, gingipains K and R. It is also stable against thermal and chemical denaturation. In conclusion, these studies reveal novel molecular properties of HmuY that are consistent with its role as a putative virulence factor during bacterial infection. PMID:19424422

  19. Involvement of an Skp-Like Protein, PGN_0300, in the Type IX Secretion System of Porphyromonas gingivalis

    PubMed Central

    Taguchi, Yuko; Sato, Keiko; Yukitake, Hideharu; Inoue, Tetsuyoshi; Nakayama, Masaaki; Naito, Mariko; Kondo, Yoshio; Kano, Konami; Hoshino, Tomonori; Nakayama, Koji; Takashiba, Shogo

    2015-01-01

    The oral Gram-negative anaerobic bacterium Porphyromonas gingivalis is an important pathogen involved in chronic periodontitis. Among its virulence factors, the major extracellular proteinases, Arg-gingipain and Lys-gingipain, are of interest given their abilities to degrade host proteins and process other virulence factors. Gingipains possess C-terminal domains (CTDs) and are translocated to the cell surface or into the extracellular milieu by the type IX secretion system (T9SS). Gingipains contribute to the colonial pigmentation of the bacterium on blood agar. In this study, Omp17, the PGN_0300 gene product, was found in the outer membrane fraction. A mutant lacking Omp17 did not show pigmentation on blood agar and showed reduced proteolytic activity of the gingipains. CTD-containing proteins were released from bacterial cells without cleavage of the CTDs in the omp17 mutant. Although synthesis of the anionic polysaccharide (A-LPS) was not affected in the omp17 mutant, the processing of and A-LPS modification of CTD-containing proteins was defective. PorU, a C-terminal signal peptidase that cleaves the CTDs of other CTD-containing proteins, was not detected in any membrane fraction of the omp17 mutant, suggesting that the defective maturation of CTD-containing proteins by impairment of Omp17 is partly due to loss of function of PorU. In the mouse subcutaneous infection experiment, the omp17 mutant was less virulent than the wild type. These results suggested that Omp17 is involved in P. gingivalis virulence. PMID:26502912

  20. Structure and Mechanism of Cysteine Peptidase Gingipain K (Kgp), a Major Virulence Factor of Porphyromonas gingivalis in Periodontitis*

    PubMed Central

    de Diego, Iñaki; Veillard, Florian; Sztukowska, Maryta N.; Guevara, Tibisay; Potempa, Barbara; Pomowski, Anja; Huntington, James A.; Potempa, Jan; Gomis-Rüth, F. Xavier

    2014-01-01

    Cysteine peptidases are key proteolytic virulence factors of the periodontopathogen Porphyromonas gingivalis, which causes chronic periodontitis, the most prevalent dysbiosis-driven disease in humans. Two peptidases, gingipain K (Kgp) and R (RgpA and RgpB), which differ in their selectivity after lysines and arginines, respectively, collectively account for 85% of the extracellular proteolytic activity of P. gingivalis at the site of infection. Therefore, they are promising targets for the design of specific inhibitors. Although the structure of the catalytic domain of RgpB is known, little is known about Kgp, which shares only 27% sequence identity. We report the high resolution crystal structure of a competent fragment of Kgp encompassing the catalytic cysteine peptidase domain and a downstream immunoglobulin superfamily-like domain, which is required for folding and secretion of Kgp in vivo. The structure, which strikingly resembles a tooth, was serendipitously trapped with a fragment of a covalent inhibitor targeting the catalytic cysteine. This provided accurate insight into the active site and suggested that catalysis may require a catalytic triad, Cys477-His444-Asp388, rather than the cysteine-histidine dyad normally found in cysteine peptidases. In addition, a 20-Å-long solvent-filled interior channel traverses the molecule and links the bottom of the specificity pocket with the molecular surface opposite the active site cleft. This channel, absent in RgpB, may enhance the plasticity of the enzyme, which would explain the much lower activity in vitro toward comparable specific synthetic substrates. Overall, the present results report the architecture and molecular determinants of the working mechanism of Kgp, including interaction with its substrates. PMID:25266723

  1. Three CoA Transferases Involved in the Production of Short Chain Fatty Acids in Porphyromonas gingivalis

    PubMed Central

    Sato, Mitsunari; Yoshida, Yasuo; Nagano, Keiji; Hasegawa, Yoshiaki; Takebe, Jun; Yoshimura, Fuminobu

    2016-01-01

    Butyryl-CoA:acetate CoA transferase, which produces butyrate and acetyl-CoA from butyryl-CoA and acetate, is responsible for the final step of butyrate production in bacteria. This study demonstrates that in the periodontopathogenic bacterium Porphyromonas gingivalis this reaction is not catalyzed by PGN_1171, previously annotated as butyryl-CoA:acetate CoA transferase, but by three distinct CoA transferases, PGN_0725, PGN_1341, and PGN_1888. Gas chromatography/mass spectrometry (GC-MS) and spectrophotometric analyses were performed using crude enzyme extracts from deletion mutant strains and purified recombinant proteins. The experiments revealed that, in the presence of acetate, PGN_0725 preferentially utilized butyryl-CoA rather than propionyl-CoA. By contrast, this preference was reversed in PGN_1888. The only butyryl-CoA:acetate CoA transferase activity was observed in PGN_1341. Double reciprocal plots revealed that all the reactions catalyzed by these enzymes follow a ternary-complex mechanism, in contrast to previously characterized CoA transferases. GC-MS analysis to determine the concentrations of short chain fatty acids (SCFAs) in culture supernatants of P. gingivalis wild type and mutant strains revealed that PGN_0725 and PGN_1888 play a major role in the production of butyrate and propionate, respectively. Interestingly, a triple deletion mutant lacking PGN_0725, PGN_1341, and PGN_1888 produced low levels of SCFAs, suggesting that the microorganism contains CoA transferase(s) in addition to these three enzymes. Growth rates of the mutant strains were mostly slower than that of the wild type, indicating that many carbon compounds produced in the SCFA synthesis appear to be important for the biological activity of this microorganism. PMID:27486457

  2. Immunoglobulin allotypes and immunoglobulin G subclass responses to Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in early-onset periodontitis.

    PubMed Central

    Choi, J I; Ha, M H; Kim, J H; Kim, S J

    1996-01-01

    The present study was performed to estimate the observed frequencies of the immunoglobulin heavy-chain (Gm) and light-chain (Km) allotypes among patients with early-onset periodontitis (EOP) and their effect on the IgG2 subclass responses against Actinobacillus actinomycetemcomitans Y4 and Porphyromonas gingivalis 381, respectively. Sixty-nine EOP patients, including 11 with localized juvenile periodontitis (LJP), 19 who had LJP, 15 with LJP-rapidly progressing periodontitis (RPP), and 24 with RPP, were examined for the Gm and Km allotypes by a hemagglutination inhibition test. Levels of immunoglobulin G2 (IgG2) antibodies against the two organisms were determined by enzyme-linked immunosorbent assay. Fifty race- and age-matched, periodontally healthy subjects were also included as a control group. The observed frequencies of the Gm haplotype afnb and Km(1) were significantly higher in the RPP and LJP groups, respectively. The G2m(n)+ group of those with RPP and the Km(1)+ group of those with LJP had significantly higher levels of IgG2 antibodies to A. actinomycetemcomitans and P. gingivalis, respectively. The results indicate that linkage disequilibrium of the G2m(n) locus in RPP patients or the Km(1) locus in LJP patients may be associated with high IgG2 antibody responses to the respective bacteria. It was reasoned that the IgG2 antibody responses are associated with the immunoglobulin allotypes. The function of IgG2 antibodies in their reaction to different bacterial antigens may be interpreted as either protective or nonprotective in the two different types of EOP (i.e., LJP and RPP). PMID:8926092

  3. Three CoA Transferases Involved in the Production of Short Chain Fatty Acids in Porphyromonas gingivalis.

    PubMed

    Sato, Mitsunari; Yoshida, Yasuo; Nagano, Keiji; Hasegawa, Yoshiaki; Takebe, Jun; Yoshimura, Fuminobu

    2016-01-01

    Butyryl-CoA:acetate CoA transferase, which produces butyrate and acetyl-CoA from butyryl-CoA and acetate, is responsible for the final step of butyrate production in bacteria. This study demonstrates that in the periodontopathogenic bacterium Porphyromonas gingivalis this reaction is not catalyzed by PGN_1171, previously annotated as butyryl-CoA:acetate CoA transferase, but by three distinct CoA transferases, PGN_0725, PGN_1341, and PGN_1888. Gas chromatography/mass spectrometry (GC-MS) and spectrophotometric analyses were performed using crude enzyme extracts from deletion mutant strains and purified recombinant proteins. The experiments revealed that, in the presence of acetate, PGN_0725 preferentially utilized butyryl-CoA rather than propionyl-CoA. By contrast, this preference was reversed in PGN_1888. The only butyryl-CoA:acetate CoA transferase activity was observed in PGN_1341. Double reciprocal plots revealed that all the reactions catalyzed by these enzymes follow a ternary-complex mechanism, in contrast to previously characterized CoA transferases. GC-MS analysis to determine the concentrations of short chain fatty acids (SCFAs) in culture supernatants of P. gingivalis wild type and mutant strains revealed that PGN_0725 and PGN_1888 play a major role in the production of butyrate and propionate, respectively. Interestingly, a triple deletion mutant lacking PGN_0725, PGN_1341, and PGN_1888 produced low levels of SCFAs, suggesting that the microorganism contains CoA transferase(s) in addition to these three enzymes. Growth rates of the mutant strains were mostly slower than that of the wild type, indicating that many carbon compounds produced in the SCFA synthesis appear to be important for the biological activity of this microorganism. PMID:27486457

  4. In-vivo effect of andrographolide on alveolar bone resorption induced by Porphyromonas gingivalis and its relation with antioxidant enzymes.

    PubMed

    Al Batran, Rami; Al-Bayaty, Fouad H; Al-Obaidi, Mazen M Jamil

    2013-01-01

    Alveolar bone resorption is one of the most important facts in denture construction. Porphyromonas gingivalis (Pg) causes alveolar bone resorption, and morphologic measurements are the most frequent methods to identify bone resorption in periodontal studies. This study has aimed at evaluating the effect of Andrographolide (AND) on alveolar bone resorption in rats induced by Pg. 24 healthy male Sprague Dawley rats were divided into four groups as follows: normal control group and three experimental groups challenged orally with Pg ATCC 33277 five times a week supplemented with 20 mg/kg and 10 mg/kg of AND for twelve weeks. Alveolar bones of the left and right sides of the mandible were assessed by a morphometric method. The bone level, that is, the distance from the alveolar bone crest to cementumenamel junction (CEJ), was measured using 6.1 : 1 zoom stereomicroscope and software. AND reduced the effect of Pg on alveolar bone resorption and decreased the serum levels of Hexanoyl-Lysine (HEL); furthermore the reduced glutathione/oxidised glutathione (GSH/GSSG) ratio in AND treated groups (10 and 20 mg/kg) significantly increased when compared with the Pg group (P < 0.05). We can conclude that AND suppresses alveolar bone resorption caused by Pg in rats.

  5. Porphyromonas gingivalis Participates in Pathogenesis of Human Abdominal Aortic Aneurysm by Neutrophil Activation. Proof of Concept in Rats

    PubMed Central

    Delbosc, Sandrine; Alsac, Jean-Marc; Journe, Clement; Louedec, Liliane; Castier, Yves; Bonnaure-Mallet, Martine; Ruimy, Raymond; Rossignol, Patrick; Bouchard, Philippe; Michel, Jean-Baptiste; Meilhac, Olivier

    2011-01-01

    Background Abdominal Aortic Aneurysms (AAAs) represent a particular form of atherothrombosis where neutrophil proteolytic activity plays a major role. We postulated that neutrophil recruitment and activation participating in AAA growth may originate in part from repeated episodes of periodontal bacteremia. Methods and Findings Our results show that neutrophil activation in human AAA was associated with Neutrophil Extracellular Trap (NET) formation in the IntraLuminal Thrombus, leading to the release of cell-free DNA. Human AAA samples were shown to contain bacterial DNA with high frequency (11/16), and in particular that of Porphyromonas gingivalis (Pg), the most prevalent pathogen involved in chronic periodontitis, a common form of periodontal disease. Both DNA reflecting the presence of NETs and antibodies to Pg were found to be increased in plasma of patients with AAA. Using a rat model of AAA, we demonstrated that repeated injection of Pg fostered aneurysm development, associated with pathological characteristics similar to those observed in humans, such as the persistence of a neutrophil-rich luminal thrombus, not observed in saline-injected rats in which a healing process was observed. Conclusions Thus, the control of periodontal disease may represent a therapeutic target to limit human AAA progression. PMID:21533243

  6. Effects of Porphyromonas gingivalis lipopolysaccharide on the expression of key genes involved in cholesterol metabolism in macrophages

    PubMed Central

    Liu, Fen; Wang, Yi; Xu, Jing; Liu, Fangqiang

    2016-01-01

    Introduction Cardiovascular diseases are positively correlated with periodontal disease. However, the molecular mechanisms linking atherosclerosis and periodontal infection are not clear. This study aimed to determine whether Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) altered the expression of genes regulating cholesterol metabolism in macrophages in the presence of low-density lipoprotein (LDL). Material and methods THP-1-derived macrophages were exposed to different concentrations (0.1, 1, 10 µg/ml) of LPS in the presence of 50 µg/ml native LDL. Macrophages were also incubated with 1 µg/ml LPS for varying times (0, 24, 48, or 72 h) in the presence of native LDL. Foam cell formation was determined by oil red O staining and cholesterol content quantification. CD36, lectin-like oxidized LDL receptor-1 (LOX-1), ATP-binding cassette G1 (ABCG1), and acetyl CoA acyltransferase 1 (ACAT1) expression levels were measured by western blot and qRT-PCR. Results Foam cell formation was induced in a time- and concentration-dependent manner as assessed by both morphological and biochemical criteria. Pg-LPS caused downregulation of CD36 and ABCG1 but upregulation of ACAT1, while LOX-1 expression was not affected (p = 0.137). Conclusions Pg-LPS appears to be an important link in the development of atherosclerosis by mechanisms targeting cholesterol homeostasis, namely, excess cholesterol ester formation via ACAT1 and reduced cellular cholesterol efflux via ABCG1.

  7. Structural and mutational analyses of dipeptidyl peptidase 11 from Porphyromonas gingivalis reveal the molecular basis for strict substrate specificity

    PubMed Central

    Sakamoto, Yasumitsu; Suzuki, Yoshiyuki; Iizuka, Ippei; Tateoka, Chika; Roppongi, Saori; Fujimoto, Mayu; Inaka, Koji; Tanaka, Hiroaki; Yamada, Mitsugu; Ohta, Kazunori; Gouda, Hiroaki; Nonaka, Takamasa; Ogasawara, Wataru; Tanaka, Nobutada

    2015-01-01

    The dipeptidyl peptidase 11 from Porphyromonas gingivalis (PgDPP11) belongs to the S46 family of serine peptidases and preferentially cleaves substrates with Asp/Glu at the P1 position. The molecular mechanism underlying the substrate specificity of PgDPP11, however, is unknown. Here, we report the crystal structure of PgDPP11. The enzyme contains a catalytic domain with a typical double β-barrel fold and a recently identified regulatory α-helical domain. Crystal structure analyses, docking studies, and biochemical studies revealed that the side chain of Arg673 in the S1 subsite is essential for recognition of the Asp/Glu side chain at the P1 position of the bound substrate. Because S46 peptidases are not found in mammals and the Arg673 is conserved among DPP11s, we anticipate that DPP11s could be utilised as targets for antibiotics. In addition, the present structure analyses could be useful templates for the design of specific inhibitors of DPP11s from pathogenic organisms. PMID:26057589

  8. Effects of Porphyromonas gingivalis lipopolysaccharide on osteoblast-osteoclast bidirectional EphB4-EphrinB2 signaling

    PubMed Central

    ZHANG, YI; WANG, XI-CHAO; BAO, XING-FU; HU, MIN; YU, WEI-XIAN

    2014-01-01

    In bone remodeling, the Eph family is involved in regulating the process of osteoclast and osteoblast coordination in order to maintain bone homeostasis. In this study, the effects of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) on the osteoblast-osteoclast bidirectional EphB4-EphrinB2 signaling were investigated. An osteoblast-osteoclast co-culture system was achieved successfully. Hence, direct contact and communication between osteoblasts and osteoclasts was permitted. Regarding the protein expression and gene expression of EphB4 and EphrinB2, it was shown that Pg-LPS increased the expression of EphB4 while inhibiting the expression of EphrinB2. Therefore, the results indicate that, when treated with Pg-LPS, the EphB4 receptor on osteoblasts and the EphrinB2 ligand on osteoclasts may generate bidirectional anti-osteoclastogenic and pro-osteoblastogenic signaling into respective cells and potentially facilitate the transition from bone resorption to bone formation. This study may contribute to the control of osteoblast differentiation and bone formation at remodeling, and possibly also modeling, sites. PMID:24348768

  9. Effects of Porphyromonas gingivalis lipopolysaccharide on osteoblast-osteoclast bidirectional EphB4-EphrinB2 signaling.

    PubMed

    Zhang, Yi; Wang, Xi-Chao; Bao, Xing-Fu; Hu, Min; Yu, Wei-Xian

    2014-01-01

    In bone remodeling, the Eph family is involved in regulating the process of osteoclast and osteoblast coordination in order to maintain bone homeostasis. In this study, the effects of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) on the osteoblast-osteoclast bidirectional EphB4-EphrinB2 signaling were investigated. An osteoblast-osteoclast co-culture system was achieved successfully. Hence, direct contact and communication between osteoblasts and osteoclasts was permitted. Regarding the protein expression and gene expression of EphB4 and EphrinB2, it was shown that Pg-LPS increased the expression of EphB4 while inhibiting the expression of EphrinB2. Therefore, the results indicate that, when treated with Pg-LPS, the EphB4 receptor on osteoblasts and the EphrinB2 ligand on osteoclasts may generate bidirectional anti-osteoclastogenic and pro-osteoblastogenic signaling into respective cells and potentially facilitate the transition from bone resorption to bone formation. This study may contribute to the control of osteoblast differentiation and bone formation at remodeling, and possibly also modeling, sites.

  10. Histatin 5 binds to Porphyromonas gingivalis hemagglutinin B (HagB) and alters HagB-induced chemokine responses

    NASA Astrophysics Data System (ADS)

    Borgwardt, Derek S.; Martin, Aaron D.; van Hemert, Jonathan R.; Yang, Jianyi; Fischer, Carol L.; Recker, Erica N.; Nair, Prashant R.; Vidva, Robinson; Chandrashekaraiah, Shwetha; Progulske-Fox, Ann; Drake, David; Cavanaugh, Joseph E.; Vali, Shireen; Zhang, Yang; Brogden, Kim A.

    2014-01-01

    Histatins are human salivary gland peptides with anti-microbial and anti-inflammatory activities. In this study, we hypothesized that histatin 5 binds to Porphyromonas gingivalis hemagglutinin B (HagB) and attenuates HagB-induced chemokine responses in human myeloid dendritic cells. Histatin 5 bound to immobilized HagB in a surface plasmon resonance (SPR) spectroscopy-based biosensor system. SPR spectroscopy kinetic and equilibrium analyses, protein microarray studies, and I-TASSER structural modeling studies all demonstrated two histatin 5 binding sites on HagB. One site had a stronger affinity with a KD1 of 1.9 μM and one site had a weaker affinity with a KD2 of 60.0 μM. Binding has biological implications and predictive modeling studies and exposure of dendritic cells both demonstrated that 20.0 μM histatin 5 attenuated (p < 0.05) 0.02 μM HagB-induced CCL3/MIP-1α, CCL4/MIP-1β, and TNFα responses. Thus histatin 5 is capable of attenuating chemokine responses, which may help control oral inflammation.

  11. Gingipains from the Periodontal Pathogen Porphyromonas gingivalis Play a Significant Role in Regulation of Angiopoietin 1 and Angiopoietin 2 in Human Aortic Smooth Muscle Cells

    PubMed Central

    Khalaf, Hazem; Sirsjö, Allan; Bengtsson, Torbjörn

    2015-01-01

    Angiopoietin 1 (Angpt1) and angiopoietin 2 (Angpt2) are the ligands of tyrosine kinase (Tie) receptors, and they play important roles in vessel formation and the development of inflammatory diseases, such as atherosclerosis. Porphyromonas gingivalis is a Gram-negative periodontal bacterium that is thought to contribute to the progression of cardiovascular disease. The aim of this study was to investigate the role of P. gingivalis infection in the modulation of Angpt1 and Angpt2 in human aortic smooth muscle cells (AoSMCs). We exposed AoSMCs to wild-type (W50 and 381), gingipain mutant (E8 and K1A), and fimbrial mutant (DPG-3 and KRX-178) P. gingivalis strains and to different concentrations of tumor necrosis factor (TNF). The atherosclerosis risk factor TNF was used as a positive control in this study. We found that P. gingivalis (wild type, K1A, DPG3, and KRX178) and TNF upregulated the expression of Angpt2 and its transcription factor ETS1, respectively, in AoSMCs. In contrast, Angpt1 was inhibited by P. gingivalis and TNF. However, the RgpAB mutant E8 had no effect on the expression of Angpt1, Angpt2, or ETS1 in AoSMCs. The results also showed that ETS1 is critical for P. gingivalis induction of Angpt2. Exposure to Angpt2 protein enhanced the migration of AoSMCs but had no effect on proliferation. This study demonstrates that gingipains are crucial to the ability of P. gingivalis to markedly increase the expressed Angpt2/Angpt1 ratio in AoSMCs, which determines the regulatory role of angiopoietins in angiogenesis and their involvement in the development of atherosclerosis. These findings further support the association between periodontitis and cardiovascular disease. PMID:26283334

  12. Interleukin-8 and Intercellular Adhesion Molecule 1 Regulation in Oral Epithelial Cells by Selected Periodontal Bacteria: Multiple Effects of Porphyromonas gingivalis via Antagonistic Mechanisms

    PubMed Central

    Huang, George T.-J.; Kim, Daniel; Lee, Jonathan K.-H.; Kuramitsu, Howard K.; Haake, Susan Kinder

    2001-01-01

    Interaction of bacteria with mucosal surfaces can modulate the production of proinflammatory cytokines and adhesion molecules produced by epithelial cells. Previously, we showed that expression of interleukin-8 (IL-8) and intercellular adhesion molecule 1 (ICAM-1) by gingival epithelial cells increases following interaction with several putative periodontal pathogens. In contrast, expression of IL-8 and ICAM-1 is reduced after Porphyromonas gingivalis ATCC 33277 challenge. In the present study, we investigated the mechanisms that govern the regulation of these two molecules in bacterially infected gingival epithelial cells. Experimental approaches included bacterial stimulation of gingival epithelial cells by either a brief challenge (1.5 to 2 h) or a continuous coculture throughout the incubation period. The kinetics of IL-8 and ICAM-1 expression following brief challenge were such that (i) secretion of IL-8 by gingival epithelial cells reached its peak 2 h following Fusobacterium nucleatum infection whereas it rapidly decreased within 2 h after P. gingivalis infection and remained decreased up to 30 h and (ii) IL-8 and ICAM-1 mRNA levels were up-regulated rapidly 2 to 4 h postinfection and then decreased to basal levels 8 to 20 h after infection with either Actinobacillus actinomycetemcomitans, F. nucleatum, or P. gingivalis. Attenuation of IL-8 secretion was facilitated by adherent P. gingivalis strains. The IL-8 secreted from epithelial cells after F. nucleatum stimulation could be down-regulated by subsequent infection with P. gingivalis or its culture supernatant. Although these results suggested that IL-8 attenuation at the protein level might be associated with P. gingivalis proteases, the Arg- and Lys-gingipain proteases did not appear to be solely responsible for IL-8 attenuation. In addition, while P. gingivalis up-regulated IL-8 mRNA expression, this effect was overridden when the bacteria were continuously cocultured with the epithelial cells. The IL-8

  13. Defining essential genes and identifying virulence factors of Porphyromonas gingivalis by massively-parallel sequencing of transposon libraries (Tn-seq)

    PubMed Central

    Klein, Brian A.; Duncan, Margaret J.; Hu, Linden T.

    2016-01-01

    Summary Porphyromonas gingivalis is a keystone pathogen in the development and progression of periodontal disease. Obstacles to the development of saturated transposon libraries have previously limited transposon mutant-based screens as well as essential gene studies. We have developed a system for efficient transposon mutagenesis of P. gingivalis using a modified mariner transposon. Tn-seq is a technique that allows for quantitative assessment of individual mutants within a transposon mutant library by sequencing the transposon-genome junctions and then compiling mutant presence by mapping to a base genome. Using Tn-seq, it is possible to quickly define all the insertional mutants in a library and thus identify non-essential genes under the conditions in which the library was produced. Identification of fitness of individual mutants under specific conditions can be performed by exposing the library to selective pressures. PMID:25636611

  14. Crystallization and preliminary X-ray analysis of the C-terminal fragment of PorM, a subunit of the Porphyromonas gingivalis type IX secretion system.

    PubMed

    Stathopulos, Julien; Cambillau, Christian; Cascales, Eric; Roussel, Alain; Leone, Philippe

    2015-01-01

    PorM is a membrane protein involved in the assembly of the type IX secretion system (T9SS) from Porphyromonas gingivalis, a major bacterial pathogen responsible for periodontal disease in humans. The periplasmic domain of PorM was overexpressed in Escherichia coli and purified. A fragment of the purified protein was obtained by limited proteolysis. Crystals of this fragment belonged to the tetragonal space group P4(3)2(1)2. Native and MAD data sets were recorded to 2.85 and 3.1 Å resolution, respectively, using synchrotron radiation. PMID:25615973

  15. Structural Insights into the PorK and PorN Components of the Porphyromonas gingivalis Type IX Secretion System

    PubMed Central

    Gorasia, Dhana G.; Veith, Paul D.; Hanssen, Eric G.; Glew, Michelle D.; Sato, Keiko; Yukitake, Hideharu; Nakayama, Koji; Reynolds, Eric C.

    2016-01-01

    The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32–36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component. PMID:27509186

  16. Structural Insights into the PorK and PorN Components of the Porphyromonas gingivalis Type IX Secretion System.

    PubMed

    Gorasia, Dhana G; Veith, Paul D; Hanssen, Eric G; Glew, Michelle D; Sato, Keiko; Yukitake, Hideharu; Nakayama, Koji; Reynolds, Eric C

    2016-08-01

    The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32-36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component. PMID:27509186

  17. Structural Insights into the PorK and PorN Components of the Porphyromonas gingivalis Type IX Secretion System.

    PubMed

    Gorasia, Dhana G; Veith, Paul D; Hanssen, Eric G; Glew, Michelle D; Sato, Keiko; Yukitake, Hideharu; Nakayama, Koji; Reynolds, Eric C

    2016-08-01

    The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32-36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component.

  18. The role of phagocytosis, oxidative burst and neutrophil extracellular traps in the interaction between neutrophils and the periodontal pathogen Porphyromonas gingivalis.

    PubMed

    Jayaprakash, K; Demirel, I; Khalaf, H; Bengtsson, T

    2015-10-01

    Neutrophils are regarded as the sentinel cells of innate immunity and are found in abundance within the gingival crevice. Discovery of neutrophil extracellular traps (NETs) within the gingival pockets prompted us to probe the nature of the interactions of neutrophils with the prominent periopathogen Porphyromonas gingivalis. Some of the noted virulence factors of this Gram-negative anaerobe are gingipains: arginine gingipains (RgpA/B) and lysine gingipain (Kgp). The aim of this study was to evaluate the role of gingipains in phagocytosis, formation of reactive oxygen species, NETs and CXCL8 modulation by using wild-type strains and isogenic gingipain mutants. Confocal imaging showed that gingipain mutants K1A (Kgp) and E8 (RgpA/B) induced extracellular traps in neutrophils, whereas ATCC33277 and W50 were phagocytosed. The viability of both ATCC33277 and W50 dwindled as the result of phagocytosis and could be salvaged by cytochalasin D, and the bacteria released high levels of lipopolysaccharide in the culture supernatant. Porphyromonas gingivalis induced reactive oxygen species and CXCL8 with the most prominent effect being that of the wild-type strain ATCC33277, whereas the other wild-type strain W50 was less effective. Quantitative real-time polymerase chain reaction revealed a significant CXCL8 expression by E8. All the tested P. gingivalis strains increased cytosolic free calcium. In conclusion, phagocytosis is the primary neutrophil response to P. gingivalis, although NETs could play an accessory role in infection control. Although gingipains do not seem to directly regulate phagocytosis, NETs or oxidative burst in neutrophils, their proteolytic properties could modulate the subsequent outcomes such as nutrition acquisition and survival by the bacteria.

  19. Evaluation of chemical composition and efficacy of Chinese propolis extract on Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans: An in vitro study

    PubMed Central

    Agarwal, Garima; Vemanaradhya, Gayathri G.; Mehta, Dhoom S.

    2012-01-01

    Background: Propolis as a natural remedy has maintained its popularity over long periods of time. The aim of this study was to determine the chemical composition in terms of total phenolic compounds and flavonoids present in Chinese propolis and to carry out an in vitro evaluation of its antimicrobial activity and the minimal inhibitory concentrations for Porphyromonas gingivalis (Pg) and Aggregatibacter actinomycetemcomitans (Aa). Materials and Methods: From the ethanolic extract of propolis (EEP), total phenol content was determined by the Folin–Ciocalteau method, flavones and flavonols by the modified aluminum chloride colorimetric method, and flavanones by the 2.4-dinitrophenylhydrazine (2,4-DNP) method. Agar well diffusion assay was used to evaluate the antimicrobial potential of propolis against Pg and Aa. The minimum inhibitory concentration of propolis against the two bacteria was determined using serial tube dilution technique. Results: The total concentration of phenol in the EEP was 19.44%, flavones and flavonols 2.616%, and flavanones 16.176%. The inhibitory zone depicting antimicrobial activity ranged from 18 to 25 mm for Pg and from 12 to 14 mm for Aa. The concentration range of Chinese propolis that is sensitive to inhibit the growth of Pg was 0.1–0.0125 μg/ml and for Aa it was 0.1–0.025 μg/ml. Conclusion: These data suggest that Chinese propolis has potent antimicrobial activity against the two periodontopathogens, suggesting its possible use as a natural alternative to the widely used synthetic antibiotics for periodontal therapy. PMID:23293477

  20. Inhibition of Sprouty2 polarizes macrophages toward an M2 phenotype by stimulation with interferon γ and Porphyromonas gingivalis lipopolysaccharide.

    PubMed

    Atomura, Ryo; Sanui, Terukazu; Fukuda, Takao; Tanaka, Urara; Toyoda, Kyosuke; Taketomi, Takaharu; Yamamichi, Kensuke; Akiyama, Hajime; Nishimura, Fusanori

    2016-03-01

    Periodontitis is a chronic inflammatory disorder caused by specific bacteria residing in the biofilm, particularly Porphyromonas gingivalis (Pg). Sprouty2 (Spry2) functions as a negative regulator of the fibroblast growth factor (FGF) signaling pathway. We previously demonstrated that sequestration of Spry2 induced proliferation and osteogenesis in osteoblastic cells by basic FGF (bFGF) and epidermal growth factor (EGF) stimulation in vitro, but diminished cell proliferation in gingival epithelial cells. In addition, Spry2 knockdown in combination with bFGF and EGF stimulation increases periodontal ligament cell proliferation and migration accompanied by prevention of osteoblastic differentiation. In this study, we investigated the mechanisms through which Spry2 depletion by interferon (IFN) γ and Pg lipopolysaccharide (LPS) stimulation affected the physiology of macrophages in vitro. Transfection of macrophages with Spry2 small-interfering RNA (siRNA) promoted the expression of genes characteristic of M2 alternative activated macrophages, induced interleukin (IL)-10 expression, and enhanced arginase activity, even in cells stimulated with IFNγ and Pg LPS. In addition, we found that phosphoinositide 3-kinase (PI3K) and AKT activation by Spry2 downregulation enhanced efferocytosis of apoptotic cells by increasing Rac1 activation and decreasing nuclear factor kappa B (NFκB) p65 phosphorylation but not signal transducer and activator of transcription 1 (STAT1) phosphorylation. Collectively, our results suggested that topical administration of Spry2 inhibitors may efficiently resolve inflammation in periodontal disease as macrophage-based anti-inflammatory immunotherapy and may create a suitable environment for periodontal wound healing. These in vitro findings provide a molecular basis for new therapeutic approaches in periodontal tissue regeneration.

  1. Biochemical characterization of the arginine-specific proteases of Porphyromonas gingivalis W50 suggests a common precursor.

    PubMed Central

    Rangarajan, M; Smith, S J; U, S; Curtis, M A

    1997-01-01

    Extracellular proteases of Porphyromonas gingivalis specific for arginyl peptide bonds are considered to be important virulence factors in periodontal disease. In order to determine the number, inter-relationship and kinetic properties of these proteases, extracellular enzymes with this peptide-bond specificity were purified and characterized from P. gingivalis W50. Three forms, which we denote RI, RI-A and RI-B, accounted for all of the activity in the supernatant. All three enzymes contain an alpha chain of approximately 54 kDa with the same N-terminal amino acid sequence. RI is a heterodimer of non-covalently linked alpha and beta chains which migrate to the same position on SDS/PAGE but which can be resolved by 8 M urea/PAGE. RI-A and RI-B are both monomeric, but the molecular mass of RI-B (70-80 kDa) is significantly increased due to post-translational modification with lipopolysaccharide. All forms show absolute specificity for peptide bonds with Arg in the P1 position and are also capable of hydrolysing N-terminal Arg and C-terminal Arg-Arg peptide bonds. Thus they show limited amino- and carboxy-peptidase activity. For the hydrolysis of Nalpha-benzoyl-L-Arg-p-nitroanilide, the pH optimum is 8.0 at 30 degrees C. The Vmax for all three enzymes is controlled by ionization of two residues with apparent pKas at 30 degrees C of 6. 5+/-0.05 and 9.7+/-0.05, and DeltaH values of approximately 29 kJ/mol and approximately 24 kJ/mol in the enzyme-substrate complex. By analogy with papain, the pKa of 6.5 could be ascribed to a Cys and the pKa of 9.7 to a His residue. E-64 [L-trans-epoxysuccinyl-leucylamide-4-(4-guanidino)butane] is a competitive inhibitor of RI, RI-A and RI-B. Based on physical properties and kinetic behaviour, RI-A appears to be analogous to gingipain from P. gingivalis HG66. However the alpha/beta structure of RI differs significantly from that of the high-molecular-mass multimeric complex of gingipain containing four haemagglutinins described by

  2. Inhibition of in vitro adhesion and virulence of Porphyromonas gingivalis by aqueous extract and polysaccharides from Rhododendron ferrugineum L. A new way for prophylaxis of periodontitis?

    PubMed

    Löhr, G; Beikler, T; Hensel, A

    2015-12-01

    The effect of an aqueous extract from the leaves of Rhododendron ferrugineum (RF) was investigated for its capacity of inhibiting the adhesion of Porphyromonas gingivalis cells to epithelial buccal KB cells. RF was characterized by HPLC (12.1% taxifolin-3-O-β-l-arabinopyranoside, 1.6% hyperoside, 0.9% isoquercitrin, 1.6% chlorogenic acid and a tannin content of 8.7%). Additionally raw polysaccharides (RPS) were obtained from the leaves of R. ferrugineum by aqueous extraction. RF and RPS interacted in a dose-dependent manner (max. 25% reduction at 1mg/ml each) with the adhesion of P. gingivalis by influencing bacterial outer membrane proteins. On protein level a time- and concentration-dependent inhibition of Arg-gingipain activity by RF was observed, while the Lys-gingipain activity remained unaltered. In addition, RF and RPS inhibited the bacterial hemagglutinin. RF affected the P. gingivalis adhesion also by interacting with KB cells in pre-incubation assays of the eukaryotic host cells, leading to reduced bacterial adhesion of about 75%. Gene expression analysis by RT-PCR indicated significant downregulation for arginine-specific gingipain rgpA by RF, while lysin-specific gingipain kgp and fimbrillinA fimA were strongly upregulated. Moreover, pre-incubation with RF abolished the P. gingivalis induced expression of IL-1β, IL-6, IL-8 and TNFα in KB cells. Results of this study indicate that an aqueous extract from R. ferrugineum combines cytoprotective and antimicrobial effects by both downregulating the expression of pro-inflammatory genes and inhibiting the adhesion of P. gingivalis. Thus RF may be potential candidate for the development of an adjunctive antimicrobial approach in the prevention of periodontal diseases.

  3. Role of gallium and silver from phosphate-based glasses on in vitro dual species oral biofilm models of Porphyromonas gingivalis and Streptococcus gordonii.

    PubMed

    Valappil, Sabeel P; Coombes, Marc; Wright, Lucy; Owens, Gareth J; Lynch, Richard J M; Hope, Christopher K; Higham, Susan M

    2012-05-01

    Phosphate-based glasses (PBGs) are excellent controlled delivery agents for antibacterial ions such as silver and gallium. The aim of this study was to assess the potential utility of novel PBGs combining both gallium and silver for use in periodontal therapy. To this end, an in vitro biofilm model with the putative periodontal pathogen, Porphyromonas gingivalis, and an initial colonizer, Streptococcus gordonii, was established. The effect of increasing calcium content in gallium-silver-doped PBG on the susceptibility of P. gingivalis was examined. A decrease in degradation rates (30.34, 25.19, 21.40 μg mm(-2) h(-1)) with increasing PBG calciumcontent (10, 11, 12 mol.% respectively) was observed, correlating well with gallium and silver ion release and antimicrobial activity against planktonic P. gingivalis (approximately 5.4log(10) colony-forming units (CFU) reduction after 24h by the C10 glass compared with controls) and S. gordonii (total growth inhibition after 32h by C10, C11 and C12 glasses compared with controls). The most potent PBG (C10) was evaluated for its ability to inhibit the biofilm growth of P. gingivalis in a newly established constant-depth film fermentor model. The simultaneous release of silver and gallium from the glass reduced P. gingivalis biofilm growth with a maximum effect (1.92log(10) CFU reduction) after 168 h. Given the emergence of antibiotic-resistant bacteria and dearth of new antibiotics in development, the glasses, especially C10, would offer effective alternatives to antibiotics or may complement current therapies through controlled, localized delivery of gallium and silver ions at infected sites in the oral cavity.

  4. Synergic phototoxic effect of visible light or Gallium-Arsenide laser in the presence of different photo-sensitizers on Porphyromonas gingivalis and Fusobacterium nucleatum

    PubMed Central

    Ghanbari, Habibollah; Mousavi, Seyed Amir; Forouzanfar, Ali; Zakeri, Mahdi; Shafaee, Hooman; Shahnaseri, Shirin

    2015-01-01

    Background: According to the development of resistant strains of pathogenic bacteria following treatment with antimicrobial chemotherapeutic agents, alternative approaches such as lethal photosensitization are being used. The aim of this study was to evaluate the effect of visible light and laser beam radiation in conjugation with three different photosensitizers on the survival of two main periodontopathogenic bacteria including Porphyromonas gingivalis and Fusobacterium nucleatum in different exposure periods. Materials and Methods: In this in vitro prospective study, strains of P. gingivalis and F. nucleatum. were exposed to visible light at wavelengths of 440 nm and diode laser light, Gallium-Arsenide, at wavelength of 830 nm in the presence of a photosensitizer (erythrosine, curcuma, or hydrogen peroxide). They were exposed 1-5 min to each light. Each experiment was repeated 3 times for each strain of bacteria. Data were analyzed by two-ways ANOVA and least significant difference post-hoc tests. P < 0.05 was considered as significant. After 4 days the colonies were counted. Results: Viability of P. gingivalis was reduced 10% and 20% subsequent to exposure to visible light and diode laser, respectively. The values were 65% and 75% for F. nucleatum in a period of 5-min, respectively. Exposure to visible light or laser beam in conjugation with the photosensitizers suspension caused significant reduction in the number of P. gingivalis in duration of 5-min, suggesting a synergic phototoxic effect. However, the survival rate of F. nucleatum following the exposure to laser with hydrogen peroxide, erythrosine and rhizome of Curcuma longa (curcumin) after 5-min was 10%, 20% and 90% respectively. Conclusion: Within the limitations of this study, the synergic phototoxic effect of visible light in combination with each of the photosensitizers on P. gingivalis and F. nucleatum. However, the synergic phototoxic effect of laser exposure and hydrogen peroxide and curcumin as

  5. Role of gallium and silver from phosphate-based glasses on in vitro dual species oral biofilm models of Porphyromonas gingivalis and Streptococcus gordonii.

    PubMed

    Valappil, Sabeel P; Coombes, Marc; Wright, Lucy; Owens, Gareth J; Lynch, Richard J M; Hope, Christopher K; Higham, Susan M

    2012-05-01

    Phosphate-based glasses (PBGs) are excellent controlled delivery agents for antibacterial ions such as silver and gallium. The aim of this study was to assess the potential utility of novel PBGs combining both gallium and silver for use in periodontal therapy. To this end, an in vitro biofilm model with the putative periodontal pathogen, Porphyromonas gingivalis, and an initial colonizer, Streptococcus gordonii, was established. The effect of increasing calcium content in gallium-silver-doped PBG on the susceptibility of P. gingivalis was examined. A decrease in degradation rates (30.34, 25.19, 21.40 μg mm(-2) h(-1)) with increasing PBG calciumcontent (10, 11, 12 mol.% respectively) was observed, correlating well with gallium and silver ion release and antimicrobial activity against planktonic P. gingivalis (approximately 5.4log(10) colony-forming units (CFU) reduction after 24h by the C10 glass compared with controls) and S. gordonii (total growth inhibition after 32h by C10, C11 and C12 glasses compared with controls). The most potent PBG (C10) was evaluated for its ability to inhibit the biofilm growth of P. gingivalis in a newly established constant-depth film fermentor model. The simultaneous release of silver and gallium from the glass reduced P. gingivalis biofilm growth with a maximum effect (1.92log(10) CFU reduction) after 168 h. Given the emergence of antibiotic-resistant bacteria and dearth of new antibiotics in development, the glasses, especially C10, would offer effective alternatives to antibiotics or may complement current therapies through controlled, localized delivery of gallium and silver ions at infected sites in the oral cavity. PMID:22314314

  6. Enhancing Specific-Antibody Production to the ragB Vaccine with GITRL That Expand Tfh, IFN-γ+ T Cells and Attenuates Porphyromonas gingivalis Infection in Mice

    PubMed Central

    Su, Zhaoliang; Kong, Fanzhi; Shi, Xiaoju; Tong, Jia; Shen, Pei; Peng, Tianqing; Wang, Shengjun; Xu, Huaxi

    2013-01-01

    The outer membrane protein RagB is one of the major virulence factors of the periodontal pathogen Porphyromonas gingivalis (P. gingivalis). In order to induce protective immune response against P. gingivalis infection, an mGITRL gene-linked ragB DNA vaccine (pIRES-ragB-mGITRL ) was constructed. Six-week-old female BALB/c mice were immunized with pIRES-ragB-mGITRL through intramuscular injection and then challenged by subcutaneous injection in the abdomen with P. gingivalis. RagB-specific antibody-forming cells were evaluated by an Enzyme-linked immunosorbent spot, and specific antibody was determined by enzyme-linked immunosorbent assay. In addition, the frequencies of Tfh and IFN-γ+ T cells in spleen were measured using flow cytometer, and the levels of IL-21 and IFN-γ mRNA or proteins were detected by real time RT-PCR or ELISA. The data showed that the mGITRL-linked ragB DNA vaccine induced higher levels of RagB-specific IgG in serum and RagB-specific antibody-forming cells in spleen. The frequencies of Tfh and IFN-γ+ T cells were obviously expanded in mice immunized by pIRES-ragB-mGITRL compared with other groups (pIRES or pIRES-ragB ). The levels of Tfh and IFN-γ+ T cells associated cytokines were also significantly increased in pIRES-ragB-mGITRL group. Therefore, the mice immunized with ragB plus mGITRL showed the stronger resistant to P. gingivalis infection and a significant reduction of the lesion size caused by P. gingivalis infection comparing with other groups. Taken together, our findings demonstrated that intramuscular injection of DNA vaccine ragB together with mGITRL induced protective immune response dramatically by increasing Tfh and IFN-γ+ T cells and antibody production to P. gingivalis. PMID:23560053

  7. Analytical performance of an immunologic-based periodontal bacterial test for simultaneous detection and differentiation of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia.

    PubMed

    Snyder, B; Ryerson, C C; Corona, H; Grogan, E A; Reynolds, H S; Contestable, P B; Boyer, B P; Mayer, J; Mangan, T; Norkus, N; Zambon, J J; Genco, R J

    1996-05-01

    The analytical performance of a membrane-based immunoassay for the simultaneous detection and differentiation of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia (including Prevotella nigrescens) was investigated. Positive reactions were observed for 71 of 71 reference strains and recent oral isolates of A. actinomycetemcomitans, P. gingivalis, and P. intermedia. No cross-reactivity was observed with 39 other common oral and environmental species. The specificity of the test was unaffected by the presence of potential oral interferents including whole blood, white blood cells, mucin, saliva, toothpastes, and oral rinses. A proficiency test by dental professionals using a standardized set of unknown simulated samples yielded a sensitivity of 97% (116/120) and a 100% specificity (240/ 240). An additional group including dental professionals and high school students was shown to be 99% proficient (1385/1397) in distinguishing proper from improper test function when processing control samples with normal test devices and devices with simulated error conditions. Comparisons to a culture standard for 104 subgingival plaque samples collected from 26 adult periodontitis patients yielded > 98% specificity for each of the test bacteria. In addition, the detection threshold for the test was determined to be equivalent to 10(4) cultivable test bacteria when compared to the culture standard. The data indicate that this membrane immunoassay is a valid and easy-to-use method for the detection of A. actinomycetemcomitans, P. gingivalis, and P. intermedia in subgingival plaque, at levels above the detection threshold of the test.

  8. Anti-Inflammatory Effect of Heme Oxygenase-1 Toward Porphyromonas gingivalis Lipopolysaccharide in Macrophages Exposed to Gomisins A, G, and J

    PubMed Central

    Ryu, Eun Yeon; Park, Sun Young; Kim, Sun Gun; Park, Da Jung; Kang, Jum Soon; Kim, Young Hun; Seetharaman, Rajaseker

    2011-01-01

    Abstract Periodontitis, a chronic inflammatory periodontal disease that develops from gingivitis, is caused by periodontal pathogenic bacteria such as Porphyromonas gingivalis. Recent studies have focused on the antioxidant, anti–human immunodeficiency virus, anticarcinogenic, and anti-inflammatory properties of gomisins. However, the anti-inflammatory activities of gomisin plants through heme oxygenase-1 (HO-1) signals remain poorly defined. We found that gomisins' anti-inflammatory activity occurs via the induction of HO-1 expression. Gomisins G and J inhibit the production of the pro-inflammatory cytokines tumor necrosis factor-α, interleukin-1β, and interleukin-6 and also block nuclear factor-κB activation in Raw264.7 cells stimulated with P. gingivalis lipopolysaccharide. Furthermore, pro-inflammatory cytokine production is inhibited through the induction of HO-1 expression. HO-1 expression is induced by all gomisins, but their anti-inflammatory activity via HO-1 signaling is observed with gomisins G and J, and not A. We found that gomisins G and J extracted from Schisandria chinensis can inhibit the P. gingivalis lipopolysaccharide induced-inflammatory responses in Raw264.7 cells. PMID:22145771

  9. Porphyromonas gingivalis Differentially Modulates Cell Death Profile in Ox-LDL and TNF-α Pre-Treated Endothelial Cells

    PubMed Central

    Bugueno, Isaac Maximiliano; Khelif, Yacine; Seelam, Narendra; Morand, David-Nicolas; Tenenbaum, Henri; Davideau, Jean-Luc; Huck, Olivier

    2016-01-01

    Objective Clinical studies demonstrated a potential link between atherosclerosis and periodontitis. Porphyromonas gingivalis (Pg), one of the main periodontal pathogen, has been associated to atheromatous plaque worsening. However, synergism between infection and other endothelial stressors such as oxidized-LDL or TNF-α especially on endothelial cell (EC) death has not been investigated. This study aims to assess the role of Pg on EC death in an inflammatory context and to determine potential molecular pathways involved. Methods Human umbilical vein ECs (HUVECs) were infected with Pg (MOI 100) or stimulated by its lipopolysaccharide (Pg-LPS) (1μg/ml) for 24 to 48 hours. Cell viability was measured with AlamarBlue test, type of cell death induced was assessed using Annexin V/propidium iodide staining. mRNA expression regarding caspase-1, -3, -9, Bcl-2, Bax-1 and Apaf-1 has been evaluated with RT-qPCR. Caspases enzymatic activity and concentration of APAF-1 protein were evaluated to confirm mRNA results. Results Pg infection and Pg-LPS stimulation induced EC death. A cumulative effect has been observed in Ox-LDL pre-treated ECs infected or stimulated. This effect was not observed in TNF-α pre-treated cells. Pg infection promotes EC necrosis, however, in infected Ox-LDL pre-treated ECs, apoptosis was promoted. This effect was not observed in TNF-α pre-treated cells highlighting specificity of molecular pathways activated. Regarding mRNA expression, Pg increased expression of pro-apoptotic genes including caspases-1,-3,-9, Bax-1 and decreased expression of anti-apoptotic Bcl-2. In Ox-LDL pre-treated ECs, Pg increased significantly the expression of Apaf-1. These results were confirmed at the protein level. Conclusion This study contributes to demonstrate that Pg and its Pg-LPS could exacerbate Ox-LDL and TNF-α induced endothelial injury through increase of EC death. Interestingly, molecular pathways are differentially modulated by the infection in function of the

  10. Morbidly obese patient with non-alcoholic steatohepatitis-related cirrhosis who died from sepsis caused by dental infection of Porphyromonas gingivalis: A case report.

    PubMed

    Omura, Yuno; Kitamoto, Mikiya; Hyogo, Hideyuki; Yamanoue, Takao; Tada, Yoshihiro; Boku, Noriko; Nishisaka, Takashi; Miyauchi, Mutsumi; Takata, Takashi; Chayama, Kazuaki

    2016-03-01

    Non-alcoholic steatohepatitis (NASH) is associated with increased risks of developing lifestyle-related diseases including type 2 diabetes, cardiovascular disease and cerebral vessel disease. While the two-hit hypothesis and, recently, multiple parallel hits hypothesis of NASH pathogenesis were proposed, further details have not emerged. Recently, dental infection of Porphyromonas gingivalis (P. gingivalis) has been reported as a critical risk factor for NASH progression, which acts as multiple parallel hits to induce inflammation and fibrogenic responses in steatosis. We describe here a 54-year-old woman who died from sepsis and was diagnosed with NASH. Briefly, her body mass index (BMI) at the age of 35 years old had been 25.6 kg/m(2) , but she became obese after withdrawing into her home at the age of 45 years. Severe obesity continued over 19 years without diabetes mellitus. She was admitted to our hospital due to a sudden disturbance of consciousness. On admission, her BMI was 48.5 kg/m(2) . Computed tomography revealed cirrhotic liver with massive ascites, and laboratory data indicated increased inflammatory responses, renal failure and C grade Child-Pugh classification, suggesting the diagnosis of sepsis. Also, severe periodontal disease was present, because the patient's front teeth fell out easily during intubation. Although the focus of infection was not specified, the oral flora Parvimonas micra, a periodontal pathogen, was detected in venous blood. In spite of intensive care including artificial respiration management and continuous hemodiafiltration, she died on the 43rd day after admission. Surprisingly, P. gingivalis was detected in her hepatocytes. This case may represent the significance of P. gingivalis in the progress to cirrhosis in NASH patients. PMID:25943712

  11. Identification of ragAB as a temperature-regulated operon of Porphyromonas gingivalis W50 using differential display of randomly primed RNA.

    PubMed

    Bonass, W A; Marsh, P D; Percival, R S; Aduse-Opoku, J; Hanley, S A; Devine, D A; Curtis, M A

    2000-07-01

    Porphyromonas gingivalis is a gram-negative, black-pigmented anaerobe that has been associated with advanced periodontal disease. The genome of P. gingivalis has the potential to produce a number of virulence determinants including proteases, hemagglutinins, hemolysin, invasion-associated proteins, and products of the pathogenicity island ragAB; however, little is known about how their expression is controlled. Periodontal pockets experience a higher temperature during inflammation, and this elevated temperature may influence the pathogenicity of P. gingivalis by changing its patterns of gene expression. In this study, RNA has been isolated from cells of P. gingivalis grown to steady state at temperatures of 37, 39, and 41 degrees C under hemin excess conditions (pH 7.0) in a chemostat. The RNA was subjected to PCR amplification following reverse transcription, using various combinations of randomly selected oligonucleotide primers. Reproducible RNA fingerprints have been obtained; however, differences were demonstrated in the RNA profiles of cells grown at the three temperatures, indicating differences in gene expression. Several PCR fragments were isolated that appeared to represent temperature-regulated genes. The nucleotide sequence of one of these has been identified as part of the ragAB locus, which codes for both a 55-kDa immunodominant antigen (RagB) and a homologue of the family of TonB-linked outer membrane receptors (RagA). These data indicate that expression of ragAB may be modulated in response to changes in temperature and that this may suggest a mechanism of evading the host response in the inflamed periodontal pocket.

  12. The GroEL protein of Porphyromonas gingivalis regulates atherogenic phenomena in endothelial cells mediated by upregulating toll-like receptor 4 expression.

    PubMed

    Huang, Chun-Yao; Shih, Chun-Ming; Tsao, Nai-Wen; Lin, Yi-Wen; Shih, Chun-Che; Chiang, Kuang-Hsing; Shyue, Song-Kun; Chang, Yu-Jia; Hsieh, Chi-Kun; Lin, Feng-Yen

    2016-01-01

    Porphyromonas gingivalis (P. gingivalis) is a bacterial species that causes periodontitis. GroEL from P. gingivalis may possess biological activity and may be involved in the destruction of periodontal tissues. However, it is unclear whether P. gingivalis GroEL enhances the appearance of atherogenic phenomena in endothelial cells and vessels. Here, we constructed recombinant GroEL from P. gingivalis to investigate its effects in human coronary artery endothelial cells (HCAECs) in vitro and on aortas of high-cholesterol (HC)-fed B57BL/6 and B57BL/6-Tlr4(lps-del) mice in vivo. The results showed that GroEL impaired tube-formation capacity under non-cytotoxic conditions in HCAECs. GroEL increased THP-1 cell/HCAEC adhesion by increasing the expression of intracellular adhesion molecule (ICAM)-1 and vascular adhesion molecule (VCAM)-1 in endothelial cells. Additionally, GroEL increased DiI-oxidized low density lipoprotein (oxLDL) uptake, which may be mediated by elevated lectin-like oxLDL receptor (LOX)-1 but not scavenger receptor expressed by endothelial cells (SREC) and scavenger receptor class B1 (SR-B1) expression. Furthermore, GroEL interacts with toll-like receptor 4 (TLR4) and plays a causal role in atherogenesis in HCAECs. Human antigen R (HuR), an RNA-binding protein with a high affinity for the 3' untranslated region (3'UTR) of TLR4 mRNA, contributes to the up-regulation of TLR4 induced by GroEL in HCAECs. In a GroEL animal administration study, GroEL elevated ICAM-1, VCAM-1, LOX-1 and TLR4 expression in the aortas of HC diet-fed wild C57BL/6 but not C57BL/6-Tlr4(lps-del) mice. Taken together, our findings suggest that P. gingivalis GroEL may contribute to cardiovascular disorders by affecting TLR4 expression. PMID:27158334

  13. The GroEL protein of Porphyromonas gingivalis regulates atherogenic phenomena in endothelial cells mediated by upregulating toll-like receptor 4 expression

    PubMed Central

    Huang, Chun-Yao; Shih, Chun-Ming; Tsao, Nai-Wen; Lin, Yi-Wen; Shih, Chun-Che; Chiang, Kuang-Hsing; Shyue, Song-Kun; Chang, Yu-Jia; Hsieh, Chi-Kun; Lin, Feng-Yen

    2016-01-01

    Porphyromonas gingivalis (P. gingivalis) is a bacterial species that causes periodontitis. GroEL from P. gingivalis may possess biological activity and may be involved in the destruction of periodontal tissues. However, it is unclear whether P. gingivalis GroEL enhances the appearance of atherogenic phenomena in endothelial cells and vessels. Here, we constructed recombinant GroEL from P. gingivalis to investigate its effects in human coronary artery endothelial cells (HCAECs) in vitro and on aortas of high-cholesterol (HC)-fed B57BL/6 and B57BL/6-Tlr4lps-del mice in vivo. The results showed that GroEL impaired tube-formation capacity under non-cytotoxic conditions in HCAECs. GroEL increased THP-1 cell/HCAEC adhesion by increasing the expression of intracellular adhesion molecule (ICAM)-1 and vascular adhesion molecule (VCAM)-1 in endothelial cells. Additionally, GroEL increased DiI-oxidized low density lipoprotein (oxLDL) uptake, which may be mediated by elevated lectin-like oxLDL receptor (LOX)-1 but not scavenger receptor expressed by endothelial cells (SREC) and scavenger receptor class B1 (SR-B1) expression. Furthermore, GroEL interacts with toll-like receptor 4 (TLR4) and plays a causal role in atherogenesis in HCAECs. Human antigen R (HuR), an RNA-binding protein with a high affinity for the 3’ untranslated region (3’UTR) of TLR4 mRNA, contributes to the up-regulation of TLR4 induced by GroEL in HCAECs. In a GroEL animal administration study, GroEL elevated ICAM-1, VCAM-1, LOX-1 and TLR4 expression in the aortas of HC diet-fed wild C57BL/6 but not C57BL/6-Tlr4lps-del mice. Taken together, our findings suggest that P. gingivalis GroEL may contribute to cardiovascular disorders by affecting TLR4 expression. PMID:27158334

  14. Myxomavirus anti-inflammatory chemokine binding protein reduces the increased plaque growth induced by chronic Porphyromonas gingivalis oral infection after balloon angioplasty aortic injury in mice.

    PubMed

    Lucas, Alexandra R; Verma, Raj K; Dai, Erbin; Liu, Liying; Chen, Hao; Kesavalu, Sheela; Rivera, Mercedes; Velsko, Irina; Ambadapadi, Sriram; Chukkapalli, Sasanka; Kesavalu, Lakshmyya

    2014-01-01

    Thrombotic occlusion of inflammatory plaque in coronary arteries causes myocardial infarction. Treatment with emergent balloon angioplasty (BA) and stent implant improves survival, but restenosis (regrowth) can occur. Periodontal bacteremia is closely associated with inflammation and native arterial atherosclerosis, with potential to increase restenosis. Two virus-derived anti-inflammatory proteins, M-T7 and Serp-1, reduce inflammation and plaque growth after BA and transplant in animal models through separate pathways. M-T7 is a broad spectrum C, CC and CXC chemokine-binding protein. Serp-1 is a serine protease inhibitor (serpin) inhibiting thrombotic and thrombolytic pathways. Serp-1 also reduces arterial inflammation and improves survival in a mouse herpes virus (MHV68) model of lethal vasculitis. In addition, Serp-1 demonstrated safety and efficacy in patients with unstable coronary disease and stent implant, reducing markers of myocardial damage. We investigate here the effects of Porphyromonas gingivalis, a periodontal pathogen, on restenosis after BA and the effects of blocking chemokine and protease pathways with M-T7 and Serp-1. ApoE-/- mice had aortic BA and oral P. gingivalis infection. Arterial plaque growth was examined at 24 weeks with and without anti-inflammatory protein treatment. Dental plaques from mice infected with P. gingivalis tested positive for infection. Neither Serp-1 nor M-T7 treatment reduced infection, but IgG antibody levels in mice treated with Serp-1 and M-T7 were reduced. P. gingivalis significantly increased monocyte invasion and arterial plaque growth after BA (P<0.025). Monocyte invasion and plaque growth were blocked by M-T7 treatment (P<0.023), whereas Serp-1 produced only a trend toward reductions. Both proteins modified expression of TLR4 and MyD88. In conclusion, aortic plaque growth in ApoE-/- mice increased after angioplasty in mice with chronic oral P. gingivalis infection. Blockade of chemokines, but not serine

  15. Determination of antibacterial activity of green coffee bean extract on periodontogenic bacteria like Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans: An in vitro study

    PubMed Central

    Bharath, Nagaraj; Sowmya, Nagur Karibasappa; Mehta, Dhoom Singh

    2015-01-01

    Background: The aim of this study was to evaluate the antibacterial activity of pure green coffee bean extract on periodonto pathogenic bacteria Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Fusobacterium nucleatum (Fn) and Aggregatibacter actinomycetemcomitans (Aa). Materials and Methods: Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBC) were used to assess the antibacterial effect of pure green coffee bean extract against periodonto pathogenic bacteria by micro dilution method and culture method, respectively. Results: MIC values of Pg, Pi and Aa were 0.2 μg/ml whereas Fn showed sensitive at concentration of 3.125 μg/ml. MBC values mirrors the values same as that of MIC. Conclusion: Antimicrobial activity of pure green coffee bean extract against Pg, Pi, Fn and Aa suggests that it could be recommended as an adjunct to mechanical therapy in the management of periodontal disease. PMID:26097349

  16. The outer-membrane export signal of Porphyromonas gingivalis type IX secretion system (T9SS) is a conserved C-terminal β-sandwich domain

    PubMed Central

    de Diego, Iñaki; Ksiazek, Miroslaw; Mizgalska, Danuta; Koneru, Lahari; Golik, Przemyslaw; Szmigielski, Borys; Nowak, Magdalena; Nowakowska, Zuzanna; Potempa, Barbara; Houston, John A.; Enghild, Jan J.; Thøgersen, Ida B.; Gao, Jinlong; Kwan, Ann H.; Trewhella, Jill; Dubin, Grzegorz; Gomis-Rüth, F. Xavier; Nguyen, Ky-Anh; Potempa, Jan

    2016-01-01

    In the recently characterized Type IX Secretion System (T9SS), the conserved C-terminal domain (CTD) in secreted proteins functions as an outer membrane translocation signal for export of virulence factors to the cell surface in the Gram-negative Bacteroidetes phylum. In the periodontal pathogen Porphyromonas gingivalis, the CTD is cleaved off by PorU sortase in a sequence-independent manner, and anionic lipopolysaccharide (A-LPS) is attached to many translocated proteins, thus anchoring them to the bacterial surface. Here, we solved the atomic structure of the CTD of gingipain B (RgpB) from P. gingivalis, alone and together with a preceding immunoglobulin-superfamily domain (IgSF). The CTD was found to possess a typical Ig-like fold encompassing seven antiparallel β-strands organized in two β-sheets, packed into a β-sandwich structure that can spontaneously dimerise through C-terminal strand swapping. Small angle X-ray scattering (SAXS) revealed no fixed orientation of the CTD with respect to the IgSF. By introducing insertion or substitution of residues within the inter-domain linker in the native protein, we were able to show that despite the region being unstructured, it nevertheless is resistant to general proteolysis. These data suggest structural motifs located in the two adjacent Ig-like domains dictate the processing of CTDs by the T9SS secretion pathway. PMID:27005013

  17. A Porphyromonas gingivalis Mutant Defective in a Putative Glycosyltransferase Exhibits Defective Biosynthesis of the Polysaccharide Portions of Lipopolysaccharide, Decreased Gingipain Activities, Strong Autoaggregation, and Increased Biofilm Formation▿ †

    PubMed Central

    Yamaguchi, Mikiyo; Sato, Keiko; Yukitake, Hideharu; Noiri, Yuichiro; Ebisu, Shigeyuki; Nakayama, Koji

    2010-01-01

    The Gram-negative anaerobic bacterium Porphyromonas gingivalis is a major pathogen in periodontal disease, one of the biofilm-caused infectious diseases. The bacterium possesses potential virulence factors, including fimbriae, proteinases, hemagglutinin, lipopolysaccharide (LPS), and outer membrane vesicles, and some of these factors are associated with biofilm formation; however, the precise mechanism of biofilm formation is still unknown. Colonial pigmentation of the bacterium on blood agar plates is related to its virulence. In this study, we isolated a nonpigmented mutant that had an insertion mutation within the new gene PGN_1251 (gtfB) by screening a transposon insertion library. The gene shares homology with genes encoding glycosyltransferase 1 of several bacteria. The gtfB mutant was defective in biosynthesis of both LPSs containing O side chain polysaccharide (O-LPS) and anionic polysaccharide (A-LPS). The defect in the gene resulted in a complete loss of surface-associated gingipain proteinases, strong autoaggregation, and a marked increase in biofilm formation, suggesting that polysaccharide portions of LPSs influence attachment of gingipain proteinases to the cell surface, autoaggregation, and biofilm formation of P. gingivalis. PMID:20624909

  18. Structure of the fimbrial protein Mfa4 from Porphyromonas gingivalis in its precursor form: implications for a donor-strand complementation mechanism.

    PubMed

    Kloppsteck, Patrik; Hall, Michael; Hasegawa, Yoshiaki; Persson, Karina

    2016-01-01

    Gingivitis and periodontitis are chronic inflammatory diseases that can lead to tooth loss. One of the causes of these diseases is the Gram-negative Porphyromonas gingivalis. This periodontal pathogen is dependent on two fimbriae, FimA and Mfa1, for binding to dental biofilm, salivary proteins, and host cells. These fimbriae are composed of five proteins each, but the fimbriae assembly mechanism and ligands are unknown. Here we reveal the crystal structure of the precursor form of Mfa4, one of the accessory proteins of the Mfa1 fimbria. Mfa4 consists of two β-sandwich domains and the first part of the structure forms two well-defined β-strands that run over both domains. This N-terminal region is cleaved by gingipains, a family of proteolytic enzymes that encompass arginine- and lysine-specific proteases. Cleavage of the N-terminal region generates the mature form of the protein. Our structural data allow us to propose that the new N-terminus of the mature protein may function as a donor strand in the polymerization of P. gingivalis fimbriae. PMID:26972441

  19. Structure of the fimbrial protein Mfa4 from Porphyromonas gingivalis in its precursor form: implications for a donor-strand complementation mechanism

    PubMed Central

    Kloppsteck, Patrik; Hall, Michael; Hasegawa, Yoshiaki; Persson, Karina

    2016-01-01

    Gingivitis and periodontitis are chronic inflammatory diseases that can lead to tooth loss. One of the causes of these diseases is the Gram-negative Porphyromonas gingivalis. This periodontal pathogen is dependent on two fimbriae, FimA and Mfa1, for binding to dental biofilm, salivary proteins, and host cells. These fimbriae are composed of five proteins each, but the fimbriae assembly mechanism and ligands are unknown. Here we reveal the crystal structure of the precursor form of Mfa4, one of the accessory proteins of the Mfa1 fimbria. Mfa4 consists of two β-sandwich domains and the first part of the structure forms two well-defined β-strands that run over both domains. This N-terminal region is cleaved by gingipains, a family of proteolytic enzymes that encompass arginine- and lysine-specific proteases. Cleavage of the N-terminal region generates the mature form of the protein. Our structural data allow us to propose that the new N-terminus of the mature protein may function as a donor strand in the polymerization of P. gingivalis fimbriae. PMID:26972441

  20. Differential quantitative proteomics of Porphyromonas gingivalis by linear ion trap mass spectrometry: Non-label methods comparison, q-values and LOWESS curve fitting

    NASA Astrophysics Data System (ADS)

    Xia, Qiangwei; Wang, Tiansong; Park, Yoonsuk; Lamont, Richard J.; Hackett, Murray

    2007-01-01

    Differential analysis of whole cell proteomes by mass spectrometry has largely been applied using various forms of stable isotope labeling. While metabolic stable isotope labeling has been the method of choice, it is often not possible to apply such an approach. Four different label free ways of calculating expression ratios in a classic "two-state" experiment are compared: signal intensity at the peptide level, signal intensity at the protein level, spectral counting at the peptide level, and spectral counting at the protein level. The quantitative data were mined from a dataset of 1245 qualitatively identified proteins, about 56% of the protein encoding open reading frames from Porphyromonas gingivalis, a Gram-negative intracellular pathogen being studied under extracellular and intracellular conditions. Two different control populations were compared against P. gingivalis internalized within a model human target cell line. The q-value statistic, a measure of false discovery rate previously applied to transcription microarrays, was applied to proteomics data. For spectral counting, the most logically consistent estimate of random error came from applying the locally weighted scatter plot smoothing procedure (LOWESS) to the most extreme ratios generated from a control technical replicate, thus setting upper and lower bounds for the region of experimentally observed random error.

  1. Pathogenesis of periodontitis: a major arginine-specific cysteine proteinase from Porphyromonas gingivalis induces vascular permeability enhancement through activation of the kallikrein/kinin pathway.

    PubMed Central

    Imamura, T; Pike, R N; Potempa, J; Travis, J

    1994-01-01

    To elucidate the mechanism of production of an inflammatory exudate, gingival crevicular fluid (GCF), from periodontal pockets in periodontitis, we examined the vascular permeability enhancement (VPE) activity induced by an arginine-specific cysteine proteinase, Arg-gingipain-1 (RGP-1), produced by a major periopathogenic bacterium, Porphyromonas gingivalis. Intradermal injections into guinea pigs of RGP-1 (> 10(-8) M), or human plasma incubated with RGP-1 (> 10(-9) M), induced VPE in a dose- and activity-dependent manner but with different time courses for the two routes of production. VPE activity induced by RGP-1 was augmented by kininase inhibitors, inhibited by a kallikrein inhibitor and unaffected by an antihistamine drug. The VPE activity in human plasma incubated with RGP-1 also correlated closely with generation of bradykinin (BK). RGP-1 induced 30-40% less VPE activity in Hageman factor-deficient plasma and no VPE in plasma deficient in either prekallikrein (PK) or high molecular weight kininogen (HMWK). After incubation with RGP-1, plasma deficient in PK or HMWK, reconstituted with each missing protein, caused VPE, as did a mixture of purified PK and HMWK, but RGP-1 induced no VPE from HMWK. The VPE of extracts of clinically isolated P. gingivalis were reduced to about 10% by anti-RGP-1-IgG, leupeptin, or tosyl-L-lysine chloromethyl ketone, which paralleled effects observed with RGP-1. These results indicate that RGP-1 is the major VPE factor of P. gingivalis, inducing this activity through PK activation and subsequent BK release, resulting in GCF production at sites of periodontitis caused by infection with this organism. Images PMID:8040277

  2. Aerosolized clindamycin is superior to aerosolized dexamethasone or clindamycin-dexamethasone combination in the treatment of severe Porphyromonas gingivalis aspiration pneumonia in an experimental murine model.

    PubMed

    Nemec, Ana; Pavlica, Zlatko; Nemec-Svete, Alenka; Eržen, Damijan; Milutinović, Aleksandra; Petelin, Milan

    2012-02-01

    Adjunctive corticosteroid treatment to reduce excessive local inflammatory response in pneumonia is controversial. To study the effects of an early local adjunct dexamethasone treatment on the course of pneumonia and inflammatory/cytokine response, mice were intratracheally inoculated with live Porphyromonas gingivalis and treated with either clindamycin (C), dexamethasone (D), C+D combination, or were not treated (Pg). Six mice from each group were euthanized at 6, 24, 72, and 168 hours after inoculation. Levels of tumor necrosis factor (TNF)-α, soluble TNF-α receptors (sTNFR1 and sTNFR2), interleukin (IL)-1β, and IL-6 in the serum and lung-homogenate supernatant were determined. Lung samples were histopathologically assessed and all findings compared to those found in 24 sham-inoculated mice (phosphate-buffered saline [PBS]). Severe P. gingivalis-induced bronchopneumonia progressed from 24 hours, peaked at 72 hours, and resolved after 168 hours with changes in local and systemic cytokine levels. Clindamycin-treated mice developed only mild bronchopneumonia that resolved fast (72 hours) with an early (6-24 hours) normalization of local and systemic cytokine levels. Similar course of pneumonia and cytokine level changes were observed in mice treated with C+D, but later. Early (6-24 hours) local elevation of sTNFRs was observed in C and C+D groups of mice, whereas nontreated (Pg) mice had increased systemic sTNFRs. Severe bronchopneumonia with delayed resolution was observed in D-group mice, with an early local and systemic decrease in sTNFR1 and persistent elevation of local TNF-α. Clindamycin or a clindamycin-dexamethasone combination treatment significantly improves the course of P. gingivalis-aspiration pneumonia, but more so if clindamycin alone is used. A favorable course of pneumonia seems to be associated with an early elevation of sTNFRs and normalization of TNF-α.

  3. Anti-HmuY antibodies specifically recognize Porphyromonas gingivalis HmuY protein but not homologous proteins in other periodontopathogens.

    PubMed

    Śmiga, Michał; Bielecki, Marcin; Olczak, Mariusz; Smalley, John W; Olczak, Teresa

    2015-01-01

    Given the emerging evidence of an association between periodontal infections and systemic conditions, the search for specific methods to detect the presence of P. gingivalis, a principal etiologic agent in chronic periodontitis, is of high importance. The aim of this study was to characterize antibodies raised against purified P. gingivalis HmuY protein and selected epitopes of the HmuY molecule. Since other periodontopathogens produce homologs of HmuY, we also aimed to characterize responses of antibodies raised against the HmuY protein or its epitopes to the closest homologous proteins from Prevotella intermedia and Tannerella forsythia. Rabbits were immunized with purified HmuY protein or three synthetic, KLH-conjugated peptides, derived from the P. gingivalis HmuY protein. The reactivity of anti-HmuY antibodies with purified proteins or bacteria was determined using Western blotting and ELISA assay. First, we found homologs of P. gingivalis HmuY in P. intermedia (PinO and PinA proteins) and T. forsythia (Tfo protein) and identified corrected nucleotide and amino acid sequences of Tfo. All proteins were overexpressed in E. coli and purified using ion-exchange chromatography, hydrophobic chromatography and gel filtration. We demonstrated that antibodies raised against P. gingivalis HmuY are highly specific to purified HmuY protein and HmuY attached to P. gingivalis cells. No reactivity between P. intermedia and T. forsythia or between purified HmuY homologs from these bacteria and anti-HmuY antibodies was detected. The results obtained in this study demonstrate that P. gingivalis HmuY protein may serve as an antigen for specific determination of serum antibodies raised against this bacterium. PMID:25658942

  4. The Daiokanzoto (TJ-84) Kampo Formulation Reduces Virulence Factor Gene Expression in Porphyromonas gingivalis and Possesses Anti-Inflammatory and Anti-Protease Activities.

    PubMed

    Fournier-Larente, Jade; Azelmat, Jabrane; Yoshioka, Masami; Hinode, Daisuke; Grenier, Daniel

    2016-01-01

    Kampo formulations used in Japan to treat a wide variety of diseases and to promote health are composed of mixtures of crude extracts from the roots, bark, leaves, and rhizomes of a number of herbs. The present study was aimed at identifying the beneficial biological properties of Daiokanzoto (TJ-84), a Kampo formulation composed of crude extracts of Rhubarb rhizomes and Glycyrrhiza roots, with a view to using it as a potential treatment for periodontal disease. Daiokanzoto dose-dependently inhibited the expression of major Porphyromonas gingivalis virulence factors involved in host colonization and tissue destruction. More specifically, Daiokanzoto reduced the expression of the fimA, hagA, rgpA, and rgpB genes, as determined by quantitative real-time PCR. The U937-3xκB-LUC monocyte cell line transfected with a luciferase reporter gene was used to evaluate the anti-inflammatory properties of Daiokanzoto. Daiokanzoto attenuated the P. gingivalis-mediated activation of the NF-κB signaling pathway. It also reduced the secretion of pro-inflammatory cytokines (IL-6 and CXCL8) by lipopolysaccharide-stimulated oral epithelial cells and gingival fibroblasts. Lastly, Daiokanzoto, dose-dependently inhibited the catalytic activity of matrix metalloproteinases (-1 and -9). In conclusion, the present study provided evidence that Daiokanzoto shows potential for treating and/or preventing periodontal disease. The ability of this Kampo formulation to act on both bacterial pathogens and the host inflammatory response, the two etiological components of periodontal disease, is of high therapeutic interest. PMID:26859747

  5. Social stress enhances IL-1beta and TNF-alpha production by Porphyromonas gingivalis lipopolysaccharide-stimulated CD11b+ cells.

    PubMed

    Bailey, Michael T; Kinsey, Steven G; Padgett, David A; Sheridan, John F; Leblebicioglu, Binnaz

    2009-09-01

    Psychological stress is associated with an increased expression of markers of peripheral inflammation, and there is a growing literature describing a link between periodontal pathogens and systemic inflammation. The hypothesis of the present work is that exposing mice to the social stressor, called social disruption (SDR), would enhance the inflammatory response to lipopolysaccharide (LPS) derived from the oral pathogen, Porphyromonas gingivalis. Mice were exposed to SDR for 2h per day on 6 consecutive days. On the morning following the last cycle of SDR, mice were tested for anxiety-like behavior in the open field test and novel object test. The mice were sacrificed the following day and their spleens harvested. Spleen cells were stimulated with LPS derived from P. gingivalis in the absence or presence of increasing doses of corticosterone. Social disruption resulted in anxiety-like behavior, and the production of IL-1beta and TNF-alpha was significantly higher in spleen cells from mice exposed to SDR in comparison to levels from non-stressed control mice. In addition, the viability of spleen cells from mice exposed to SDR was significantly greater than the viability of cells from non-stressed control mice, even in the presence of high doses of corticosterone. The use of cultures enriched for CD11b+ cells indicated that the stressor was affecting the activity of splenic myeloid cells. This study demonstrates that social stress enhances the inflammatory response to an oral pathogen and could provide a critical clue in the reported associations between stress, inflammation, and oral pathogens.

  6. Porphyromonas gingivalis-specific serum IgG and IgA antibodies originate from immunoglobulin-secreting cells in inflamed gingiva.

    PubMed Central

    Ogawa, T; Kono, Y; McGhee, M L; McGhee, J R; Roberts, J E; Hamada, S; Kiyono, H

    1991-01-01

    Patients with adult periodontitis (AP) exhibit elevated serum antibody levels to Porphyromonas (Bacteroides) gingivalis; however, it is not known whether these antibodies originate from plasma cells in the local disease site or from peripheral lymphoid tissues. We studied the isotype and subclass levels and origin of antibodies to P. gingivalis fimbriae, since elevated serum anti-fimbriae responses were seen when compared with sera of healthy controls. IgG anti-fibriae titres were dominant and the subclass response was IgG3 much greater than IgG1 greater than IgG2 much greater than IgG4; however, some IgA anti-fimbriae antibodies were also seen. The IgA subclass fimbriae-specific response was mainly IgA1; however, significant IgA2 anti-fimbrae antibodies were seen. We also assessed numbers of anti-fimbriae antibody producing cells from peripheral blood mononuclear cells (PMBC) and from either healthy or inflamed gingiva of AP subjects. Gingival mononuclear cells (GMC) of AP patients exhibited high numbers of immunoglobulin-producing (spot-forming) cells (SFC) including fimbriae-specific antibody secreting cells in a pattern of IgG greater than IgA greater than greater than greater than IgM. However, low numbers of SFC were seen in GMC from healthy gingiva; further, no anti-fimbriae SFC responses were noted in healthy GMC. Although no fimbriae-specific immunoglobulin-producing cells were seen in PBMC, low numbers of antigen-specific SFC were found in pokeweed mitogen-triggered PBMC from AP subjects. Treatment of AP patients for plaque and surgical removal of inflamed gingiva resulted in significant reductions in serum anti-fimbriae responses. These studies show that AP patients exhibit brisk serum IgG and IgA subclass anti-fimbriae antibodies, whose origin appear to be the plasma cells present in the localized inflamed tissues. PMID:1671564

  7. Effects of immunization with Porphyromonas gingivalis and Prevotella intermedia on progression of ligature-induced periodontitis in the nonhuman primate Macaca fascicularis.

    PubMed Central

    Ebersole, J L; Brunsvold, M; Steffensen, B; Wood, R; Holt, S C

    1991-01-01

    The nonhuman primate (Nhp) has proven to be a useful model of human periodontitis. This study describes the immunological characteristics of this model and the ability of active immunization to interfere with ecological changes in the microbiota and its associated disease symptoms. Nhps were parenterally immunized with whole-cell antigens of Porphyromonas gingivalis and Prevotella intermedia. The immunization elicited an approximate 2-log increase in serum immunoglobulin G (IgG), IgM, and IgA isotype antibody that was highly specific for these immunogens. Postimmunization and postligation, there was minimal change in the levels of specific antibody. P. gingivalis immunization significantly inhibited the emergence of this species during disease progression. In contrast, induction of anti-P. intermedia antibody had a minimal effect on this species within the subgingival plaque. Plaque indices showed few changes that could be attributed to active immunization. Both bleeding on probing and loss of attachment were higher in ligated sites of immunized animals than in the placebo-treated group. A significant increase in bone density loss was observed in the ligated teeth from immunized versus control animals. These findings indicate that active immunization of Nhps can elicit a substantial systemic immune response; however, while this response may effect the emergence of an individual microorganism, it appears that other ecological considerations are critical in disease progression. It is also possible that the induction of a broad-based immune response to multiple bacterial antigens can result in increased disease, potentially associated with hypersensitivity reactions to the bacteria in the subgingival plaque. PMID:1894349

  8. Genome of the pathogen Porphyromonas gingivalis recovered from a biofilm in a hospital sink using a high-throughput single-cell genomics platform

    PubMed Central

    McLean, Jeffrey S.; Lombardo, Mary-Jane; Ziegler, Michael G.; Novotny, Mark; Yee-Greenbaum, Joyclyn; Badger, Jonathan H.; Tesler, Glenn; Nurk, Sergey; Lesin, Valery; Brami, Daniel; Hall, Adam P.; Edlund, Anna; Allen, Lisa Z.; Durkin, Scott; Reed, Sharon; Torriani, Francesca; Nealson, Kenneth H.; Pevzner, Pavel A.; Friedman, Robert; Venter, J. Craig; Lasken, Roger S.

    2013-01-01

    Although biofilms have been shown to be reservoirs of pathogens, our knowledge of the microbial diversity in biofilms within critical areas, such as health care facilities, is limited. Available methods for pathogen identification and strain typing have some inherent restrictions. In particular, culturing will yield only a fraction of the species present, PCR of virulence or marker genes is mainly focused on a handful of known species, and shotgun metagenomics is limited in the ability to detect strain variations. In this study, we present a single-cell genome sequencing approach to address these limitations and demonstrate it by specifically targeting bacterial cells within a complex biofilm from a hospital bathroom sink drain. A newly developed, automated platform was used to generate genomic DNA by the multiple displacement amplification (MDA) technique from hundreds of single cells in parallel. MDA reactions were screened and classified by 16S rRNA gene PCR sequence, which revealed a broad range of bacteria covering 25 different genera representing environmental species, human commensals, and opportunistic human pathogens. Here we focus on the recovery of a nearly complete genome representing a novel strain of the periodontal pathogen Porphyromonas gingivalis (P. gingivalis JCVI SC001) using the single-cell assembly tool SPAdes. Single-cell genomics is becoming an accepted method to capture novel genomes, primarily in the marine and soil environments. Here we show for the first time that it also enables comparative genomic analysis of strain variation in a pathogen captured from complex biofilm samples in a healthcare facility. PMID:23564253

  9. The Daiokanzoto (TJ-84) Kampo Formulation Reduces Virulence Factor Gene Expression in Porphyromonas gingivalis and Possesses Anti-Inflammatory and Anti-Protease Activities

    PubMed Central

    Fournier-Larente, Jade; Azelmat, Jabrane; Yoshioka, Masami; Hinode, Daisuke; Grenier, Daniel

    2016-01-01

    Kampo formulations used in Japan to treat a wide variety of diseases and to promote health are composed of mixtures of crude extracts from the roots, bark, leaves, and rhizomes of a number of herbs. The present study was aimed at identifying the beneficial biological properties of Daiokanzoto (TJ-84), a Kampo formulation composed of crude extracts of Rhubarb rhizomes and Glycyrrhiza roots, with a view to using it as a potential treatment for periodontal disease. Daiokanzoto dose-dependently inhibited the expression of major Porphyromonas gingivalis virulence factors involved in host colonization and tissue destruction. More specifically, Daiokanzoto reduced the expression of the fimA, hagA, rgpA, and rgpB genes, as determined by quantitative real-time PCR. The U937-3xκB-LUC monocyte cell line transfected with a luciferase reporter gene was used to evaluate the anti-inflammatory properties of Daiokanzoto. Daiokanzoto attenuated the P. gingivalis-mediated activation of the NF-κB signaling pathway. It also reduced the secretion of pro-inflammatory cytokines (IL-6 and CXCL8) by lipopolysaccharide-stimulated oral epithelial cells and gingival fibroblasts. Lastly, Daiokanzoto, dose-dependently inhibited the catalytic activity of matrix metalloproteinases (-1 and -9). In conclusion, the present study provided evidence that Daiokanzoto shows potential for treating and/or preventing periodontal disease. The ability of this Kampo formulation to act on both bacterial pathogens and the host inflammatory response, the two etiological components of periodontal disease, is of high therapeutic interest. PMID:26859747

  10. Kinetic Parameters and Cytotoxic Activity of Recombinant Methionine γ-Lyase from Clostridium tetani, Clostridium sporogenes, Porphyromonas gingivalis and Citrobacter freundii.

    PubMed

    Morozova, E A; Kulikova, V V; Yashin, D V; Anufrieva, N V; Anisimova, N Y; Revtovich, S V; Kotlov, M I; Belyi, Y F; Pokrovsky, V S; Demidkina, T V

    2013-07-01

    The steady-state kinetic parameters of pyridoxal 5'-phosphate-dependent recombinant methionine γ -lyase from three pathogenic bacteria, Clostridium tetani, Clostridium sporogenes, and Porphyromonas gingivalis, were determined in β- and γ-elimination reactions. The enzyme from C. sporogenes is characterized by the highest catalytic efficiency in the γ-elimination reaction of L-methionine. It was demonstrated that the enzyme from these three sources exists as a tetramer. The N-terminal poly-histidine fragment of three recombinant enzymes influences their catalytic activity and facilitates the aggregation of monomers to yield dimeric forms under denaturing conditions. The cytotoxicity of methionine γ-lyase from C. sporogenes and C. tetani in comparison with Citrobacter freundii was evaluated using K562, PC-3, LnCap, MCF7, SKOV-3, and L5178y tumor cell lines. K562 (IC50=0.4-1.3 U/ml), PC-3 (IC50=0.1-0.4 U/ml), and MCF7 (IC50=0.04-3.2 U/ml) turned out to be the most sensitive cell lines.

  11. CCL3 and CXCL12 production in vitro by dental pulp fibroblasts from permanent and deciduous teeth stimulated by Porphyromonas gingivalis LPS

    PubMed Central

    SIPERT, Carla Renata; MORANDINI, Ana Carolina de Faria; MODENA, Karin Cristina da Silva; DIONÍSIO, Thiago José; MACHADO, Maria Aparecida Andrade Moreira; de OLIVEIRA, Sandra Helena Penha; CAMPANELLI, Ana Paula; SANTOS, Carlos Ferreira

    2013-01-01

    Objective: The aim of this study was to compare the production of the chemokines CCL3 and CXCL12 by cultured dental pulp fibroblasts from permanent (PDPF) and deciduous (DDPF) teeth under stimulation by Porphyromonas gingivalis LPS (PgLPS). Material and Methods: Primary culture of fibroblasts from permanent (n=3) and deciduous (n=2) teeth were established using an explant technique. After the fourth passage, fibroblasts were stimulated by increasing concentrations of PgLPS (0 - 10 µg/mL) at 1, 6 and 24 h. The cells were tested for viability through MTT assay, and production of the chemokines CCL3 and CXCL12 was determined through ELISA. Comparisons among samples were performed using One-way ANOVA for MTT assay and Two-way ANOVA for ELISA results. Results: Cell viability was not affected by the antigen after 24 h of stimulation. PgLPS induced the production of CCL3 by dental pulp fibroblasts at similar levels for both permanent and deciduous pulp fibroblasts. Production of CXCL12, however, was significantly higher for PDPF than DDPF at 1 and 6 h. PgLPS, in turn, downregulated the production of CXCL12 by PDPF but not by DDPF. Conclusion: These data suggest that dental pulp fibroblasts from permanent and deciduous teeth may present a differential behavior under PgLPS stimulation. PMID:23739851

  12. Structural Significance of the β1K396 Residue Found in the Porphyromonas gingivalis Sialidase β-Propeller Domain: A Computational Study with Implications for Novel Therapeutics Against Periodontal Disease

    PubMed Central

    Kamio, Noriaki; Imai, Kenichi; Ohya, Manabu; Tamura, Muneaki

    2014-01-01

    Abstract Porphyromonas gingivalis sialidase activity is associated with virulence and initiated by sialic acid (SA) binding to the β-propeller domain (BPD). Sialidase BPD is structurally conserved in various bacterial species and the protein binding interfaces have the tendency to form salt bridges, whereas uncommitted charged residues may affect binding and protein structure. However, it is not clear whether the sialidase BPD of varying strains of the same bacterial species differ, particularly with regards to salt bridge formation. Here, we determined the P. gingivalis ATCC 33277 and W50 sialidase homology models and sialidase activities, while the putative salt bridge residues found in the sialidase BPDs were compared. We established that both ATCC 33277 and W50 have different sialidase homology models and activities, whereas, the BPD (β1–6) is structurally conserved with most salt bridge-forming residues following a common orientation. Moreover, β2D444–β6K338 distance measurement in ATCC 33277 (5.99 Å) and W50 (3.09 Å) differ, while β1K396A substitution alters the β2D444–β6K338 distance measurements in ATCC 33277 (3.09 Å) and W50 (3.01 Å) consequentially affecting each model. P. gingivalis plays a major role in periodontitis induction and its virulence is greatly influenced by the sialidase enzyme wherein the sialidase BPD is highly conserved. Our results suggest that alterations in the salt bridge formation within the BPD interface may affect the P. gingivalis sialidase structure. This would imply that disrupting the salt bridge formation within the P. gingivalis sialidase BPD could serve as a potential therapeutic strategy for the treatment of P. gingivalis-related periodontitis. PMID:25000206

  13. Comparison of the benzoyl-DL-arginine-naphthylamide (BANA) test, DNA probes, and immunological reagents for ability to detect anaerobic periodontal infections due to Porphyromonas gingivalis, Treponema denticola, and Bacteroides forsythus.

    PubMed Central

    Loesche, W J; Lopatin, D E; Giordano, J; Alcoforado, G; Hujoel, P

    1992-01-01

    Most forms of periodontal disease are associated with the presence or overgrowth of anaerobic species that could include Treponema denticola, Porphyromonas gingivalis, and Bacteroides forsythus among others. These three organisms are among the few cultivable plaque species that can hydrolyze the synthetic trypsin substrate benzoyl-DL-arginine-naphthylamide (BANA). In turn, BANA hydrolysis by the plaque can be associated with periodontal morbidity and with the presence of these three BANA-positive organisms in the plaque. In this investigation, the results of the BANA test, which simultaneously detects one or more of these organisms, were compared with the detection of these organisms by (i) highly specific antibodies to P. gingivalis, T. denticola, and B. forsythus; (ii) whole genomic DNA probes to P. gingivalis and T. denticola; and (iii) culturing or microscopic procedures. The BANA test, the DNA probes, and an enzyme-linked immunosorbent assay or an indirect immunofluorescence assay procedure exhibited high sensitivities, i.e., 90 ot 96%, and high accuracies, i.e., 83 to 92%, in their ability to detect combinations of these organisms in over 200 subgingival plaque samples taken from the most periodontally diseased sites in 67 patients. This indicated that if P. gingivalis, T. denticola, and B. forsythus are appropriate marker organisms for an anaerobic periodontal infection, then the three detection methods are equally accurate in their ability to diagnose this infection. The same statement could not be made for the culturing approach, where accuracies of 50 to 62% were observed. PMID:1311335

  14. Detection of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans after Systemic Administration of Amoxicillin Plus Metronidazole as an Adjunct to Non-surgical Periodontal Therapy: A Systematic Review and Meta-Analysis

    PubMed Central

    Dakic, Aleksandar; Boillot, Adrien; Colliot, Cyrille; Carra, Maria-Clotilde; Czernichow, Sébastien; Bouchard, Philippe

    2016-01-01

    Objective: To evaluate the variations in the detection of Porphyromonas gingivalis and/or Aggregatibacter actinomycetemcomitans before and after systemic administration of amoxicillin plus metronidazole in association with non-surgical periodontal therapy (NSPT). Background: The adjunctive use of antibiotics has been advocated to improve the clinical outcomes of NSPT. However, no systematic review has investigated the microbiological benefit of this combination. Materials and Methods: An electronic search was conducted up to December 2015. Randomized clinical trials comparing the number of patients testing positive for P. gingivalis and/or A. actinomycetemcomitans before and after NSPT with (test group) or without (control group) amoxicillin plus metronidazole were included. The difference between groups in the variation of positive patients was calculated using the inverse variance method with a random effects model. Results: The frequency of patients positive for A. actinomycetemcomitans was decreased by 30% (p = 0.002) and by 25% (p = 0.01) in the test group compared to the control group at 3- and 6-month follow-up, respectively. Similar findings were observed when considering the frequency of patients positive for Porphyromonas gingivalis, with a reduction by 28% (p < 0.0001), 32% (p < 0.0001), and 34% (p = 0.03) in the test group compared to the control group at 3-, 6-, and 12-month follow-up, respectively. Conclusion: The systemic administration of amoxicillin plus metronidazole as an adjunct to NSPT significantly decreased the number of patients positive for P. gingivalis and A. actinomycetemcomitans compared with periodontal therapy alone or with a placebo. PMID:27594851

  15. Detection of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans after Systemic Administration of Amoxicillin Plus Metronidazole as an Adjunct to Non-surgical Periodontal Therapy: A Systematic Review and Meta-Analysis

    PubMed Central

    Dakic, Aleksandar; Boillot, Adrien; Colliot, Cyrille; Carra, Maria-Clotilde; Czernichow, Sébastien; Bouchard, Philippe

    2016-01-01

    Objective: To evaluate the variations in the detection of Porphyromonas gingivalis and/or Aggregatibacter actinomycetemcomitans before and after systemic administration of amoxicillin plus metronidazole in association with non-surgical periodontal therapy (NSPT). Background: The adjunctive use of antibiotics has been advocated to improve the clinical outcomes of NSPT. However, no systematic review has investigated the microbiological benefit of this combination. Materials and Methods: An electronic search was conducted up to December 2015. Randomized clinical trials comparing the number of patients testing positive for P. gingivalis and/or A. actinomycetemcomitans before and after NSPT with (test group) or without (control group) amoxicillin plus metronidazole were included. The difference between groups in the variation of positive patients was calculated using the inverse variance method with a random effects model. Results: The frequency of patients positive for A. actinomycetemcomitans was decreased by 30% (p = 0.002) and by 25% (p = 0.01) in the test group compared to the control group at 3- and 6-month follow-up, respectively. Similar findings were observed when considering the frequency of patients positive for Porphyromonas gingivalis, with a reduction by 28% (p < 0.0001), 32% (p < 0.0001), and 34% (p = 0.03) in the test group compared to the control group at 3-, 6-, and 12-month follow-up, respectively. Conclusion: The systemic administration of amoxicillin plus metronidazole as an adjunct to NSPT significantly decreased the number of patients positive for P. gingivalis and A. actinomycetemcomitans compared with periodontal therapy alone or with a placebo.

  16. CD36/SR-B2-TLR2 Dependent Pathways Enhance Porphyromonas gingivalis Mediated Atherosclerosis in the Ldlr KO Mouse Model.

    PubMed

    Brown, Paul M; Kennedy, David J; Morton, Richard E; Febbraio, Maria

    2015-01-01

    There is strong epidemiological association between periodontal disease and cardiovascular disease but underlying mechanisms remain ill-defined. Because the human periodontal disease pathogen, Porphyromonas gingivalis (Pg), interacts with innate immune receptors Toll-like Receptor (TLR) 2 and CD36/scavenger receptor-B2 (SR-B2), we studied how CD36/SR-B2 and TLR pathways promote Pg-mediated atherosclerosis. Western diet fed low density lipoprotein receptor knockout (Ldlr°) mice infected orally with Pg had a significant increase in lesion burden compared with uninfected controls.This increase was entirely CD36/SR-B2-dependent, as there was no significant change in lesion burden between infected and uninfected Cd36o/Ldlro mice [corrected]. Western diet feeding promoted enhanced CD36/SR-B2-dependent IL1β generation and foam cell formation as a result of Pg lipopolysaccharide (PgLPS) exposure. CD36/SR-B2 and TLR2 were necessary for inflammasome activation and optimal IL1ß generation, but also resulted in LPS induced lethality (pyroptosis). Modified forms of LDL inhibited Pg-mediated IL1ß generation in a CD36/SR-B2-dependent manner and prevented pyroptosis, but promoted foam cell formation. Our data show that Pg infection in the oral cavity can lead to significant TLR2-CD36/SR-B2 dependent IL1ß release. In the vessel wall, macrophages encountering systemic release of IL1ß, PgLPS and modified LDL have increased lipid uptake, foam cell formation, and release of IL1ß, but because pyroptosis is inhibited, this enables macrophage survival and promotes increased plaque development. These studies may explain increased lesion burden as a result of periodontal disease, and suggest strategies for development of therapeutics.

  17. Serum Immunoglobulin G Levels to Porphyromonas gingivalis Peptidylarginine Deiminase Affect Clinical Response to Biological Disease-Modifying Antirheumatic Drug in Rheumatoid Arthritis

    PubMed Central

    Kobayashi, Tetsuo; Ito, Satoshi; Kobayashi, Daisuke; Shimada, Atsushi; Narita, Ichiei; Murasawa, Akira; Nakazono, Kiyoshi; Yoshie, Hiromasa

    2016-01-01

    Objectives To determine whether serum immunity to Porphyromonas gingivalis peptidylarginine deiminase (PPAD) affects the clinical response to biological disease-modifying antirheumatic drug (bDMARD) in patients with rheumatoid arthritis (RA). Methods In a retrospective study, rheumatologic and periodontal conditions of 60 patients with RA who had been treated with conventional synthetic DMARD were evaluated before (baseline) and after 3 and 6 months of bDMARD therapy. After serum levels of anti-PPAD immunoglobulin G (IgG) were determined at baseline, the patients were respectively divided into two groups for high and low anti-PPAD IgG titers according to the median measurements. Genotypes at 8 functional single nucleotide polymorphisms (SNPs) related to RA were also determined. Results After 3 and 6 months of therapy, patients with low anti-PPAD IgG titers showed a significantly greater decrease in changes in the Disease Activity Score including 28 joints using C-reactive protein (DAS28-CRP) (P = 0.04 for both) and anti-cyclic citrullinated peptide (CCP) IgG levels (P = 0.03 and P = 0.04) than patients with high anti-PPAD IgG titers, although these parameter values were comparable at baseline. The anti-PPAD IgG titers were significantly positively correlated with changes in the DAS28-CRP (P = 0.01 for both) and the anti-CCP IgG levels (P = 0.02 for both) from baseline to 3 and 6 months later. A multiple regression analysis revealed a significantly positive association between the anti-PPAD IgG titers and changes in the DAS28-CRP after 6 months of bDMARD therapy (P = 0.006), after adjusting for age, gender, smoking, periodontal condition, and RA-related SNPs. Conclusion The serum IgG levels to PPAD affect the clinical response to bDMARD in patients with RA. PMID:27111223

  18. Revised sequence of the Porphyromonas gingivalis prtT cysteine protease/hemagglutinin gene: homology with streptococcal pyrogenic exotoxin B/streptococcal proteinase.

    PubMed Central

    Madden, T E; Clark, V L; Kuramitsu, H K

    1995-01-01

    The prtT gene from Porphyromonas gingivalis ATCC 53977 was previously isolated from an Escherichia coli clone possessing trypsinlike protease activity upstream of a region encoding hemagglutinin activity (J. Otogoto and H. Kuramitsu, Infect. Immun. 61;117-123, 1993). Subsequent molecular analysis of this gene has revealed that the PrtT protein is larger than originally reported, encompassing the hemagglutination region. Results of primer extension experiments indicate that the translation start site was originally misidentified. An alternate open reading frame of nearly 2.7 kb, which encodes a protein in the size range of 96 to 99 kDa, was identified. In vitro transcription-translation experiments confirm this size, and Northern (RNA) blot experiments indicate that the protease is translated from a 3.3-kb mRNA. Searching the EMBL protein database revealed that the amino acid sequence of the revised PrtT is similar to sequences of two related proteins from Streptococcus pyogenes. PrtT is 31% identical and 73% similar over 401 amino acids to streptococcal pyrogenic exotoxin B. In addition, it is 36% identical and 74% similar over 244 amino acids with streptococcal proteinase, which is closely related to streptococcal pyrogenic exotoxin B. The similarity is particularly high at the putative active site of streptococcal proteinase, which is similar to the active sites of the family of cysteine proteases. Thus, we conclude that PrtT is a 96- to 99-kDa cysteine protease and hemagglutinin with significant similarity to streptococcal enzymes. PMID:7806362

  19. Constitutive nitric oxide synthase-mediated caspase-3 S-nitrosylation in ghrelin protection against Porphyromonas gingivalis-induced salivary gland acinar cell apoptosis.

    PubMed

    Slomiany, B L; Slomiany, A

    2010-06-01

    Recent advances in identifying the salivary constituents capable of influencing the oral mucosal inflammatory responses have brought to focus the importance of a peptide hormone, ghrelin. Here, we report on the involvement of ghrelin in controlling the apoptotic processes induced in sublingual salivary gland acinar cells by the lipopolysaccharide (LPS) of a periodontopathic bacterium, Porphyromonas gingivalis. We show that the countering effect of ghrelin on the LPS-induced acinar cell apoptosis was associated with the increase in constitutive nitric oxide synthase (cNOS) activity, and the reduction in caspase-3 and inducible nitric oxide synthase (iNOS). The loss in countering effect of ghrelin on the LPS-induced changes in apoptosis and caspase-3 activity was attained with Src kinase inhibitor, PP2, as well as Akt inhibitor, SH-5, and cNOS inhibitor, L-NAME, but not the iNOS inhibitor, 1400W. The effect of ghrelin on the LPS-induced changes in cNOS activity, moreover, was reflected in the increased cNOS phosphorylation that was sensitive to PP2 as well as SH-5. Furthermore, the ghrelin-induced up-regulation in cNOS activity was associated with the increase in caspase-3 S-nitrosylation that was susceptible to the blockage by SH-5 and L-NAME. The findings point to the involvement of ghrelin in Src/Akt kinase-mediated cNOS activation and the apoptogenic signal inhibition through the NO-induced caspase-3 S-nitrosylation.

  20. Periodontal pathogens Porphyromonas gingivalis and Fusobacterium nucleatum promote tumor progression in an oral-specific chemical carcinogenesis model.

    PubMed

    Binder Gallimidi, Adi; Fischman, Stuart; Revach, Brurya; Bulvik, Raanan; Maliutina, Alina; Rubinstein, Ariel M; Nussbaum, Gabriel; Elkin, Michael

    2015-09-01

    Oral squamous cell carcinoma (OSCC) is a lethal disease whose incidence is increasing. Epidemiologic studies demonstrate an association between periodontitis and oral cancer, and periodontal pathogens are implicated in the pathogenesis of numerous disorders, including rheumatoid arthritis, cardiovascular diseases, diabetes and gastrointestinal malignancies. Nevertheless, a causal role for periodontal pathogens in OSCC has not been shown, partly due to the lack of an appropriate animal model. Here, utilizing a newly-established murine model of periodontitis-associated oral tumorigenesis, we report that chronic bacterial infection promotes OSCC, and that augmented signaling along the IL-6-STAT3 axis underlies this effect. Our results indicate that periodontal pathogens P. gingivalis and F. nucleatum stimulate tumorigenesis via direct interaction with oral epithelial cells through Toll-like receptors. Furthermore, oral pathogens stimulate human OSCC proliferation and induce expression of key molecules implicated in tumorigenesis. To the best of our knowledge, these findings represent the first demonstration of a mechanistic role for oral bacteria in chemically induced OSCC tumorigenesis. These results are highly relevant for the design of effective prevention and treatment strategies for OSCC.

  1. A novel approach for purification and selective capture of membrane vesicles of the periodontopathic bacterium, Porphyromonas gingivalis: membrane vesicles bind to magnetic beads coated with epoxy groups in a noncovalent, species-specific manner.

    PubMed

    Nakao, Ryoma; Kikushima, Kenji; Higuchi, Hideo; Obana, Nozomu; Nomura, Nobuhiko; Bai, Dongying; Ohnishi, Makoto; Senpuku, Hidenobu

    2014-01-01

    Membrane vesicles (MVs) of Porphyromonas gingivalis are regarded as an offensive weapon of the bacterium, leading to tissue deterioration in periodontal disease. Therefore, isolation of highly purified MVs is indispensable to better understand the pathophysiological role of MVs in the progression of periodontitis. MVs are generally isolated by a conventional method based on ultracentrifugation of the bacterial culture supernatant. However, the resulting MVs are often contaminated with co-precipitating bacterial appendages sheared from the live bacteria. Here, we report an intriguing property of P. gingivalis MVs--their ability to bind superparamagnetic beads coated with epoxy groups (SB-Epoxy). Analysis of fractions collected during the purification revealed that all MVs of five tested P. gingivalis stains bound to SB-Epoxy. In contrast, free fimbriae in the crude MV preparation did not bind to the SB-Epoxy. The SB-Epoxy-bound MVs were easily dissociated from the SB-Epoxy using a mild denaturation buffer. These results suggest that the surface chemistry conferred by epoxy on the beads is responsible for the binding, which is mediated by noncovalent bonds. Both the structural integrity and purity of the isolated MVs were confirmed by electron microscopy. The isolated MVs also caused cell detachment from culture dishes at a physiologically relevant concentration. Assays of competitive binding between the SB-Epoxy and mixtures of MVs from five bacterial species demonstrated that only P. gingivalis MVs could be selectively eliminated from the mixtures. We suggest that this novel approach enables efficient purification and selective elimination of P. gingivalis MVs. PMID:24830438

  2. Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Treponema denticola / Prevotella intermedia Co-Infection Are Associated with Severe Periodontitis in a Thai Population.

    PubMed

    Torrungruang, Kitti; Jitpakdeebordin, Supawadee; Charatkulangkun, Orawan; Gleebbua, Yingampa

    2015-01-01

    Periodontitis is a polymicrobial infection of tooth-supporting tissues. This cross-sectional study aimed to examine the associations between five target species and severe periodontitis in a Thai population. Using the CDC/AAP case definition, individuals diagnosed with no/mild and severe periodontitis were included. Quantitative analyses of Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf), Treponema denticola (Td), and Prevotella intermedia (Pi) in subgingival plaque were performed using real-time polymerase chain reaction. The association between target species and severe periodontitis was examined using logistic regression analysis. The study subjects comprised 479 individuals with no/mild periodontitis and 883 with severe periodontitis. Bacterial prevalence and quantity were higher in subjects with severe periodontitis than in those with no/mild disease. In the fully adjusted model, all species except Tf showed a dose-dependent relationship with periodontitis. The mere presence of Pg, even in low amount, was significantly associated with severe periodontitis, while the amount of Aa, Td, and Pi had to reach the critical thresholds to be significantly associated with disease. Compared to individuals with low levels of both Td and Pi, high colonization by either Td or Pi alone significantly increased the odds of having severe periodontitis by 2.5 (95%CI 1.7-3.5) folds. The odds ratio was further increased to 14.8 (95%CI 9.2-23.8) in individuals who were highly colonized by both species. Moreover, the presence of Pg and high colonization by Aa were independently associated with severe periodontitis with odds ratios of 5.6 (95%CI 3.4-9.1) and 2.2 (95%CI 1.5-3.3), respectively. Our findings suggest that the presence of Pg and high colonization by Aa, Td, and Pi play an important role in severe periodontitis in this study population. We also demonstrate for the first time that individuals co-infected with Td and Pi

  3. Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Treponema denticola / Prevotella intermedia Co-Infection Are Associated with Severe Periodontitis in a Thai Population

    PubMed Central

    Torrungruang, Kitti; Jitpakdeebordin, Supawadee; Charatkulangkun, Orawan; Gleebbua, Yingampa

    2015-01-01

    Periodontitis is a polymicrobial infection of tooth-supporting tissues. This cross-sectional study aimed to examine the associations between five target species and severe periodontitis in a Thai population. Using the CDC/AAP case definition, individuals diagnosed with no/mild and severe periodontitis were included. Quantitative analyses of Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf), Treponema denticola (Td), and Prevotella intermedia (Pi) in subgingival plaque were performed using real-time polymerase chain reaction. The association between target species and severe periodontitis was examined using logistic regression analysis. The study subjects comprised 479 individuals with no/mild periodontitis and 883 with severe periodontitis. Bacterial prevalence and quantity were higher in subjects with severe periodontitis than in those with no/mild disease. In the fully adjusted model, all species except Tf showed a dose-dependent relationship with periodontitis. The mere presence of Pg, even in low amount, was significantly associated with severe periodontitis, while the amount of Aa, Td, and Pi had to reach the critical thresholds to be significantly associated with disease. Compared to individuals with low levels of both Td and Pi, high colonization by either Td or Pi alone significantly increased the odds of having severe periodontitis by 2.5 (95%CI 1.7–3.5) folds. The odds ratio was further increased to 14.8 (95%CI 9.2–23.8) in individuals who were highly colonized by both species. Moreover, the presence of Pg and high colonization by Aa were independently associated with severe periodontitis with odds ratios of 5.6 (95%CI 3.4–9.1) and 2.2 (95%CI 1.5–3.3), respectively. Our findings suggest that the presence of Pg and high colonization by Aa, Td, and Pi play an important role in severe periodontitis in this study population. We also demonstrate for the first time that individuals co-infected with Td

  4. The Tla protein of Porphyromonas gingivalis W50: a homolog of the RI protease precursor (PrpRI) is an outer membrane receptor required for growth on low levels of hemin.

    PubMed Central

    Aduse-Opoku, J; Slaney, J M; Rangarajan, M; Muir, J; Young, K A; Curtis, M A

    1997-01-01

    The prpR1 gene of Porphyromonas gingivalis W50 encodes the polyprotein precursor (PrpRI) of an extracellular arginine-specific protease. PrpRI is organized into four distinct domains (pro, alpha, beta, and gamma) and is processed to a heterodimeric protease (RI) which comprises the alpha and beta components in a noncovalent association. The alpha component contains the protease active site, whereas the beta component appears to have a role in adherence and hemagglutination processes. DNA sequences homologous to the coding region for the RI beta component are present at multiple loci on the P. gingivalis chromosome and may represent a family of related genes. In this report, we describe the cloning, sequence analysis, and characterization of one of these homologous loci isolated in plasmid pJM7. The 6,041-bp P. gingivalis DNA fragment in pJM7 contains a major open reading frame of 3,291 bp with coding potential for a protein with an Mr 118,700. An internal region of the deduced sequence (V304 to N768) shows 98% identity to the beta domain of PrpRI, and the recombinant product of pJM7 is immunoreactive with an antibody specific to the RI beta component. The N terminus of the deduced sequence has regional similarity to TonB-linked receptors which are frequently involved in periplasmic translocation of hemin, iron, colicins, or vitamin B12 in other bacteria. We have therefore designated this gene tla (TonB-linked adhesin). In contrast to the parent strain, an isogenic mutant of P. gingivalis W50 in which the tla was insertionally inactivated was unable to grow in medium containing low concentrations of hemin (<2.5 mg liter(-1)), and hemin-depleted cells of this mutant failed to respond to hemin in an agar diffusion plate assay. These data suggest a role for this gene product in hemin acquisition and utilization. Furthermore, the mutant produced significantly less arginine- and lysine-specific protease activities than the parent strain, indicating that there may be a

  5. The PorX Response Regulator of the Porphyromonas gingivalis PorXY Two-Component System Does Not Directly Regulate the Type IX Secretion Genes but Binds the PorL Subunit

    PubMed Central

    Vincent, Maxence S.; Durand, Eric; Cascales, Eric

    2016-01-01

    The Type IX secretion system (T9SS) is a versatile multi-protein complex restricted to bacteria of the Bacteriodetes phylum and responsible for the secretion or cell surface exposition of diverse proteins that participate to S-layer formation, gliding motility or pathogenesis. The T9SS is poorly characterized but a number of proteins involved in the assembly of the secretion apparatus in the oral pathogen Porphyromonas gingivalis have been identified based on genome substractive analyses. Among these proteins, PorY, and PorX encode typical two-component system (TCS) sensor and CheY-like response regulator respectively. Although the porX and porY genes do not localize at the same genetic locus, it has been proposed that PorXY form a bona fide TCS. Deletion of porX in P. gingivalis causes a slight decrease of the expression of a number of other T9SS genes, including sov, porT, porP, porK, porL, porM, porN, and porY. Here, we show that PorX and the soluble cytoplasmic domain of PorY interact. Using electrophoretic mobility shift, DNA-protein co-purification and heterologous host expression assays, we demonstrate that PorX does not bind T9SS gene promoters and does not directly regulate expression of the T9SS genes. Finally, we show that PorX interacts with the cytoplasmic domain of PorL, a component of the T9SS membrane core complex and propose that the CheY-like PorX protein might be involved in the dynamics of the T9SS. PMID:27630829

  6. The PorX Response Regulator of the Porphyromonas gingivalis PorXY Two-Component System Does Not Directly Regulate the Type IX Secretion Genes but Binds the PorL Subunit.

    PubMed

    Vincent, Maxence S; Durand, Eric; Cascales, Eric

    2016-01-01

    The Type IX secretion system (T9SS) is a versatile multi-protein complex restricted to bacteria of the Bacteriodetes phylum and responsible for the secretion or cell surface exposition of diverse proteins that participate to S-layer formation, gliding motility or pathogenesis. The T9SS is poorly characterized but a number of proteins involved in the assembly of the secretion apparatus in the oral pathogen Porphyromonas gingivalis have been identified based on genome substractive analyses. Among these proteins, PorY, and PorX encode typical two-component system (TCS) sensor and CheY-like response regulator respectively. Although the porX and porY genes do not localize at the same genetic locus, it has been proposed that PorXY form a bona fide TCS. Deletion of porX in P. gingivalis causes a slight decrease of the expression of a number of other T9SS genes, including sov, porT, porP, porK, porL, porM, porN, and porY. Here, we show that PorX and the soluble cytoplasmic domain of PorY interact. Using electrophoretic mobility shift, DNA-protein co-purification and heterologous host expression assays, we demonstrate that PorX does not bind T9SS gene promoters and does not directly regulate expression of the T9SS genes. Finally, we show that PorX interacts with the cytoplasmic domain of PorL, a component of the T9SS membrane core complex and propose that the CheY-like PorX protein might be involved in the dynamics of the T9SS. PMID:27630829

  7. The PorX Response Regulator of the Porphyromonas gingivalis PorXY Two-Component System Does Not Directly Regulate the Type IX Secretion Genes but Binds the PorL Subunit.

    PubMed

    Vincent, Maxence S; Durand, Eric; Cascales, Eric

    2016-01-01

    The Type IX secretion system (T9SS) is a versatile multi-protein complex restricted to bacteria of the Bacteriodetes phylum and responsible for the secretion or cell surface exposition of diverse proteins that participate to S-layer formation, gliding motility or pathogenesis. The T9SS is poorly characterized but a number of proteins involved in the assembly of the secretion apparatus in the oral pathogen Porphyromonas gingivalis have been identified based on genome substractive analyses. Among these proteins, PorY, and PorX encode typical two-component system (TCS) sensor and CheY-like response regulator respectively. Although the porX and porY genes do not localize at the same genetic locus, it has been proposed that PorXY form a bona fide TCS. Deletion of porX in P. gingivalis causes a slight decrease of the expression of a number of other T9SS genes, including sov, porT, porP, porK, porL, porM, porN, and porY. Here, we show that PorX and the soluble cytoplasmic domain of PorY interact. Using electrophoretic mobility shift, DNA-protein co-purification and heterologous host expression assays, we demonstrate that PorX does not bind T9SS gene promoters and does not directly regulate expression of the T9SS genes. Finally, we show that PorX interacts with the cytoplasmic domain of PorL, a component of the T9SS membrane core complex and propose that the CheY-like PorX protein might be involved in the dynamics of the T9SS.

  8. The PorX Response Regulator of the Porphyromonas gingivalis PorXY Two-Component System Does Not Directly Regulate the Type IX Secretion Genes but Binds the PorL Subunit

    PubMed Central

    Vincent, Maxence S.; Durand, Eric; Cascales, Eric

    2016-01-01

    The Type IX secretion system (T9SS) is a versatile multi-protein complex restricted to bacteria of the Bacteriodetes phylum and responsible for the secretion or cell surface exposition of diverse proteins that participate to S-layer formation, gliding motility or pathogenesis. The T9SS is poorly characterized but a number of proteins involved in the assembly of the secretion apparatus in the oral pathogen Porphyromonas gingivalis have been identified based on genome substractive analyses. Among these proteins, PorY, and PorX encode typical two-component system (TCS) sensor and CheY-like response regulator respectively. Although the porX and porY genes do not localize at the same genetic locus, it has been proposed that PorXY form a bona fide TCS. Deletion of porX in P. gingivalis causes a slight decrease of the expression of a number of other T9SS genes, including sov, porT, porP, porK, porL, porM, porN, and porY. Here, we show that PorX and the soluble cytoplasmic domain of PorY interact. Using electrophoretic mobility shift, DNA-protein co-purification and heterologous host expression assays, we demonstrate that PorX does not bind T9SS gene promoters and does not directly regulate expression of the T9SS genes. Finally, we show that PorX interacts with the cytoplasmic domain of PorL, a component of the T9SS membrane core complex and propose that the CheY-like PorX protein might be involved in the dynamics of the T9SS.

  9. Suppression by Ghrelin of Porphyromonas gingivalis-Induced Constitutive Nitric Oxide Synthase S-Nitrosylation and Apoptosis in Salivary Gland Acinar Cells.

    PubMed

    Slomiany, Bronislaw L; Slomiany, Amalia

    2010-01-01

    Oral mucosal inflammatory responses to periodontopathic bacterium, P. gingivalis, and its key virulence factor, LPS, are characterized by a massive rise in epithelial cell apoptosis and the disturbances in NO signaling pathways. Here, we report that the LPS-induced enhancement in rat sublingual salivary gland acinar cell apoptosis and NO generation was associated with the suppression in constitutive nitric oxide synthase (cNOS) activity and a marked increase in the activity of inducible nitric oxide synthase (iNOS). We demonstrate that the detrimental effect of the LPS on cNOS was manifested by the enzyme protein S-nitrosylation, that was susceptible to inhibition by iNOS inhibitor, 1400 W. Further, we show that a peptide hormone, ghrelin, countered the LPS-induced changes in apoptosis and cNOS activity. This effect of ghrelin was reflected in the decrease in cNOS S-nitrosylation and the increase in phosphorylation. Our findings imply that P. gingivalis-induced disturbances in the acinar cell NO signaling pathways result from upregulation in iNOS-derived NO that causes cNOS S-nitrosylation that interferes with its activation through phosphorylation. We also show that ghrelin protection against P. gingivalis-induced disturbances involves cNOS activation associated with a decrease in its S-nitrosylation and the increase in phosphorylation.

  10. Sulfonamide inhibition study of the carbonic anhydrases from the bacterial pathogen Porphyromonas gingivalis: the β-class (PgiCAb) versus the γ-class (PgiCA) enzymes.

    PubMed

    Prete, Sonia Del; Vullo, Daniela; Osman, Sameh M; Scozzafava, Andrea; AlOthman, Zeid; Capasso, Clemente; Supuran, Claudiu T

    2014-09-01

    The oral pathogenic bacterium Porphyromonas gingivalis, encodes for two carbonic anhydrases (CAs, EC 4.2.1.1) one belonging to the γ-class (PgiCA) and another one to the β-class (PgiCAb). This last enzyme has been cloned and characterized here for its inhibition profile with the main class of CA inhibitors, the sulfonamides. Many of the clinically used sulfonamides as well as simple aromatic/heterocyclic sulfonamides were ineffective as PgiCAb inhibitors whereas better inhibition was observed with simple derivatives such as sulfanilamide, metanilamide, 4-aminoalkylbenzenesulfonamides (KIs of 364-475nM). The halogenosulfanilamides incorporating heavy halogens, 4-hydroxy- and 4-hydroxyalkyl-benzenesulfonamides, were also micromolar, ineffective PgiCAb inhibitors. The best inhibitors of the β-class enzyme were acetazolamide and ethoxzolamide, with KIs of 214-280nM. Interestingly, the γ-class enzyme was much more sensitive to sulfonamide inhibitors compared to the β-class one, PgiCAb. Identification of potent and possibly selective inhibitors of PgiCAb/PgiCA may lead to pharmacological tools useful for understanding the physiological role(s) of these enzymes, since this bacterium is the main causative agent of periodontitis and few treatment options are presently available. PMID:25129169

  11. Extract from Rumex acetosa L. for Prophylaxis of Periodontitis: Inhibition of Bacterial In Vitro Adhesion and of Gingipains of Porphyromonas gingivalis by Epicatechin-3-O-(4β→8)-Epicatechin-3-O-Gallate (Procyanidin-B2-Di-Gallate)

    PubMed Central

    Schmuch, Jana; Beckert, Sabine; Brandt, Simone; Löhr, Gesine; Hermann, Fabian; Schmidt, Thomas J.; Beikler, Thomas; Hensel, Andreas

    2015-01-01

    Background The aerial parts of Rumex acetosa L. have been used in traditional European medicine for inflammatory diseases of the mouth epithelial tissue. The following study aimed to investigate the influence of a proanthocyanidin-enriched extract from R. acetosa extract against the adhesion of Porphyromonas gingivalis (P. gingivalis), a pathogen strongly involved in chronic and aggressive periodontitis. A further goal was to define the bioactive lead structures responsible for a potential antiadhesive activity and to characterize the underlying molecular mechanisms of the antiadhesive effects. Methodology An extract of R. acetosa (RA1) with a defined mixture of flavan-3-ols, oligomeric proanthocyanidins and flavonoids, was used. Its impact on P. gingivalis adhesion to KB cells was studied by flow cytometry, confocal laser scanning microscopy and in situ adhesion assay using murine buccal tissue. RA1 and its compounds 1 to 15 were further investigated for additional effects on gingipain activity, hemagglutination and gene expression by RT-PCR. Principal Findings RA1 (5 to 15 μg/mL) reduced P. gingivalis adhesion in a dose-dependent manner to about 90%. Galloylated proanthocyanidins were confirmed to be responsible for this antiadhesive effect with epicatechin-3-O-gallate-(4β,8)-epicatechin-3’-O-gallate (syn. procyanidin B2-di-gallate) being the lead compound. Ungalloylated flavan-3-ols and oligomeric proanthocyanidins were inactive. RA1 and the galloylated proanthocyanidins strongly interact with the bacterial virulence factor Arg-gingipain, while the corresponding Lys-gingipain was hardly influenced. RA1 inhibited also hemagglutination. In silico docking studies indicated that epicatechin-3-O-gallate-(4β,8)-epicatechin-3’-O-gallate interacts with the active side of Arg-gingipain and hemaglutinin from P. gingivalis; the galloylation of the molecule seems to be responsible for fixation of the ligand to the protein. In conclusion, the proanthocyanidin

  12. Culture-based identification of pigmented Porphyromonas and Prevotella species in primary endodontic infections

    PubMed Central

    Rajaram, Anuradha; Kotrashetti, Vijayalakshmi S.; Somannavar, Pradeep D.; Ingalagi, Preeti; Bhat, Kishore

    2016-01-01

    Background. The most common species isolated from primary endodontic infections are black-pigmented bacteria. These species are implicated in apical abscess formation due to their proteolytic activity and are fastidious in nature. Therefore, the present study was carried out to evaluate the presence and identification of various pigmented Porphyromonas and Prevotella species in the infected root canal through culture-based techniques. Methods. Thirty-one patients with primary endodontic infections were selected. Using sterile paper points, samples were collected from the root canals after access opening and prior to obturation, which were cultured using blood and kanamycin blood agar. Subsequently, biochemical test was used to identify the species and the results were analyzed using percentage comparison analysis, McNemar and chi-squared tests, Wilcoxon match pair test and paired t-test. Results. Out of 31 samples 26 were positive for black-pigmented organisms; the predominantly isolated species were Prevotella followed by Porphyromonas. In Porphyromonas only P. gingivalis was isolated. One of the interesting features was isolation of P. gingivalis through culture, which is otherwise very difficult to isolate through culture. Conclusion. The presence of Prevotella and Porphyromonas species suggests that a significant role is played by these organisms in the pathogenesis of endodontic infections.

  13. Culture-based identification of pigmented Porphyromonas and Prevotella species in primary endodontic infections

    PubMed Central

    Rajaram, Anuradha; Kotrashetti, Vijayalakshmi S.; Somannavar, Pradeep D.; Ingalagi, Preeti; Bhat, Kishore

    2016-01-01

    Background. The most common species isolated from primary endodontic infections are black-pigmented bacteria. These species are implicated in apical abscess formation due to their proteolytic activity and are fastidious in nature. Therefore, the present study was carried out to evaluate the presence and identification of various pigmented Porphyromonas and Prevotella species in the infected root canal through culture-based techniques. Methods. Thirty-one patients with primary endodontic infections were selected. Using sterile paper points, samples were collected from the root canals after access opening and prior to obturation, which were cultured using blood and kanamycin blood agar. Subsequently, biochemical test was used to identify the species and the results were analyzed using percentage comparison analysis, McNemar and chi-squared tests, Wilcoxon match pair test and paired t-test. Results. Out of 31 samples 26 were positive for black-pigmented organisms; the predominantly isolated species were Prevotella followed by Porphyromonas. In Porphyromonas only P. gingivalis was isolated. One of the interesting features was isolation of P. gingivalis through culture, which is otherwise very difficult to isolate through culture. Conclusion. The presence of Prevotella and Porphyromonas species suggests that a significant role is played by these organisms in the pathogenesis of endodontic infections. PMID:27651878

  14. Culture-based identification of pigmented Porphyromonas and Prevotella species in primary endodontic infections.

    PubMed

    Rajaram, Anuradha; Kotrashetti, Vijayalakshmi S; Somannavar, Pradeep D; Ingalagi, Preeti; Bhat, Kishore

    2016-01-01

    Background. The most common species isolated from primary endodontic infections are black-pigmented bacteria. These species are implicated in apical abscess formation due to their proteolytic activity and are fastidious in nature. Therefore, the present study was carried out to evaluate the presence and identification of various pigmented Porphyromonas and Prevotella species in the infected root canal through culture-based techniques. Methods. Thirty-one patients with primary endodontic infections were selected. Using sterile paper points, samples were collected from the root canals after access opening and prior to obturation, which were cultured using blood and kanamycin blood agar. Subsequently, biochemical test was used to identify the species and the results were analyzed using percentage comparison analysis, McNemar and chi-squared tests, Wilcoxon match pair test and paired t-test. Results. Out of 31 samples 26 were positive for black-pigmented organisms; the predominantly isolated species were Prevotella followed by Porphyromonas. In Porphyromonas only P. gingivalis was isolated. One of the interesting features was isolation of P. gingivalis through culture, which is otherwise very difficult to isolate through culture. Conclusion . The presence of Prevotella and Porphyromonas species suggests that a significant role is played by these organisms in the pathogenesis of endodontic infections. PMID:27651878

  15. Bacteroides gingivalis and Bacteroides intermedius recognize different sites on human fibrinogen

    SciTech Connect

    Lantz, M.S.; Allen, R.D.; Bounelis, P.; Switalski, L.M.; Hook, M. )

    1990-02-01

    Bacteroides (Porphyromonas) gingivalis and Bacteroides (Porphyromonas) intermedius have been implicated in the etiology of human periodontal diseases. These organisms are able to bind and degrade human fibrinogen, and these interactions may play a role in the pathogenesis of periodontal disease. In attempts to map the bacterial binding sites along the fibrinogen molecule, we have found that strains of B. gingivalis and B. intermedius, respectively, recognize spatially distant and distinct sites on the fibrinogen molecule. Isolated reduced and alkylated alpha-, beta-, and gamma-fibrinogen chains inhibited binding of 125I-fibrinogen to both Bacteroides species in a concentration-dependent manner. Plasmin fragments D and to some extent fragment E, however, produced a concentration-dependent inhibition of 125I-fibrinogen binding to B. intermedius strains but did not affect binding of 125I-fibrinogen to B. gingivalis strains. Radiolabeled fibrinogen chains and fragments were compared with 125I-fibrinogen with respect to specificity and reversibility of binding to bacteria. According to these criteria, gamma chain most closely resembled the native fibrinogen molecule in behavior toward B. gingivalis strains and fragments D most closely resembled fibrinogen in behavior toward B. intermedius strains. The ability of anti-human fibrinogen immunoglobulin G (IgG) to inhibit binding of 125I-fibrinogen to B. intermedius strains was greatly reduced by absorbing the IgG with fragments D. Absorbing the IgG with fragments D had no effect on the ability of the antibody to inhibit binding of 125I-fibrinogen to B. gingivalis strains. A purified staphylococcal fibrinogen-binding protein blocked binding of 125I-fibrinogen to B. intermedius strains but not to B. gingivalis strains.

  16. Bacteroides gingivalis and Bacteroides intermedius recognize different sites on human fibrinogen.

    PubMed Central

    Lantz, M S; Allen, R D; Bounelis, P; Switalski, L M; Hook, M

    1990-01-01

    Bacteroides (Porphyromonas) gingivalis and Bacteroides (Porphyromonas) intermedius have been implicated in the etiology of human periodontal diseases. These organisms are able to bind and degrade human fibrinogen, and these interactions may play a role in the pathogenesis of periodontal disease. In attempts to map the bacterial binding sites along the fibrinogen molecule, we have found that strains of B. gingivalis and B. intermedius, respectively, recognize spatially distant and distinct sites on the fibrinogen molecule. Isolated reduced and alkylated alpha-, beta-, and gamma-fibrinogen chains inhibited binding of 125I-fibrinogen to both Bacteroides species in a concentration-dependent manner. Plasmin fragments D and to some extent fragment E, however, produced a concentration-dependent inhibition of 125I-fibrinogen binding to B. intermedius strains but did not affect binding of 125I-fibrinogen to B. gingivalis strains. Radiolabeled fibrinogen chains and fragments were compared with 125I-fibrinogen with respect to specificity and reversibility of binding to bacteria. According to these criteria, gamma chain most closely resembled the native fibrinogen molecule in behavior toward B. gingivalis strains and fragments D most closely resembled fibrinogen in behavior toward B. intermedius strains. The ability of anti-human fibrinogen immunoglobulin G (IgG) to inhibit binding of 125I-fibrinogen to B. intermedius strains was greatly reduced by absorbing the IgG with fragments D. Absorbing the IgG with fragments D had no effect on the ability of the antibody to inhibit binding of 125I-fibrinogen to B. gingivalis strains. A purified staphylococcal fibrinogen-binding protein blocked binding of 125I-fibrinogen to B. intermedius strains but not to B. gingivalis strains. PMID:2404954

  17. The Haemoglobins of Algae.

    PubMed

    Johnson, Eric A; Lecomte, Juliette T J

    2015-01-01

    In the last few years, advances in algal research have identified the participation of haemoglobins in nitrogen metabolism and the management of reactive nitrogen and oxygen species. This chapter summarises the state of knowledge concerning algal haemoglobins with a focus on the most widely used model system, namely, Chlamydomonas reinhardtii. Genetic, physiologic, structural, and chemical information is compiled to provide a framework for further studies.

  18. The Haemoglobins of Algae.

    PubMed

    Johnson, Eric A; Lecomte, Juliette T J

    2015-01-01

    In the last few years, advances in algal research have identified the participation of haemoglobins in nitrogen metabolism and the management of reactive nitrogen and oxygen species. This chapter summarises the state of knowledge concerning algal haemoglobins with a focus on the most widely used model system, namely, Chlamydomonas reinhardtii. Genetic, physiologic, structural, and chemical information is compiled to provide a framework for further studies. PMID:26616518

  19. Comparative Genomics of the Genus Porphyromonas Identifies Adaptations for Heme Synthesis within the Prevalent Canine Oral Species Porphyromonas cangingivalis.

    PubMed

    O'Flynn, Ciaran; Deusch, Oliver; Darling, Aaron E; Eisen, Jonathan A; Wallis, Corrin; Davis, Ian J; Harris, Stephen J

    2015-12-01

    Porphyromonads play an important role in human periodontal disease and recently have been shown to be highly prevalent in canine mouths. Porphyromonas cangingivalis is the most prevalent canine oral bacterial species in both plaque from healthy gingiva and plaque from dogs with early periodontitis. The ability of P. cangingivalis to flourish in the different environmental conditions characterized by these two states suggests a degree of metabolic flexibility. To characterize the genes responsible for this, the genomes of 32 isolates (including 18 newly sequenced and assembled) from 18 Porphyromonad species from dogs, humans, and other mammals were compared. Phylogenetic trees inferred using core genes largely matched previous findings; however, comparative genomic analysis identified several genes and pathways relating to heme synthesis that were present in P. cangingivalis but not in other Porphyromonads. Porphyromonas cangingivalis has a complete protoporphyrin IX synthesis pathway potentially allowing it to synthesize its own heme unlike pathogenic Porphyromonads such as Porphyromonas gingivalis that acquire heme predominantly from blood. Other pathway differences such as the ability to synthesize siroheme and vitamin B12 point to enhanced metabolic flexibility for P. cangingivalis, which may underlie its prevalence in the canine oral cavity. PMID:26568374

  20. Comparative Genomics of the Genus Porphyromonas Identifies Adaptations for Heme Synthesis within the Prevalent Canine Oral Species Porphyromonas cangingivalis

    PubMed Central

    O’Flynn, Ciaran; Deusch, Oliver; Darling, Aaron E.; Eisen, Jonathan A.; Wallis, Corrin; Davis, Ian J.; Harris, Stephen J.

    2015-01-01

    Porphyromonads play an important role in human periodontal disease and recently have been shown to be highly prevalent in canine mouths. Porphyromonas cangingivalis is the most prevalent canine oral bacterial species in both plaque from healthy gingiva and plaque from dogs with early periodontitis. The ability of P. cangingivalis to flourish in the different environmental conditions characterized by these two states suggests a degree of metabolic flexibility. To characterize the genes responsible for this, the genomes of 32 isolates (including 18 newly sequenced and assembled) from 18 Porphyromonad species from dogs, humans, and other mammals were compared. Phylogenetic trees inferred using core genes largely matched previous findings; however, comparative genomic analysis identified several genes and pathways relating to heme synthesis that were present in P. cangingivalis but not in other Porphyromonads. Porphyromonas cangingivalis has a complete protoporphyrin IX synthesis pathway potentially allowing it to synthesize its own heme unlike pathogenic Porphyromonads such as Porphyromonas gingivalis that acquire heme predominantly from blood. Other pathway differences such as the ability to synthesize siroheme and vitamin B12 point to enhanced metabolic flexibility for P. cangingivalis, which may underlie its prevalence in the canine oral cavity. PMID:26568374

  1. Phylogeny of Bacteroides, Prevotella, and Porphyromonas spp. and related bacteria.

    PubMed

    Paster, B J; Dewhirst, F E; Olsen, I; Fraser, G J

    1994-02-01

    The phylogenetic structure of the bacteroides subgroup of the cytophaga-flavobacter-bacteroides (CFB) phylum was examined by 16S rRNA sequence comparative analysis. Approximately 95% of the 16S rRNA sequence was determined for 36 representative strains of species of Prevotella, Bacteroides, and Porphyromonas and related species by a modified Sanger sequencing method. A phylogenetic tree was constructed from a corrected distance matrix by the neighbor-joining method, and the reliability of tree branching was established by bootstrap analysis. The bacteroides subgroup was divided primarily into three major phylogenetic clusters which contained most of the species examined. The first cluster, termed the prevotella cluster, was composed of 16 species of Prevotella, including P. melaninogenica, P. intermedia, P. nigrescens, and the ruminal species P. ruminicola. Two oral species, P. zoogleoformans and P. heparinolytica, which had been recently placed in the genus Prevotella, did not fall within the prevotella cluster. These two species and six species of Bacteroides, including the type species B. fragilis, formed the second cluster, termed the bacteroides cluster. The third cluster, termed the porphyromonas cluster, was divided into two subclusters. The first contained Porphyromonas gingivalis, P. endodontalis, P. asaccharolytica, P. circumdentaria, P. salivosa, [Bacteroides] levii (the brackets around genus are used to indicate that the species does not belong to the genus by the sensu stricto definition), and [Bacteroides] macacae, and the second subcluster contained [Bacteroides] forsythus and [Bacteroides] distasonis. [Bacteroides] splanchnicus fell just outside the three major clusters but still belonged within the bacteroides subgroup. With few exceptions, the 16 S rRNA data were in overall agreement with previously proposed reclassifications of species of Bacteroides, Prevotella, and Porphyromonas. Suggestions are made to accommodate those species which do not

  2. Haem degradation in abnormal haemoglobins.

    PubMed Central

    Brown, S B; Docherty, J C

    1978-01-01

    The coupled oxidation of certain abnormal haemoglobins leads to different bile-pigment isomer distributions from that of normal haemoglobin. The isomer pattern may be correlated with the structure of the abnormal haemoglobin in the neighbourhood of the haem pocket. This is support for haem degradation by an intramolecular reaction. PMID:708385

  3. P. gingivalis in Periodontal Disease and Atherosclerosis – Scenes of Action for Antimicrobial Peptides and Complement

    PubMed Central

    Hussain, Mehak; Stover, Cordula M.; Dupont, Aline

    2015-01-01

    According to the NHS, it is estimated that over 50% of the adult population are, to some extent, affected by gum disease and approximately 15% of UK population have been diagnosed with severe periodontitis. Periodontitis, a chronic polymicrobial disease of the gums, causes inflammation in its milder form, whereas in its severe form affects the surrounding tissues and can result in tooth loss. During periodontitis, plaque accumulates and sits between the junctional epithelium and the tooth itself, resulting in inflammation and the formation of a periodontal pocket. An interface is formed directly between the subgingival bacteria and the junctional epithelial cells. Bacterial pathogens commonly associated with periodontal disease are, among others, Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola, together known as the “red complex.” This review will mostly concentrate on the role of P. gingivalis, a Gram-negative anaerobic bacterium and one of the major and most studied contributors of this disease. Because periodontal disease is associated with the development of atherosclerosis, it is important to understand the local immune response to P. gingivalis. Innate immune players, in particular, complement and antimicrobial peptides and their effects with regard to P. gingivalis during periodontitis and in the development of atherosclerosis will be presented. PMID:25713575

  4. Soluble CD14 Enhances the Response of Periodontal Ligament Stem Cells to P. gingivalis Lipopolysaccharide

    PubMed Central

    Andrukhov, Oleh; Andrukhova, Olena; Özdemir, Burcu; Haririan, Hady; Müller-Kern, Michael; Moritz, Andreas; Rausch-Fan, Xiaohui

    2016-01-01

    Periodontal ligament stem cells (PDLSCs) are lacking membrane CD14, which is an important component of lipopolysaccharide (LPS) signaling through toll-like receptor (TLR) 4. In the present study we investigated the effect of soluble CD14 on the response of human PDLSCs to LPS of Porphyromonas (P.) gingivalis. Human PDLSCs (hPDLSCs) were stimulated with P. gingivalis LPS in the presence or in the absence of soluble CD14 (sCD14) and the production of interleukin (IL)-6, chemokine C-X-C motif ligand 8 (CXCL8), and chemokine C-C motif ligand 2 (CCL2) was measured. The response to P. gingivalis LPS was compared with that to TLR4 agonist Escherichia coli LPS and TLR2-agonist Pam3CSK4. The response of hPDLSCs to both P. gingivalis LPS and E. coli LPS was significantly enhanced by sCD14. In the absence of sCD14, no significant difference in the hPDLSCs response to two kinds of LPS was observed. These responses were significantly lower compared to that to Pam3CSK4. In the presence of sCD14, the response of hPdLSCs to P. gingivalis LPS was markedly higher than that to E. coli LPS and comparable with that to Pam3CSK4. The response of hPdLSCs to bacterial LPS is strongly augmented by sCD14. Local levels of sCD14 could be an important factor for modulation of the host response against periodontal pathogens. PMID:27504628

  5. Haemoglobin scavenging after subarachnoid haemorrhage.

    PubMed

    Durnford, A; Dunbar, J; Galea, J; Bulters, D; Nicoll, J A R; Boche, D; Galea, I

    2015-01-01

    Rapid and effective clearance of cell-free haemoglobin after subarachnoid haemorrhage (SAH) is important to prevent vasospasm and neurotoxicity and improve long-term outcome. Haemoglobin is avidly bound by haptoglobin, and the complex is cleared by CD163 expressed on the membrane surface of macrophages. We studied the kinetics of haemoglobin and haptoglobin in cerebrospinal fluid after SAH. We show that haemoglobin levels rise gradually after SAH. Haptoglobin levels rise acutely with aneurysmal rupture as a result of injection of blood into the subarachnoid space. Although levels decline as haemoglobin scavenging occurs, complete depletion of haptoglobin does not occur and levels start rising again, indicating saturation of CD163 sites available for haptoglobin-haemoglobin clearance. In a preliminary neuropathological study we demonstrate that meningeal CD163 expression is upregulated after SAH, in keeping with a proinflammatory state. However, loss of CD163 occurs in meningeal areas with overlying blood compared with areas without overlying blood. Becauses ADAM17 is the enzyme responsible for shedding membrane-bound CD163, its inhibition may be a potential therapeutic strategy after SAH.

  6. Entamoeba gingivalis in sputum smears.

    PubMed

    Dao, A H

    1985-01-01

    Entamoeba gingivalis is a common parasite of the human buccal cavity whose rare appearance in Papanicolaou-stained sputum smears may be missed. Two such cases are described, including the morphologic features of this ameba. The trophozoites were seen to phagocytize leukocytes as well as red blood cells, in distinction to E. histiolytica, which phagocytizes only red blood cells and also can cause pulmonary abscesses. The concomitant finding of Actinomyces sp. organisms in one patient reinforces the possible symbiotic relationship between the two organisms, as has been suggested for their appearance in other extraoral sites, such as the female genital tract. PMID:3861055

  7. Structure of the haptoglobin-haemoglobin complex.

    PubMed

    Andersen, Christian Brix Folsted; Torvund-Jensen, Morten; Nielsen, Marianne Jensby; de Oliveira, Cristiano Luis Pinto; Hersleth, Hans-Petter; Andersen, Niels Højmark; Pedersen, Jan Skov; Andersen, Gregers Rom; Moestrup, Søren Kragh

    2012-09-20

    Red cell haemoglobin is the fundamental oxygen-transporting molecule in blood, but also a potentially tissue-damaging compound owing to its highly reactive haem groups. During intravascular haemolysis, such as in malaria and haemoglobinopathies, haemoglobin is released into the plasma, where it is captured by the protective acute-phase protein haptoglobin. This leads to formation of the haptoglobin-haemoglobin complex, which represents a virtually irreversible non-covalent protein-protein interaction. Here we present the crystal structure of the dimeric porcine haptoglobin-haemoglobin complex determined at 2.9 Å resolution. This structure reveals that haptoglobin molecules dimerize through an unexpected β-strand swap between two complement control protein (CCP) domains, defining a new fusion CCP domain structure. The haptoglobin serine protease domain forms extensive interactions with both the α- and β-subunits of haemoglobin, explaining the tight binding between haptoglobin and haemoglobin. The haemoglobin-interacting region in the αβ dimer is highly overlapping with the interface between the two αβ dimers that constitute the native haemoglobin tetramer. Several haemoglobin residues prone to oxidative modification after exposure to haem-induced reactive oxygen species are buried in the haptoglobin-haemoglobin interface, thus showing a direct protective role of haptoglobin. The haptoglobin loop previously shown to be essential for binding of haptoglobin-haemoglobin to the macrophage scavenger receptor CD163 (ref. 3) protrudes from the surface of the distal end of the complex, adjacent to the associated haemoglobin α-subunit. Small-angle X-ray scattering measurements of human haptoglobin-haemoglobin bound to the ligand-binding fragment of CD163 confirm receptor binding in this area, and show that the rigid dimeric complex can bind two receptors. Such receptor cross-linkage may facilitate scavenging and explain the increased functional affinity of

  8. "Race", ethnicity and haemoglobin disorders.

    PubMed

    Dyson, S M

    1998-07-01

    The new genetics has brought forth concerns that such developments as screening for genetic diseases will accentuate the oppression of minority ethnic groups [Bradby (1996) Genetics and racism. In The Troubled Helix: social and psychological aspects of the new human genetics, ed. T. Marteau and M. Richards, pp. 295-316. Cambridge University Press, Cambridge]. Haemoglobin disorders primarily affect minority ethnic groups in the U.K. but have been the subject of protest regarding lack of services as much as the unwelcome advent of them. This paper examines various conceptions of "race", from biological reductionism, through notions of ethnicity, racialized groups, sociological conceptions of "race", political and analytical uses of the term "Black" and so-called "new ethnicities" such as situational and plastic ethnicity in order to examine the consequences of these competing conceptions of race for a social analysis of sickle cell anaemia and beta-thalassaemia. The paper concludes that any group of people associated with the haemoglobin disorders are subject both to constraints upon their actions and opportunities for re-interpreting their social world. In conclusion it is proposed that no nomenclature classifies the phenomenon unproblematically. The notion of race as a political construct [Goldberg (1993) Racist Culture: Philosophy and the Politics of Meaning. Blackwell, Oxford] is used to suggest that attempts to construct all-embracing definitions themselves signal the potential abuses which may be attendant upon programmatic or mechanistic conceptions of the relationship between race and haemoglobin disorders.

  9. Specific cell components of Bacteroides gingivalis mediate binding and degradation of human fibrinogen

    SciTech Connect

    Lantz, M.S.; Allen, R.D.; Vail, T.A.; Switalski, L.M.; Hook, M. )

    1991-01-01

    Bacteroides (Porphyromonas) gingivalis, which has been implicated as an etiologic agent in human periodontal diseases, has been shown to bind and degrade human fibrinogen. B. gingivalis strains bind fibrinogen reversibly and with high affinity and bind to a specific region of the fibrinogen molecule that appears to be located between the D and E domains. The authors now report that human fibrinogen is bound and then degraded by specific B. gingivalis components that appear to be localized at the cell surface. Fibrinogen binding to bacterial cells occurred at 4, 22, and 37{degree}C. A functional fibrinogen-binding component (M{sub r}, 150 000) was identified when sodium dodecyl sulfate-solubilized bacteria were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and probed with {sup 125}I-fibrinogen. Fibrinogen degradation did not occur at 4{degree}C but did occur at 22 and 37{degree}C. When bacteria and iodinated fibrinogen were incubated at 37{degree}C, two major fibrinogen fragments (M{sub r}, 97 000 and 50 000) accumulated in incubation mixture supernatant fractions. Two major fibrinogen-degrading components (M{sub r}, 120 000 and 150 000) have been identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in substrate-containing gels. Fibrinogen degradation by the M{sub r}-120 000 and -150 000 proteases was enhanced by reducing agents, completely inhibited by N-{alpha}-p-tosyl-L-lysyl chloromethyl ketone, and partially inhibited by n-ethyl maleimide, suggesting that these enzymes are thiol-dependent proteases with trypsinlike substrate specificity. The fibrinogen-binding component could be separated from the fibrinogen-degrading components by selective solubilization of bacteria in sodium deoxycholate.

  10. Specific cell components of Bacteroides gingivalis mediate binding and degradation of human fibrinogen.

    PubMed Central

    Lantz, M S; Allen, R D; Vail, T A; Switalski, L M; Hook, M

    1991-01-01

    Bacteroides (Porphyromonas) gingivalis, which has been implicated as an etiologic agent in human periodontal diseases, has been shown to bind and degrade human fibrinogen. B. gingivalis strains bind fibrinogen reversibly and with high affinity and bind to a specific region of the fibrinogen molecule that appears to be located between the D and E domains (M. S. Lantz, R. D. Allen, P. Bounelis, L. M. Switalski, and M. Hook, J. Bacteriol. 172:716-726, 1990). We now report that human fibrinogen is bound and then degraded by specific B. gingivalis components that appear to be localized at the cell surface. Fibrinogen binding to bacterial cells occurred at 4, 22, and 37 degrees C. A functional fibrinogen-binding component (Mr, 150,000) was identified when sodium dodecyl sulfate-solubilized bacteria were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and probed with 125I-fibrinogen. Fibrinogen degradation did not occur at 4 degrees C but did occur at 22 and 37 degrees C. When bacteria and iodinated fibrinogen were incubated at 37 degrees C, two major fibrinogen fragments (Mr, 97,000 and 50,000) accumulated in incubation mixture supernatant fractions. Two major fibrinogen-degrading components (Mr, 120,000 and 150,000) have been identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in substrate-containing gels. Fibrinogen degradation by the Mr-120,000 and -150,000 proteases was enhanced by reducing agents, completely inhibited by N-alpha-p-tosyl-L-lysyl chloromethyl ketone, and partially inhibited by n-ethyl maleimide, suggesting that these enzymes are thiol-dependent proteases with trypsinlike substrate specificity. The fibrinogen-binding component could be separated from the fibrinogen-degrading components by selective solubilization of bacteria in sodium deoxycholate. Images PMID:1987144

  11. Laser antisepsis of Phorphyromonas gingivalis in vitro with dental lasers

    NASA Astrophysics Data System (ADS)

    Harris, David M.

    2004-05-01

    It has been shown that both pulsed Nd:YAG (1064nm) and continuous diode (810nm) dental lasers kill pathogenic bacteria (laser antisepsis), but a quantitative method for determining clinical dosimetry does not exist. The purpose of this study was to develop a method to quantify the efficacy of ablation of Porphyromonas gingivalis (Pg) in vitro for two different lasers. The ablation thresholds for the two lasers were compared in the following manner. The energy density was measured as a function of distance from the output of the fiber-optic delivery system. Pg cultures were grown on blood agar plates under standard anaerobic conditions. Blood agar provides an approximation of gingival tissue for the wavelengths tested in having hemoglobin as a primary absorber. Single pulses (Nd:YAG: 100- Œs diode: 100-msec) of laser energy were delivered to Pg colonies and the energy density was increased until the appearance of a small plume was observed coincident with a laser pulse. The energy density at this point defines the ablation threshold. Ablation thresholds to a single pulse were determined for both Pg and for blood agar alone. The large difference in ablation thresholds between the pigmented pathogen and the host matrix for pulsed-Nd:YAG represented a significant therapeutic ratio and Pg was ablated without visible effect on the blood agar. Near threshold the 810-nm diode laser destroyed both the pathogen and the gel. Clinically, the pulsed Nd:YAG may selectively destroy pigmented pathogens leaving the surrounding tissue intact. The 810-nm diode laser may not demonstrate this selectivity due to its longer pulse length and greater absorption by hemoglobin.

  12. Haem disorder in reconstituted human haemoglobin

    PubMed Central

    Docherty, John C.; Brown, Stanley B.

    1982-01-01

    Degradation of the haem of haemoglobin (used as a chemical probe of the haem–protein relationship), suggests that reconstituted human haemoglobin contains significant haem disorder. This results from the insertion of haem into globin with an orientation 180° different from the natural orientation. Haem disorder also slowly occurs in methaemoglobin solutions. PMID:7165711

  13. Molecular evolution of plant haemoglobin: two haemoglobin genes in Nymphaeaceae Euryale ferox.

    PubMed

    Guldner, E; Desmarais, E; Galtier, N; Godelle, B

    2004-01-01

    We isolated and sequenced two haemoglobin genes from the early-branching angiosperm Euryale ferox (Nymphaeaceae). The two genes belong to the two known classes of plant haemoglobin. Their existence in Nymphaeaceae supports the theory that class 1 haemoglobin was ancestrally present in all angiosperms, and is evidence for class 2 haemoglobin being widely distributed. These sequences allowed us to unambiguously root the angiosperm haemoglobin phylogeny, and to corroborate the hypothesis that the class 1/class 2 duplication event occurred before the divergence between monocots and eudicots. We addressed the molecular evolution of plant haemoglobin by comparing the synonymous and nonsynonymous substitution rates in various groups of genes. Class 2 haemoglobin genes of legumes (functionally involved in a symbiosis with nitrogen-fixing bacteria) show a higher nonsynonymous substitution rate than class 1 (nonsymbiotic) haemoglobin genes. This suggests that a change in the selective forces applying to plant haemoglobins has occurred during the evolutionary history of this gene family, potentially in relation with the evolution of symbiosis.

  14. Mucosal Langerhans Cells Promote Differentiation of Th17 Cells in a Murine Model of Periodontitis but Are Not Required for Porphyromonas gingivalis–Driven Alveolar Bone Destruction

    PubMed Central

    Bittner-Eddy, Peter D.; Fischer, Lori A.; Kaplan, Daniel H.; Thieu, Kathleen

    2016-01-01

    Periodontitis is a chronic oral inflammatory disease affecting one in five individuals that can lead to tooth loss. CD4+ Th cells activated by a microbial biofilm are thought to contribute to the destruction of alveolar bone surrounding teeth by influencing osteoclastogenesis through IL-17A and receptor activator for NF-κB ligand effects. The relative roles of mucosal Ag presentation cells in directing Th cell immune responses against oral pathogens and their contribution to destruction of alveolar bone remain unknown. We tested the contribution of mucosal Langerhans cells (LCs) to alveolar bone homeostasis in mice following oral colonization with a well-characterized human periodontal pathogen, Porphyromonas gingivalis. We found that oral mucosal LCs did not protect from or exacerbate crestal alveolar bone destruction but were responsible for promoting differentiation of Th17 cells specific to P. gingivalis. In mice lacking LCs the Th17 response was suppressed and a Th1 response predominated. Bypassing LCs with systemic immunization of P. gingivalis resulted in a predominantly P. gingivalis–specific Th1 response regardless of whether LCs were present. Interestingly, we find that in vivo clonal expansion of P. gingivalis–specific Th cells and induced regulatory T cells does not depend on mucosal LCs. Furthermore, destruction of crestal alveolar bone induced by P. gingivalis colonization occurred regardless of the presence of mucosal LCs or P. gingivalis–specific Th17 cells. Our data indicate that both LCs and Th17 cells are redundant in contributing to alveolar bone destruction in a murine model of periodontitis. PMID:27402698

  15. Oral Administration of P. gingivalis Induces Dysbiosis of Gut Microbiota and Impaired Barrier Function Leading to Dissemination of Enterobacteria to the Liver

    PubMed Central

    Nakajima, Mayuka; Arimatsu, Kei; Kato, Tamotsu; Matsuda, Yumi; Minagawa, Takayoshi; Takahashi, Naoki; Ohno, Hiroshi; Yamazaki, Kazuhisa

    2015-01-01

    Although periodontitis has been implicated as a risk factor for various systemic diseases, the precise mechanisms by which periodontitis induces systemic disease remain to be elucidated. We have previously revealed that repeated oral administration of Porphyromonas gingivalis elicits endotoxemia via changes in the gut microbiota of the ileum, and thereby induces systemic inflammation and insulin resistance. However, it is not clear to what extent a single administration of P. gingivalis could affect gut microbiota composition, gut barrier function, and subsequent influx of gut microbiota into the liver. Therefore, in the present study, C57BL/6 mice were orally administered P. gingivalis (strain W83) once and compared to sham-inoculated mice. The phylogenetic structure and diversity of microbial communities in the gut and liver were analyzed by pyrosequencing the 16S ribosomal RNA genes. Serum endotoxin activity was determined by a Limulus amebocyte lysate test. Gene expression in the intestine and expression of 16S rRNA genes in the blood and liver were examined by quantitative polymerase chain reaction. Administration of P. gingivalis significantly altered gut microbiota, with an increased proportion of phylum Bacteroidetes, a decreased proportion of phylum Firmicutes, and increased serum endotoxin levels. In the intestinal tissues, gene expression of tjp-1 and occludin, which are involved in intestinal permeability, were downregulated. Higher amounts of bacterial DNA were detected in the liver of infected mice. Importantly, changes in gut microbiota preceded systemic inflammatory changes. These results further support the idea that disturbance of the gut microbiota composition by orally derived periodontopathic bacteria may be a causal mechanism linking periodontitis and systemic disease. PMID:26218067

  16. Oral Administration of P. gingivalis Induces Dysbiosis of Gut Microbiota and Impaired Barrier Function Leading to Dissemination of Enterobacteria to the Liver.

    PubMed

    Nakajima, Mayuka; Arimatsu, Kei; Kato, Tamotsu; Matsuda, Yumi; Minagawa, Takayoshi; Takahashi, Naoki; Ohno, Hiroshi; Yamazaki, Kazuhisa

    2015-01-01

    Although periodontitis has been implicated as a risk factor for various systemic diseases, the precise mechanisms by which periodontitis induces systemic disease remain to be elucidated. We have previously revealed that repeated oral administration of Porphyromonas gingivalis elicits endotoxemia via changes in the gut microbiota of the ileum, and thereby induces systemic inflammation and insulin resistance. However, it is not clear to what extent a single administration of P. gingivalis could affect gut microbiota composition, gut barrier function, and subsequent influx of gut microbiota into the liver. Therefore, in the present study, C57BL/6 mice were orally administered P. gingivalis (strain W83) once and compared to sham-inoculated mice. The phylogenetic structure and diversity of microbial communities in the gut and liver were analyzed by pyrosequencing the 16S ribosomal RNA genes. Serum endotoxin activity was determined by a Limulus amebocyte lysate test. Gene expression in the intestine and expression of 16S rRNA genes in the blood and liver were examined by quantitative polymerase chain reaction. Administration of P. gingivalis significantly altered gut microbiota, with an increased proportion of phylum Bacteroidetes, a decreased proportion of phylum Firmicutes, and increased serum endotoxin levels. In the intestinal tissues, gene expression of tjp-1 and occludin, which are involved in intestinal permeability, were downregulated. Higher amounts of bacterial DNA were detected in the liver of infected mice. Importantly, changes in gut microbiota preceded systemic inflammatory changes. These results further support the idea that disturbance of the gut microbiota composition by orally derived periodontopathic bacteria may be a causal mechanism linking periodontitis and systemic disease. PMID:26218067

  17. Synthesis of Human Haemoglobin by Plants

    ERIC Educational Resources Information Center

    Onyesom, I.

    2006-01-01

    Haemoglobin, Hb is the red, protein pigment in blood that transports oxygen round the body. Decreased quantity could lead to anaemia, and when the anaemic condition turns severe, blood transfusion becomes inevitable. However, the safety of human source has become questionable in recent times, and this has aroused the interest of scientists to…

  18. Evolutionary diversification of the trypanosome haptoglobin-haemoglobin receptor from an ancestral haemoglobin receptor

    PubMed Central

    Lane-Serff, Harriet; MacGregor, Paula; Peacock, Lori; Macleod, Olivia JS; Kay, Christopher; Gibson, Wendy; Higgins, Matthew K; Carrington, Mark

    2016-01-01

    The haptoglobin-haemoglobin receptor of the African trypanosome species, Trypanosoma brucei, is expressed when the parasite is in the bloodstream of the mammalian host, allowing it to acquire haem through the uptake of haptoglobin-haemoglobin complexes. Here we show that in Trypanosoma congolense this receptor is instead expressed in the epimastigote developmental stage that occurs in the tsetse fly, where it acts as a haemoglobin receptor. We also present the structure of the T. congolense receptor in complex with haemoglobin. This allows us to propose an evolutionary history for this receptor, charting the structural and cellular changes that took place as it adapted from a role in the insect to a new role in the mammalian host. DOI: http://dx.doi.org/10.7554/eLife.13044.001 PMID:27083048

  19. Analysis of Mitochondrial haemoglobin in Parkinson's disease brain.

    PubMed

    Shephard, Freya; Greville-Heygate, Oliver; Liddell, Susan; Emes, Richard; Chakrabarti, Lisa

    2016-07-01

    Mitochondrial dysfunction is an early feature of neurodegeneration. We have shown there are mitochondrial haemoglobin changes with age and neurodegeneration. We hypothesised that altered physiological processes are associated with recruitment and localisation of haemoglobin to these organelles. To confirm a dynamic localisation of haemoglobin we exposed Drosophila melanogaster to cyclical hypoxia with recovery. With a single cycle of hypoxia and recovery we found a relative accumulation of haemoglobin in the mitochondria compared with the cytosol. An additional cycle of hypoxia and recovery led to a significant increase of mitochondrial haemoglobin (p<0.05). We quantified ratios of human mitochondrial haemoglobin in 30 Parkinson's and matched control human post-mortem brains. Relative mitochondrial/cytosolic quantities of haemoglobin were obtained for the cortical region, substantia nigra and cerebellum. In age matched post-mortem brain mitochondrial haemoglobin ratios change, decreasing with disease duration in female cerebellum samples (n=7). The change is less discernible in male cerebellum (n=18). In cerebellar mitochondria, haemoglobin localisation in males with long disease duration shifts from the intermembrane space to the outer membrane of the organelle. These new data illustrate dynamic localisation of mitochondrial haemoglobin within the cell. Mitochondrial haemoglobin should be considered in the context of gender differences characterised in Parkinson's disease. It has been postulated that cerebellar circuitry may be activated to play a protective role in individuals with Parkinson's. The changing localisation of intracellular haemoglobin in response to hypoxia presents a novel pathway to delineate the role of the cerebellum in Parkinson's disease. PMID:27181046

  20. Frequency of Entamoeba gingivalis in human gingival scrapings.

    PubMed

    Dao, A H; Robinson, D P; Wong, S W

    1983-09-01

    A survey was made of gingival scrapings stained by the Papanicolaou method to assess the occurrence of Entamoeba gingivalis, a nonpathogenic-oral amoeba. Positive findings were recorded in 59% of 113 dental patients, and 32% of 96 healthy controls. These figures showed no significant changes during the last 20 years when compared with data published in 1960 and 1963. The existence of E. gingivalis and its rare appearance in the sputum should be known to cytologists because of the morphologic resemblance to Entamoeba histolytica, a pathogenic amoeba. Morphologic features are described to differentiate E. gingivalis from similar structures found in sputum. PMID:6881102

  1. Investigate the correlation between clinical sign and symptoms and the presence of P. gingivalis, T. denticola, and T. forsythia individually or as a “Red complex” by a multiplex PCR method

    PubMed Central

    Sanghavi, Tulsi Hasmukhrai; Shah, Nimisha; Shah, Ruchi Rani; Sanghavi, Akta

    2014-01-01

    Aim: The aim of this study was to investigate the correlation between endodontic clinical signs and symptoms and the presence of Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia or their association by Multiplex polymerase chain reaction assay. Materials and Methods: Microbial samples were taken from 30 cases with necrotic pulp tissues in primary infections. DNA was extracted from the samples, which were analyzed for the presence of three endodontic pathogens by using species-specific primers. Results: P. gingivalis, T. denticola, T. forsythia, and Red Complex were present in 11, 17, 4, and 2 canals, respectively. Clinical and statistically significant relationships were found between T. forsythia and mobility and between T. denticola and swelling. (P < 0.05). Presence of other Red complex bacteria shows clinical association with presence of signs and symptoms but no statistically significant relationship. Conclusion: The high prevalence of P. gingivalis, T. denticola, and T. forsythia in the examined samples suggests that these bacteria are related to the etiology of symptomatic periradicular diseases. PMID:25506144

  2. First Human Case of Fatal Halicephalobus gingivalis Meningoencephalitis in Australia

    PubMed Central

    Crawford, April; Moore, Casey V.; Gasser, Robin B.; Nelson, Renjy; Koehler, Anson V.; Bradbury, Richard S.; Speare, Rick; Dhatrak, Deepak; Weldhagen, Gerhard F.

    2015-01-01

    Halicephalobus gingivalis (previously Micronema deletrix) is a free-living nematode known to cause opportunistic infections, mainly in horses. Human infections are very rare, but all cases described to date involved fatal meningoencephalitis. Here we report the first case of H. gingivalis infection in an Australian human patient, confirmed by nematode morphology and sequencing of ribosomal DNA. The implications of this case are discussed, particularly, the need to evaluate real-time PCR as a diagnostic tool. PMID:25694532

  3. A simple and reliable method for estimating haemoglobin.

    PubMed Central

    Stott, G. J.; Lewis, S. M.

    1995-01-01

    A new colour scale has been advised for estimating haemoglobin levels by matching the blood samples with ten levels of haemoglobin (3, 4, 5, 6, 7, 8, 9, 10, 12, and 14 g/dl) on the scale. Preliminary results show good correlations with spectrophotometric readings. The new device is being field tested and if the initial promise is confirmed, will provide a simple and reliable method for estimating haemoglobin where laboratory facilities are not available. Images Fig. 2 PMID:7614669

  4. Haemoglobin Porto Alegre in a Cuban family.

    PubMed Central

    Martínez, G; Lima, F; Wade, M; Estrada, M; Colombo, B; Heredero, L; Granda, H

    1977-01-01

    During a screening programme for abnormal haemoglobins in Habana, one case of Hb Porto Alegre was found in 23 000 cases analysed. The ability of this variant to polymerise in vitro and the absence of clinical features in the carriers have been confirmed. These observations are now explained by the findings of high levels of glutathione in the red cells of subjects heterozygous for Hb Porto Alegre: it is suggested that the increase of glutathione is responsible for the absence of in vivo polymerisation and accounts for the lack of clinical symptoms. Images PMID:604493

  5. Evaluation of the Haemoglobin Colour Scale and comparison with the HemoCue haemoglobin assay.

    PubMed Central

    Paddle, J. J.

    2002-01-01

    OBJECTIVE: To evaluate the Haemoglobin Colour Scale developed by WHO for estimating haemoglobin concentration and to compare the results obtained using it and the HemoCue assay with those determined using a reference method, the Technicon H3 analyser. METHODS: The Colour Scale and HemoCue assay were used to test 408 blood samples. Subsequently, Bland-Altman plots were determined and the proximity of the test results to those obtained using the reference method was determined. FINDINGS: The mean difference between the Haemoglobin Colour Scale and the reference method was 0.19 g/dl (95% confidence interval: 3.50 g/dl below to 3.11 g/dl above); the corresponding value for the HemoCue assay was 0.50 g/dl (1.16 g/dl below to 0.16 g/dl above). Only 46.08% of the results obtained by means of the Colour Scale were within 1.0 g/dl of the reference method, whereas 95.34% of the HemoCue results fell within this limit; 22.79% of the Colour Scale results but none of the HemoCue results lay more than 2.0 g/dl from the reference method. CONCLUSION: The Haemoglobin Colour Scale test is too inaccurate for general use, particularly if devices such as the HemoCue are available. PMID:12471402

  6. A molecular survey of S. mutans and P. gingivalis oral microbial burden in human saliva using Relative Endpoint Polymerase Chain Reaction (RE-PCR) within the population of a Nevada dental school revealed disparities among minorities

    PubMed Central

    2012-01-01

    Background The University of Nevada, Las Vegas School of Dental Medicine recently opened an orthodontic treatment clinic to address the needs of the racially and ethnically diverse population of Southern Nevada, primarily focusing on the treatment and care of low-income and minority patients. Although orthodontic treatment and therapy has been shown to induce changes in the oral cavity, much of this evidence was collected from traditional White, teenage orthodontic clinic populations. The primary goal of this study was to describe the microbial burden of the cariogenic and periodontal pathogens, Streptococcus mutans and Porphyromonas gingivalis within the UNLV-SDM patient population. Methods Representative saliva samples were collected from healthy adult patients for DNA isolation. Relative endpoint polymerase chain reaction (RE-PCR) was performed to ascertain the presence and relative microbial burden of these oral pathogens. Results Nearly one quarter (13/56) or 23.3% of these patients had elevated levels of S. mutans, while (10/56) and 17.8% of these samples were found to have elevated levels of P. gingivalis, - with (90%) of P. gingivalis-positive samples from minority patients (X2 = 17.921, d.f. = 1; p < 0.0001). Conclusions These findings of elevated P. gingivalis levels, primarily among minority patients, may suggest underlying oral health practices contributing to adverse oral health conditions within this population. Oral health knowledge and practices among minority patients may be strongly influenced by other factors, including education and socioeconomic status, suggesting additional research may be needed to accurately determine the most appropriate standards for care and oral health education within this patient population. PMID:22925755

  7. Haemoglobin, a new major allergen of Anisakis simplex.

    PubMed

    González-Fernández, Juan; Daschner, Alvaro; Nieuwenhuizen, Natalie E; Lopata, Andreas L; Frutos, Consolación De; Valls, Ana; Cuéllar, Carmen

    2015-05-01

    Gastro-allergic anisakiasis and Anisakis sensitisation associated chronic urticaria are diseases which differ in their IgE and IgG4 responses against both crude extract and specific allergens. Anisakis and Ascaris are closely related nematodes that usually cause problems with specificity in immunodiagnostics. In this study we measured IgE and IgG4 antibodies against Anisakis simplex sensu lato (s. l.) and Ascaris suum haemoglobins in sera of 21 gastro-allergic anisakiasis and 23 chronic urticaria patients. We used a capture ELISA with the anti-Anisakis haemoglobin monoclonal antibody 4E8g, which also recognises Ascaris haemoglobin. In addition, we determined specific IgE and IgG4 to both nematodes by indirect ELISA and immunoblotting. Anti-A. simplex s. l. haemoglobin IgE and IgG4 levels were higher in gastro-allergic anisakiasis than in chronic urticaria patients (P=0.002 and 0.026, respectively). Surprisingly, no patient had detectable IgE levels against A. suum haemoglobin. Finally, we carried out an in silico study of the B-cell epitopes of both haemoglobin molecules. Five epitopes were predicted in Anisakis pegreffii and four in A. suum haemoglobin. The epitope propensity values of Anisakis haemoglobin in the equivalent IgE binding region of the allergenic haemoglobin Chi t 1 from Chironomus thummi, were higher those of the Ascaris haemoglobin. In conclusion, we describe A. simplex haemoglobin as a new major allergen (Ani s 13), being recognised by a large number (64.3%) of sensitised patients and up to 80.9% in patients with gastro-allergic anisakiasis. The presence of a specific epitope and the different values of epitope propensity between Anisakis and Ascaris haemoglobin could explain the lack of cross-reactivity between the two molecules. The absence of IgE reactivity to Ascaris haemoglobin in Anisakis patients makes Anisakis haemoglobin (Ani s 13) a potential candidate for developing more specific diagnosis tools. PMID:25683373

  8. An outbreak of bovine meningoencephalomyelitis with identification of Halicephalobus gingivalis.

    PubMed

    Enemark, Heidi Larsen; Hansen, Mette Sif; Jensen, Tim Kåre; Larsen, Gitte; Al-Sabi, Mohammad Nafi Solaiman

    2016-03-15

    Halicephalobus gingivalis is an opportunistic parasite which is known to cause fatal meningoencephalomyelitis primarily in equines but sporadically also in humans. In April 2014, laboratory examination of the head of a young dairy calf, euthanized due to severe central nervous system symptoms, revealed the presence of granulomatous to necrotizing encephalitis and myriads of nematodes in the brain lesion. Morphologically the parasites were identified as H. gingivalis. The diagnosis was confirmed by molecular analysis of the large subunit (LSU) rRNA and the small subunit (SSU) rRNA genes, revealing genetic variations of 0.5-4.4% and 0.7-8.6%, respectively, between the H. gingivalis isolated from the Danish calf and published isolates, collected worldwide from free-living and parasitic stages of the nematode. Clinical symptoms and histological changes indicated infection with H. gingivalis from another three calves in the herd. This is the first scientific publication of H. gingivalis induced meningoencephalomyelitis in ruminants. As ante mortem diagnosis is a major challenge, the infection may easily remain undiagnosed in cattle. PMID:26872932

  9. Digestion of haemoglobin by schistosomes: 35 years on.

    PubMed

    Dalton, J P; Smith, A M; Clough, K A; Brindley, P J

    1995-08-01

    Adult schistosomes obtain nutrients by digesting haemoglobin, which they obtain from ingested host red blood cells. Here John Dalton, Angela Smith, Karen Clough and Paul Brindley argue that a cathepsin L proteinase recently identified in their laboratory as the predominant cysteine proteinase activity of Schistosoma mansoni may play the leading role in haemoglobin digestion. They discuss the importance of elucidating the roles of both cathepsin B and cathepsin L in the digestion of haemoglobin, since both should be considered important targets to which novel schistosomicidal agents could be directed.

  10. N-terminal amino acid sequence of the deep-sea tube worm haemoglobin remarkably resembles that of annelid haemoglobin.

    PubMed Central

    Suzuki, T; Takagi, T; Ohta, S

    1988-01-01

    The deep-sea giant tube worm Lamellibrachia, belonging to the phylum Vestimentifera, contains two extracellular haemoglobins, an Mr 3,000,000 haemoglobin and an Mr 440,000 haemoglobin. The former has a hexagonal bilayer structure and consists of six polypeptide chains (AI-VI); a study of its haem content shows that not all of the chains contain haem. The Mr 440,000 haemoglobin consists of four haem-containing chains (BI-IV). We isolated most of the chains by reverse-phase chromatography and determined the amino acid sequences of the 21-45 N-terminal residues. Eight chains (AI-IV and BI-IV) showed significant homology with haem-containing chains of annelid giant haemoglobin. The highest homology was found between Lamellibrachia chain AI and Tylorrhynchus chain I; surprisingly, 18 out of the 20 N-terminal residues are identical. On the other hand, chain AV, with an unusual Mr of 32,000, showed a rather different sequence and is likely to be a non-haem chain which might act as a linker protein in the assembly of the haem-containing chains. From these results, we conclude that the tube worm Mr 3,000,000 haemoglobin is highly homologous with annelid haemoglobin. Images Fig. 2. PMID:3202832

  11. Haemoglobin binding of a musk xylene metabolite in man.

    PubMed

    Riedel, J; Birner, G; van Dorp, C; Neumann, H G; Dekant, W

    1999-06-01

    1. Musk xylene (1-tert-butyl-3,5-dimethyl-2,4,6-trinitrobenzene) is used as a fragrance component in toiletries, detergents and skin care products. Musk xylene is widely distributed in the environment and has been identified as a persistent contaminant in fish and in mothers' milk. Experimental data in man indicate a slow elimination of musk xylene and a potential for accumulation. Nitroarenes may be biotransformed to the respective amines. Some aromatic amines are known to be tumorigenic in animals and in man. Quantitation of the binding of those aromatic amines to haemoglobin has been proposed as a biomarker of internal exposure. 2. To determine bioavailability, metabolic reduction and haemoglobin binding of musk xylene in man, we investigated the presence of musk xylene metabolites bound to haemoglobin in blood samples from rat and from 10 human volunteers not knowingly exposed to musk xylene. 3. Haemoglobin from the blood samples was isolated, and bound metabolites were liberated as amines by alkaline hydrolysis. In haemoglobin samples from all individuals, 1-tert-butyl-3,5-dimethyl-4-amino-2,6-dinitrobenzene and, after chemical derivatization, the corresponding N-perfluoropropyl amide were identified by GC/MS using electron-impact and electron-capture mass spectrometry. 4. The amounts of 1-tert-butyl-3,5-dimethyl-4-amino-2,6-dinitrobenzene bound to haemoglobin in the human blood samples ranged from 13 to 46 fmol/mg haemoglobin. 5. These data demonstrate that musk xylene is bioavailable in man. The use of haemoglobin binding as a biomarker for nitromusk exposure in the general population warrants further studies.

  12. Modulation of oxidative stability of haemoglobin inside liposome-encapsulated haemoglobin

    PubMed Central

    Awasthi, Vibhudutta; Yadav, Vivek R; Goins, Beth; Phillips, William T

    2013-01-01

    The major hurdle in the formulation of liposome-encapsulated haemoglobin (LEH) is the oxidation of haemoglobin (Hb) into methaemoglobin during storage and after administration. In order to reduce this oxidative degradation, we tested various reducing conditions in the presence of catalase. We found that at 37°C more than 50% of Hb oxidized to methaemoglobin within 24 h, whereas in presence of catalase, the oxidation was significantly reduced. The effect of catalase was further enhanced by a reduction mixture containing β-NAD, d-glucose, adenine, inosine, MgCl2, KCl, KH2PO4 and Na2HPO4−, only 14% methaemoglobin was generated in the presence of catalase and reduction mixture (CRM). Contrary to the expectation, glutathione, deferoxamine and homocysteine enhanced Hb oxidation. The presence of CRM inside liposomes (250 nm) significantly decreased Hb oxidation. The results suggest that catalase and a well-defined mixture of co-factors may help control Hb oxidation for improvement in the functional life of LEH. PMID:23231644

  13. AHSP (α-haemoglobin-stabilizing protein) stabilizes apo-α-haemoglobin in a partially folded state.

    PubMed

    Krishna Kumar, Kaavya; Dickson, Claire F; Weiss, Mitchell J; Mackay, Joel P; Gell, David A

    2010-12-01

    To produce functional Hb (haemoglobin), nascent α-globin (αo) and β-globin (βo) chains must each bind a single haem molecule (to form αh and βh) and interact together to form heterodimers. The precise sequence of binding events is unknown, and it has been suggested that additional factors might enhance the efficiency of Hb folding. AHSP (α-haemoglobin-stabilizing protein) has been shown previously to bind αh and regulate redox activity of the haem iron. In the present study, we used a combination of classical and dynamic light scattering and NMR spectroscopy to demonstrate that AHSP forms a heterodimeric complex with αo that inhibits αo aggregation and promotes αo folding in the absence of haem. These findings indicate that AHSP may function as an αo-specific chaperone, and suggest an important role for αo in guiding Hb assembly by stabilizing βo and inhibiting off-pathway self-association of βh.

  14. Alpha-haemoglobin stabilizing protein (AHSP) stabilizes apo-α-haemoglobin in a partially folded state

    PubMed Central

    Krishna Kumar, Kaavya; Dickson, Claire F.; Weiss, Mitchell J.; Mackay, Joel P.; Gell, David A.

    2015-01-01

    SYNOPSIS To produce functional haemoglobin, nascent α-globin (αo) and β-globin (βo) chains must each bind a single haem molecule (to form αh and βh) and interact together to form heterodimers. The precise sequence of binding events is unknown, and it has been suggested that additional factors might enhance the efficiency of Hb folding. The α-haemoglobin stabilizing protein (AHSP) has previously been shown to bind αh and regulate redox activity of the haem iron. Here, we use a combination of classical and dynamic light scattering and NMR spectroscopy to demonstrate that AHSP forms a heterodimeric complex with αo that inhibits αo aggregation and promotes αo folding in the absence of haem. These findings indicate that AHSP may function as an αo-specific chaperone, and suggest an important role for αo in guiding Hb assembly by stabilizing βo and inhibiting off-pathway self-association of βh. PMID:20860551

  15. Aberrantly high glycated haemoglobin measurement due to the haemoglobin variant Hb Santa Juana.

    PubMed

    Ng, H L; Koh, C K

    2011-04-01

    Various laboratory and patient-related factors can result in falsely high or low glycated haemoglobin (HbA1c) measurements, and haemoglobin (Hb) variants that interfere with laboratory readings is an important cause of this. We report a case of a rare Hb variant, Hb Santa Juana, manifesting as a falsely high HbA1c in a 62-year-old patient with type 2 diabetes mellitus. The patient presented with high HbA1c values that persisted despite the intensification of anti-diabetic treatment. His home blood glucose levels were incongruently low compared to his HbA1c values. Further investigations revealed a family history of the variant Hb Santa Juana. This was confirmed in the patient when his blood was sent for DNA analysis. It is important for clinicians to be aware of the factors that can influence laboratory HbA1c measurements, as clinical decisions on treatment are often based on these measurements.

  16. C-Terminal Domain Residues Important for Secretion and Attachment of RgpB in Porphyromonas gingivalis▿

    PubMed Central

    Slakeski, Nada; Seers, Christine A.; Ng, Kaiting; Moore, Caroline; Cleal, Steven M.; Veith, Paul D.; Lo, Alvin W.; Reynolds, Eric C.

    2011-01-01

    Porphyromonas gingivalis, a periodontal pathogen, expresses a group of surface proteins with a common C-terminal domain (CTD) that are exported by a novel secretion system to the surface, where they are covalently attached. Using RgpB as a model CTD protein, we have produced a series of site-directed mutations in the CTD sequence at conserved residues and at residues that may be modified and, hence, surface attached. The mutant RgpB proteins were expressed in a P. gingivalis host lacking functional RgpB and RgpA Arg-specific proteases. The RgpB mutants produced were Y674F, Y674F Y718F, T675Q S679Q T682Q T684Q, T693Q, F695A, D696A, N698A, G699P, G716P, T724Q, T728Q T730Q, and K732Q and a protein with a deletion of residues 692 to 702 (Δ692-702). The mutants were characterized for cell-associated Arg-specific protease activity and for cellular distribution using anti-Rgp antibodies and Western blotting of culture fractions. All the mutants exhibited cell-associated Arg-specific activity similar to that of the positive control except for the D696A and Δ692-702 mutants. For all mutants, except D696A and Δ692-702, the RgpB proteins were found modified and attached to the cell surface, which was the same profile found in the positive-control strain. Only trace amounts of the precursor form of the Δ692-702 mutant were detected in the outer membrane, with none detected in the periplasm or culture fluid although cell transcript levels were normal. The results suggest that residues 692 to 702 of the CTD, in particular, residue D696, have an important role in the attachment of RgpB at the cell surface and that without attachment secretion does not occur. PMID:20971915

  17. Haemoglobin-E in the presence of oxidative substances from fava bean may be protective against Plasmodium falciparum malaria.

    PubMed

    Kitayaporn, D; Nelson, K E; Charoenlarp, P; Pholpothi, T

    1992-01-01

    A case-control study was carried out at a community hospital in eastern Thailand in order to study the association between haemoglobin E and Plasmodium falciparum malaria; 271 P. falciparum cases and 271 controls were enrolled. After adjusting for age, sex, time since last malaria attack, history of mosquito net use, and history of fava bean consumption in the previous month, neither heterozygous nor homozygous haemoglobin E provided significant protection against P. falciparum infection, with odds ratios (OR) = 0.91 (95% confidence limits = 0.61, 1.36) and 0.78 (0.34, 1.82) respectively when compared to persons with haemoglobin A who were not consumers of fava beans. However, haemoglobin E carriers who ate fava beans were significantly protected against P. falciparum malaria with OR = 0.26 (0.09, 0.76) and OR = 0.001 (0.00, 1120.59) for subjects with heterozygous and homozygous haemoglobin E, respectively. The study suggests a possible synergistic protective effect of haemoglobin E on the risk of P. falciparum malaria in subjects who have consumed fava beans.

  18. Haemoglobin-E in the presence of oxidative substances from fava bean may be protective against Plasmodium falciparum malaria.

    PubMed

    Kitayaporn, D; Nelson, K E; Charoenlarp, P; Pholpothi, T

    1992-01-01

    A case-control study was carried out at a community hospital in eastern Thailand in order to study the association between haemoglobin E and Plasmodium falciparum malaria; 271 P. falciparum cases and 271 controls were enrolled. After adjusting for age, sex, time since last malaria attack, history of mosquito net use, and history of fava bean consumption in the previous month, neither heterozygous nor homozygous haemoglobin E provided significant protection against P. falciparum infection, with odds ratios (OR) = 0.91 (95% confidence limits = 0.61, 1.36) and 0.78 (0.34, 1.82) respectively when compared to persons with haemoglobin A who were not consumers of fava beans. However, haemoglobin E carriers who ate fava beans were significantly protected against P. falciparum malaria with OR = 0.26 (0.09, 0.76) and OR = 0.001 (0.00, 1120.59) for subjects with heterozygous and homozygous haemoglobin E, respectively. The study suggests a possible synergistic protective effect of haemoglobin E on the risk of P. falciparum malaria in subjects who have consumed fava beans. PMID:1412643

  19. Detection and identification of Entamoeba gingivalis by specific amplification of rRNA gene.

    PubMed

    Kikuta, N; Yamamoto, A; Goto, N

    1996-12-01

    A pair of oligonucleotide primers were designed from the nucleotide sequence of the gene encoding the small subunit ribosomal RNA (SrRNA) of the oral protozoan parasite Entamoeba gingivalis. The primers amplified a 1.4-kb DNA fragment by polymerase chain reaction and were specific for Entamoeba gingivalis but not for other protozoa, oral protists and bacteria, or human leukocytes. With this method, the DNA from as few as 30 cells of Entamoeba gingivalis could be detected. These results suggest that this approach is applicable to the detection and identification of Entamoeba gingivalis in the human oral cavity.

  20. Variation of haemoglobin extinction coefficients can cause errors in the determination of haemoglobin concentration measured by near-infrared spectroscopy

    NASA Astrophysics Data System (ADS)

    Kim, J. G.; Liu, H.

    2007-10-01

    Near-infrared spectroscopy or imaging has been extensively applied to various biomedical applications since it can detect the concentrations of oxyhaemoglobin (HbO2), deoxyhaemoglobin (Hb) and total haemoglobin (Hbtotal) from deep tissues. To quantify concentrations of these haemoglobin derivatives, the extinction coefficient values of HbO2 and Hb have to be employed. However, it was not well recognized among researchers that small differences in extinction coefficients could cause significant errors in quantifying the concentrations of haemoglobin derivatives. In this study, we derived equations to estimate errors of haemoglobin derivatives caused by the variation of haemoglobin extinction coefficients. To prove our error analysis, we performed experiments using liquid-tissue phantoms containing 1% Intralipid in a phosphate-buffered saline solution. The gas intervention of pure oxygen was given in the solution to examine the oxygenation changes in the phantom, and 3 mL of human blood was added twice to show the changes in [Hbtotal]. The error calculation has shown that even a small variation (0.01 cm-1 mM-1) in extinction coefficients can produce appreciable relative errors in quantification of Δ[HbO2], Δ[Hb] and Δ[Hbtotal]. We have also observed that the error of Δ[Hbtotal] is not always larger than those of Δ[HbO2] and Δ[Hb]. This study concludes that we need to be aware of any variation in haemoglobin extinction coefficients, which could result from changes in temperature, and to utilize corresponding animal's haemoglobin extinction coefficients for the animal experiments, in order to obtain more accurate values of Δ[HbO2], Δ[Hb] and Δ[Hbtotal] from in vivo tissue measurements.

  1. Transplanting a unique allosteric effect from crocodile into human haemoglobin.

    PubMed

    Komiyama, N H; Miyazaki, G; Tame, J; Nagai, K

    1995-01-19

    Crocodiles are able to remain under water for more than one hour without surfacing to breathe and often kill their prey by drowning it. How do crocodiles stay under water for a long time? When they hold their breath, bicarbonate ions, the final product of respiration, accumulate and drastically reduce the oxygen affinity of haemoglobin, releasing a large fraction of haemoglobin-bound oxygen into the tissues. We have now located the bicarbonate-ion-binding site at the alpha 1 beta 2-subunit interface by making various human-crocodile chimaeric haemoglobins. Furthermore, we have been able to transplant the bicarbonate effect into human haemoglobin by replacing only a few residues, even though the amino-acid sequence identity between crocodile (Crocodylus niloticus) and human haemoglobins is only 68% for the alpha- and 51% for the beta-subunit. These results indicate that an entirely new function which enables species to adapt to a new environment could evolve in a protein by a relatively small number of amino-acid substitutions in key positions.

  2. Characterization of a polysaccharide antigen from Bacteroides gingivalis.

    PubMed

    Schifferle, R E; Reddy, M S; Zambon, J J; Genco, R J; Levine, M J

    1989-11-01

    A polysaccharide Ag (PS) was isolated from the phenol-water extract of Bacteroides gingivalis strain A7A1-28 and separated from LPS by Sephacryl S-400 HR chromatography. The PS was composed of glucose, glucosamine, galactosamine, and galactosaminuronic acid, while the LPS contained rhamnose, mannose, galactose, glucose, glucosamine, galactosamine, phosphate, and lipid, but not galactosaminuronic acid. The PS and LPS were immunochemically distinct by immunoelectrophoresis in agarose with homologous rabbit antiserum. The phenol-water extract from strain A7A1-28 was immunoreactive by immunoelectrophoresis against antisera to three additional strains of B. gingivalis, however, the PS was only reactive with homologous serum. Immunochemical characterization of decarboxylated and deacetylated PS derivatives suggest that the acetylation of the amino sugars, but not the presence of the carboxylate residue on galactosaminuronic acid contributes to major immunodeterminant expression.

  3. Association between glutathione, haemoglobin and transferrin in finnsheep.

    PubMed

    Atroshi, F; Osterberg, S; Lindström, U B

    1980-04-01

    Haemoglobin (Hb) and transferrin (Tf) types were determined in 760 Finnsheep and correlated with the reduced glutathione (GSH) levels of packed erythrocytes. The gene frequencies of the two haemoglobin alleles A and B were: HbA = 0.748 and HbB = 0.252. Five transferrin alleles were found: A, B, C, D and E, with gene frequencies of 0.056, 0.226, 0.620, 0.075 and 0.023, respectively. The Hb B group had significantly higher GSH levels and lower haematocrit values than heterozygotes (Hb AB) and homozygote (Hb AA). There was no significant difference in GSH concentration among the haemoglobin types in lambs. Erythrocyte glutathione levels of Tf BC ewes were significantly higher than the levels in sheep with other transferrin types, whereas young lambs of Tf AB types had the highest GSH levels. The significance of these findings is discussed.

  4. Haemoglobin biosynthesis site in rabbit embryo erythroid cells.

    PubMed

    Cianciarullo, Aurora M; Bertho, Alvaro L; Soares, Maurilio J; Hosoda, Tânia M; Nogueira-Silva, Simone; Beçak, Willy

    2003-01-01

    Properly metabolized globin synthesis and iron uptake are indispensable for erythroid cell differentiation and maturation. Mitochondrial participation is crucial in the process of haeme synthesis for cytochromes and haemoglobin. We studied the final biosynthesis site of haemoglobin using an ultrastructural approach, with erythroid cells obtained from rabbit embryos, in order to compare these results with those of animals treated with saponine or phenylhydrazine. Our results are similar to those obtained in assays with adult mammals, birds, amphibians, reptiles and fish, after induction of haemolytic anaemia. Therefore, the treatment did not interfere with the process studied, confirming our previous findings. Immunoelectron microscopy showed no labelling of mitochondria or other cellular organelles supposedly involved in the final biosynthesis of haemoglobin molecules, suggesting instead that it occurs free in the cytoplasm immediately after the liberation of haeme from the mitochondria, by electrostatic attraction between haeme and globin chains. PMID:12972280

  5. Detection of the amoeba Entamoeba gingivalis in periodontal pockets

    PubMed Central

    Bonner, Mark; Amard, Véronique; Bar-Pinatel, Charlotte; Charpentier, Frédéric; Chatard, Jean-Michel; Desmuyck, Yvan; Ihler, Serge; Rochet, Jean-Pierre; Roux de La Tribouille, Véronique; Saladin, Luc; Verdy, Marion; Gironès, Núria; Fresno, Manuel; Santi-Rocca, Julien

    2014-01-01

    Periodontitis is a public health issue, being one of the most prevalent diseases worldwide. However, the aetiology of the disease is still unclear: genetics of patients cannot explain the dispersed or isolated localisation of gingival pockets, while bacteria-based models are insufficient to distinguish gingivitis and periodontitis. The possible role of parasites in the establishment of periodontitis has been poorly studied until now. The aim of this project was to study a potential link between colonisation of gingival crevices by the amoeba Entamoeba gingivalis and periodontitis. In eight different dental clinics in France, samples were taken in periodontal pockets (72) or healthy sites (33), and submitted to microscopic observation and molecular identification by PCR with a new set of primers designed to specifically detect E. gingivalis. This blind sample analysis showed the strong sensitivity of PCR compared with clinical diagnosis (58/72 = 81%), and microscopy (51/65 = 78%). The results of this work show that the parasites detected by microscopy mainly – if not exclusively – belong to the species E. gingivalis and that the presence of the parasite is correlated with periodontitis. PMID:24983705

  6. Detection of the amoeba Entamoeba gingivalis in periodontal pockets.

    PubMed

    Bonner, Mark; Amard, Véronique; Bar-Pinatel, Charlotte; Charpentier, Frédéric; Chatard, Jean-Michel; Desmuyck, Yvan; Ihler, Serge; Rochet, Jean-Pierre; Roux de La Tribouille, Véronique; Saladin, Luc; Verdy, Marion; Gironès, Núria; Fresno, Manuel; Santi-Rocca, Julien

    2014-01-01

    Periodontitis is a public health issue, being one of the most prevalent diseases worldwide. However, the aetiology of the disease is still unclear: genetics of patients cannot explain the dispersed or isolated localisation of gingival pockets, while bacteria-based models are insufficient to distinguish gingivitis and periodontitis. The possible role of parasites in the establishment of periodontitis has been poorly studied until now. The aim of this project was to study a potential link between colonisation of gingival crevices by the amoeba Entamoeba gingivalis and periodontitis. In eight different dental clinics in France, samples were taken in periodontal pockets (72) or healthy sites (33), and submitted to microscopic observation and molecular identification by PCR with a new set of primers designed to specifically detect E. gingivalis. This blind sample analysis showed the strong sensitivity of PCR compared with clinical diagnosis (58/72 = 81%), and microscopy (51/65 = 78%). The results of this work show that the parasites detected by microscopy mainly - if not exclusively - belong to the species E. gingivalis and that the presence of the parasite is correlated with periodontitis.

  7. Unusual pattern in haemoglobin electrophoresis in Croatian population: a case report

    PubMed Central

    Segulja, Dragana; Matisic, Danica; Honovic, Lorena; Batinic, Josip; Rogic, Dunja

    2016-01-01

    Haemoglobinopathies are hereditary disorders of globin chain synthesis and are the most common inherited diseases worldwide. Haemoglobin E is a structural haemoglobin variant characteristic for South East Asian population. We present a rare and unusual finding of haemoglobin E detected in University Hospital Centre Zagreb by capillary zone electrophoresis. Detection of haemoglobin structural variant helped to avoid misdiagnosis of sideropenic anemia and thus potentially harmful therapeutic intervention. In today’s European multiethnic population haemoglobinopathies are a public health issue and Croatian laboratory professionals should be aware of a possibility of finding an unusual haemoglobin pattern. PMID:27812312

  8. Extracellular vesicles in the circulation: are erythrocyte microvesicles a confounder in the plasma haemoglobin assay?

    PubMed

    de Vooght, Karen M K; Lau, Cedric; de Laat, Pim P M; van Wijk, Richard; van Solinge, Wouter W; Schiffelers, Raymond M

    2013-02-01

    Blood contains a mixture of extracellular vesicles from different cell types, primarily platelets, endothelial cells, leucocytes and erythrocytes. Erythrocytes are the most abundant cell type in blood and could, especially in certain pathologies, represent an important source of vesicles. Since erythrocytes contain the haemoglobin components iron and haem, which are potentially toxic, it is important to investigate the contribution of vesicle-associated haemoglobin to total cell-free haemoglobin levels. To our knowledge, this is the first time that cell-free plasma haemoglobin has been differentiated into vesicle-associated and molecular species. We investigated the contribution of vesicle-associated haemoglobin in residual patient material that was routinely analysed for total cell-free plasma haemoglobin. All patient samples included in the study were haemolytic with total cell-free haemoglobin concentration ranging from 80 to 2500 mg/l. In the majority of the samples, total cell-free haemoglobin concentration was between 100 and 200 mg/l. No haemoglobin could be detected in the vesicle fraction, indicating that the contribution of vesicle-associated haemoglobin to total cell free-haemoglobin levels in plasma is negligible. It is important to investigate whether erythrocyte vesicles are not formed in blood or that their production is not increased during pathologies associated with haemolysis or that the clearance rate of the vesicles surpasses the formation rate.

  9. Structural basis for the specific interaction of chicken haemoglobin with bromophenol blue: a computational analysis

    NASA Astrophysics Data System (ADS)

    Yu, Hui; Wang, Zhanli; Qin, Wenbin; Zhang, Liangren

    2010-01-01

    It has been observed that bromophenol blue interacted specifically with chicken haemoglobin but not with carp haemoglobin during electrophoresis, but the mechanism of interaction is still not well understood. In this computational study, the binding of bromophenol blue to chicken haemoglobin has been investigated using sequence alignment, homology modelling, electrostatic potential distribution and flexible docking methods. Molecular modelling studies reveal that bromophenol blue-binding site, formed by residues Val1α, Leu2α, Ala131α, Thr134α, Ala138α and Arg141α, is located between two α chains of chicken haemoglobin, and the binding is dominated by hydrophobic interactions. Moreover, comparison of chicken and carp haemoglobin structural models provides a structural rationale for the recognition of bromophenol blue by chicken haemoglobin. These principles can in turn be used to study the molecular recognition mechanism and design a mimic of bromophenol blue for the development of new haemoglobin binders.

  10. Glycated haemoglobin (HbA1c): today and tomorrow.

    PubMed

    Roszyk, L; Faye, B; Sapin, V; Somda, F; Tauveron, I

    2007-10-01

    The assay of glycated haemoglobin (HbA1c) is a gold standard in bioanalysis, and is essential to ensure the optimal care of diabetic patients. Accordingly, the principal scientific societies in diabetology and clinical chemistry have made efforts to standardize this assay in order to select and validate certain analytical methods and achieve consistency in the results obtained therewith. However, clinicians have to be aware of the caution required when interpreting HbA1c assay results owing to modified lifetime and (or) abnormal synthesis of haemoglobin. Although this biological examination has now become an essential part of diabetes monitoring, its status as a screening tool is still controversial, even after 30 years of debate. Other uses of HbA1c assay are currently being assessed in cardiology (coronary syndromes), vascular diseases (arteriopathy), nephrology (renal insufficiency), haematology (anaemia) and oncology (factors of predisposition). PMID:17904515

  11. Inherited haemoglobin disorders: an increasing global health problem.

    PubMed Central

    Weatherall, D. J.; Clegg, J. B.

    2001-01-01

    Despite major advances in our understanding of the molecular pathology, pathophysiology, and control and management of the inherited disorders of haemoglobin, thousands of infants and children with these diseases are dying through lack of appropriate medical care. This problem will undoubtedly increase over the next 20 years because, as the result of a reduction in childhood mortality due to infection and malnutrition, more babies with haemoglobin disorders will survive to present for treatment. Although WHO and various voluntary agencies have tried to disseminate information about these diseases, they are rarely mentioned as being sufficiently important to be included in setting health care priorities for the future. It takes considerable time to establish expertise in developing programmes for the control and management of these conditions, and the lessons learned in developed countries will need to be transmitted to those countries in which they occur at a high frequency. PMID:11545326

  12. Anaemia in the sow: a cohort study to assess factors with an impact on haemoglobin concentration, and the influence of haemoglobin concentration on the reproductive performance.

    PubMed

    Normand, V; Perrin, H; Auvigne, V; Robert, N; Laval, A

    2012-10-01

    The aim of this study was to conduct a descriptive study of haemoglobin concentration found on high-prolificacy sows, to study the relationship between the concentration of haemoglobin and body reserves, and to determine whether anaemia is a risk factor for reproductive performance. A cohort of 308 sows from seven farms was followed from the last third of gestation to the confirmation of the following gestation. Haemoglobin concentration was assessed at four stages of the reproductive cycle: seven and four weeks before farrowing, a few days and three weeks after farrowing. Backfat thickness (BFT) was measured at parturition. The results were analysed using linear mixed-effect models. The mean haemoglobin concentration was 108.4 g/l. The mean modellised haemoglobin concentration of parity 1 sows with a BFT of 16 mm, sampled seven weeks before farrowing, was 118 g/l. Haemoglobin concentration of sows of parity 6 or higher was 8.0 g/l lower than those of parity 1 sows (95% confidence interval -11.0 to -5.1). Haemoglobin concentration is lower in sows with a lower BFT, whatever parity rank. There is no evidence of a relation between haemoglobin concentration and the number of total born, stillborn or number of piglets alive at three weeks and the next breeding performance.

  13. Phosphate-haemoglobin interaction. The primary structure of the haemoglobin of the African elephant (Loxodonta africana, Proboscidea): asparagine in position 2 of the beta-chain.

    PubMed

    Braunitzer, G; Stangl, A; Schrank, B; Krombach, C; Wiesner, H

    1984-07-01

    The primary structure of the haemoglobin of the African Elephant (Loxodonta africana) is reported. The sequence was determined by means of a sequenator. The haemoglobin differs in 26 amino acids in the alpha-chains and in 27 in the beta-chains from that of adult human haemoglobin. The haemoglobin of the African Elephant, like that of the Indian Elephant and Ilama, has only 5 binding sites for polyphosphate. This finding explains the low p(O2)50 value in whole blood as a result of the lower 2,3-bisphosphoglycerate-haemoglobin interaction. This is discussed in relation to aspects of respiratory physiology; some points are also of interest with regard to the Second Punic War and Hannibal's crossing of the Alps.

  14. Allium cepa L. and Quercetin Inhibit RANKL/Porphyromonas gingivalis LPS-Induced Osteoclastogenesis by Downregulating NF-κB Signaling Pathway.

    PubMed

    Oliveira, Tatiane; Figueiredo, Camila A; Brito, Carlos; Stavroullakis, Alexander; Ferreira, Ana Carolina; Nogueira-Filho, Getulio; Prakki, Anuradha

    2015-01-01

    Objectives. We evaluated the in vitro modulatory effects of Allium cepa L. extract (AcE) and quercetin (Qt) on osteoclastogenesis under inflammatory conditions (LPS-induced). Methods. RAW 264.7 cells were differentiated with 30 ng/mL of RANKL, costimulated with PgLPS (1 µg/mL), and treated with AcE (50-1000 µg/mL) or Qt (1.25, 2.5, or 5 µM). Cell viability was determined by alamarBlue and protein assays. Nuclei morphology was analysed by DAPI staining. TRAP assays were performed as follows: p-nitrophenyl phosphate was used to determine the acid phosphatase activity of the osteoclasts and TRAP staining was used to evaluate the number and size of TRAP-positive multinucleated osteoclast cells. Von Kossa staining was used to measure osteoclast resorptive activity. Cytokine levels were measured on osteoclast precursor cell culture supernatants. Using western blot analysis, p-IκBα and IκBα degradation, inhibitor of NF-kappaB, were evaluated. Results. Both AcE and Qt did not affect cell viability and significantly reduced osteoclastogenesis compared to control. We observed lower production of IL-6 and IL-1α and an increased production of IL-3 and IL-4. AcE and Qt downregulated NF-κB pathway. Conclusion. AcE and Qt may be inhibitors of osteoclastogenesis under inflammatory conditions (LPS-induced) via attenuation of RANKL/PgLPS-induced NF-κB activation. PMID:26273314

  15. Allium cepa L. and Quercetin Inhibit RANKL/Porphyromonas gingivalis LPS-Induced Osteoclastogenesis by Downregulating NF-κB Signaling Pathway

    PubMed Central

    Oliveira, Tatiane; Figueiredo, Camila A.; Brito, Carlos; Stavroullakis, Alexander; Ferreira, Ana Carolina; Nogueira-Filho, Getulio; Prakki, Anuradha

    2015-01-01

    Objectives. We evaluated the in vitro modulatory effects of Allium cepa L. extract (AcE) and quercetin (Qt) on osteoclastogenesis under inflammatory conditions (LPS-induced). Methods. RAW 264.7 cells were differentiated with 30 ng/mL of RANKL, costimulated with PgLPS (1 µg/mL), and treated with AcE (50–1000 µg/mL) or Qt (1.25, 2.5, or 5 µM). Cell viability was determined by alamarBlue and protein assays. Nuclei morphology was analysed by DAPI staining. TRAP assays were performed as follows: p-nitrophenyl phosphate was used to determine the acid phosphatase activity of the osteoclasts and TRAP staining was used to evaluate the number and size of TRAP-positive multinucleated osteoclast cells. Von Kossa staining was used to measure osteoclast resorptive activity. Cytokine levels were measured on osteoclast precursor cell culture supernatants. Using western blot analysis, p-IκBα and IκBα degradation, inhibitor of NF-kappaB, were evaluated. Results. Both AcE and Qt did not affect cell viability and significantly reduced osteoclastogenesis compared to control. We observed lower production of IL-6 and IL-1α and an increased production of IL-3 and IL-4. AcE and Qt downregulated NF-κB pathway. Conclusion. AcE and Qt may be inhibitors of osteoclastogenesis under inflammatory conditions (LPS-induced) via attenuation of RANKL/PgLPS-induced NF-κB activation. PMID:26273314

  16. The use of haemoglobin concentrations to assess physiological condition in birds: a review

    PubMed Central

    Minias, Piotr

    2015-01-01

    Total blood haemoglobin concentration is increasingly being used to assess physiological condition in wild birds, although it has not been explicitly recognized how reliably this parameter reflects different components of individual quality. Thus, I reviewed over 120 published studies linking variation in haemoglobin concentrations to different measures of condition and other phenotypic or ecological traits. In most of the studied avian species, haemoglobin concentrations were positively correlated with other commonly used indices of condition, such as body mass and fat loads, as well as with quality of the diet. Also, chick haemoglobin concentrations reliably reflected the intensity of nest infestation by parasitic arthropods, and haemoglobin was suggested to reflect parasitism by haematophagous ectoparasites much more precisely than haematocrit. There was also some evidence for the negative effect of helminths on haemoglobin levels in adult birds. Finally, haemoglobin concentrations were found to correlate with such fitness-related traits as timing of arrival at breeding grounds, timing of breeding, egg size, developmental stability and habitat quality, although these relationships were not always consistent between species. In consequence, I recommend the total blood haemoglobin concentration as a relatively robust indicator of physiological condition in birds, although this parameter is also strongly affected by age, season and the process of moult. Thus, researchers are advised to control fully for these confounding effects while using haemoglobin concentrations as a proxy of physiological condition in both experimental and field studies on birds. PMID:27293692

  17. First evidence of genetic intraspecific variability and occurrence of Entamoeba gingivalis in HIV(+)/AIDS.

    PubMed

    Cembranelli, Sibeli B S; Souto, Fernanda O; Ferreira-Paim, Kennio; Richinho, Túlio T; Nunes, Poliana L; Nascentes, Gabriel A N; Ferreira, Thatiana B; Correia, Dalmo; Lages-Silva, Eliane

    2013-01-01

    Entamoeba gingivalis is considered an oral commensal but demonstrates a pathogenic potential associated with periodontal disease in immunocompromised individuals. Therefore, this study evaluated the occurrence, opportunistic conditions, and intraspecific genetic variability of E. gingivalis in HIV(+)/AIDS patients. Entamoeba gingivalis was studied using fresh examination (FE), culture, and PCR from bacterial plaque samples collected from 82 HIV(+)/AIDS patients. Genetic characterization of the lower ribosomal subunit of region 18S (18S-SSU rRNA) was conducted in 9 positive samples using low-stringency single specific primer PCR (LSSP-PCR) and sequencing analysis. Entamoeba gingivalis was detected in 63.4% (52/82) of the samples. No association was detected between the presence of E. gingivalis and the CD4(+) lymphocyte count (≤200 cells/mm(3) (p = 0.912) or viral load (p = 0.429). The LSSP-PCR results helped group E. gingivalis populations into 2 polymorphic groups (68.3% similarity): group I, associated with 63.6% (7/11) of the samples, and group II, associated with 36.4% (4/11) of the samples, which shared 74% and 83.7% similarity and association with C and E isolates from HIV(-) individuals, respectively. Sequencing of 4 samples demonstrated 99% identity with the reference strain ATCC 30927 and also showed 2 divergent clusters, similar to those detected by LSSP-PCR. Opportunistic behavior of E. gingivalis was not detected, which may be related to the use of highly active antiretroviral therapy by all HIV(+)/AIDS patients. The high occurrence of E. gingivalis in these patients can be influenced by multifactorial components not directly related to the CD4(+) lymphocyte counts, such as cholesterol and the oral microbiota host, which could mask the potential opportunistic ability of E. gingivalis. The identification of the 18S SSU-rRNA polymorphism by LSSP-PCR and sequencing analysis provides the first evidence of genetic variability in E. gingivalis

  18. First Evidence of Genetic Intraspecific Variability and Occurrence of Entamoeba gingivalis in HIV(+)/AIDS

    PubMed Central

    Cembranelli, Sibeli B. S.; Souto, Fernanda O.; Ferreira-Paim, Kennio; Richinho, Túlio T.; Nunes, Poliana L.; Nascentes, Gabriel A. N.; Ferreira, Thatiana B.; Correia, Dalmo; Lages-Silva, Eliane

    2013-01-01

    Entamoeba gingivalis is considered an oral commensal but demonstrates a pathogenic potential associated with periodontal disease in immunocompromised individuals. Therefore, this study evaluated the occurrence, opportunistic conditions, and intraspecific genetic variability of E. gingivalis in HIV(+)/AIDS patients. Entamoeba gingivalis was studied using fresh examination (FE), culture, and PCR from bacterial plaque samples collected from 82 HIV(+)/AIDS patients. Genetic characterization of the lower ribosomal subunit of region 18S (18S-SSU rRNA) was conducted in 9 positive samples using low-stringency single specific primer PCR (LSSP-PCR) and sequencing analysis. Entamoeba gingivalis was detected in 63.4% (52/82) of the samples. No association was detected between the presence of E. gingivalis and the CD4+ lymphocyte count (≤200 cells/mm3 (p = 0.912) or viral load (p = 0.429). The LSSP-PCR results helped group E. gingivalis populations into 2 polymorphic groups (68.3% similarity): group I, associated with 63.6% (7/11) of the samples, and group II, associated with 36.4% (4/11) of the samples, which shared 74% and 83.7% similarity and association with C and E isolates from HIV(−) individuals, respectively. Sequencing of 4 samples demonstrated 99% identity with the reference strain ATCC 30927 and also showed 2 divergent clusters, similar to those detected by LSSP-PCR. Opportunistic behavior of E. gingivalis was not detected, which may be related to the use of highly active antiretroviral therapy by all HIV(+)/AIDS patients. The high occurrence of E. gingivalis in these patients can be influenced by multifactorial components not directly related to the CD4+ lymphocyte counts, such as cholesterol and the oral microbiota host, which could mask the potential opportunistic ability of E. gingivalis. The identification of the 18S SSU-rRNA polymorphism by LSSP-PCR and sequencing analysis provides the first evidence of genetic variability in E. gingivalis isolated

  19. α-Thalassemia with Haemoglobin Adana mutation: prenatal diagnosis.

    PubMed

    Zainal, N Z; Alauddin, H; Ahmad, S; Hussin, N H

    2014-12-01

    Thalassaemia carriers are common in the Asian region including Malaysia. Asymptomatic patients can be undiagnosed until they present for their antenatal visits. Devastating obstetric outcome may further complicate the pregnancy if both parents are thalassaemia carriers leading to hydrophic fetus due to haemoglobin Bart's disease. However in certain cases where unexplained hydrops fetalis occur in parents with heterozygous thalassaemia carrier,mutated α genes should be suspected. We report a twenty-nine year old woman in her third pregnancy with two previous pregnancies complicated by early neonatal death at 21 and 28 weeks of gestation due to hydrops fetalis. DNA analysis revealed the patient to have heterozygous (--SEA) α-gene deletion, while her husband has a compound heterozygosity for α(3.7) deletion and codon 59 (GGC → GAC) mutation of the α-gene. This mutation, also known as hemoglobin Adana, can explain hydrops fetalis resulting from two alpha gene deletions from the patient (mother) and a single alpha gene deletion with mutation from the father. The third pregnancy resulted in a grossly normal baby boy with 3 α-gene deletions (HbH disease). We postulate that, in view of heterogenisity of the α-thalassaemia in this patient with severely unstable haemoglobin Adana chains from her husband, there will be a 25% possibility of fetal hydrops in every pregnancy. PMID:25500521

  20. The structure of the giant haemoglobin from Glossoscolex paulistus.

    PubMed

    Ruggiero Bachega, José Fernando; Vasconcelos Maluf, Fernando; Andi, Babak; D'Muniz Pereira, Humberto; Falsarella Carazzollea, Marcelo; Orville, Allen M; Tabak, Marcel; Brandão-Neto, José; Garratt, Richard Charles; Horjales Reboredo, Eduardo

    2015-06-01

    The sequences of all seven polypeptide chains from the giant haemoglobin of the free-living earthworm Glossoscolex paulistus (HbGp) are reported together with the three-dimensional structure of the 3.6 MDa complex which they form. The refinement of the full particle, which has been solved at 3.2 Å resolution, the highest resolution reported to date for a hexagonal bilayer haemoglobin composed of 12 protomers, is reported. This has allowed a more detailed description of the contacts between subunits which are essential for particle stability. Interpretation of features in the electron-density maps suggests the presence of metal-binding sites (probably Zn(2+) and Ca(2+)) and glycosylation sites, some of which have not been reported previously. The former appear to be important for the integrity of the particle. The crystal structure of the isolated d chain (d-HbGp) at 2.1 Å resolution shows different interchain contacts between d monomers compared with those observed in the full particle. Instead of forming trimers, as seen in the complex, the isolated d chains associate to form dimers across a crystallographic twofold axis. These observations eliminate the possibility that trimers form spontaneously in solution as intermediates during the formation of the dodecameric globin cap and contribute to understanding of the possible ways in which the particle self-assembles.

  1. microRNAs responsive to A. actinomycetemcomitans and P. gingivalis LPS modulate expression of genes regulating innate immunity in human macrophages

    PubMed Central

    Naqvi, Afsar R.; Fordham, Jezrom B.; Khan, Asma; Nares, Salvador

    2013-01-01

    microRNAs (miRNA) are a class of small noncoding RNAs that regulate post-transcriptional expression of their respective target genes and are responsive to various stimuli, including lipopolysaccharide (LPS). Here we examined the early (4h) miRNA responses of THP1-differentiated macrophages challenged with LPS derived from the periodontal pathogens, Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg) or environmentally modified LPS obtained from Pg grown in cigarette smoke extract. Predicted miRNA-gene target interactions for LPS-responsive miR-29b and let-7f were confirmed using dual-luciferase assays and by transfection experiments using miRNA mimics and inhibitors. Convergent and divergent miRNA profiles were observed in treated samples where differences in miRNA levels related to the type, concentration and incubation times of LPS challenge. Dual-luciferase experiments revealed miR-29b targeting of IL-6Rα and IFN-γ inducible protein 30 (IFI30) and let-7f targeting of suppressor of cytokine signaling 4 (SOCS4) and Thrombospondin-1 (TSP-1). Transfection experiments confirmed miR-29b and let-7f modulation of IL-6R and SOCS4 protein expression levels, respectively. Thus, we demonstrate convergent/divergent miRNA responses to wild type and its environmentally-modified LPS and demonstrate miRNA targeting of key genes linked to inflammation and immunity. Our data indicate that these LPS-responsive miRNAs may play a key role in fine-tuning the host response to periodontal pathogens. PMID:24062196

  2. Periodontitis‐associated pathogens P. gingivalis and A. actinomycetemcomitans activate human CD14+ monocytes leading to enhanced Th17/IL‐17 responses

    PubMed Central

    Cheng, Wan‐Chien; van Asten, Saskia D.; Burns, Lachrissa A.; Evans, Hayley G.; Walter, Gina J.; Hashim, Ahmed; Hughes, Francis J.

    2016-01-01

    The Th17/IL‐17 pathway is implicated in the pathogenesis of periodontitis (PD), however the mechanisms are not fully understood. We investigated the mechanism by which the periodontal pathogens Porphyromonas gingivalis (Pg) and Aggregatibacter actinomycetemcomitans (Aa) promote a Th17/IL‐17 response in vitro, and studied IL‐17+ CD4+ T‐cell frequencies in gingival tissue and peripheral blood from patients with PD versus periodontally healthy controls. Addition of Pg or Aa to monocyte/CD4+ T‐cell co‐cultures promoted a Th17/IL‐17 response in vitro in a dose‐ and time‐dependent manner. Pg or Aa stimulation of monocytes resulted in increased CD40, CD54 and HLA‐DR expression, and enhanced TNF‐α, IL‐1β, IL‐6 and IL‐23 production. Mechanistically, IL‐17 production in Pg‐stimulated co‐cultures was partially dependent on IL‐1β, IL‐23 and TLR2/TLR4 signalling. Increased frequencies of IL‐17+ cells were observed in gingival tissue from patients with PD compared to healthy subjects. No differences were observed in IL‐17+ CD4+ T‐cell frequencies in peripheral blood. In vitro, Pg induced significantly higher IL‐17 production in anti‐CD3 mAb‐stimulated monocyte/CD4+ T‐cell co‐cultures from patients with PD compared to healthy controls. Our data suggest that periodontal pathogens can activate monocytes, resulting in increased IL‐17 production by human CD4+ T cells, a process that appears enhanced in patients with PD. PMID:27334899

  3. Bacteroides gingivalis-Actinomyces viscosus cohesive interactions as measured by a quantitative binding assay

    SciTech Connect

    Schwarz, S.; Ellen, R.P.; Grove, D.A.

    1987-10-01

    There is limited evidence, mostly indirect, to suggest that the adherence of Bacteroides gingivalis to teeth may be enhanced by the presence of gram-positive dental plaque bacteria like Actinomyces viscosus. The purpose of this study was to carry out direct quantitative assessments of the cohesion of B gingivalis and A. viscosus by using an in vitro assay modeled on the natural sequence in which these two species colonize the teeth. The assay allowed comparisons to be made of the adherence of /sup 3/H-labeled B. gingivalis 2561 and 381 to saliva-coated hydroxyapatite beads (S-HA) and A. viscosus WVU627- or T14V-coated S-HA (actinobeads) in equilibrium and kinetics binding studies. A series of preliminary binding studies with 3H-labeled A. viscosus and parallel studies by scanning electron microscopy with unlabeled A. viscosus were conducted to establish a protocol by which actinobeads suitable for subsequent Bacteroides adherence experiments could be prepared. By scanning electron microscopy, the actinobeads had only small gaps of exposed S-HA between essentially irreversibly bound A. viscosus cells. Furthermore, B. gingivalis cells appeared to bind preferentially to the Actinomyces cells instead of the exposed S-HA. B. gingivalis binding to both S-HA and actinobeads was saturable with at least 2 X 10(9) to 3 X 10(9) cells per ml, and equilibrium with saturating concentrations was reached within 10 to 20 min. B. gingivalis always bound in greater numbers to the actinobeads than to S-HA. These findings provide direct measurements supporting the concept that cohesion with dental plaque bacteria like A. viscosus may foster the establishment of B. gingivalis on teeth by enhancing its adherence.

  4. α-Haemoglobin regulates sympathoadrenal cell metabolism to maintain a catecholaminergic phenotype.

    PubMed

    Marcos-Almaraz, María T; Rodríguez-Gómez, José A; López-Barneo, José; Pascual, Alberto

    2012-02-01

    Discovery of haemoglobin A expression outside of the erythroid cell lineage suggests that oxygen transport is the main, but not the unique, function of adult haemoglobin chains in mammals. The contribution of haemoglobin A to antioxidant defences has been proposed in the territories where it is expressed. Catecholaminergic cells rely on an active oxidative metabolism to accomplish their biological function, but are exposed to strong oxidative stress due to metabolism of catecholaminergic transmitters. We show in the present study that peripheral catecholaminegic cells express the α- and not the β-haemoglobin A chains, and that α-haemoglobin expression could modulate the antioxidant capabilities of these cells. We also show that α-haemoglobin overexpression in PC12 cells leads to a selective increase of tyrosine hydroxylase synthesis and activity. This is achieved by means of a reorganization of antioxidant defences, decreasing cytoplasmic glutathione peroxidase and superoxide dismutase, and increasing mitochondrial peroxidase. Moreover, α-haemoglobin induces a decrease in lipogenesis and increase in lipid degradation, situations that help save NAD(P)H and favour supply of acetyl-CoA to the tricarboxylic acid cycle and production of reducing equivalents in the cell. All of these results point to a role for α-haemoglobin as a regulator of catecholaminergic cell metabolism required for phenotype acquisition and maintenance. PMID:22060312

  5. Acid Bohr effect of a monomeric haemoglobin from Dicrocoelium dendriticum. Mechanism of the allosteric conformation transition.

    PubMed

    Smit, J D; Sick, H; Peterhans, A; Gersonde, K

    1986-03-01

    The dioxygen affinity of Dicrocoelium dendriticum haemoglobin was determined as a function of pH with a thin-layer diffusion technique. From the oxygen dissociation and association curves Hill coefficients h equal 1 were obtained throughout. Ultracentrifugation studies prove this haemoglobin to be monomeric irrespective of pH and ligation state. Thus, Dicrocoelium haemoglobin is a non-cooperative monomer. It has the highest O2 affinity so far known for any monomeric haemoglobin: its half-saturation pressure, p50 value, ranges at 25 degrees C from 0.016 mm Hg to 0.15 mm Hg (2.13-20.0 Pa) dependent on pH. Dicrocoelium haemoglobin shows an acid Bohr effect only and as such it constitutes a new class of haemoglobins. Its log p50 versus pH plot (Bohr effect curve) is characterized by a large amplitude, delta log p50 = 0.96, and an inflection point (Bohr effect pK) at pH 5.0. A model for the acid Bohr effect of D. dendriticum haemoglobin is proposed. By generalization, both the alkaline and the acid Bohr effect in various monomeric haemoglobins may arise from a single Bohr group complex (salt bridge).

  6. α-Haemoglobin regulates sympathoadrenal cell metabolism to maintain a catecholaminergic phenotype.

    PubMed

    Marcos-Almaraz, María T; Rodríguez-Gómez, José A; López-Barneo, José; Pascual, Alberto

    2012-02-01

    Discovery of haemoglobin A expression outside of the erythroid cell lineage suggests that oxygen transport is the main, but not the unique, function of adult haemoglobin chains in mammals. The contribution of haemoglobin A to antioxidant defences has been proposed in the territories where it is expressed. Catecholaminergic cells rely on an active oxidative metabolism to accomplish their biological function, but are exposed to strong oxidative stress due to metabolism of catecholaminergic transmitters. We show in the present study that peripheral catecholaminegic cells express the α- and not the β-haemoglobin A chains, and that α-haemoglobin expression could modulate the antioxidant capabilities of these cells. We also show that α-haemoglobin overexpression in PC12 cells leads to a selective increase of tyrosine hydroxylase synthesis and activity. This is achieved by means of a reorganization of antioxidant defences, decreasing cytoplasmic glutathione peroxidase and superoxide dismutase, and increasing mitochondrial peroxidase. Moreover, α-haemoglobin induces a decrease in lipogenesis and increase in lipid degradation, situations that help save NAD(P)H and favour supply of acetyl-CoA to the tricarboxylic acid cycle and production of reducing equivalents in the cell. All of these results point to a role for α-haemoglobin as a regulator of catecholaminergic cell metabolism required for phenotype acquisition and maintenance.

  7. Oxygen-binding characteristics of three extracellular haemoglobins of Artemia salina.

    PubMed

    D'Hondt, J; Moens, L; Heip, J; D'Hondt, A; Kondo, M

    1978-06-01

    The oxygen-binding characteristics of the three extracellular haemoglobins of brine shrimp (Artemia salina) were studied in vitro by using highly purified preparations. Haemoglobin I is induced last in the development of brine shrimps when functional gills are formed. It has the lowest oxygen affinity (p(50) 5.34mmHg), an intermediate Bohr effect (ø -0.09 at 20 degrees C) above pH8 and a temperature-sensitivity (DeltaH -44.8 to -45.6kJ/mol at pH8-9) comparable with those observed with other invertebrate haemoglobins [Weber & Heidemann (1977) Comp. Biochem. Physiol. A57, 151-155]. Haemoglobin II, which is the first to be induced, soon after hatching of nauplius larvae, persists generally throughout the whole adult life. It has an intermediate oxygen affinity (p(50) 3.7mmHg), the highest Bohr effect (ø -0.21 at 20 degrees C) above pH8 and a similar temperature-sensitivity (DeltaH -46.0 to -54.8kJ/mol at pH8-9) as haemoglobin I. However, haemoglobin III, which is induced second several hours after the induction of haemoglobin II but disappearing from the haemolymph in the middle of adult life, has the highest oxygen affinity (p(50) 1.8mmHg), the lowest Bohr effect (ø -0.03 at 20 degrees C) above pH8.5 and a high resistance against temperature variation between 10 and 25 degrees C at pH8.5-9 (DeltaH -22.6 to -23.0kJ/mol). At pH7.5-8, haemoglobin III exhibits a similar temperature-sensitivity under 30 degrees C as do other haemoglobins. All three haemoglobins have a rather low co-operativity, with Hill coefficients (h 1.6-1.9 at pH8.5), which are dependent on both pH and temperature. The highest co-operativity was observed at 20 degrees C and pH9 for haemoglobins I and II, whereas it was at 27 degrees C and pH8.5 for haemoglobin III. Thus the oxygen-binding behaviour of haemoglobin III in vitro is significantly different from those of haemoglobins I and II and indicates possibly its specific physiological role in vivo in the adaptive process in the natural

  8. Large scale screening for haemoglobin disorders in southern Vietnam: implications for avoidance and management.

    PubMed

    O'Riordan, Sean; Hien, Tran Tinh; Miles, Katie; Allen, Angela; Quyen, Nguyen Ngoc; Hung, Nguyen Quoc; Anh, Do Quang; Tuyen, Luc Nguyen; Khoa, Dao Bach; Thai, Cao Quang; Triet, Dao Minh; Phu, Nguyen Hoan; Dunstan, Sarah; Peto, Tim; Clegg, John; Farrar, Jeremy; Weatherall, David

    2010-08-01

    In order to obtain an approximate assessment of the public health burden that will be posed by the inherited disorders of haemoglobin in southern Vietnam, several thousand individuals were screened for these conditions. A smaller sample was screened for glucose-6-phosphate dehydrogenase (G6PD) deficiency. The important haemoglobin disorders identified were beta thalassaemia, haemoglobin E and a variety of different forms of alpha thalassaemia. There were sufficient G6PD-deficient individuals to materially affect malaria control programme design. The most remarkable finding was wide variation in the gene frequencies of these conditions among the ethnic groups sampled. The approximate number of babies expected to be born with clinically significant haemoglobin disorders in Vietnam was estimated from the gene-frequency data. This study emphasizes the importance of wide-scale population screening, including ethnic subgroups, to establish the requirements for inherited haemoglobin disorder programmes in resource-limited settings.

  9. Structural analysis of haemoglobin binding by HpuA from the Neisseriaceae family

    PubMed Central

    Wong, Chi T.; Xu, Yingqi; Gupta, Akshari; Garnett, James A.; Matthews, Steve J.; Hare, Stephen A.

    2015-01-01

    The Neisseriaceae family of bacteria causes a range of diseases including meningitis, septicaemia, gonorrhoea and endocarditis, and extracts haem from haemoglobin as an important iron source within the iron-limited environment of its human host. Herein we report crystal structures of apo- and haemoglobin-bound HpuA, an essential component of this haem import system. The interface involves long loops on the bacterial receptor that present hydrophobic side chains for packing against the surface of haemoglobin. Interestingly, our structural and biochemical analyses of Kingella denitrificans and Neisseria gonorrhoeae HpuA mutants, although validating the interactions observed in the crystal structure, show how Neisseriaceae have the fascinating ability to diversify functional sequences and yet retain the haemoglobin binding function. Our results present the first description of HpuA's role in direct binding of haemoglobin. PMID:26671256

  10. Structural analysis of haemoglobin binding by HpuA from the Neisseriaceae family.

    PubMed

    Wong, Chi T; Xu, Yingqi; Gupta, Akshari; Garnett, James A; Matthews, Steve J; Hare, Stephen A

    2015-12-16

    The Neisseriaceae family of bacteria causes a range of diseases including meningitis, septicaemia, gonorrhoea and endocarditis, and extracts haem from haemoglobin as an important iron source within the iron-limited environment of its human host. Herein we report crystal structures of apo- and haemoglobin-bound HpuA, an essential component of this haem import system. The interface involves long loops on the bacterial receptor that present hydrophobic side chains for packing against the surface of haemoglobin. Interestingly, our structural and biochemical analyses of Kingella denitrificans and Neisseria gonorrhoeae HpuA mutants, although validating the interactions observed in the crystal structure, show how Neisseriaceae have the fascinating ability to diversify functional sequences and yet retain the haemoglobin binding function. Our results present the first description of HpuA's role in direct binding of haemoglobin.

  11. The structure of α-haemoglobin in complex with a haemoglobin-binding domain from Staphylococcus aureus reveals the elusive α-haemoglobin dimerization interface.

    PubMed

    Kumar, Kaavya Krishna; Jacques, David A; Guss, J Mitchell; Gell, David A

    2014-08-01

    Adult haemoglobin (Hb) is made up of two α and two β subunits. Mutations that reduce expression of the α- or β-globin genes lead to the conditions α- or β-thalassaemia, respectively. Whilst both conditions are characterized by anaemia of variable severity, other details of their pathophysiology are different, in part owing to the greater stability of the β chains that is conferred through β self-association. In contrast, α subunits interact weakly, and in the absence of stabilizing quaternary interactions the α chain (α) is prone to haem loss and denaturation. The molecular contacts that confer weak self-association of α have not been determined previously. Here, the first structure of an α2 homodimer is reported in complex with one domain of the Hb receptor from Staphylococcus aureus. The α2 dimer interface has a highly unusual, approximately linear, arrangement of four His side chains within hydrogen-bonding distance of each other. Some interactions present in the α1β1 dimer interface of native Hb are preserved in the α2 dimer. However, a marked asymmetry is observed in the α2 interface, suggesting that steric factors limit the number of stabilizing interactions that can form simultaneously across the interface.

  12. The structure of α-haemoglobin in complex with a haemoglobin-binding domain from Staphylococcus aureus reveals the elusive α-haemoglobin dimerization interface

    PubMed Central

    Krishna Kumar, Kaavya; Jacques, David A.; Guss, J. Mitchell; Gell, David A.

    2014-01-01

    Adult haemoglobin (Hb) is made up of two α and two β subunits. Mutations that reduce expression of the α- or β-globin genes lead to the conditions α- or β-thalassaemia, respectively. Whilst both conditions are characterized by anaemia of variable severity, other details of their pathophysiology are different, in part owing to the greater stability of the β chains that is conferred through β self-association. In contrast, α subunits interact weakly, and in the absence of stabilizing quaternary interactions the α chain (α) is prone to haem loss and denaturation. The molecular contacts that confer weak self-association of α have not been determined previously. Here, the first structure of an α2 homodimer is reported in complex with one domain of the Hb receptor from Staphylococcus aureus. The α2 dimer interface has a highly unusual, approximately linear, arrangement of four His side chains within hydrogen-bonding distance of each other. Some interactions present in the α1β1 dimer interface of native Hb are preserved in the α2 dimer. However, a marked asymmetry is observed in the α2 interface, suggesting that steric factors limit the number of stabilizing interactions that can form simultaneously across the interface. PMID:25084376

  13. Care of patients with haemoglobin abnormalities: nursing management.

    PubMed

    Khattab, Ahmed D; Rawlings, Barry; Ali, Ibtisam S

    This second of two articles on sickle cell disease (SCD) aims to provide advice on nursing care for patients. All health professionals should be aware of the various manifestations of the disease, the life-threatening complications and their optimal management. Prophylactic strategy is of paramount importance as it may avert many possible adverse outcomes. Recurrent episodes of acute, severe pain require frequent hospital admissions. The care of individuals with SCD must extend into the community and take account of domestic, employment and educational issues. Affected pregnant women should be looked after by units experienced in the care of women with this condition. The diagnosis of haemoglobin type is simple and inexpensive and a comprehensive national screening programme to detect SCD in pregnant women and newborn babies was started in April 2002 as part of the Government's broader aims to tackle inequalities in health and community.

  14. Reticulum vs Inclusions: A Learning Experience in Haemoglobin H Disease

    PubMed Central

    Sridevi, Hanaganahalli B; Hegde, Anupama; Balanthimogru, Prashantha; Khadilkar, Urmila N.

    2015-01-01

    Haemoglobin H disease, also known as the alpha-thalassaemia is characterized by the presence of HbH inclusions in red blood cells, detectable on supra-vital stain. We present a case of a previously asymptomatic 31-year-old male, who insidiously developed anaemia and had prominent splenomegaly. Peripheral smear examination revealed microcytic hypochromic anaemia with numerous spherocytes and moderate polychromasia. In reticulocyte preparation with Brilliant cresyl blue, HbH inclusions were mistakenly identified as granulofilamentous reticulum of reticulocytes, giving a spuriously high reticulocyte percentage. After the literature review, repeat assessment was performed and with the aid of high performance liquid chromatography result, it was possible to delineate the HbH inclusions. PMID:26557534

  15. Diet and glycosylated haemoglobin in the 1946 British birth cohort

    PubMed Central

    Prynne, CJ; Mander, A; Wadsworth, MEJ; Stephen, AM

    2009-01-01

    Objectives: Raised glycosylated haemoglobin (HbA1c) concentration is a recognised risk factor for diabetes the incidence of which is rising world-wide. The intake of certain foods has been related to HbA1c concentration . The aim of this study was to investigate whether nutrient intake, sourced by these foods, was predictive of raised glycosylated haemoglobin (HbA1c) concentration in a British cohort. Subjects/methods: The subjects were 495 men and 570 women who were members of the Medical Research Council National Survey of Health and Development, 1946 birth cohort. Diet was assessed from 5-day records in 1982, 1989 and 1999. HbA1c was measured in blood samples collected in 1999. Individuals in whom concentration of HbA1c was ≥ 6.3% were identified as being “at risk” and their nutrient intake was compared to those whose concentration of HbA1c was within the normal range (≤6.2%). Results: Lower intakes of protein, carbohydrate, non-starch polysaccharide, iron, folate, vitamin B12 and a higher percentage energy from fat in 1989 were significantly predictive of high HbA1c status in 1999. In 1999 there were no nutrient intakes that were predictive of HbA1c status. Global tests of whether the intakes of energy, carbohydrate, sodium, iron, riboflavin and vitamin B12 at all three time-points were related to HbA1c status in 1999, were significant. Conclusion: An increased intake of energy, carbohydrate, sodium, iron, riboflavin and vitamin B12 over 10 years was predictive of raised HbA1c status. Increased energy intake may have resulted in the increase in body weight that is a risk factor for diabetes. PMID:19550434

  16. Foetal haemoglobin and the dynamics of paediatric malaria

    PubMed Central

    2012-01-01

    Background Although 80% of malaria occurs in children under five years of age, infants under six months of age are known to have low rates of infection and disease. It is not clear why this youngest age group is protected; possible factors include maternal antibodies, unique nutrition (breast milk), and the presence of foetal haemoglobin (HbF). This work aims to gain insight into possible mechanisms of protection, and suggest pathways for focused empirical work, by modelling a range of possible effects of foetal haemoglobin and other red blood cell (RBC) developmental changes on parasite dynamics in infants. Methods A set of ordinary differential equations was created to investigate the leading hypotheses about the possible protective mechanisms of HbF-containing red blood cells, in particular whether HbF suppresses parasite population growth because parasite multiplication in individual RBCs is lower, slower or absent. The model also incorporated the intrinsic changes in blood volume and haematocrit that occur with age, and the possibility of parasite affinities for HbF-containing RBCs or reticulocytes. Results The model identified several sets of conditions in which the infant remained protected, or displayed a much slower growth of parasitaemia in the first few months of life, without any intervening immune response. The most protective of the hypothesized mechanisms would be the inhibition of schizont division in foetal RBCs so that fewer merozoites are produced. The model showed that a parasite preference for HbF-containing RBCs increases protective effects for the host, while a preference for reticulocytes has little effect. Conclusions The results from this simple model of haematological changes in infants and their effects on Plasmodium falciparum infection dynamics emphasize the likely importance of HbF and RBC number as an explanatory factor in paediatric malaria, and suggest a framework for organizing related empirical research. PMID:23190739

  17. Differences between horse and human haemoglobins in effects of organic and inorganic anions on oxygen binding.

    PubMed Central

    Giardina, B; Brix, O; Clementi, M E; Scatena, R; Nicoletti, B; Cicchetti, R; Argentin, G; Condo, S G

    1990-01-01

    Despite the fact that the horse is one of the more common domesticated animals, there are few reports dealing with the properties of its blood, and no comprehensive study has been performed on the reactivity of horse haemoglobin towards organic and inorganic ions. Here we report data on the effects of the organic phosphates D-glycerate-2,3-bisphosphate (2,3-DPG) and InsP6, and of chloride on the properties of horse haemoglobin. Thus the effect of saturating concentrations of 2,3-DPG on the oxygen affinity of horse haemoglobin is about 60% lower than with human adult haemoglobin under the same experimental conditions. The same applies also to InsP6, whose effect on oxygen binding to horse haemoglobin is decreased by about 55% compared with human adult haemoglobin. On the whole, horse haemoglobin appears to be much less sensitive to organic phosphates than previously believed. These results are discussed in the light of the primary structure of the molecule. PMID:2327974

  18. Draft genome sequences of 26 porphyromonas strains isolated from the canine oral microbiome.

    PubMed

    Coil, David A; Alexiev, Alexandra; Wallis, Corrin; O'Flynn, Ciaran; Deusch, Oliver; Davis, Ian; Horsfall, Alexander; Kirkwood, Nicola; Jospin, Guillaume; Eisen, Jonathan A; Harris, Stephen; Darling, Aaron E

    2015-01-01

    We present the draft genome sequences for 26 strains of Porphyromonas (P. canoris, P. gulae, P. cangingavalis, P. macacae, and 7 unidentified) and an unidentified member of the Porphyromonadaceae family. All of these strains were isolated from the canine oral cavity, from dogs with and without early periodontal disease. PMID:25858832

  19. Determination of the effect of pH and foetal haemoglobin fraction on haemoglobin oxygen saturation readings by the OSM3 hemoximeter.

    PubMed

    Mackay, A R; Dooley, K C; Whyte, R K

    1999-05-01

    The visible absorption spectrum of oxyhaemoglobin depends on its content of foetal haemoglobin and on the pH of the blood. These variables are known to affect oxygen saturation readings measured by the OSM3 Hemoximeter. As the hemoximeter estimates foetal haemoglobin content from the absorption spectrum of oxyhaemoglobin, this estimation is affected by pH. We quantified the effect of pH on oxyhaemoglobin reading in order to calculate a correction factor to adjust for pH. We manipulated a pool of fresh heparinized cord blood samples to produce a range of pH values from 6.99-7.53 in fully oxygenated blood with a carbon dioxide tension of 5.3 kPa. We measured oxygen saturation and percent foetal haemoglobin using an OSM3 Hemoximeter and pH with a linked blood gas analyser. The effect of pH on readings of saturation and fractional foetal haemoglobin was confirmed and a correction factor was calculated. The value of KHbF, the coefficient used in the OSM3 Hemoximeter to estimate the amount of fractional foetal haemoglobin present in a sample, was determined to be 18.3, which slightly but significantly differed (p = 0.04) from the 18.6 used by the OSM3 Hemoximeter. An equation was derived to correct the errors made by pH on the saturation measurement of oxygenated blood. PMID:10400168

  20. Identification of a rare variant haemoglobin (Hb Sinai-Baltimore) causing spuriously low haemoglobin A(1c) values on ion exchange chromatography.

    PubMed

    Smith, Geoff; Murray, Heather; Brennan, Stephen O

    2013-01-01

    Commonly used methods for assay of haemoglobin A(1c) (HbA(1c)) are susceptible to interference from the presence of haemoglobin variants. In many systems, the common variants can be identified but scientists and pathologists must remain vigilant for more subtle variants that may result in spuriously high or low HbA(1c) values. It is clearly important to recognize these events whether HbA(1c) is being used as a monitoring tool or, as is increasingly the case, for diagnostic purposes. We report a patient with a rare haemoglobin variant (Hb Sinai-Baltimore) that resulted in spuriously low values of HbA(1c) when assayed using ion exchange chromatography, and the steps taken to elucidate the nature of the variant.

  1. Raman and SERS recognition of β-carotene and haemoglobin fingerprints in human whole blood

    NASA Astrophysics Data System (ADS)

    Casella, Michele; Lucotti, Andrea; Tommasini, Matteo; Bedoni, Marzia; Forvi, Elena; Gramatica, Furio; Zerbi, Giuseppe

    2011-09-01

    The present work reports on Raman and Surface Enhanced Raman Scattering (SERS) vibrational fingerprints of β-carotene and haemoglobin in fresh whole blood (i.e. right after blood test) with different laser excitations, i.e. visible (514 nm) and near-infrared (NIR, 785 nm). The use of colloidal silver nanoparticles significantly increases the Raman signal, thus providing a clear SERS spectrum of blood. The collected spectra have been examined and marker bands of β-carotene and of the haem prosthetic group of haemoglobin have been found. In particular, the fundamental features of β-carotene (514 nm excitation), blood proteins and haem molecules (785 nm excitation) were recognized and assigned. Moreover haemoglobin SERS signals can be identified and related with its oxygenation state (oxy-haemoglobin). The data reported show the prospects of Raman and SERS techniques to detect important bio-molecules in a whole blood sample with no pre-treatment.

  2. Raman and SERS recognition of β-carotene and haemoglobin fingerprints in human whole blood.

    PubMed

    Casella, Michele; Lucotti, Andrea; Tommasini, Matteo; Bedoni, Marzia; Forvi, Elena; Gramatica, Furio; Zerbi, Giuseppe

    2011-09-01

    The present work reports on Raman and Surface Enhanced Raman Scattering (SERS) vibrational fingerprints of β-carotene and haemoglobin in fresh whole blood (i.e. right after blood test) with different laser excitations, i.e. visible (514 nm) and near-infrared (NIR, 785 nm). The use of colloidal silver nanoparticles significantly increases the Raman signal, thus providing a clear SERS spectrum of blood. The collected spectra have been examined and marker bands of β-carotene and of the haem prosthetic group of haemoglobin have been found. In particular, the fundamental features of β-carotene (514 nm excitation), blood proteins and haem molecules (785 nm excitation) were recognized and assigned. Moreover haemoglobin SERS signals can be identified and related with its oxygenation state (oxy-haemoglobin). The data reported show the prospects of Raman and SERS techniques to detect important bio-molecules in a whole blood sample with no pre-treatment.

  3. The Use of Haemoglobin as a Model for Teaching the Relationship Between Structure and Function

    ERIC Educational Resources Information Center

    Diggins, F. W. E.

    1974-01-01

    Presents information about an atomic model of haemoglobin and describes the oxygenation mechanism as a teaching principle to illustrate the relationship between structure and function at the molecular level. (Author/PEB)

  4. Modification by simetryn sulphoxide of a specific thiol group in rat haemoglobin.

    PubMed Central

    Hughes, G J; de Jong, C; Fischer, R W; Winterhalter, K H; Wilson, K J

    1981-01-01

    Native rat haemoglobins were found to bind simetryn sulphoxide to an extent 40-fold greater than human haemoglobin. This specific behaviour was studied by using only high-pressure ('performance') liquid chromatography for the preparative separation of globin chains and the isolation of peptides resulting from chemical and enzymic degradation. High recoveries (greater than 80%) of peptides throughout the procedures in combination with microsequence techniques, allow a definitive assignment of the residue undergoing modification. The haemoglobin beta-chain cystine-125 residue, with a stoichiometry of one per tetramer of rat haemoglobin, was found to be modified. Stereochemical implications of this finding are discussed. Simetryn sulphoxide would appear to be useful as a specific reagent for the mapping of exposed thiol residues in proteins. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:7337714

  5. Total haemoglobin mass, maximal and submaximal power in elite rowers.

    PubMed

    Treff, G; Schmidt, W; Wachsmuth, N; Völzke, C; Steinacker, J M

    2014-06-01

    Elite rowers are highly endurance trained and present with a large lean body mass (LBM), which is closely related to total haemoglobin mass (tHbmass), a major determinant of blood O2-transport. This study aims to determine the magnitude of tHbmass in elite rowers and its relation to performance parameters that are common in rowing worldwide. 13 rowers (3 lightweight) performed a 2000 m test to evaluate maximal performance on the rowing ergometer (P2k) and an incremental test to evaluate power output at lactate 2 and 4 mmol/l (N=15). tHbmass was measured by CO-rebreathing. tHbmass amounted to 1285±123 g (open weight) and 1059±48 g (lightweight). Coefficients of correlation between tHbmass and power output increased with intensity, being highest for P2k (r=0.80). An increase of 100 g tHbmass is associated with an increase of 24 W in P2k between subjects. The ratio between tHbmass/LBM amounted to approximately 16 g/kg. Absolute tHbmass in elite rowers of open weight class is very high. In relation to body mass or LBM, data is similar to other endurance athletes. The relation between P2k performance and tHbmass is very large. However, it is partly mediated by body composition.

  6. Endothelial cell expression of haemoglobin α regulates nitric oxide signalling.

    PubMed

    Straub, Adam C; Lohman, Alexander W; Billaud, Marie; Johnstone, Scott R; Dwyer, Scott T; Lee, Monica Y; Bortz, Pamela Schoppee; Best, Angela K; Columbus, Linda; Gaston, Benjamin; Isakson, Brant E

    2012-11-15

    Models of unregulated nitric oxide (NO) diffusion do not consistently account for the biochemistry of NO synthase (NOS)-dependent signalling in many cell systems. For example, endothelial NOS controls blood pressure, blood flow and oxygen delivery through its effect on vascular smooth muscle tone, but the regulation of these processes is not adequately explained by simple NO diffusion from endothelium to smooth muscle. Here we report a new model for the regulation of NO signalling by demonstrating that haemoglobin (Hb) α (encoded by the HBA1 and HBA2 genes in humans) is expressed in human and mouse arterial endothelial cells and enriched at the myoendothelial junction, where it regulates the effects of NO on vascular reactivity. Notably, this function is unique to Hb α and is abrogated by its genetic depletion. Mechanistically, endothelial Hb α haem iron in the Fe(3+) state permits NO signalling, and this signalling is shut off when Hb α is reduced to the Fe(2+) state by endothelial cytochrome b5 reductase 3 (CYB5R3, also known as diaphorase 1). Genetic and pharmacological inhibition of CYB5R3 increases NO bioactivity in small arteries. These data reveal a new mechanism by which the regulation of the intracellular Hb α oxidation state controls NOS signalling in non-erythroid cells. This model may be relevant to haem-containing globins in a broad range of NOS-containing somatic cells. PMID:23123858

  7. Immeasurable glycosylated haemoglobin: a marker for severe haemolysis.

    PubMed

    Aggarwal, Nidhi; Rai, Anand Kumar; Kupfer, Yizhak; Tessler, Sidney

    2013-08-08

    Glycosylated haemoglobin (HbA1c) is a measurement commonly performed in patients with diabetes. Factors causing a change in the life span of the red blood cell (RBC) can affect the measurement of HbA1c. Thus haemolysis is an important factor that may affect the HbA1c level determination. Haemolysis has been shown to cause a falsely low HbA1c. A 62-year-old man with a history of autoimmune haemolytic anaemia was admitted for severe haemolytic anaemia and an Hb of 2.9 g/dL. HbA1c tested during hospitalisation was unrecordable due to the extremely low Hb. The patient was treated with intravenous steroids, immunoglobulin, fluids and RBC transfusions but continued to haemolyse and eventually expired. We emphasise that an extremely low HbA1c level can serve as a marker of haemolysis and an unrecordable HbA1c level may point towards fatal haemolysis.

  8. The structure of haemoglobin bound to the haemoglobin receptor IsdH from Staphylococcus aureus shows disruption of the native α-globin haem pocket.

    PubMed

    Dickson, Claire F; Jacques, David A; Clubb, Robert T; Guss, J Mitchell; Gell, David A

    2015-06-01

    Staphylococcus aureus is a common and serious cause of infection in humans. The bacterium expresses a cell-surface receptor that binds to, and strips haem from, human haemoglobin (Hb). The binding interface has previously been identified; however, the structural changes that promote haem release from haemoglobin were unknown. Here, the structure of the receptor-Hb complex is reported at 2.6 Å resolution, which reveals a conformational change in the α-globin F helix that disrupts the haem-pocket structure and alters the Hb quaternary interactions. These features suggest potential mechanisms by which the S. aureus Hb receptor induces haem release from Hb.

  9. Physical, biochemical and functional characterization of haemoglobin from three strains of Artemia.

    PubMed

    Sugumar, Vasudevan; Munuswamy, Natesan

    2007-02-01

    The brine shrimp, Artemia, an inhabitant of coastal and inland salterns, encounter fluctuations in the salinity which in turn influences the oxygen availability of their habitat. Hence, experiments were performed to analyze variations in haemoglobin structure and patterns of three strains of Artemia from South India and also to reflect the effect of varying oxygen levels in their habitat. Haemoglobins were purified on a DEAE-Sephadex column and haemoglobin types were analyzed by comparing their relative mobility on a non-denaturing medium. Furthermore, their molecular masses were determined by gel filtration in Sepharose column and by dodecylsulfate polyacrylamide gel electrophoresis. Results clearly reveal the presence of three distinct extracellular haemoglobins Hb I, Hb II and Hb III in Tuticorin strain while the other strains displayed only trails or the complete absence of Hb III and Hb II. Estimated molecular masses of these haemoglobins are 235,000-250,000 Da. Denaturation of the reduced and alkylated haemoglobins revealed apparently one polypeptide chain with a molecular mass of 124,000 Da. Upon denaturing gel electrophoresis of native haemoglobin Hb II, it was found that the 124,000 Da, polypeptide was cleaved specifically into two unequally-sized fragments of 50,400 and 79,800 Da. With regard to oxygen affinity, Hb III has a very high affinity for oxygen, an almost negligible Bohr effect and a good physiological adaptation to temperature changes. By combining the three haemoglobins in different proportions Artemia strains must be able to withstand diverging environmental conditions. In particular, the absence of Hb III in Puthalam and its occurrence as a faint band in Thamaraikulam could be correlated to the oxygen levels of their habitats. PMID:17185017

  10. Evaluation of haemoglobin in blister fluid as an indicator of paediatric burn wound depth.

    PubMed

    Tanzer, Catherine; Sampson, Dayle L; Broadbent, James A; Cuttle, Leila; Kempf, Margit; Kimble, Roy M; Upton, Zee; Parker, Tony J

    2015-08-01

    The early and accurate assessment of burns is essential to inform patient treatment regimens; however, this first critical step in clinical practice remains a challenge for specialist burns clinicians worldwide. In this regard, protein biomarkers are a potential adjunct diagnostic tool to assist experienced clinical judgement. Free circulating haemoglobin has previously shown some promise as an indicator of burn depth in a murine animal model. Using blister fluid collected from paediatric burn patients, haemoglobin abundance was measured using semi-quantitative Western blot and immunoassays. Although a trend was observed in which haemoglobin abundance increased with burn wound severity, several patient samples deviated significantly from this trend. Further, it was found that haemoglobin concentration decreased significantly when whole cells, cell debris and fibrinous matrix was removed from the blister fluid by centrifugation; although the relationship to depth was still present. Statistical analyses showed that haemoglobin abundance in the fluid was more strongly related to the time between injury and sample collection and the time taken for spontaneous re-epithelialisation. We hypothesise that prolonged exposure to the blister fluid microenvironment may result in an increased haemoglobin abundance due to erythrocyte lysis, and delayed wound healing.

  11. Incorporation of haemoglobin haem into the rat hepatic haemoproteins tryptophan pyrrolase and cytochrome P-450

    SciTech Connect

    Wyman, J.F.; Gollan, J.L.; Settle, W.; Farrell, G.C.; Correia, M.A.

    1986-01-01

    After its administration to intact rats, haemoglobin haem was incorporated into hepatic tryptophan pyrrolase as shown by the marked increase in functional constitution of this enzyme. Incorporation of haemoglobin haem into cytochrome P-450 was demonstrated in intact rats and in the isolated rat liver perfused with haemoglogin-free medium. In both systems, haemoglobin haem restored cytochrome P-450 content and its dependent mixed-function-oxidase activity after substrate-induced destruction of the cytochrome P-450 haem moiety. Further confirmation that heamoglobin haem could be incorporated prosthetically into cytochrome P-450 was achieved by administration of (tritium) haemoglobin to rats and subsequent isolation and characterization of radiolabelled substrate-alkylated products of cytochrome P-450 haem. Findings indicate that, although hepatic uptake of parenteral haemoglobin is slower than that of haem, it appears to serve as an effective haem donor to the intrahepatic free haem pool. Thus parenteral haemoglobin may warrant consideration as a therapeutic alternative to haem in the acute hepatic porphyrias.

  12. Life in the extreme environment at a hydrothermal vent: haemoglobin in a deep-sea copepod.

    PubMed

    Sell, A F

    2000-11-22

    This is the first study, to my knowledge, quantifying the respiratory pigment haemoglobin discovered in a deep-sea copepod. Haemoglobin in copepods has previously been documented in only one other species from the deep water of an Italian lake. Specimens of the siphonostomatoid Scotoecetes introrsus Humes were collected during submersible dives at 2500 m depth near a hydrothermal vent at the East Pacific Rise (9 degrees N). The haemoglobin content in the copepods' haemolymph was 4.3 +/- 0.6 micrograms per individual female (n = 6) and 1.8 +/- 0.1 micrograms per individual male (n = 6). Weight-specific concentrations of haemoglobin were identical for females and males (0.25 +/- 0.04 and 0.26 +/- 0.02 microgram per microgram dry weight, respectively). These haemoglobin concentrations are higher than those found in other small crustaceans. Activity of the electron transport system indicated that the respiration rates in S. introrsus (13.7 +/- 7.7 microliters O2 per milligram dry weight per hour) were similar to those in the shallow-water copepod Acartia tonsa (9.1 +/- 1.3 microliters O2 per milligram dry weight per hour). It was concluded that the possession of highly concentrated haemoglobin allows S. introrsus to colonize a geologically young, thermally active site such as the vicinity of a hydrothermal vent, despite the prevailing oxygen depletion.

  13. Oxygen binding properties of backswimmer (Notonectidae, Anisops) haemoglobin, determined in vivo.

    PubMed

    Matthews, Philip G D; Seymour, Roger S

    2011-12-01

    Aquatic backswimmers (Anisops spp.) collect oxygen from the atmosphere in order to breathe underwater, carrying it within a bubble of air on the ventral surface of their body and bound within haemoglobin-filled cells inside their abdomen. These oxygen stores are interconnected via the abdominal spiracles and the tracheal system. Fibre optic oxygen probes were used to measure PO(2) changes within the air bubbles of submerged backswimmers (Anisops deanei) and these measurements were transformed into in vivo haemoglobin-oxygen equilibrium curves (OECs) using a biotonometric approach. The haemoglobin displayed a triphasic, highly sigmoid OEC with a P(50) of 3.90 kPa. Comparisons made with a previous in vitro analysis of Anisops haemoglobin demonstrate that while the apparent cooperativity and oxygen affinity are considerably higher in vivo, both measurements share unusual Hb-O(2) binding characteristics. The affinity and cooperativity of the backswimmers' haemoglobin appears adaptive as it lengthens dives and promotes neutral buoyancy. While there are limitations associated with biotonometry, the in vivo OEC accurately represents the loading and unloading of biologically available oxygen within the backswimmers' haemoglobin cells. Potential errors associated with determining the OEC are small, as evaluated with sensitivity analyses in numerical models.

  14. Haemoglobin Vanderbilt (alpha2beta289Ser leads to Arg): a new haemoglobin with high oxygen affinity and compensatory erythrocytosis.

    PubMed

    Paniker, N V; Lin, K T; Krantz, S B; Flexner, J M; Wasserman, B K; Puett, D

    1978-06-01

    Haemolysates of family members from three generations, all of whom had polycythaemia, were analysed by polyacrylamide gel electrophoresis at pH 8.8. Two closely spaced major bands were observed, one of which corresponded to Hb A and the other to a new mutant designated Hb Vanderbilt. Whole blood from a heterozygote for Hb Vanderbilt was analysed for oxygen affinity which was found to be much higher than that of normal subjects. Haemoglobin Vanderbilt was separated from Hb A using anion exchange chromatography. Cation exchange chromatography yielded a variant beta chain from which a mutant peptide was identified with a structure corresponding to residues beta83--89 with a Ser leads to Arg replacement at position 89. The oxygen affinity of 'stripped' haemolysates from the heterozygote was found to be much less sensitive to added organic phosphates than haemolysates from normal subjects. In while blood, the decreased sensitivity to 2,3-diphosphoglycerate results in an increased oxygen affinity, thus explaining the clinical observations of tissue hypoxia and compensatory polycythaemia.

  15. Non-invasive in vivo infrared laser spectroscopy to analyse endogenous oxy-haemoglobin, deoxy-haemoglobin, and blood volume in the rat CNS.

    PubMed

    Crespi, Francesco; Bandera, Andrea; Donini, Maurizio; Heidbreder, Christian; Rovati, Luigi

    2005-06-30

    Oxy-haemoglobin (HbO2) and deoxy-haemoglobin (Hb) are chromophores present in biological tissues. Near infrared spectroscopy (NIRS) is a non-invasive methodology based on the low extinction coefficient of tissue in the near infrared region. NIRS can be used to measure changes in the concentration of these chromophores, i.e., haemoglobin, in muscular tissue. In the present work, NIRS has been used for the non-invasive monitoring of HbO2, Hb, and blood volume (V: representing total haemoglobin, i.e., HbO2+Hb) in vivo in the whole rat brain. This has been performed by means of prototype instrumentation based on optic fibre probes placed in contact with the head of anaesthetised rats held in a stereotaxic frame. A preliminary test of the instrument has been performed on human muscle, i.e., lateral gastrocnemius, in order to evaluate the ability of the instrument to detect oxygenation changes. Afterwards, the effects of pharmacological treatments, such as systemic amphetamine and nicotine treatments on the CNS have been detected.

  16. First report of fatal systemic Halicephalobus gingivalis infection in two Lipizzaner horses from Romania: clinical, pathological, and molecular characterization.

    PubMed

    Taulescu, Marian A; Ionicã, Angela M; Diugan, Eva; Pavaloiu, Alexandra; Cora, Roxana; Amorim, Irina; Catoi, Cornel; Roccabianca, Paola

    2016-03-01

    Halicephalobus gingivalis (H. gingivalis) causes a rare and fatal infection in horses and humans. Despite the zoonotic potential and severity of the disease, the epidemiology and pathogenesis of halicephalobiasis are still poorly understood. Several European cases of equine halicephalobiasis have been documented; however, in South-Eastern European countries, including Romania, equine neurohelminthiasis caused by H. gingivalis has not been previously described. Two Lipizzaner horses with a clinical history of progressive neurological signs were referred to the Pathology Department of the Cluj-Napoca (Romania) for necropsy. Both horses died with severe neurological signs. Gross examination and cytological, histological, and molecular analyses were performed. The stallions came from two different breeding farms. No history of traveling outside Romania was recorded. At necropsy, granulomatous and necrotizing lesions were observed in the kidneys, lymph nodes, brain, retroperitoneal adipose tissue, and lungs, indicating a systemic infection. Parasitological and histopathological analyses evidenced larval and adult forms of rhabditiform nematodes consistent with Halicephalobus species. Parasites were observed in both lymph and blood vessels of different organs and were also identified in urine samples. A subunit of the large-subunit ribosomal RNA gene (LSU rDNA) of H. gingivalis (673 bp) was amplified from lesions in both horses.To the authors' knowledge, this is the first report of equine systemic H. gingivalis infection in Romania and in South-Eastern Europe. Our findings provide new insights into the geographic distribution of specific genetic lineages of H. gingivalis, while also raising public health awareness, as the parasite is zoonotic.

  17. Occurrence and antimicrobial susceptibility of Porphyromonas spp. and Fusobacterium spp. in dogs with and without periodontitis.

    PubMed

    Senhorinho, Gerusa N A; Nakano, Viviane; Liu, Chengxu; Song, Yuli; Finegold, Sydney M; Avila-Campos, Mario J

    2012-08-01

    The occurrence of Porphyromonas gulae, Porphyromonas macacae, Fusobacterium nucleatum and Fusobacterium canifelinum in subgingival plaque from dogs with and without periodontitis as well as their antimicrobial susceptibility were evaluated. From 50 dogs with periodontitis were identified 38 P. gulae, 8 P. macacae, 26 F. nucleatum and 15 F. canifelinum, and from 50 dogs without periodontitis were identified 15 P. gulae, 12 F. nucleatum and 11 F. canifelinum. All strains were susceptible to most of the antibiotics tested, however, different resistance rates to clarithromycin, erythromycin and metronidazole among strains were observed. The role of P. gulae, P. macacae, F. nucleatum and F. canifelinum in periodontal disease of household pets needs to be defined to a better prevention and treatment of the canine periodontitis.

  18. Within-subject haemoglobin variation in elite athletes: a longitudinal investigation of 13 887 haemoglobin concentration readings.

    PubMed

    Lobigs, Louisa M; Knight, Emma J; Schumacher, Yorck Olaf; Gore, Christopher J

    2016-02-01

    The Athlete Biological Passport (ABP) estimates individualized reference ranges for key blood markers, such as haemoglobin concentration ([Hb]), using predetermined population mean, between- and within-subject variances. Here, we aim to reassess previously published estimates for within-subject [Hb] variance and determine whether sex-, analyzer-, sport-, or season-specific values are required. Our reference population contains 7723 male (mean ± SD, 22.3 ± 4.6 years of age) and 6164 female (21.6 ± 4.3) athlete observations from 49 sports. [Hb] was calculated using one of three cytometers; Bayer-H3 (1997-1999, n = 4554), ADVIA-120 (1999-2010, n = 8636) and Sysmex XT-2000i (2010-2012, n = 697). The final model was a linear mixed model for [Hb] with analyzer (H3, ADVIA, Sysmex), sex (male, female), sport (power-endurance, endurance, skill, team, disabled and non-athletes), season (summer, winter), and the interaction between sex and sport as fixed effects and athlete as a random effect. The model included an exponential correlation structure to allow for within-subject autocorrelation, and allowed different within-subject variances for each sport. Within-subject [Hb] variance (g(2) /L(2) ) was significantly less for power endurance (35.09, 95% CI 33.50 to 36.76), disabled (25.82, 95% CI 21.71 to 35.28) and non-athletes (34.30, 95% CI 28.53 to 35.87) than for endurance (40.35, 95% CI 39.62 to 47.22) and team sports (38.70, 95% CI 37.68 to 39.76) athletes. No new evidence was found to justify adjusting the current within-subject [Hb] variance estimate.

  19. Glycosylated haemoglobin for screening and diagnosis of gestational diabetes mellitus

    PubMed Central

    Phuah, Eileen; Al-Barazan, Abdul Majeed; Nikakis, Irena; Radford, Andrea; Clarkson, Wade; Trevett, Clinton; Brain, Terry; Gebski, Val; Corbould, Anne

    2016-01-01

    Objectives The oral glucose tolerance test (OGTT) is a cumbersome test that is time consuming, labour intensive and often poorly tolerated by pregnant women. To date, glycosylated haemoglobin (HbA1c) is the most accepted measure of chronic glycaemia outside of pregnancy. HbA1c is an uncomplicated test, less time consuming, does not require any specific patient preparation and is considered straightforward compared with the OGTT. Therefore, we prospectively tested the utility of the HbA1c when used as a screening tool in pregnancy for gestational diabetes mellitus (GDM). Settings Primary health care. Single tertiary referral centre, Tasmania, Australia. Participants A direct comparison between HbA1c levels and the OGTT results in pregnant women, tested concurrently at the 24–28 gestational week, was undertaken. A full profile of 480 pregnant women during the period from September 2012 to July 2014 was completed. Median and mean age of participants was 29 years (range 18–47 years). Interventions A simultaneous prospective assessment of HbA1c versus standard OGTT in a cohort of consecutive pregnant women presenting to our institute was performed. Results The number of women who had GDM according to OGTT criteria was 57, representing 11.9% of the evaluated 480 pregnant women. Using a cut-off value for HbA1c at 5.1% (32 mmol/mol) for detecting GDM showed sensitivity of 61% and specificity of 68% with negative predictive value (NPV) of 93%, versus sensitivity of 27% and specificity of 95% with NPV of 91% when using HbA1c cut-off value of 5.4% (36 mmol/mol). Conclusions Our results suggest that pregnant women with an HbA1c of≥5.4% (36 mmol/mol) should proceed with an OGTT. This may result in a significant reduction in the burden of testing on both patients and testing facility staff and resources. Further investigations are required to integrate and optimise the HbA1c as a single, non-fasting, screening tool for GDM. Trial registration number ACTRN

  20. Spermine uptake is necessary to induce haemoglobin synthesis in murine erythroleukaemia cells.

    PubMed Central

    Clément, S; Delcros, J G; Feuerstein, B G

    1995-01-01

    To determine whether intracellular uptake of spermine is necessary to induce haemoglobin synthesis in murine erythroleukaemia (MEL) DS 19 cells, we used single-step selection for resistance to N1,N12-bis(ethyl)spermine (BESM), a cytotoxic spermine analogue, to isolate clones deficient in polyamine transport. The cells were approximately 500-fold more resistant to BESM than parental cells and were unable to accumulate BESM, putrescine, spermidine or spermine. Addition of spermine to the polyamine-transport-deficient cells failed to induce haemoglobin synthesis. Hexamethylene-1,6-bisacetamide, a well-known differentiating agent, induced haemoglobin synthesis in both parental and resistant cells. Polyamine-transport-deficient cells transfected with DNA purified from the parental cell line were further selected for their ability to grow in the presence of alpha-difluoromethylornithine and putrescine. The transfectants had an active transport system for polyamines, and spermine added to their culture medium accumulated inside the cells and induced haemoglobin production. These findings indicate that intracellular spermine uptake is required to induce haemoglobin production in MEL cells. PMID:8554541

  1. Haemoglobin polymorphisms affect the oxygen-binding properties in Atlantic cod populations.

    PubMed

    Andersen, Oivind; Wetten, Ola Frang; De Rosa, Maria Cristina; Andre, Carl; Carelli Alinovi, Cristiana; Colafranceschi, Mauro; Brix, Ole; Colosimo, Alfredo

    2009-03-01

    A major challenge in evolutionary biology is to identify the genes underlying adaptation. The oxygen-transporting haemoglobins directly link external conditions with metabolic needs and therefore represent a unique system for studying environmental effects on molecular evolution. We have discovered two haemoglobin polymorphisms in Atlantic cod populations inhabiting varying temperature and oxygen regimes in the North Atlantic. Three-dimensional modelling of the tetrameric haemoglobin structure demonstrated that the two amino acid replacements Met55beta1Val and Lys62beta1Ala are located at crucial positions of the alpha1beta1 subunit interface and haem pocket, respectively. The replacements are proposed to affect the oxygen-binding properties by modifying the haemoglobin quaternary structure and electrostatic feature. Intriguingly, the same molecular mechanism for facilitating oxygen binding is found in avian species adapted to high altitudes, illustrating convergent evolution in water- and air-breathing vertebrates to reduction in environmental oxygen availability. Cod populations inhabiting the cold Arctic waters and the low-oxygen Baltic Sea seem well adapted to these conditions by possessing the high oxygen affinity Val55-Ala62 haplotype, while the temperature-insensitive Met55-Lys62 haplotype predominates in the southern populations. The distinct distributions of the functionally different haemoglobin variants indicate that the present biogeography of this ecologically and economically important species might be seriously affected by global warming.

  2. Haemoglobin polymorphisms affect the oxygen-binding properties in Atlantic cod populations

    PubMed Central

    Andersen, Øivind; Wetten, Ola Frang; De Rosa, Maria Cristina; Andre, Carl; Carelli Alinovi, Cristiana; Colafranceschi, Mauro; Brix, Ole; Colosimo, Alfredo

    2008-01-01

    A major challenge in evolutionary biology is to identify the genes underlying adaptation. The oxygen-transporting haemoglobins directly link external conditions with metabolic needs and therefore represent a unique system for studying environmental effects on molecular evolution. We have discovered two haemoglobin polymorphisms in Atlantic cod populations inhabiting varying temperature and oxygen regimes in the North Atlantic. Three-dimensional modelling of the tetrameric haemoglobin structure demonstrated that the two amino acid replacements Met55β1Val and Lys62β1Ala are located at crucial positions of the α1β1 subunit interface and haem pocket, respectively. The replacements are proposed to affect the oxygen-binding properties by modifying the haemoglobin quaternary structure and electrostatic feature. Intriguingly, the same molecular mechanism for facilitating oxygen binding is found in avian species adapted to high altitudes, illustrating convergent evolution in water- and air-breathing vertebrates to reduction in environmental oxygen availability. Cod populations inhabiting the cold Arctic waters and the low-oxygen Baltic Sea seem well adapted to these conditions by possessing the high oxygen affinity Val55–Ala62 haplotype, while the temperature-insensitive Met55–Lys62 haplotype predominates in the southern populations. The distinct distributions of the functionally different haemoglobin variants indicate that the present biogeography of this ecologically and economically important species might be seriously affected by global warming. PMID:19033139

  3. Direct In Vivo Electrochemical Detection of Haemoglobin in Red Blood Cells

    NASA Astrophysics Data System (ADS)

    Toh, Rou Jun; Peng, Weng Kung; Han, Jongyoon; Pumera, Martin

    2014-08-01

    The electrochemical behavior of iron ion in haemoglobin provides insight to the chemical activity in the red blood cell which is important in the field of hematology. Herein, the detection of haemoglobin in human red blood cells on glassy carbon electrode (GC) was demonstrated. Red blood cells or raw blood cells was immobilized on a glassy carbon electrode surface with Nafion films employed to sandwich the layer of biological sample firmly on the electrode surface. Cyclic voltammetry (CV) analyses revealed a well-defined reduction peak for haemoglobin at about -0.30 V (vs. Ag/AgCl) at the red blood cell (GC-Nf-RBC-3Nf) and blood (GC-Nf-B-3Nf) film modified GCE in a pH 3.5 phosphate buffer solution. We further demonstrated that the complex biological conditions of a human red blood cell displayed no interference with the detection of haemoglobin. Such findings shall have an implication on the possibilities of studying the electrochemical behaviour of haemoglobin directly from human blood, for various scientific and clinical purposes.

  4. Assessing diagnostic accuracy of Haemoglobin Colour Scale in real-life setting.

    PubMed

    Shah, Pankaj P; Desai, Shrey A; Modi, Dhiren K; Shah, Shobha P

    2014-03-01

    The study was undertaken to determine diagnostic accuracy of Haemoglobin Colour Scale (HCS) in hands of village-based community health workers (CHWs) in real-life community setting in India. Participants (501 women) were randomly selected from 8 villages belonging to a project area of SEWA-Rural, a voluntary organization located in India. After receiving a brief training, CHWs and a research assistant obtained haemoglobin readings using HCS and HemoCue (reference) respectively. Sensitivity, specificity, positive and negative predictive-values, and likelihood ratios were calculated. Bland-Altman plot was constructed. Mean haemoglobin value, using HCS and HemoCue were 11.02 g/dL (CI 10.9-11.2) and 11.07 g/dL (CI 10.9-11.2) respectively. Mean difference between haemoglobin readings was 0.95 g/dL. Sensitivity of HCS was 0.74 (CI 0.65-0.81) and 0.84 (CI 0.8-0.87) whereas specificity was 0.84 (CI:0.51-0.98) and 0.99 (CI:0.97-0.99) using haemoglobin cutoff limits of 10 g/dL and 7 g/dL respectively. CHWs can accurately diagnose severe and moderately-severe anaemia by using HCS in real-life field condition after a brief training.

  5. Degradation of the human proteinase inhibitors alpha-1-antitrypsin and alpha-2-macroglobulin by Bacteroides gingivalis.

    PubMed Central

    Carlsson, J; Herrmann, B F; Höfling, J F; Sundqvist, G K

    1984-01-01

    Various strains of black-pigmented Bacteroides species were grown on horse blood agar and suspended in human serum. After various times of incubation the effect of the bacteria on the serum was evaluated by polyacrylamide gel electrophoresis and "rocket" immunoelectrophoresis. The formation of trichloroacetic acid-soluble material in the suspensions and the capacity of the treated sera to inhibit the activity of trypsin were also determined. The two tested strains of Bacteroides gingivalis (W83, H185) degraded most serum proteins, including the plasma proteinase inhibitors alpha-1-antitrypsin and alpha-2-macroglobulin. They did not, however, degrade alpha-1-antichymotrypsin. Bacteroides intermedius NCTC 9336, Bacteroides asaccharolyticus NCTC 9337, and an asaccharolytic oral strain different from B. gingivalis (BN11a-f) did not degrade the plasma proteinase inhibitors. These strains were, however, able to inactivate the capacity of serum to inhibit the activity of trypsin. Images PMID:6198282

  6. Formation of bile pigments by coupled oxidation of cobalt-substituted haemoglobin and myoglobin.

    PubMed Central

    Vernon, D I; Brown, S B

    1984-01-01

    Treatment of cobalt-substituted haemoglobin and myoglobin with ascorbate and molecular O2 (coupled oxidation) resulted in biliverdin formation from the cobalt(II) derivatives but not from the cobalt(III) derivatives. This was apparently due to the inability of ascorbate to reduce cobalt(III) haemoproteins. Isomer analysis of the biliverdins produced from coupled oxidation of cobalt(II) oxyhaemoglobin suggested that the orientation of the cobalt protoporphyrin IX in the haem pocket differed slightly from that of the haem in native haemoglobin. PMID:6497839

  7. Haemoglobin recovery among HIV-1 infected patients on zidovudine-based antiretroviral therapy and other regimens in north-central Nigeria.

    PubMed

    Parrish, Deidra D; Blevins, Meridith; Megazzini, Karen M; Shepherd, Bryan E; Mohammed, Mukhtar Y; Wester, C William; Vermund, Sten H; Aliyu, Muktar H

    2014-04-01

    We conducted a study to assess trends in haemoglobin recovery among HIV-infected patients initiated on zidovudine-based combination antiretroviral therapy (cART) stratified by baseline haemoglobin level. Haemoglobin data from non-pregnant adult patients initiating cART in rural north-central Nigeria between June 2009 and May 2011 were analysed using a linear mixed effects model to assess the interaction between time, zidovudine-containing regimen and baseline haemoglobin level on the outcome of subsequent haemoglobin level. Best-fit curves were created for baseline haemoglobin in the 10th, 25th, 75th and 90th percentiles. We included 313 patients with 736 measures of haemoglobin in the analysis (239 on zidovudine and 74 on non-zidovudine-containing regimens). Median haemoglobin increased over time in both groups, with differences in haemoglobin response over time related to baseline haemoglobin levels and zidovudine use (p = 0.003). The groups of patients on zidovudine at the 10th and 90th percentiles had downward sloping curves while all other groups had upward trending haemoglobin levels. Although haemoglobin levels increased overall for patients on zidovudine-containing regimens, for those in the 10th and 90th percentiles haemoglobin levels trended downward over time. These results have implications for decisions regarding when to initiate, switch from or avoid the use of zidovudine.

  8. Pro-oxidant effects of cross-linked haemoglobins explored using liposome and cytochrome c oxidase vesicle model membranes.

    PubMed Central

    Rogers, M S; Patel, R P; Reeder, B J; Sarti, P; Wilson, M T; Alayash, A I

    1995-01-01

    The therapeutic use of cell-free haemoglobin as a blood substitute has been hampered by toxicological effects. A model asolectin (phosphatidylcholine/phosphatidylethanolamine) liposome system was utilized to study the pro-oxidant efficiency of several chemically modified haemoglobins on biological membranes. Lipid peroxidation, resulting from the interactions between haemoglobin and liposomes, was measured by conjugated diene formation and the maximal rates of oxygen uptake. Spectral changes gave insight into the occurrence of the ferryl iron species. The residual reactivity of oxidatively damaged haemoglobins with ligands during incubation with liposomes was assessed from rapid kinetic carbon monoxide-binding experiments. Liposomes in which cytochrome c oxidase was embedded show both haemoglobin and the enzyme to be oxidatively damaged during incubation. The functional state of cytochrome c oxidase was monitored in the presence and absence of a free radical scavenger. Once in contact, both unmodified and modified haemoglobins triggered and maintained severe radical-mediated membrane damage. Differences in the pro-oxidant activities among haemoglobins may be explained by either the differential population of their ferryl intermediates or disparate dimerization and transfer of haem into the membrane with subsequent haem degradation. This study may contribute to a better understanding of the molecular determinants of haemoglobin interactions with a variety of biological membranes. Images Figure 2 PMID:7575415

  9. The calcium, copper and zinc content of some annelid extracellular haemoglobins.

    PubMed Central

    Standley, P R; Mainwaring, M G; Gotoh, T; Vinogradov, S N

    1988-01-01

    The extracellular haemoglobins of Lumbricus and Tylorrhynchus contain 50 and 61 tightly bound calcium atoms per molecule, respectively. In addition, they contain one to four tightly bound copper and zinc atoms. Although the role of the latter is unknown, that of calcium is probably structural, assisting in the maintenance of the native hexagonal bilayer structure. PMID:3355504

  10. Establishing traceability of photometric absorbance values for accurate measurements of the haemoglobin concentration in blood

    NASA Astrophysics Data System (ADS)

    Witt, K.; Wolf, H. U.; Heuck, C.; Kammel, M.; Kummrow, A.; Neukammer, J.

    2013-10-01

    Haemoglobin concentration in blood is one of the most frequently measured analytes in laboratory medicine. Reference and routine methods for the determination of the haemoglobin concentration in blood are based on the conversion of haeme, haemoglobin and haemiglobin species into uniform end products. The total haemoglobin concentration in blood is measured using the absorbance of the reaction products. Traceable absorbance measurement values on the highest metrological level are a prerequisite for the calibration and evaluation of procedures with respect to their suitability for routine measurements and their potential as reference measurement procedures. For this purpose, we describe a procedure to establish traceability of spectral absorbance measurements for the haemiglobincyanide (HiCN) method and for the alkaline haematin detergent (AHD) method. The latter is characterized by a higher stability of the reaction product. In addition, the toxic hazard of cyanide, which binds to the iron ion of the haem group and thus inhibits the oxygen transport, is avoided. Traceability is established at different wavelengths by applying total least-squares analysis to derive the conventional quantity values for the absorbance from the measured values. Extrapolation and interpolation are applied to get access to the spectral regions required to characterize the Q-absorption bands of the HiCN and AHD methods, respectively. For absorbance values between 0.3 and 1.8, the contributions of absorbance measurements to the total expanded uncertainties (95% level of confidence) of absorbance measurements range from 1% to 0.4%.

  11. Haemoglobin as a buoyancy regulator and oxygen supply in the backswimmer (Notonectidae, Anisops).

    PubMed

    Matthews, Philip G D; Seymour, Roger S

    2008-12-01

    Unlike all other diving insects, backswimmers of the genus Anisops can exploit the pelagic zone by temporarily achieving near-neutral buoyancy during the course of a dive. They begin a dive positively buoyant due to the large volume of air carried in their ventral air-stores, but rapidly enter a protracted period of near-neutral buoyancy before becoming negatively buoyant. This dive profile is due to haemoglobin found in large tracheated cells in the abdomen. Fibre optic oxygen probes placed in the air-stores of submerged bugs revealed that oxygen partial pressure (P(O(2))) dropped in a sigmoid curve, where a linear decline preceded a plateau between 5.1 and 2.0 kPa, before a final drop. Buoyancy measurements made by attaching backswimmers to a sensitive electronic balance showed the same three phases. Inactivating the haemoglobin by fumigating backswimmers with 15% CO eliminated both buoyancy and P(O(2)) plateaus. Oxygen unloaded from the haemoglobin stabilises the air-store during the neutrally buoyant phase after a decrease in volume of between 16% and 19%. Using measurements of air-store P(O(2)) and volume, it was calculated that during a dive the haemoglobin and air-store contribute 0.25 and 0.26 microl of oxygen, respectively.

  12. Close tetrapod relationships of the coelacanth Latimeria indicated by haemoglobin sequences.

    PubMed

    Gorr, T; Kleinschmidt, T; Fricke, H

    1991-05-30

    The origin of tetrapods has been debated for many years. In traditional systematics, the extinct lobe-finned bony fish (Rhipidistia) are regarded as the closest relatives of tetrapods. Among living fish, the coelacanth Latimeria chalumnae (Actinistia), which is the only recent representative of the Crossopterygii (Actinistia and Rhipidistia), the lungfish (Dipnoi) and ray-finned fish (Actinopterygii), have each been considered as sister-groups of the tetrapods. We have now determined the sequence of the alpha- and beta-globin chains of coelacanth haemoglobin and compared them with all known haemoglobins of bony and cartilaginous fish as well as those of tadpoles and adult amphibians. Haemoglobins of bony fish match more closely those of larval than adult amphibians. The beta chains of Latimeria match those of tadpoles more closely (54%) than do those of any other fish, whereas the alpha chains of Latimeria (45.4%), and especially of teleosts (49.2%), are closer to those of larval amphibians than are those of lungfish (39.8%). If only synapomorphous sequence matches (those at derived positions shared by one bony fish and tadpoles but not by any other bony fish) are considered, both Latimeria globin chains have distinctly more identities with phase of tadpoles than do those of any bony fish. Thus the primary structure of Latimeria haemoglobin indicates that the coelacanth is the closest living relative of tetrapods.

  13. The relationship between juvenile and non-juvenile periodontitis, ABO blood groups and haemoglobin types.

    PubMed

    Arowojolu, M O; Dosmu, E B; Adingbola, T S

    2002-09-01

    This study was carried out to investigate the relationship between juvenile and non-juvenile peridontitis (JP, non-JP), ABO blood groups and haemoglobin type. The heamoglobin electrophoresis was determined by routine technique using cellulose acetate paper and tris buffer at pH 8.5. Tile blood grouping was carried out on all specimens. Forty Nigerian adolescent individuals were investigated, twenty of which were diagnosed as having JP while the remaining 20 were diagnosed a having plaque-induced chronic periodontitis (non JP). This latter group was used as the control group. All the JP patients were either of blood group B/AB, rhesus positive while the non-JP subjects had B rhesus positive/negative, O rhesus positive/negative or AB rhesus positive. The differences between the results of the test and the control groups were statistically significant P < 0.05. All the forty subjects (JP and non-JP) had the haemoglobin type A and none of them exhibited the S and C haemoglobin types. There is a need to further investigate the relationship between juvenile periodontitis, ABO blood group and the common haemoglobin types (A, AS, S, C, and SS) at molecular level.

  14. Use of a topical haemoglobin spray for oxygenating pressure ulcers: healing outcomes.

    PubMed

    Tickle, Joy; Bateman, Sharon Dawn

    2015-12-01

    A published evaluation ( Tickle, 2015 ) of the use of a topical haemoglobin spray plus standard care in 18 patients with pressure ulcers showed that, following 4 weeks of treatment, the wound size reduced in 17 wounds and there was a progression toward healing in all 18. All but one of the wounds were over 2 months in duration at baseline. This article reports the results of the healing rates at 3 months of the 11 patients who continued to be treated with the haemoglobin spray. Nine of the 11 wounds healed, and 2 reduced in size by week 12 (i.e. 1 wound reduced from 30 cm(2) at baseline to 7 cm(2), while the other reduced from 6 cm(2) to 4 cm(2)). Of the 10 patients who were experiencing wound pain at baseline, 9 were pain free by week 8. Rapid elimination of slough was observed in all patients. The 82% healing rate achieved at 3 months and the fact that most patients continued to receive the same standard care as they had in the 4 weeks before recruitment into the evaluation increases the likelihood that the clinical outcomes observed here can be attributed to the haemoglobin spray. Topical haemoglobin shows promise in terms of its ability to accelerate healing in chronic pressure ulcers.

  15. Characterization of haemoglobin from actinorhizal plants--an in silico approach.

    PubMed

    Bhattacharya, Sanghati; Sen, Arnab; Thakur, Subarna; Tisa, Louis S

    2013-11-01

    Plant haemoglobins (Hbs), found in both symbiotic and non-symbiotic plants, are heme proteins and members of the globin superfamily. Hb genes of actinorhizal Fagales mostly belong to the non-symbiotic type of haemoglobin; however, along with the non-symbiotic Hb, Casuarina sp. posses a symbiotic one (symCgHb), which is expressed specifically in infected cells of nodules. A thorough sequence analysis of 26 plant Hb proteins, currently available in public domain, revealed a consensus motif of 29 amino acids. This motif is present in all the members of symbiotic class II Hbs including symCgHb and non-symbiotic Class II Hbs, but is totally absent in Class I symbiotic and non-symbiotic Hbs. Further, we constructed 3D structures of Hb proteins from Alnus and Casuarina through homology modelling and peeped into their structural properties. Structure-based studies revealed that the Casuarina symbiotic haemoglobin protein shows distinct stereochemical properties from that of the other Casuarina and Alnus Hb proteins. It also showed considerable structural similarities with leghemoglobin structure from yellow lupin (pdb id 1GDI). Therefore, sequence and structure analyses point to the fact that symCgHb protein shows significant resemblance to symbiotic haemoglobin found in legumes and may thus eventually play a similar role in shielding the nitrogenase from oxygen as seen in the case of leghemoglobin. PMID:24287657

  16. Selective disappearance of individuals with high levels of glycated haemoglobin in a free-living bird.

    PubMed

    Récapet, Charlotte; Sibeaux, Adélaïde; Cauchard, Laure; Doligez, Blandine; Bize, Pierre

    2016-08-01

    Although disruption of glucose homeostasis is a hallmark of ageing in humans and laboratory model organisms, we have little information on the importance of this process in free-living animals. Poor control of blood glucose levels leads to irreversible protein glycation. Hence, levels of protein glycation are hypothesized to increase with age and to be associated with a decline in survival. We tested these predictions by measuring blood glycated haemoglobin in 274 adult collared flycatchers of known age and estimating individual probability of recapture in the following 2 years. Results show a strong decrease in glycated haemoglobin from age 1 to 5 years and an increase thereafter. Individuals with high levels of glycated haemoglobin had a lower probability of recapture, even after controlling for effects of age and dispersal. Altogether, our findings suggest that poor control of glucose homoeostasis is associated with lower survival in this free-living bird population, and that the selective disappearance of individuals with the highest glycation levels could account for the counterintuitive age-related decline in glycated haemoglobin in the early age categories. PMID:27555645

  17. Fetal haemoglobin in sickle-cell disease: from genetic epidemiology to new therapeutic strategies.

    PubMed

    Lettre, Guillaume; Bauer, Daniel E

    2016-06-18

    Sickle-cell disease affects millions of individuals worldwide, but the global incidence is concentrated in Africa. The burden of sickle-cell disease is expected to continue to rise over the coming decades, adding to stress on the health infrastructures of many countries. Although the molecular cause of sickle-cell disease has been known for more than half a century, treatment options remain greatly limited. Allogeneic haemopoietic stem-cell transplantation is the only existing cure but is limited to specialised clinical centres and remains inaccessible for most patients. Induction of fetal haemoglobin production is a promising strategy for the treatment of sickle-cell disease. In this Series paper, we review scientific breakthroughs in epidemiology, genetics, and molecular biology that have brought reactivation of fetal haemoglobin to the forefront of sickle-cell disease research. Improved knowledge of the regulation of fetal haemoglobin production in human beings and the development of genome editing technology now support the design of innovative therapies for sickle-cell disease that are based on fetal haemoglobin.

  18. The Prevalence of Self-Reported Health Problems and Haemoglobin Status of Sudanese Adolescents

    ERIC Educational Resources Information Center

    Moukhyer, M. E.; de Vries, N. K.; Bosma, H.; van Eijk, J. Th. M.

    2006-01-01

    In this paper we describe self-reported health problems and haemoglobin status among 1200 Sudanese adolescents (53.2% females, 46.8% males). Many adolescents report their general health as excellent and good (84%). A large number, however, report separate physical and psychological complaints. Report of psychological complaints is equal for both…

  19. Fetal haemoglobin in sickle-cell disease: from genetic epidemiology to new therapeutic strategies.

    PubMed

    Lettre, Guillaume; Bauer, Daniel E

    2016-06-18

    Sickle-cell disease affects millions of individuals worldwide, but the global incidence is concentrated in Africa. The burden of sickle-cell disease is expected to continue to rise over the coming decades, adding to stress on the health infrastructures of many countries. Although the molecular cause of sickle-cell disease has been known for more than half a century, treatment options remain greatly limited. Allogeneic haemopoietic stem-cell transplantation is the only existing cure but is limited to specialised clinical centres and remains inaccessible for most patients. Induction of fetal haemoglobin production is a promising strategy for the treatment of sickle-cell disease. In this Series paper, we review scientific breakthroughs in epidemiology, genetics, and molecular biology that have brought reactivation of fetal haemoglobin to the forefront of sickle-cell disease research. Improved knowledge of the regulation of fetal haemoglobin production in human beings and the development of genome editing technology now support the design of innovative therapies for sickle-cell disease that are based on fetal haemoglobin. PMID:27353686

  20. Purification, crystallisation and preliminary X-ray study of haemoglobin from Crocodilis palustris and Crocodilis porosus.

    PubMed

    Deepthi, S; Johnson, A; Sathish, R; Pattabhi, V

    2000-07-14

    The unsolved three-dimensional structure of crocodile haemoglobin and its prospects as a blood substitute have led us to initiate the purification and crystallisation of haemoglobin molecules from crocodile species (Crocodilis palustris or mugger and Crocodilis porosus or salt water crocodile). The work has resulted in the prevention of polymerisation of naked haemoglobin molecules using N-ethylmaleimide or iodoacetamide. The purified monomeric haemoglobin molecule of C. porosus was crystallised in two different forms and X-ray diffraction data were collected up to 2 A resolution for both forms. Form I: a=53.62, b=53.55, c=103.77 A; beta=93.35 degrees, space group P2(1), Z=2. Form II: a=71.30, b=54.70, c=80.00 A; beta=106.4 degrees, space group P2(1), Z=2. Structure solution and rigid body refinement of form I data resulted in a model with R(free)=0.42 and R=0.35. PMID:11004575

  1. Haemoglobin decreases in NSAID users over time: an analysis of two large outcome trials

    PubMed Central

    Goldstein, J L; Chan, F K L; Lanas, A; Wilcox, C M; Peura, D; Sands, G H; Berger, M F; Nguyen, H; Scheiman, J M

    2011-01-01

    Background Nonsteroidal anti-inflammatory drugs (NSAIDs) have been associated with clinically significant decreases in haemoglobin dependent and independent of acute bleeding events. Aim To evaluate the incidence and time to a clinically meaningful decrease in haemoglobin in two double-blind, prospective randomised clinical trials comparing NSAIDs in patients with osteoarthritis (OA) or rheumatoid arthritis (RA). Methods In CLASS, patients with OA/RA who were aged ≥18 years and required continuous NSAID treatment were included; patients who were Helicobacter pylori positive and/or using aspirin were not excluded. In contrast, in the CONDOR trial, comparing celecoxib alone to diclofenac sustained release (plus omeprazole), patients were aged ≥60 years or ≥18 years with a history of gastroduodenal ulcer and were H. pylori negative; aspirin or other anti-platelet users were excluded. To make a parallel post hoc analysis we limited our study to 6 months and the populations to only the non-aspirin users in CLASS and those patients receiving either celecoxib or diclofenac. A decrease in haemoglobin of ≥2 g/dL defined the primary end point. Results At 6 months, in the CLASS and CONDOR trials, 1.9% and 2.0% of patients treated with celecoxib and 3.3% and 5.7% of patients treated with diclofenac developed a ≥2 g/dL decrease in haemoglobin, respectively, [CLASS: odds ratio (OR) 1.80 (95% confidence interval (CI), 1.22–2.65) and CONDOR: OR 2.93 (95% CI, 2.06–4.15), respectively]. Conclusion In these two large, independent trials, clinically-meaningful decreases in haemoglobin ≥2 g/dL occurred in a relatively similar fashion over time despite differences in trial designs. PMID:21810115

  2. New insights into bioprotective effectiveness of disaccharides: an FTIR study of human haemoglobin aqueous solutions exposed to static magnetic fields.

    PubMed

    Magazù, Salvatore; Calabrò, Emanuele; Campo, Salvatore; Interdonato, Salvatore

    2012-01-01

    The aim of this study was the investigation of static magnetic field effects on haemoglobin secondary structure and the bioprotective effectiveness of two disaccharides, sucrose and trehalose. Samples of haemoglobin aqueous solutions, in the absence and in the presence of sucrose and trehalose, were exposed to a uniform magnetic field at 200 mT, which is the exposure limit established by the ICNIRP recommendation for occupational exposure. Spectral analysis by FTIR spectroscopy after 3 and 7 h of exposure revealed a decrease in the amide A vibration band for haemoglobin in bi-distilled water solution. Analogue exposures did not produce any appreciable change of amide A for haemoglobin in sucrose and trehalose solutions. Otherwise, no relative increase of [Formula: see text]-sheet contents in amide I and II regions was detected for haemoglobin aqueous solutions, leading us to exclude the hypothesis that static magnetic fields can induce the formation of aggregates in the protein. In addition, a decrease in CH(3) stretching linkages occurred for haemoglobin in bi-distilled water solution after exposure, which was not observed for haemoglobin in sucrose and trehalose aqueous solutions, providing further evidence of a bioprotective compensatory mechanism of such disaccharides. PMID:23277670

  3. Studies on iron metabolism in sickle cell anaemia, sickle cell haemoglobin C disease, and haemoglobin C disease using a large volume liquid scintillation counter

    PubMed Central

    Ringelhann, Bela; Konotey-Ahulu, Felix; Dodu, Silas R. A.

    1970-01-01

    Iron absorption as measured by a faecal recovery method in young adult males living in a tropical zone was high, even in the absence of anaemia. There was an inverse relation between the iron absorption and the packed cell volume. The highest absorption was found in sickle cell anaemia patients, where the packed cell volume is the lowest. The incorporation of iron was also the fastest and greatest in this group. In the controls the iron absorbed accumulated in the marrow and the spleen on the first day; in the sickle cell anaemia group the spleen has an insignificant role in iron storage. The growing radioactivity in the liver parallels that of the heart in the group of sickle cell anaemia patients; however, it remains low in the spleen in the same group, implying a diminution of splenic blood flow. In the sickle cell haemoglobin C and the haemoglobin C patients, the liver and spleen have an intermediate position between that of the sickle cell anaemia group and the control group. PMID:5423947

  4. β-Thalassaemia and its Co-existence with Haemoglobin E and Haemoglobin S in Upper Assam Region of North Eastern India: A Hospital Based Study

    PubMed Central

    Deori, Rumi; Saikia, Sidhartha Protim; Pathak, Kalyani; Panyang, Rita; Rajkakati, Rashmi

    2016-01-01

    Introduction β-Thalassaemias are common genetic disorders in the Indian subcontinent and its status has not been well studied in the Upper Assam region of North Eastern India. Aim The aim of the study was to show the prevalence of β- thalassaemias and its co-existence with Haemoglobin E and Haemoglobin S in the Upper Assam region of North Eastern India. Materials and Methods A total of 1200 anaemic patients were investigated for β- thalassaemias. Complete Blood Count (CBC) and High Performance Liquid Chromatography (HPLC) were done for screening. Results Out of 1200 patients screened, 5.83% β-thalassaemia trait, 2.33% compound Hb E/β-Thalassaemia, 1.33% β-thalassaemia major and 0.42% compound Hb S/β- thalassaemia were detected. A high incidence of thalassaemia is found among the people of Upper Assam region of North Eastern India. Conclusion The only way to prevent the disease is carrier detection and awareness among the people about it. PMID:27190829

  5. A rare haemoglobin variant (Hb Phnom Penh) manifesting as a falsely high haemoglobin A1c value on ion-exchange chromatography.

    PubMed

    Chen, Chi-Fen; Tai, Yen-Kuang

    2014-08-01

    Most haemoglobin (Hb) variants are clinically silent. However, some Hb variants may interfere with the measurement of haemoglobin A1c (HbA1c), resulting in spurious values depending on the assays used. We herein report the case of a 53-year-old Taiwanese man with type 2 diabetes mellitus, who presented with an abnormal HbA1c peak on ion-exchange chromatography. Additional investigations, including intensified self-monitored blood glucose tests, an alternative HbA1c assay, and a glycaemic indicator based on a different method, revealed that the HbA1c values were falsely elevated. Subsequent DNA analysis confirmed that the patient was heterozygous for the insertion of an isoleucine residue at codons 117/118 of the a1-globin gene, Hb Phnom Penh. Clinical laboratorians should be aware of the interfering factors in their HbA1c analysis. Cautious inspection of the chromatogram may provide a valuable clue to the presence of an Hb variant. PMID:25189312

  6. Changes in haemoglobin and antigenic constitution of erythrocytes during embryonic and postembryonic chick development.

    PubMed

    Godet, J; Blanchet, J P; Nigon, V

    1976-01-01

    Between the 18th and 20th day of incubation, the proportion of haemoglobin F (Hb F) synthesis decreases in the embryo bone marrow from the value found in the yolk sac (10% of the total haemoglobin synthesis) to the value found in the bone marrow of young chicks (2%). A similar drop in Hb F synthesis is observed with a delay of 48 hours in the blood. Two membrane antigens each specific of either embryo or adult erythrocytes have been detected. The study by immunofluorescence of their cellular distribution shows that 3 erythrocyte populations are successively produced: a population E bearing the embryonic antigen only, a population EA bearing both antigens and a population A bearing the adult antigen only. The presence of a relatively high proportion of Hb F in population E is strongly suggested by the similar kinetics of Hb F and E cells disappearance within the first posthatching month.

  7. Fetal anaemia from red blood cell membrane defect and co-inherited haemoglobin Constant Spring.

    PubMed

    Srisupundit, Kasemsri; Charoenkwan, Pimlak; Traisrisilp, Kuntharee; Tongsong, Theera

    2015-07-27

    The case presented here is an example of hereditary red blood cell membrane defect with a co-inherited haemoglobin Constant Spring. This case is of an anaemic fetus that presented with isolated ascites at 18 weeks of gestation. Fetal blood analysis revealed abnormal shaped red blood cells. The same pattern of red blood cell morphology was also seen on paternal peripheral blood smear. Intrauterine blood transfusions were given twice to correct fetal anaemia. The fetus showed a good response to the transfusions and was delivered at term with mild anaemia and did not need blood transfusion after birth. This report describes a natural course of red blood cell membrane defect with co-inherited haemoglobin Constant Spring, indicating that the course of disease was more severe during fetal life. Intrauterine transfusion supported the transition of the fetus through the critical period in utero to a healthier life after birth.

  8. The effect of 2,3-diphosphoglycerate on the oxygen dissociation curve of human haemoglobin.

    PubMed Central

    Goodford, P J; Norrington, F E; Paterson, R A; Wootton, R

    1977-01-01

    1. Oxygen dissociation curves for concentrated human haemoglobin solutions (1.6 mmol dm-3 in haem) have been measured by mixing known quantities of oxy- and deoxyhaemoglobin solutions and measuring the resulting partial pressure of oxygen with an oxygen electrode. 2. Observations in the presence of 2,3-diphosphoglycerate support previous conclusions derived from experiments at low haemoglobin concentrations, the validity of which has been questioned. 3. The two affinity state model of Monod, Wyman & Changeux (1965) does not fully describe the actions of 2,3-diphosphoglycerate and a model in which this allosteric effector not only binds preferentially to the T state but also lowers the oxygen affinity of this state gives an improved fit to the data. PMID:604451

  9. Growth inhibitory effects of endotoxins from Bacteroides gingivalis and intermedius on human gingival fibroblasts in vitro

    SciTech Connect

    Layman, D.L.; Diedrich, D.L.

    1987-06-01

    Purified endotoxin or lipopolysaccharide from Bacteroides gingivalis and Bacteroides intermedius caused a similar dose-dependent inhibition of growth of cultured human gingival fibroblasts as determined by /sup 3/H-thymidine incorporation and direct cell count. Approximately 200 micrograms/ml endotoxin caused a 50% reduction in /sup 3/H-thymidine uptake of logarithmically growing cells. Inhibition of growth was similar in cultures of fibroblasts derived from either healthy or diseased human gingiva. When examining the change in cell number with time of exposure in culture, the rate of proliferation was significantly suppressed during the logarithmic phase of growth. However, the cells recovered so that the rate of proliferation, although reduced, was sufficient to produce a cell density similar to the control cells with prolonged culture. The endotoxins were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The profiles of the Bacteroides endotoxins were different. B. gingivalis endotoxin showed a wide range of distinct bands indicating a heterogeneous distribution of molecular species. Endotoxin from B. intermedius exhibited a few discrete low molecular weight bands, but the majority of the lipopolysaccharides electrophoresed as a diffuse band of high molecular weight material. The apparent heterogeneity of the two Bacteroides endotoxins and the similarity in growth inhibitory capacity suggest that growth inhibitory effects of these substances cannot be attributed to any polysaccharide species of endotoxin.

  10. Distribution of ABO, Rhesus blood and haemoglobin electrophoresis among the undergraduate students of Niger Delta State University, Nigeria.

    PubMed

    Egesie, U G; Egesie, O J; Usar, I; Johnbull, T O

    2008-01-01

    The distribution of ABO, Rhesus blood groups and haemoglobin electrophoresis among 200 undergraduate students of Niger Delta University, Bayelsa State, Nigeria randomly selected were studied. Blood samples were collected by venepuncture from the antecubital vein. The blood sample were transferred into EDTA bottle and mixed. The determination of the ABO, Rhesus (RhD) blood groups and haemoglobin electrophoresis was done. The results showed that blood group O had the highest percentage distribution of 49% followed by blood groups A and B with 22% respectively and the least percentage distribution of 7% was blood group AB. Rh-D positive rate was 98% and it was 2% for Rh-D negative. The percentage distribution for the haemoglobin electrophoresis pattern for HbAA, HbAS, HbSS, HbAC and HbSC were 66%, 26%, 2%, 2%, and 4% respectively. HbAA and HbAS occurred more frequently than other haemoglobin variants in this study.

  11. Growth and change in blood haemoglobin concentration among underweight Malawian infants receiving fortified spreads for 12 weeks: a preliminary trial

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fortified spreads (FSs) have proven effective in the rehabilitation of severely malnourished children. We examined acceptability, growth and change in blood haemoglobin (Hb) concentration among moderately underweight ambulatory infants given FS. This was a randomised, controlled, parallel-group, inv...

  12. The Effect of Nonsurgical Periodontal Therapy on Trichomonas Tenax and Entamoeba Gingivalis in Patients with Chronic Periodontitis

    PubMed Central

    Rashidi Maybodi, Fahimeh; Haerian Ardakani, Ahmad; Fattahi Bafghi, Ali; Haerian Ardakani, Alireza; Zafarbakhsh, Akram

    2016-01-01

    Statement of the Problem Trichomonas tenax and Entamoeba gingivalis are commensal protozoa which inhabit the human oral cavity. These parasites are found in patients with poor oral hygiene and might be a reason for progressive periodontal diseases. Purpose The aim of this study was to evaluate the effect of nonsurgical periodontal treatment on the frequency of these protozoa in saliva and plaque samples. Materials and Method In this clinical trial, samples of saliva and dental plaque were collected from 46 patients with moderate to severe chronic periodontitis before and after periodontal therapy. The samples were assessed for the frequency of parasites. Results The frequency of Entamoeba gingivalis was reduced in saliva (p= 0.007) and plaque (p= 0.027) three weeks after the treatment. Likewise, the frequency of Trichomonas tenax reduced in saliva (p= 0.030); however, the decrease was not significant in plaque (p= 0.913). Trichomonas tenax frequency in dental plaque directly related to the severity of periodontitis (r= 0.565, p≤ 0.000). In contrast, the number of Entamoeba gingivalis in both saliva (r= -0.405, p≤ 0.005) and plaque (r= -0.304, p= 0.040) was inversely related with the severity of the periodontal disease. Conclusion Nonsurgical periodontal treatment could reduce the number of Trichomonas Tenax and Entamoeba gingivalis in the oral environment of patients with chronic periodontitis. PMID:27602391

  13. The Effect of Nonsurgical Periodontal Therapy on Trichomonas Tenax and Entamoeba Gingivalis in Patients with Chronic Periodontitis

    PubMed Central

    Rashidi Maybodi, Fahimeh; Haerian Ardakani, Ahmad; Fattahi Bafghi, Ali; Haerian Ardakani, Alireza; Zafarbakhsh, Akram

    2016-01-01

    Statement of the Problem Trichomonas tenax and Entamoeba gingivalis are commensal protozoa which inhabit the human oral cavity. These parasites are found in patients with poor oral hygiene and might be a reason for progressive periodontal diseases. Purpose The aim of this study was to evaluate the effect of nonsurgical periodontal treatment on the frequency of these protozoa in saliva and plaque samples. Materials and Method In this clinical trial, samples of saliva and dental plaque were collected from 46 patients with moderate to severe chronic periodontitis before and after periodontal therapy. The samples were assessed for the frequency of parasites. Results The frequency of Entamoeba gingivalis was reduced in saliva (p= 0.007) and plaque (p= 0.027) three weeks after the treatment. Likewise, the frequency of Trichomonas tenax reduced in saliva (p= 0.030); however, the decrease was not significant in plaque (p= 0.913). Trichomonas tenax frequency in dental plaque directly related to the severity of periodontitis (r= 0.565, p≤ 0.000). In contrast, the number of Entamoeba gingivalis in both saliva (r= -0.405, p≤ 0.005) and plaque (r= -0.304, p= 0.040) was inversely related with the severity of the periodontal disease. Conclusion Nonsurgical periodontal treatment could reduce the number of Trichomonas Tenax and Entamoeba gingivalis in the oral environment of patients with chronic periodontitis.

  14. The structure of Artemia sp. (brine shrimp) haemoglobins. Purification of a structural unit to homogeneity.

    PubMed

    Moens, L; Van Hauwaert, M L; Wolf, G

    1985-05-01

    The extracellular haemoglobins (Mr 260 000) of the brine shrimp Artemia sp. were cleaved by limited digestion with subtilisin. Structural units of Mr 16 000, which can bind dioxygen reversibly, were isolated. Analysis of the 16 000-Mr fraction (E) reveals the presence of a limited number of structural units. A single type of structural unit, E1 (Mr 15 800; pI4.8), was purified to homogeneity and characterized. PMID:4004806

  15. Erythrocyte morphology and haemoglobin types of neonatal roan antelopes (Hippotragus equinus) with hypochromic poikilocytic anaemia.

    PubMed

    Parsons, S D C; Penzhorn, B L; Reyers, F; Steyl, J C A; Becker, P J

    2006-01-01

    Neonatal, poikilocytic anaemia in some members of the Hippotragini has previously been documented but not fully investigated. This study was undertaken to describe the erythrocyte morphology of roan antelopes (Hippotragus equinus) during the first 4 weeks after birth and to identify aspects of haemoglobin (Hb) production that might be implicated in this syndrome. Twenty-nine roan antelope calves were sampled on, or close to, 1, 7, 14 and 28 days after birth. Erythrocyte morphology was characterized, and microhaematocrit values and Hb parameters determined, for each sampling occasion. Findings indicated a significant change in erythrocyte morphology during the neonatal period and two haemoglobin types, fetal and adult, were identified. The perinatal onset of adult Hb synthesis was delayed relative to the termination of fetal Hb production, resulting in the observed anaemia. Haemoglobin concentration and erythrocyte morphology were significantly correlated. These findings suggest an intimate relationship between Hb synthesis and the observed poikilocytosis. An imbalance in the synthesis of the alpha- and beta-globin chains of Hb (a thalassaemia) may prove to be the underlying pathophysiology of this syndrome.

  16. Structure of oxidized alpha-haemoglobin bound to AHSP reveals a protective mechanism for haem.

    PubMed

    Feng, Liang; Zhou, Suiping; Gu, Lichuan; Gell, David A; Mackay, Joel P; Weiss, Mitchell J; Gow, Andrew J; Shi, Yigong

    2005-06-01

    The synthesis of haemoglobin A (HbA) is exquisitely coordinated during erythrocyte development to prevent damaging effects from individual alpha- and beta-subunits. The alpha-haemoglobin-stabilizing protein (AHSP) binds alpha-haemoglobin (alphaHb), inhibits the ability of alphaHb to generate reactive oxygen species and prevents its precipitation on exposure to oxidant stress. The structure of AHSP bound to ferrous alphaHb is thought to represent a transitional complex through which alphaHb is converted to a non-reactive, hexacoordinate ferric form. Here we report the crystal structure of this ferric alphaHb-AHSP complex at 2.4 A resolution. Our findings reveal a striking bis-histidyl configuration in which both the proximal and the distal histidines coordinate the haem iron atom. To attain this unusual conformation, segments of alphaHb undergo drastic structural rearrangements, including the repositioning of several alpha-helices. Moreover, conversion to the ferric bis-histidine configuration strongly and specifically inhibits redox chemistry catalysis and haem loss from alphaHb. The observed structural changes, which impair the chemical reactivity of haem iron, explain how AHSP stabilizes alphaHb and prevents its damaging effects in cells.

  17. The effects of residual pump blood on patient plasma free haemoglobin levels post cardiac surgery.

    PubMed

    H, Schotola; Aj, Wetz; Af, Popov; I, Bergmann; Bc, Danner; Fa, Schöndube; M, Bauer; A, Bräuer

    2016-09-01

    At the end of cardiopulmonary bypass, there are invariably several hundred millilitres of residual pump blood in the reservoir, which can either be re-transfused or discarded. The objective of this prospective observational study was to investigate the quality of the residual pump blood, focusing on plasma free haemoglobin (pfHb) and blood cell counts. Fifty-one consecutive patients were included in the study. Forty-nine units of residual pump blood and 58 units of transfused red blood cell (RBC) concentrates were analysed. The mean preoperative pfHb of the patients was 0.057 ± 0.062 g/l, which increased gradually to 0.55 ± 0.36 g/l on arrival in the intensive care unit postoperatively. On the first postoperative day, the mean pfHb had returned to within the normal range. Our data showed that haemoglobin, haematocrit, and erythrocyte counts of residual pump blood were approximately 40% of the values in standardised RBC concentrates. Plasma free haemoglobin was significantly higher in residual pump blood compared to RBC concentrates, and nearly twice as high as the pfHb in patient blood samples taken contemporaneously. Our findings indicate that residual pump blood pfHb levels are markedly higher compared to patients' blood and RBC concentrates, but that its administration does not significantly increase patients' pfHb levels. PMID:27608341

  18. Capillary crystallization and molecular-replacement solution of haemoglobin II from the clam Lucina pectinata

    SciTech Connect

    Gavira, José A.; Jesus, Walleska de; Camara-Artigas, Ana; López-Garriga, Juan; García-Ruiz, Juan M.

    2006-03-01

    The haemoglobin II from the clam L. pectinata has been crystallized using counter-diffusion in single capillary in the presence of agarose to improve crystal quality. Initial phases have been obtained by molecular replacement. Haemoglobin II is one of three haemoglobins present in the cytoplasm of the Lucina pectinata mollusc that inhabits the Caribbean coast. Using HBII purified from its natural source, crystallization screening was performed using the counter-diffusion method with capillaries of 0.2 mm inner diameter. Crystals of HbII suitable for data collection and structure determination were grown in the presence of agarose at 0.1%(w/v) in order to improve their quality. The crystals belong to the tetragonal space group P4{sub 2}2{sub 1}2, with unit-cell parameters a = b = 73.92, c = 152.35 Å, and diffracted X-rays to a resolution of better than 2.0 Å. The asymmetric unit is a homodimer with a corresponding Matthews coefficient (V{sub M}) of 3.15 Å{sup 3} Da{sup −1} and a solvent content of 61% by volume.

  19. Structural basis for ligand and innate immunity factor uptake by the trypanosome haptoglobin-haemoglobin receptor.

    PubMed

    Lane-Serff, Harriet; MacGregor, Paula; Lowe, Edward D; Carrington, Mark; Higgins, Matthew K

    2014-12-12

    The haptoglobin-haemoglobin receptor (HpHbR) of African trypanosomes allows acquisition of haem and provides an uptake route for trypanolytic factor-1, a mediator of innate immunity against trypanosome infection. In this study, we report the structure of Trypanosoma brucei HpHbR in complex with human haptoglobin-haemoglobin (HpHb), revealing an elongated ligand-binding site that extends along its membrane distal half. This contacts haptoglobin and the β-subunit of haemoglobin, showing how the receptor selectively binds HpHb over individual components. Lateral mobility of the glycosylphosphatidylinositol-anchored HpHbR, and a ∼50° kink in the receptor, allows two receptors to simultaneously bind one HpHb dimer. Indeed, trypanosomes take up dimeric HpHb at significantly lower concentrations than monomeric HpHb, due to increased ligand avidity that comes from bivalent binding. The structure therefore reveals the molecular basis for ligand and innate immunity factor uptake by trypanosomes and identifies adaptations that allow efficient ligand uptake in the context of the complex trypanosome cell surface.

  20. Haemoglobin concentrations and infection by Giardia intestinalis in children: effect of treatment with secnidazole.

    PubMed

    Jiménez, J C; Rodríguez, N; Di Prisco, M C; Lynch, N R; Costa, V

    1999-12-01

    The blood concentrations of haemoglobin were investigated in 82 children aged 2-9 years. Fifty-seven (31 boys and 26 girls) were stool-positive for Giardia intestinalis but the other 25, used as controls, were negative. The mean (S.D.) haemoglobin concentration among the infected children was significantly lower pre-treatment than that for the control group [11.6 (1.2) v. 12.6 (1.5) g/dl; P < 0.05]. Treatment of the infected children with a single oral dose of secnidazole (30 mg/kg) led to a significant increase in their mean haemoglobin level 15 days later, from 11.6 (1.2) g/dl pre-treatment to 12.4 (1.2) g/dl post-treatment (P < 0.05). The results indicate that the therapeutic control of giardiasis could be important in programmes to combat anaemia in children living in endemic areas. PMID:10715676

  1. Structural basis for ligand and innate immunity factor uptake by the trypanosome haptoglobin-haemoglobin receptor

    PubMed Central

    Lane-Serff, Harriet; MacGregor, Paula; Lowe, Edward D; Carrington, Mark; Higgins, Matthew K

    2014-01-01

    The haptoglobin-haemoglobin receptor (HpHbR) of African trypanosomes allows acquisition of haem and provides an uptake route for trypanolytic factor-1, a mediator of innate immunity against trypanosome infection. In this study, we report the structure of Trypanosoma brucei HpHbR in complex with human haptoglobin-haemoglobin (HpHb), revealing an elongated ligand-binding site that extends along its membrane distal half. This contacts haptoglobin and the β-subunit of haemoglobin, showing how the receptor selectively binds HpHb over individual components. Lateral mobility of the glycosylphosphatidylinositol-anchored HpHbR, and a ∼50o kink in the receptor, allows two receptors to simultaneously bind one HpHb dimer. Indeed, trypanosomes take up dimeric HpHb at significantly lower concentrations than monomeric HpHb, due to increased ligand avidity that comes from bivalent binding. The structure therefore reveals the molecular basis for ligand and innate immunity factor uptake by trypanosomes and identifies adaptations that allow efficient ligand uptake in the context of the complex trypanosome cell surface. DOI: http://dx.doi.org/10.7554/eLife.05553.001 PMID:25497229

  2. Continuous monitoring of haemoglobin concentration after in-vivo adjustment in patients undergoing surgery with blood loss.

    PubMed

    Frasca, D; Mounios, H; Giraud, B; Boisson, M; Debaene, B; Mimoz, O

    2015-07-01

    Non-invasive monitoring of haemoglobin concentration provides real-time measurement of haemoglobin concentration (SpHb) using multi-wavelength pulse co-oximetry. We hypothesised that in-vivo adjustment using the mean of three haemoglobinometer (HemoCue®) measurements from an arterial blood sample at the first SpHb measurement (HCueART) would increase the accuracy of the monitor. The study included 41 adults for a total of 173 measurements of haemoglobin concentration. In-vivo adjusted SpHb was automatically calculated by the following formula: in-vivo adjusted SpHb = unadjusted SpHb - (SpHb - HCueART). The accuracy of in-vivo adjusted SpHb was compared with SpHb retrospectively adjusted using the same formula, except for haemoglobin level which was assessed at the central laboratory and then compared with all other available invasive methods of haemoglobin measurement (co-oximetry, HbSAT; arterial HemoCue, HCueART; capillary HemoCue, HCueCAP). Compared with laboratory measurement of haemoglobin concentration, bias (precision) for unadjusted SpHb, in-vivo adjusted SpHb, retrospectively adjusted SpHb, HbSAT, HCueART and HCueCAP were -0.4 (1.4), -0.3 (1.1), -0.3 (1.1), -0.6 (0.7), 0.0 (0.4) and -0.5 (1.2) g.dl(-1) , respectively. In-vivo adjustment of SpHb values using the mean of three arterial HemoCue measurements improved the accuracy of the device similar to those observed after a retrospective adjustment using central laboratory haemoglobin level. PMID:25676902

  3. Continuous monitoring of haemoglobin concentration after in-vivo adjustment in patients undergoing surgery with blood loss.

    PubMed

    Frasca, D; Mounios, H; Giraud, B; Boisson, M; Debaene, B; Mimoz, O

    2015-07-01

    Non-invasive monitoring of haemoglobin concentration provides real-time measurement of haemoglobin concentration (SpHb) using multi-wavelength pulse co-oximetry. We hypothesised that in-vivo adjustment using the mean of three haemoglobinometer (HemoCue®) measurements from an arterial blood sample at the first SpHb measurement (HCueART) would increase the accuracy of the monitor. The study included 41 adults for a total of 173 measurements of haemoglobin concentration. In-vivo adjusted SpHb was automatically calculated by the following formula: in-vivo adjusted SpHb = unadjusted SpHb - (SpHb - HCueART). The accuracy of in-vivo adjusted SpHb was compared with SpHb retrospectively adjusted using the same formula, except for haemoglobin level which was assessed at the central laboratory and then compared with all other available invasive methods of haemoglobin measurement (co-oximetry, HbSAT; arterial HemoCue, HCueART; capillary HemoCue, HCueCAP). Compared with laboratory measurement of haemoglobin concentration, bias (precision) for unadjusted SpHb, in-vivo adjusted SpHb, retrospectively adjusted SpHb, HbSAT, HCueART and HCueCAP were -0.4 (1.4), -0.3 (1.1), -0.3 (1.1), -0.6 (0.7), 0.0 (0.4) and -0.5 (1.2) g.dl(-1) , respectively. In-vivo adjustment of SpHb values using the mean of three arterial HemoCue measurements improved the accuracy of the device similar to those observed after a retrospective adjustment using central laboratory haemoglobin level.

  4. Using longitudinal data from the Health Survey for England to resolve discrepancies in thresholds for haemoglobin in older adults.

    PubMed

    Mindell, Jennifer; Moody, Alison; Ali, Ayesha; Hirani, Vasant

    2013-02-01

    Anaemia increases with age and is common among older people. Due to its relationship with morbidity and mortality, accurate diagnosis is important. Thresholds defining the diagnosis of anaemia have been the subject of considerable scientific debate, with both higher and lower cut-offs proposed. High haemoglobin is also a health risk in some but not all studies. Using nationally representative data of 5,329 adults aged 65 + years (Health Survey for England 1998, 2005, 2006), linked to administrative mortality data, this paper describes the relationship between haemoglobin levels and mortality, adjusted for age and other confounders. Among men, a reverse J shaped relationship was observed: relative to the modal group (140-149 g/l), those with 'mild anaemia' of 120-129 g/l haemoglobin had a 56% (95% confidence interval 24-96%) greater mortality hazard, and those with 'severe anaemia', haemoglobin <120 g/l, had an 87% (39-153%) greater hazard. At the other end of the range, those with haemoglobin ≥160 g/l had 32% (2-70%) greater mortality hazard. Haemoglobin levels in women showed a similar but smaller, non-significant pattern: hazard ratio 1·32 (0·91-1·92) for severe anaemia (<110 g/l), and 1·30 (0·95-1·79) for high haemoglobin (≥150 g/l). This research supports the use of the World Health Organization thresholds (130 g/l for men, 120 g/l for women).

  5. Molecular cloning and sequencing of the gene encoding the fimbrial subunit protein of Bacteroides gingivalis.

    PubMed Central

    Dickinson, D P; Kubiniec, M A; Yoshimura, F; Genco, R J

    1988-01-01

    The gene encoding the fimbrial subunit protein of Bacteroides gingivalis 381, fimbrilin, has been cloned and sequenced. The gene was present as a single copy on the bacterial chromosome, and the codon usage in the gene conformed closely to that expected for an abundant protein. The predicted size of the mature protein was 35,924 daltons, and the secretory form may have had a 10-amino-acid, hydrophilic leader sequence similar to the leader sequences of the MePhe fimbriae family. The protein sequence had no marked similarity to known fimbrial sequences, and no homologous sequences could be found in other black-pigmented Bacteroides species, suggesting that fimbrillin represents a class of fimbrial subunit protein of limited distribution. Images PMID:2895100

  6. Effect of environmental pH on enzyme activity and growth of Bacteroides gingivalis W50.

    PubMed Central

    McDermid, A S; McKee, A S; Marsh, P D

    1988-01-01

    Since the pH of the gingival crevice increases from below neutrality in health to above pH 8 in disease, we decided to investigate the effect of environmental pH on the growth and enzyme activity of Bacteroides gingivalis W50. Cells were grown in a chemostat under hemin-excess conditions over a range of pH values; stable growth was observed only between pH 6.7 and 8.3, with the maximum yields obtained between pH 7.0 and 8.0. The enzyme profile of cells varied markedly with pH. Enzymes with a specificity for gingival connective tissue (collagenase, hyaluronidase) were produced optimally at or below neutral pH, whereas trypsinlike activity increased with the growth pH and was maximal at pH 8.0. Chymotrypsinlike activity was generally low, although its activity was highest at the extremes of growth pH, i.e., at pH 6.7 and 8.3. Inhibitor studies provided evidence that the breakdown of collagen involved the concerted action of both a collagenase and the trypsinlike enzyme. The ratio of trypsin to collagenolytic activity rose from 1:1 during growth at neutral pH and below to almost 7:1 during growth at pH 8.3. Thus B. gingivalis appears to be uniquely adapted as a periodontopathic organism in that under environmental conditions likely to prevail during the initial stages of pocket development it produces maximally those enzymes with a tissue-damaging potential. Then, as the pH of the pocket rises during the host inflammatory response, the activity of the trypsinlike enzyme increases markedly, which may enable cells to inactivate key components of the host defenses such as immunoglobulins and complement. PMID:3281900

  7. Crystallization and diffraction patterns of the oxy and cyano forms of the Lucina pectinata haemoglobins complex

    PubMed Central

    Ruiz-Martínez, Carlos R.; Nieves-Marrero, Carlos A.; Estremera-Andújar, Rafael A.; Gavira, José A.; González-Ramírez, Luis A.; López-Garriga, Juan; García-Ruiz, Juan M.

    2009-01-01

    The native oxygen-carrier haemoglobins complex (HbII–III) is composed of haemoglobin II (HbII) and haemoglobin III (HbIII), which are found in the ctenidia tissue of the bivalve mollusc Lucina pectinata. This protein complex was isolated and purified from its natural source and crystallized using the vapour-diffusion and capillary counter-diffusion methods. Oxy and cyano derivatives of the complex crystallized using several conditions, but the best crystals in terms of quality and size were obtained from sodium formate pH 5 using the counter-diffusion method in a single capillary. Crystals of the oxy and cyano complexes, which showed a ruby-red colour and nonsingular prismatic shapes, scattered X-­rays to resolution limits of 2.15 and 2.20 Å, respectively, using a 0.886 Å synchrotron-radiation source. The crystals belonged to the tetragonal system, space group P42212, with unit-cell parameters a = b = 74.07, c = 152.07 and a = b = 73.83, c = 152.49 Å for the oxy and cyano complexes, respectively. The asymmetric unit of both crystals is composed of a single copy of the heterodimer, with Matthew coefficients (V M) of 3.08 and 3.06 Å3 Da−1 for the oxy and cyano complexes, respectively, which correspond to a solvent content of approximately 60.0% by volume. PMID:19153450

  8. [A double immunochemical method for detecting faecal haemoglobin and albumin in rectal screening].

    PubMed

    Tarpay, Adám; Szabadosné Németh, Mária; Orosz, Enikõ; Kásler, Miklós; Burai, Mária; Pap, Akos; Ottó, Szabolcs

    2011-11-01

    Undoubtedly, colonoscopy is the "gold standard" in the diagnosis of colorectal cancers. Sophisticated bowel preparation and risk of bowel perforation and bleeding, as well as the patient's discomfort during examination lead to low compliance in screening. Therefore, alternative non-invasive screening methods tend to come into the fore. In this study we compared the sensitivity and specificity of the double immunochemical FECA test for the haemoglobin + albumin content of the faeces with those of control colonoscopy in the detection of colorectal neoplasms. In a 3-year period 154 patients (69 males and 85 females) were scheduled for colonoscopy with previously collected stool samples. The sensitivity and specificity of the double immunochemical test for faecal haemoglobin + albumin content were determined in colorectal neoplasms of different severity. Colonoscopy served as a control examination. Colonoscopy identified in 77 cases benign lesions, and in 10 cases malignant tumours. The double immunochemical test for faecal blood and protein successfully used in model screening population showed in our present study 52.7% sensitivity and 92.3% specificity for significant neoplastic lesions (high-risk polyps and tumours). When the evaluation was limited to the high-risk polyps, the sensitivity was modified to 45.5% and the specificity to 92.3% and in case of invasive tumours to 90% and 100%, respectively. If only faecal haemoglobin content was measured, the overall sensitivity for polyps of any size and sort was 15.7% which, however, increased to 27.63% if faecal albumin was also measured. Based on relevant literature, the sensitivity of the FECA test for colorectal polyp and cancer is more favourable than that of other FITs. However, the increased sensitivity of the double faecal protein test falls short of the standard colonoscopy. Therefore, in certain cases the latter might be considered as a primary screening method.

  9. The structure of Artemia sp. (brine shrimp) haemoglobins. Purification of a structural unit to homogeneity.

    PubMed Central

    Moens, L; Van Hauwaert, M L; Wolf, G

    1985-01-01

    The extracellular haemoglobins (Mr 260 000) of the brine shrimp Artemia sp. were cleaved by limited digestion with subtilisin. Structural units of Mr 16 000, which can bind dioxygen reversibly, were isolated. Analysis of the 16 000-Mr fraction (E) reveals the presence of a limited number of structural units. A single type of structural unit, E1 (Mr 15 800; pI4.8), was purified to homogeneity and characterized. Images Fig. 1. Fig. 3. Fig. 4. Fig. 5. PMID:4004806

  10. New developments in anti-sickling agents: can drugs directly prevent the polymerization of sickle haemoglobin in vivo?

    PubMed

    Oder, Esther; Safo, Martin K; Abdulmalik, Osheiza; Kato, Gregory J

    2016-10-01

    The hallmark of sickle cell disease is the polymerization of sickle haemoglobin due to a point mutation in the β-globin gene (HBB). Under low oxygen saturation, sickle haemoglobin assumes the tense (T-state) deoxygenated conformation that can form polymers, leading to rigid erythrocytes with impaired blood vessel transit, compounded or initiated by adhesion of erythrocytes to endothelium, neutrophils and platelets. This process results in vessel occlusion and ischaemia, with consequent acute pain, chronic organ damage, morbidity and mortality. Pharmacological agents that stabilize the higher oxygen affinity relaxed state (R-state) and/or destabilize the lower oxygen affinity T-state of haemoglobin have the potential to delay the sickling of circulating red cells by slowing polymerization kinetics. Relevant classes of agents include aromatic aldehydes, thiol derivatives, isothiocyanates and acyl salicylates derivatives. The aromatic aldehyde, 5-hydroxymethylfurfural (5-HMF) increases oxygen affinity of sickle haemoglobin and reduces hypoxia-induced sickling in vitro and protects sickle cell mice from effects of hypoxia. It has completed pre-clinical testing and has entered clinical trials as treatment for sickle cell disease. A related molecule, GBT440, has shown R-state stabilization and increased oxygen affinity in preclinical testing. Allosteric modifiers of haemoglobin as direct anti-sickling agents target the fundamental pathophysiological mechanism of sickle cell disease. PMID:27605087

  11. Clinical evaluation of a novel technology for non-invasive and continuous measurement of plasma haemoglobin concentration.

    PubMed

    Broderick, A J; Desmond, F; Leen, G; Shorten, G

    2015-10-01

    We undertook the first clinical evaluation of a novel, non-invasive device for the continuous measurement of plasma haemoglobin concentration in 25 patients undergoing elective cardiac surgery. At four pre-determined intervals, samples of blood were taken for plasma haemoglobin estimation on a blood gas analyser and a laboratory device and were compared with the plasma haemoglobin estimation on the novel device using the Bland-Altman method. The 95% limits of agreement for estimation of plasma haemoglobin concentration for the device vs. laboratory, the device vs. the blood gas analyser and the blood gas analyser vs. the laboratory were 101.3 g.l(-1) , 103.1 g.l(-1) and 14.5 g.l(-1) , respectively. The bias (mean difference) in each case was 27.4 g.l(-1) , 25.1 g.l(-1) and 2.4 g.l(-1) , respectively. We conclude that the novel device in its current form is not a suitable replacement for more invasive methods of determining plasma haemoglobin concentration in patients in the setting of cardiac surgery; however, lessons learnt from the study will help to improve the device's future performance.

  12. New developments in anti-sickling agents: can drugs directly prevent the polymerization of sickle haemoglobin in vivo?

    PubMed

    Oder, Esther; Safo, Martin K; Abdulmalik, Osheiza; Kato, Gregory J

    2016-10-01

    The hallmark of sickle cell disease is the polymerization of sickle haemoglobin due to a point mutation in the β-globin gene (HBB). Under low oxygen saturation, sickle haemoglobin assumes the tense (T-state) deoxygenated conformation that can form polymers, leading to rigid erythrocytes with impaired blood vessel transit, compounded or initiated by adhesion of erythrocytes to endothelium, neutrophils and platelets. This process results in vessel occlusion and ischaemia, with consequent acute pain, chronic organ damage, morbidity and mortality. Pharmacological agents that stabilize the higher oxygen affinity relaxed state (R-state) and/or destabilize the lower oxygen affinity T-state of haemoglobin have the potential to delay the sickling of circulating red cells by slowing polymerization kinetics. Relevant classes of agents include aromatic aldehydes, thiol derivatives, isothiocyanates and acyl salicylates derivatives. The aromatic aldehyde, 5-hydroxymethylfurfural (5-HMF) increases oxygen affinity of sickle haemoglobin and reduces hypoxia-induced sickling in vitro and protects sickle cell mice from effects of hypoxia. It has completed pre-clinical testing and has entered clinical trials as treatment for sickle cell disease. A related molecule, GBT440, has shown R-state stabilization and increased oxygen affinity in preclinical testing. Allosteric modifiers of haemoglobin as direct anti-sickling agents target the fundamental pathophysiological mechanism of sickle cell disease.

  13. Haemoglobin synthesis in K562 erythroleukaemia cells is affected by intimate contact with monolayers of various human cell types.

    PubMed

    Zuhrie, S R; Pearson, J D; Wickramasinghe, S N

    1988-01-01

    The haemoglobin content of K562 erythroleukaemia cells was affected by co-culture over monolayers of various human cell types. Haemoglobin synthesis was increased after co-culture with umbilical-cord-derived endothelial cells and most monolayers of bone-marrow-derived macrophages, and inhibited after co-culture with two fibroblast lines, blood-monocyte-derived macrophages, a neuroglial cell line (U-251 MG) and most monolayers of bone-marrow-derived stromal cells. These effects were modified when a thin layer of agar was placed over the monolayers. Cell-free culture media conditioned by all but two of the seven types of monolayer studied inhibited haemoglobin synthesis by K562 cells; those conditioned by blood-monocyte-derived macrophages and two of 11 monolayers of bone-marrow-derived macrophages stimulated haemoglobin synthesis. Thus, the haemoglobin content of K562 cells appeared to be influenced both by intimate contact between K562 cells and the cells of the monolayers and by humoral factors released by the monolayers. The data support the concept that erythroid differentiation is partly dependent on intimate contact between erythroid progenitor cells and microenvironmental cells.

  14. Purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies of great cormorant (Phalacrocorax carbo) haemoglobin.

    PubMed

    Jagadeesan, G; Malathy, P; Gunasekaran, K; Harikrishna Etti, S; Aravindhan, S

    2014-11-01

    Haemoglobin is the iron-containing oxygen-transport metalloprotein that is present in the red blood cells of all vertebrates. In recent decades, there has been substantial interest in attempting to understand the structural basis and functional diversity of avian haemoglobins. Towards this end, purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies have been carried out on cormorant (Phalacrocorax carbo) haemoglobin. Crystals were grown by the hanging-drop vapour-diffusion method using PEG 3350, NaCl and glycerol as precipitants. The crystals belonged to the trigonal system P3₁21, with unit-cell parameters a=b=55.64, c=153.38 Å, β=120.00°; a complete data set was collected to a resolution of 3.5 Å. Matthews coefficient analysis indicated that the crystals contained a half-tetramer in the asymmetric unit. PMID:25372822

  15. Adaptive phylogeography: functional divergence between haemoglobins derived from different glacial refugia in the bank vole

    PubMed Central

    Kotlík, Petr; Marková, Silvia; Vojtek, Libor; Stratil, Antonín; Šlechta, Vlastimil; Hyršl, Pavel; Searle, Jeremy B.

    2014-01-01

    Over the years, researchers have used presumptively neutral molecular variation to infer the origins of current species' distributions in northern latitudes (especially Europe). However, several reported examples of genic and chromosomal replacements suggest that end-glacial colonizations of particular northern areas may have involved genetic input from different source populations at different times, coupled with competition and selection. We investigate the functional consequences of differences between two bank vole (Clethrionomys glareolus) haemoglobins deriving from different glacial refugia, one of which partially replaced the other in Britain during end-glacial climate warming. This allows us to examine their adaptive divergence and hence a possible role of selection in the replacement. We determine the amino acid substitution Ser52Cys in the major expressed β-globin gene as the allelic difference. We use structural modelling to reveal that the protein environment renders the 52Cys thiol a highly reactive functional group and we show its reactivity in vitro. We demonstrate that possessing the reactive thiol in haemoglobin increases the resistance of bank vole erythrocytes to oxidative stress. Our study thus provides striking evidence for physiological differences between products of genic variants that spread at the expense of one another during colonization of an area from different glacial refugia. PMID:24827438

  16. Antioxidant status in haemoglobin E carriers after acute and chronic strenuous exercises.

    PubMed

    Palasuwan, Attakorn; Soogarun, Suphan; Suksom, Daroonwan; Pitaksathienkul, Chatchadaporn; Rousseau, Anne-Sophie

    2015-01-01

    Haemoglobin E (HbE), an unstable haemoglobin, is highly susceptible to oxidative damages. We examined how acute or chronic physiological challenge induced by exercise affects antioxidant response in HbE carriers. Two independent studies were conducted in individuals with HbE trait and paired normal Hb. In study 1, sedentary participants were tested in a graded maximal exercise and blood samples were collected before, immediately after, and 45 minutes after an acute exercise. Our data showed that erythrocyte glutathione peroxidase (GPx) activity failed to recover in HbE carriers after 45 minutes of rest. In study 2, athletes were trained in a 10-week strenuous training and blood samples were collected before and after training period. We found that athletes with HbE carriers showed a larger increase in plasma GPx activity compared to those with normal Hb. These data suggest that HbE carriers could cope with exercise-induced oxidative stress by adjusting endogenous antioxidant markers.

  17. Adaptive phylogeography: functional divergence between haemoglobins derived from different glacial refugia in the bank vole.

    PubMed

    Kotlík, Petr; Marková, Silvia; Vojtek, Libor; Stratil, Antonín; Slechta, Vlastimil; Hyršl, Pavel; Searle, Jeremy B

    2014-07-01

    Over the years, researchers have used presumptively neutral molecular variation to infer the origins of current species' distributions in northern latitudes (especially Europe). However, several reported examples of genic and chromosomal replacements suggest that end-glacial colonizations of particular northern areas may have involved genetic input from different source populations at different times, coupled with competition and selection. We investigate the functional consequences of differences between two bank vole (Clethrionomys glareolus) haemoglobins deriving from different glacial refugia, one of which partially replaced the other in Britain during end-glacial climate warming. This allows us to examine their adaptive divergence and hence a possible role of selection in the replacement. We determine the amino acid substitution Ser52Cys in the major expressed β-globin gene as the allelic difference. We use structural modelling to reveal that the protein environment renders the 52Cys thiol a highly reactive functional group and we show its reactivity in vitro. We demonstrate that possessing the reactive thiol in haemoglobin increases the resistance of bank vole erythrocytes to oxidative stress. Our study thus provides striking evidence for physiological differences between products of genic variants that spread at the expense of one another during colonization of an area from different glacial refugia.

  18. Haem-binding-site heterogeneity and haem Cotton effects of Glycera dibranchiata monomeric haemoglobins.

    PubMed Central

    DiFeo, T J; Addison, A W

    1989-01-01

    The five major components of the monomeric haemoglobin from Glycera dibranchiata were separated and characterized by absorption spectroscopy, isoelectric focusing, azide-binding affinities and nitrosyl autoreduction kinetics. The differences found among the components are discussed in terms of haem-pocket variations. In addition, the Fourier-transform i.r. spectra of pooled monomeric haemoglobin carbonyl (HbmCO) and the major component carbonyl are reported. The c.d. spectra of the carbonyl and azide derivatives of the five components are compared and found to be similar. The c.d. spectra of myoglobin(II) carbonyl [Mb(II)CO] and of apomyoglobin (apoMb) reconstituted with a symmetric synthetic iron porphyrin carbonyl, meso-tetra-(p-carboxyphenyl)porphinatoiron(II) carbonyl [TCPPFe(II)CO], are compared with the c.d. spectra of pooled HbmCO and its TCPPFe(II)CO analogue. HbmTCPPFe(II)CO shows a negative Soret c.d. band whereas MbTCPPFe(II)CO produces both a negative and a positive Soret c.d. band. Displacement of the symmetric porphyrin by 8-anilinonaphthalene-1-sulphonate and the resulting fluorescence emission are reported. PMID:2764907

  19. Effect of heavy metal exposure on blood haemoglobin concentration and methemoglobin percentage in Lumbricus terrestris.

    PubMed

    Calisi, A; Lionetto, M G; Sanchez-Hernandez, J C; Schettino, T

    2011-06-01

    The earthworm haemoglobin (Hb) is a large extracellular hemoprotein flowing in a closed circulatory system. In spite of the fundamental role of this respiratory pigment in earthworm physiology, little is known about its sensitivity to environmental pollutants. The aim of the present work was to investigate the possible effect of heavy metal (cadmium, copper, mercury) exposure on Hb concentration and oxidation state (methemoglobin formation) in the earthworm Lumbricus terrestris. In addition, the tissue concentration of metallothioneins, a well-known biomarker of heavy metal exposure, was determined as an indicator of metal uptake. The animals were exposed to increasing concentrations of Cd, Cu and Hg utilizing the standard acute toxicity test, "Filter paper test" for 48 h. Exposure to heavy metals (10(-5)-10(-3) M for Cd, 10(-4)-10(-3) M for Hg, and 10(-4)-10(-2) M for Cu) was found to increase haemoglobin concentration in L. terrestris, although the magnitude of such an increase was dependent on the metal. In addition, metal exposure led to the formation of methemoglobin. Compared to other known biological responses to heavy metals, such as metallothionein induction, methemoglobin increase showed a higher sensitivity and a higher percentage variation in exposed organisms, showing to be a possible suitable biomarker of exposure/effect to be included in a multi biomarker strategy in earthworm in soil monitoring assessment. PMID:21424722

  20. A topical haemoglobin spray for oxygenating pressure ulcers: a pilot study.

    PubMed

    Tickle, Joy

    2015-03-01

    The effect of pressure ulcers on patient quality of life have been recognised as a real problem for many years, and the need for robust and effective management of pressure ulcers is now a prominent national health-care issue. Myriad different interventions exist for the treatment of pressure ulcers, including clinically effective dressings and pressure-relieving devices, yet many pressure ulcers still do not heal and often become a chronic wound. This is the second of a series of articles (Norris, 2014) discussing the clinical evaluation of a topical oxygen therapy in practice. It describes a small evaluation involving 18 patients with pressure ulcers. The study set out to determine the effect of a topical oxygen therapy on wound size. The therapy comprises a canister that sprays pure haemoglobin in a water solution into or onto the wound. The haemoglobin spray needs to be used at least once every 3 days, does not require training on its use and can be used in any care setting. Overall, results identified wound healing progression in all 18 wounds and wound size reduction in 17 of the 18 wounds.

  1. Haemoglobin degradation underpins the sensitivity of early ring stage Plasmodium falciparum to artemisinins

    PubMed Central

    Xie, Stanley C.; Dogovski, Con; Hanssen, Eric; Chiu, Francis; Yang, Tuo; Crespo, Maria P.; Stafford, Che; Batinovic, Steven; Teguh, Silvia; Charman, Susan; Klonis, Nectarios; Tilley, Leann

    2016-01-01

    ABSTRACT Current first-line artemisinin antimalarials are threatened by the emergence of resistant Plasmodium falciparum. Decreased sensitivity is evident in the initial (early ring) stage of intraerythrocytic development, meaning that it is crucial to understand the action of artemisinins at this stage. Here, we examined the roles of iron (Fe) ions and haem in artemisinin activation in early rings using Fe ion chelators and a specific haemoglobinase inhibitor (E64d). Quantitative modelling of the antagonism accounted for its complex dependence on the chemical features of the artemisinins and on the drug exposure time, and showed that almost all artemisinin activity in early rings (>80%) is due to haem-mediated activation. The surprising implication that haemoglobin uptake and digestion is active in early rings is supported by identification of active haemoglobinases (falcipains) at this stage. Genetic down-modulation of the expression of the two main cysteine protease haemoglobinases, falcipains 2 and 3, renders early ring stage parasites resistant to artemisinins. This confirms the important role of haemoglobin-degrading falcipains in artemisinin activation, and shows that changes in the rate of artemisinin activation could mediate high-level artemisinin resistance. PMID:26675237

  2. Haemoglobin fortified cereal: a source of available iron to breast-fed infants.

    PubMed

    Hertrampf, E; Olivares, M; Pizarro, F; Walter, T; Cayazzo, M; Heresi, G; Llaguno, S; Chadud, P; Stekel, A

    1990-11-01

    We tested in the field an extruded rice flour, fortified with a bovine haemoglobin concentrate (Fe:14 mg/100 g of powder). This cereal has a high iron bioavailability, good protein quality and amino acid score. Healthy, term breast-fed infants were prospectively studied. One group (n = 92) received the fortified cereal (from 4 to 12 months of age). As control, 96 infants received regular solid foods (cooked vegetables and meat) from age 4 months. At the end of the field trial, a subsample of infants in both groups was supplemented with 45 mg Fe during 90 d. Iron nutrition status was determined at 9, 12 and 15 months. At 12 months, iron deficiency anaemia was present in 17 per cent of controls, in 10 per cent of fortified infants as a whole, but only in 6 per cent of the babies who consumed over 30 g of cereal/d. In addition, this latter group did not show any significant changes in iron nutrition status after the supplementation trial. Results demonstrate that the consumption of a haemoglobin fortified cereal is effective in markedly reducing the incidence of iron deficiency in breast-fed infants. PMID:2086208

  3. Haemoglobin adducts as biomarkers of occupational exposure to 1,3-butadiene.

    PubMed

    Osterman-Golkar, S; Peltonen, K; Anttinen-Klemetti, T; Landin, H H; Zorcec, V; Sorsa, M

    1996-03-01

    Adducts of 1,2-epoxy-3-butene (EB) with haemoglobin were monitored in 17 workers from the 1,3-butadiene (BD) production unit of a petrochemical plant and in nine referents employed at the same factory but not exposed to BD. The air concentrations of BD were determined using stationary and personal monitoring. The ambient level of exposure of the plant workers handling butadiene containers (sampling and voiding) was on average 11.2 +/- 18.6 (mean +/- SD) mg/m3. Maintenance and laboratory workers were exposed to levels < or = 1.2 mg/m3. The particular haemoglobin adduct measured was 2-hydroxy-3-butenylvaline, formed by reaction of N-terminal valine with carbon 1 in EB. The adduct levels were increased (0.16 +/- 0.099 pmol/g; n = 10) in plant workers compared with the levels in maintenance and laboratory workers and controls (approximately 0.05 pmol/g; seven laboratory workers and nine controls evaluated). Thus, the method used for adduct determination--derivatization of 200-300 mg globin samples with penta-fluorophenyl isothiocyanate according to the N-alkyl Edman method and detection of the thiohydantoin derivatives by tandem mass spectrometry--is sufficiently sensitive to allow monitoring of exposure to BD down to the p.p.m. level. PMID:8671730

  4. A topical haemoglobin spray for oxygenating chronic venous leg ulcers: a pilot study.

    PubMed

    Norris, Ray

    2014-11-01

    Acute wounds will generally heal independently of any interventions, whereas chronic wounds are chronic for a reason and are unlikely to successfully heal without intervention. In the treatment of venous leg ulcers, the gold standard will always be compression therapy. However, many wounds still do not heal despite best practice. Therefore, the use of adjunct therapies alongside standard care become the priority for healing. This article describes a small evaluation, involving 17 patients with chronic venous leg ulcers, that set out to determine the effect of a topical oxygen therapy on wound size. The therapy comprises a canister that sprays pure haemoglobin in a water solution into the wound. The haemoglobin spray needs to be used at least once every 3 days, and no training is required on its use. Results showed the device helped promote wound healing in 14 out of 17 wounds treated for more than 2 weeks. These patients had previously been shown to be non-healing during a 4-week run-in period where they received standard care, including compression therapy.

  5. From tissue iron retention to low systemic haemoglobin levels, new pathophysiological biomarkers of human abdominal aortic aneurysm.

    PubMed

    Martinez-Pinna, R; Lindholt, J S; Madrigal-Matute, J; Blanco-Colio, L M; Esteban-Salan, M; Torres-Fonseca, M M; Lefebvre, T; Delbosc, S; Laustsen, J; Driss, F; Vega de Ceniga, M; Gouya, L; Weiss, G; Egido, J; Meilhac, O; Michel, J-B; Martin-Ventura, J

    2014-07-01

    Iron deposits are observed in tissue of abdominal aortic aneurysm (AAA) patients, although the underlying mechanisms are not completely elucidated. Therefore we explored circulating markers of iron metabolism in AAA patients, and tested if they could serve as biomarkers of AAA. Increased red blood cell (RBC)-borne iron retention and transferrin, transferrin receptor and ferritin expression was observed in AAA tissue compared to control aorta (immunohistochemistry and western blot). In contrast, decreased circulating iron, transferrin, mean corpuscular haemoglobin concentration (MCHC) and haemoglobin concentration, along with circulating RBC count, were observed in AAA patients (aortic diameter >3 cm, n=114) compared to controls (aortic diameter <3 cm, n=88) (ELISA), whereas hepcidin concentrations were increased in AAA subjects (MS/MS assay). Moreover, iron, transferrin and haemoglobin levels were negatively, and hepcidin positively, correlated with aortic diameter in AAA patients. The association of low haemoglobin with AAA presence or aortic diameter was independent of specific risk factors. Moreover, MCHC negatively correlated with thrombus area in another cohort of AAA patients (aortic diameter 3-5 cm, n=357). We found that anaemia was significantly more prevalent in AAA patients (aortic diameter >5 cm, n=8,912) compared to those in patients with atherosclerotic aorto-iliac occlusive disease (n=17,737) [adjusted odds ratio=1.77 (95% confidence interval: 1.61;1.93)]. Finally, the mortality risk among AAA patients with anaemia was increased by almost 30% [adjusted hazard ratio: 1.29 (95% confidence interval: 1.16;1.44)] as compared to AAA subjects without anaemia. In conclusion, local iron retention and altered iron recycling associated to high hepcidin and low transferrin systemic concentrations could lead to reduced circulating haemoglobin levels in AAA patients. Low haemoglobin levels are independently associated to AAA presence and clinical outcome.

  6. Haplotype Analyses of Haemoglobin C and Haemoglobin S and the Dynamics of the Evolutionary Response to Malaria in Kassena-Nankana District of Ghana

    PubMed Central

    Ghansah, Anita; Rockett, Kirk A.; Clark, Taane G.; Wilson, Michael D.; Koram, Kwadwo A.; Oduro, Abraham R.; Amenga-Etego, Lucas; Anyorigiya, Thomas; Hodgson, Abraham; Milligan, Paul; Rogers, William O.; Kwiatkowski, Dominic P.

    2012-01-01

    Background Haemoglobin S (HbS) and C (HbC) are variants of the HBB gene which both protect against malaria. It is not clear, however, how these two alleles have evolved in the West African countries where they co-exist at high frequencies. Here we use haplotypic signatures of selection to investigate the evolutionary history of the malaria-protective alleles HbS and HbC in the Kassena-Nankana District (KND) of Ghana. Methodology/Principal Findings The haplotypic structure of HbS and HbC alleles was investigated, by genotyping 56 SNPs around the HBB locus. We found that, in the KND population, both alleles reside on extended haplotypes (approximately 1.5 Mb for HbS and 650 Kb for HbC) that are significantly less diverse than those of the ancestral HbA allele. The extended haplotypes span a recombination hotspot that is known to exist in this region of the genome Significance Our findings show strong support for recent positive selection of both the HbS and HbC alleles and provide insights into how these two alleles have both evolved in the population of northern Ghana. PMID:22506028

  7. Serendipity: A Rare Discovery of Haemoglobin D-Iran in An Indian Female During Routine Antenatal Screening for β-Thalassemia.

    PubMed

    Chandel, Rittu Surjit; Roy, Abhishek; Abichandani, Leela Gul

    2015-07-01

    Haemoglobin D is a rare form of haemoglobinopathy in homozygous form. However, the heterozygous form of the disease is clinically silent and relatively easier to find in North-West India, Pakistan and Iran. Haemoglobin D is sometimes found to be coexistent with Haemoglobin S and/or Thalassaemia leading to clinically significant conditions like sickle cell anaemia with mild to moderate splenomegaly. In India the more prevalent form is Haemoglobin D-Punjab (also known as Hb D- Los Angeles) which has a prevalence of 2% in Punjab and around 1% in Gujarat. However, the variant, Haemoglobin D- Iran is very rare in India in heterozygous as well as homozygous forms. This report is of a 36-year-old female, who visited for an antenatal check up. On analysing the blood sample using Agarose Gel Electrophoresis in Alkaline media, the migration of abnormal haemoglobin to haemoglobin S/D/G region was observed. Sickle cell solubility test was negative. On capillary electrophoresis, peak in the Haemoglobin D Zone was seen. PMID:26393119

  8. Nucleotide sequence of the SrRNA gene of Entamoeba gingivalis: applications for construction of a species-specific DNA probe and phylogenetic analysis.

    PubMed

    Yamamoto, A; Kikuta, N; Hashimoto, T; Oyaizu, H; Goto, N

    1995-01-01

    The small subunit ribosomal RNA (SrRNA) gene of Entamoeba gingivalis was amplified by PCR and the product of 1.9-kbp sequence was cloned into a plasmid vector pUC18. Four clones were isolated and sequenced. The insert DNAs were 1918- to 1921-bp long and A+T rich (65.5%). The four SrRNA sequences of E. gingivalis were found to be aligned with those of nine related protozoans while searching for E. gingivalis-specific sequences. A sequence of 28 oligonucleotides was chosen, chemically synthesized, and labeled with digoxigenin for use as a DNA probe. The probe thus constructed was shown to hybridize only with either the SrRNA-coding DNAs or the cells of the two E. gingivalis strains and not with those of other protozons or oral fungi tested. A representative SrRNA-sequence was analyzed and a phylogenetic tree was constructed by the neighbor-joining (NJ) method. Among the protists examined, E. gingivalis was placed next to Entamoeba histolytica as expected from the traditional taxonomy.

  9. Paradoxical correlation between signal in functional magnetic resonance imaging and deoxygenated haemoglobin content in capillaries: a new theoretical explanation

    NASA Astrophysics Data System (ADS)

    Yamamoto, Toru; Kato, Toshinori

    2002-04-01

    Signal increases in functional magnetic resonance imaging (fMRI) are believed to be a result of decreased paramagnetic deoxygenated haemoglobin (deoxyHb) content in the neural activation area. However, discrepancies in this canonical blood oxygenation level dependent (BOLD) theory have been pointed out in studies using optical techniques, which directly measure haemoglobin changes. To explain the discrepancies, we developed a new theory bridging magnetic resonance (MR) signal and haemoglobin changes. We focused on capillary influences, which have been neglected in most previous fMRI studies and performed a combined fMRI and near-infrared spectroscopy (NIRS) study using a language task. Paradoxically, both the MR signal and deoxyHb content increased in Broca's area. On the other hand, fMRI activation in the auditory area near large veins correlated with a mirror-image decrease in deoxyHb and increase in oxygenated haemoglobin (oxyHb), in agreement with canonical BOLD theory. All fMRI signal changes correlated consistently with changes in oxyHb, the diamagnetism of which is insensitive to MR. We concluded that the discrepancy with the canonical BOLD theory is caused by the fact that the BOLD theory ignores the effect of the capillaries. Our theory explains the paradoxical phenomena of the oxyHb and deoxyHb contributions to the MR signal and gives a new insight into the precise haemodynamics of activation by analysing fMRI and NIRS data.

  10. Microwaving Blood as a Non-Destructive Technique for Haemoglobin Measurements on Microlitre Samples

    PubMed Central

    Basey-Fisher, Toby H.; Guerra, Nadia; Triulzi, Chiara; Gregory, Andrew; Hanham, Stephen M.; Stevens, Molly M.; Maier, Stefan A.; Klein, Norbert

    2016-01-01

    The non-destructive ex vivo determination of haemoglobin (Hgb) concentration offers the capability to conduct multiple red blood cell haematological measurements on a single sample, an advantage that current optical techniques are unable to offer. Here, a microwave method and device for the accurate and non-destructive determination of Hgb concentration in microlitre blood samples are described. Using broadband microwave spectroscopy, a relationship is established between the dielectric properties of murine blood and Hgb concentration that is utilized to create a technique for the determination of Hgb concentration. Subsequently, a microwave dielectric resonator-microfluidic system is implemented in the analysis of 52 murine samples with microlitre volumes and Hgb concentrations ranging from 0 to 17 g dL−1. Using the characterized relationship, independent and minimally invasive Hgb measurements are made on nine healthy mice as well as seven with mutations in the Adenomatous polyposis coli (APC) gene that leads to colorectal cancer and consequently anaemia. PMID:24002989

  11. Comparison of erythrocyte antioxidative enzyme activities between two types of haemoglobin H disease.

    PubMed Central

    Prasartkaew, S; Bunyaratvej, A; Fucharoen, S; Wasi, P

    1986-01-01

    The activities of erythrocyte antioxidative enzymes were measured in two groups of patients with different genotypes of haemoglobin (Hb) H disease: 21 with alpha-thalassaemia 1 or alpha-thalassaemia 2 (alpha-thalassaemia 1/2) and 21 with alpha-thalassaemia 1/Hb Constant Spring (HbCS). They were compared with 21 normal subjects. Both genotypes of Hb H disease had increased activities of erythrocyte superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase when compared with those of controls. Comparison of the two genotypes showed that subjects with alpha-thalassaemia 1/Hb CS, the more severe disease, had higher SOD and GSH-Px activities but lower catalase activity than those with alpha-thalassaemia 1/2. This indicates that there are compensatory mechanisms in Hb H erythrocytes to cope with increased generation of oxygen free radicals as a result of increased excess beta chain. PMID:3805316

  12. Fetal haemoglobin production and the sickle gene in the oases of Eastern Saudi Arabia.

    PubMed

    Pembrey, M E; Wood, W G; Weatherall, D J; Perrine, R P

    1978-11-01

    Fetal haemoglobin (HbF) levels have been measured in 137 normal (AA) subjects, 109 with the sickle-cell trait (AS) and 237 with sickle-cell anaemia (SS) from the oasis population of Eastern Saudi Arabia. In addition the proportion of F-cells has been estimated in 71 AA, 51 AS and 34 SS subjects. The mean HbF% (and the range of F-cells %) were: AA 0.77 (0.3--18), AS 1.38 (2.3--43) and SS 25.56 (33--98). The distribution of Hb F was always heterocellular. The influence of pregnancy accounts for most of the excess female subjects with sickle-cell trait showing raised Hb F and F-cells. Whilst the normal Arabs and those with sickle-cell trait did not differ from comparable groups of American blacks, both the % Hb F and % F-cells in Saudi Arabian patients with sickle-cell anaemia were much higher than in Blacks. The high Hb F levels in individuals with sickle-cell anaemia are not due to coexistent glucose-6-phosphate dehydrogenase deficiency or alpha-thalassaemia trait, and the Hb F level showed an inverse correlation with the degree of haemolysis. These findings indicate that the unusually elevated levels of Hb F are not due to an associated high frequency of a gene for hetero-cellular hereditary persistence of fetal haemoglobin in the oasis population, but rather from a genetically determined absolute increase in Hb F production related in some way to the SS genotype.

  13. A mitochondrial location for haemoglobins--dynamic distribution in ageing and Parkinson's disease.

    PubMed

    Shephard, Freya; Greville-Heygate, Oliver; Marsh, Oliver; Anderson, Susan; Chakrabarti, Lisa

    2014-01-01

    Haemoglobins are iron-containing proteins that transport oxygen in the blood of most vertebrates. The mitochondrion is the cellular organelle which consumes oxygen in order to synthesise ATP. Mitochondrial dysfunction is implicated in neurodegeneration and ageing. We find that α and β haemoglobin (Hba and Hbb) proteins are altered in their distribution in mitochondrial fractions from degenerating brain. We demonstrate that both Hba and Hbb are co-localised with the mitochondrion in mammalian brain. The precise localisation of the Hbs is within the inner membrane space and associated with inner mitochondrial membrane. Relative mitochondrial to cytoplasmic ratios of Hba and Hbb show changing distributions of these proteins during the process of neurodegeneration in the pcd(5j) mouse brain. A significant difference in mitochondrial Hba and Hbb content in the mitochondrial fraction is seen at 31 days after birth, this corresponds to a stage when dynamic neuronal loss is measured to be greatest in the Purkinje Cell Degeneration mouse. We also report changes in mitochondrial Hba and Hbb levels in ageing brain and muscle. Significant differences in mitochondrial Hba and Hbb can be seen when comparing aged brain to muscle, suggesting tissue specific functions of these proteins in the mitochondrion. In muscle there are significant differences between Hba levels in old and young mitochondria. To understand whether the changes detected in mitochondrial Hbs are of clinical significance, we examined Parkinson's disease brain, immunohistochemistry studies suggest that cell bodies in the substantia nigra accumulate mitochondrial Hb. However, western blotting of mitochondrial fractions from PD and control brains indicates significantly less Hb in PD brain mitochondria. One explanation could be a specific loss of cells containing mitochondria loaded with Hb proteins. Our study opens the door to an examination of the role of Hb function, within the context of the mitochondrion

  14. Post-surgical wound management of pilonidal cysts with a haemoglobin spray: a case series.

    PubMed

    Mustafi, N; Engels, P

    2016-04-01

    Painful acute cysts in the natal cleft or lower back, known as pilonidal sinus disease, are a severe burden to many younger patients. Although surgical intervention is the preferred first line treatment, postsurgical wound healing disturbances are frequently reported due to infection or other complications. Different treatment options of pilonidal cysts have been discussed in the literature, however, no standardised guideline for the postsurgical wound treatment is available. After surgery, a common recommended treatment to patients is rinsing the wound with clean water and dressing with a sterile compress. We present a case series of seven patients with wounds healing by secondary intention after surgical intervention of a pilonidal cyst. The average age of the patients was 40 years old. Of the seven patients, three had developed a wound healing disturbance, one wound had started to develop a fibrin coating and three were in a good condition. The applied wound care regimens comprised appropriate mechanical or autolytic debridement, rinsing with an antimicrobial solution, haemoglobin application, and primary and secondary dressings. In all seven cases a complete wound closure was achieved within an average of 76 days with six out of seven wounds achieving wound closure within 23-98 days. Aesthetic appearance was deemed excellent in five out of seven cases excellent and acceptable in one. Treatment of one case with a sustained healing disturbance did result in wound closure but with a poor aesthetic outcome and an extensive cicatrisation of the new tissue. Based on these results we recommend that to avoid healing disturbances of wounds healing by secondary intention after surgical pilonidal cyst intervention, an adequate wound care regime comprising appropriate wound debridement, rinsing, topically applied haemoglobin and adequate wound dressing is recommendable as early as possible after surgery. PMID:27064368

  15. Post-surgical wound management of pilonidal cysts with a haemoglobin spray: a case series.

    PubMed

    Mustafi, N; Engels, P

    2016-04-01

    Painful acute cysts in the natal cleft or lower back, known as pilonidal sinus disease, are a severe burden to many younger patients. Although surgical intervention is the preferred first line treatment, postsurgical wound healing disturbances are frequently reported due to infection or other complications. Different treatment options of pilonidal cysts have been discussed in the literature, however, no standardised guideline for the postsurgical wound treatment is available. After surgery, a common recommended treatment to patients is rinsing the wound with clean water and dressing with a sterile compress. We present a case series of seven patients with wounds healing by secondary intention after surgical intervention of a pilonidal cyst. The average age of the patients was 40 years old. Of the seven patients, three had developed a wound healing disturbance, one wound had started to develop a fibrin coating and three were in a good condition. The applied wound care regimens comprised appropriate mechanical or autolytic debridement, rinsing with an antimicrobial solution, haemoglobin application, and primary and secondary dressings. In all seven cases a complete wound closure was achieved within an average of 76 days with six out of seven wounds achieving wound closure within 23-98 days. Aesthetic appearance was deemed excellent in five out of seven cases excellent and acceptable in one. Treatment of one case with a sustained healing disturbance did result in wound closure but with a poor aesthetic outcome and an extensive cicatrisation of the new tissue. Based on these results we recommend that to avoid healing disturbances of wounds healing by secondary intention after surgical pilonidal cyst intervention, an adequate wound care regime comprising appropriate wound debridement, rinsing, topically applied haemoglobin and adequate wound dressing is recommendable as early as possible after surgery.

  16. Anaemia: can we define haemoglobin thresholds for impaired oxygen homeostasis and suggest new strategies for treatment?

    PubMed

    Hare, Gregory M T; Tsui, Albert K Y; Ozawa, Sherri; Shander, Aryeh

    2013-03-01

    Observational clinical studies in perioperative medicine have defined a progressive increase in mortality that is proportional to both chronic preoperative anaemia and acute interpretative reductions in haemoglobin concentration (Hb). However, this knowledge has not yet helped to define the critical Hb threshold for organ injury and mortality in specific patient populations or in individual patients. Nor has this knowledge enabled us to develop effective treatment strategies for anaemia, as evident from the lack of a demonstrable improvement in survival in patients randomised to higher Hb levels by various treatment strategies including allogeneic red blood cell transfusion, erythropoiesis-stimulating agents (ESAs) and haemoglobin-based oxygen carriers (HBOCs). These findings emphasise the need for a clearer understanding of the mechanism of anaemia-induced mortality. Towards achieving this goal, experimental studies have defined adaptive mechanism by which oxygen homeostasis is maintained during acute anaemia. The mechanisms include: (1) effective sensing of anaemia-induced tissue hypoxia; (2) adaptive cardiovascular responses to maintain adequate tissue oxygen delivery; (3) heterogeneity of organ-specific oxygen delivery to preferentially sustain vital organs which are essential for acute survival (heart and brain); (4) evidence of increased vital organ injury with interruption of cardiovascular responses to anaemia and (5) evidence of activation of adaptive cellular responses to maintain oxygen homeostasis and support survival during acute anaemia. Understanding these mechanisms may allow us to define treatment thresholds and novel treatment strategies for acute anaemia based on biological markers of tissue hypoxia. The overall goal of these approaches is to improve patient outcomes, including event-free perioperative survival.

  17. Influence of altitude training modality on performance and total haemoglobin mass in elite swimmers.

    PubMed

    Gough, Clare E; Saunders, Philo U; Fowlie, John; Savage, Bernard; Pyne, David B; Anson, Judith M; Wachsmuth, Nadine; Prommer, Nicole; Gore, Christopher J

    2012-09-01

    We compared changes in performance and total haemoglobin mass (tHb) of elite swimmers in the weeks following either Classic or Live High:Train Low (LHTL) altitude training. Twenty-six elite swimmers (15 male, 11 female, 21.4 ± 2.7 years; mean ± SD) were divided into two groups for 3 weeks of either Classic or LHTL altitude training. Swimming performances over 100 or 200 m were assessed before altitude, then 1, 7, 14 and 28 days after returning to sea-level. Total haemoglobin mass was measured twice before altitude, then 1 and 14 days after return to sea-level. Changes in swimming performance in the first week after Classic and LHTL were compared against those of Race Control (n = 11), a group of elite swimmers who did not complete altitude training. In addition, a season-long comparison of swimming performance between altitude and non-altitude groups was undertaken to compare the progression of performances over the course of a competitive season. Regardless of altitude training modality, swimming performances were substantially slower 1 day (Classic 1.4 ± 1.3% and LHTL 1.6 ± 1.6%; mean ± 90% confidence limits) and 7 days (0.9 ± 1.0% and 1.9 ± 1.1%) after altitude compared to Race Control. In both groups, performances 14 and 28 days after altitude were not different from pre-altitude. The season-long comparison indicated that no clear advantage was obtained by swimmers who completed altitude training. Both Classic and LHTL elicited ~4% increases in tHb. Although altitude training induced erythropoeisis, this physiological adaptation did not transfer directly into improved competitive performance in elite swimmers.

  18. Isolation and some properties of exohemagglutinin from the culture medium of Bacteroides gingivalis 381.

    PubMed Central

    Inoshita, E; Amano, A; Hanioka, T; Tamagawa, H; Shizukuishi, S; Tsunemitsu, A

    1986-01-01

    Exohemagglutinin was found in the culture medium of Bacteroides gingivalis 381. Exohemagglutinin was purified 3,150-fold from culture fluid by ultracentrifugation followed by gel filtration on Sepharose CL-4B and by affinity chromatography on arginine-agarose. Examination of the final preparation of exohemagglutinin by biochemical analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the isolated exohemagglutinin contained three major proteins but not a detectable lipopolysaccharide. Hemagglutination inhibition experiments showed that the activity of exohemagglutinin was inhibited by L-arginine and the arginine-containing peptides, although the activity was unaffected by the sugars tested. Some protein and glycoproteins that were examined also exhibited the inhibitory activity. When the bovine submaxillary mucin was chemically modified by beta-elimination and bovine serum albumin was modified by guanidination, the inhibitory effects on hemagglutination were significantly enhanced. These results suggest that the hemagglutination of the isolated exohemagglutinin may be involved in arginine residues as components of ligand-binding sites on erythrocytes. Images PMID:3699890

  19. Glycated haemoglobin and metabolic control of diabetes mellitus: external versus locally established clinical targets for primary care.

    PubMed Central

    Butler, C.; Peters, J.; Stott, N.

    1995-01-01

    OBJECTIVES--To examine current targets for glycated haemoglobin as a marker for metabolic control in diabetes mellitus in relation to datasets from several areas, and to consider whether target setting could be improved. DESIGN--Data collected from enhanced care records of general practices for a representative community based sample of people with diabetes. SETTING AND SUBJECTS--3022 people with diabetes on the lists of 37 general practices (total list size 222,550) in South Glamorgan in 1992; samples of glycated haemoglobin had been processed at two laboratories with different methodologies and reference ranges. MAIN OUTCOME MEASURES--Last glycated haemoglobin level measured in subjects for 1992 and published data from other studies considered in relation to existing goals and standards for the metabolic control of diabetes. RESULTS--An ascertainment rate for people with diabetes of 1.36% was obtained. The rate of data capture for haemoglobin A1 was 75.7%, and the mean level for study samples was 10.5% at one laboratory and 10.0% at the other (similar values to those of comparable studies). These mean levels of haemoglobin A1 in representative populations of people with diabetes are poor or very poor according to published standards, including those of the British Diabetic Association. These findings are set in the context of the psychology of goal setting and performance in complex clinical situations. CONCLUSION--Targets for clinical care that are set in the absence of normative data and local feasibility assessments should be treated with caution. Targets are more likely to enhance health care if target setters recognise the importance of psychological aspects of goal setting and motivation. PMID:7677834

  20. Correlation between Either Cupriavidus or Porphyromonas and Primary Pulmonary Tuberculosis Found by Analysing the Microbiota in Patients’ Bronchoalveolar Lavage Fluid

    PubMed Central

    Zhou, Yuhua; Lin, Feishen; Cui, Zelin; Zhang, Xiangrong; Hu, Chunmei; Shen, Tian; Chen, Chunyan; Zhang, Xia; Guo, Xiaokui

    2015-01-01

    Pulmonary tuberculosis (TB) has gained attention in recent decades because of its rising incidence trend; simultaneously, increasing numbers of studies have identified the relationship between microbiota and chronic infectious diseases. In our work, we enrolled 32 patients with primary TB characterised by unilateral TB lesion formation diagnosed by chest radiographic exam. Bronchoalveolar lavage fluid was taken from both lungs. Twenty-four healthy people were chosen as controls. Pyrosequencing was performed on the V3 hypervariable region of 16S rDNA in all bacterial samples and used as a culture-independent method to describe the phylogenetic composition of the microbiota. Through pyrosequencing, 271,764 amplicons were detected in samples and analysed using tools in the Ribosomal Database Project (RDP) and bioinformatics. These analyses revealed significant differences in the microbiota in the lower respiratory tract (LRT) of TB patients compared with healthy controls; in contrast, the microbiota of intra/extra-TB lesions were similar. These results showed that the dominant bacterial genus in the LRT of TB patients was Cupriavidus and not Streptococcus, which resulted in a significant change in the microbiota in TB patients. The abundance of Mycobacteria and Porphyromonas significantly increased inside TB lesions when compared with non-lesion-containing contralateral lungs. From these data, it can be concluded that Cupriavidus plays an important role in TB’s secondary infection and that in addition to Mycobacteria, Porphyromonas may also be a co-factor in lesion formation. The mechanisms underlying this connection warrant further research. PMID:26000957

  1. Monoclonal antibody against Porphyromonas (Bacteroides) endodontalis lipopolysaccharide and application of the antibody for direct identification of the species.

    PubMed Central

    Hanazawa, S; Sagiya, T; Kitami, H; Ohta, K; Nishikawa, H; Kitano, S

    1991-01-01

    The aim of the present study was to develop a monoclonal antibody that recognizes the shared antigen of Porphyromonas endodontalis so that we could use the antibody in direct identification and detection of P. endodontalis in infectious material from apical periodontal patients. We established a hybridoma cell line producing monoclonal antibody (BEB5) specific for P. endodontalis. BEB5 antibody reacted with all of the P. endodontalis strains tested, but not with any of the other black-pigmented Porphyromonas and Bacteroides spp. The antibody reacted specifically with the lipopolysaccharide (LPS) of three P. endodontalis strains of different serotypes (O1K1, O1K2, and O1K-). Western blotting (immunoblotting) analysis confirmed the specificity of the antibody to these LPSs, because the antibody recognized the typical "repetitive ladder" pattern characteristic of LPS on sodium dodecyl sulfate-polyacrylamide electrophoretic gels. These observations demonstrate that P. endodontalis LPS is the shared antigen of this species. The antibody can specifically identify P. endodontalis on nitrocellulose membrane blots of bacterial colonies grown on agar. The antibody is also capable of directly detecting the presence of P. endodontalis in infectious material by immunoslot blot assay. These results indicate that LPS is the shared antigen of P. endodontalis and that BEB5 antibody against LPS is a useful one for direct identification and detection of the organisms in samples from apical periodontal patients. Images PMID:1774262

  2. Altitude Exposure at 1800 m Increases Haemoglobin Mass in Distance Runners

    PubMed Central

    Garvican-Lewis, Laura A.; Halliday, Iona; Abbiss, Chris R.; Saunders, Philo U.; Gore, Christopher J.

    2015-01-01

    The influence of low natural altitudes (< 2000 m) on erythropoietic adaptation is currently unclear, with current recommendations indicating that such low altitudes may be insufficient to stimulate significant increases in haemoglobin mass (Hbmass). As such, the purpose of this study was to determine the influence of 3 weeks of live high, train high exposure (LHTH) at low natural altitude (i.e. 1800 m) on Hbmass, red blood cell count and iron profile. A total of 16 elite or well-trained runners were assigned into either a LHTH (n = 8) or CONTROL (n = 8) group. Venous blood samples were drawn prior to, at 2 weeks and at 3 weeks following exposure. Hbmass was measured in duplicate prior to exposure and at 2 weeks and at 3 weeks following exposure via carbon monoxide rebreathing. The percentage change in Hbmass from baseline was significantly greater in LHTH, when compared with the CONTROL group at 2 (3.1% vs 0.4%; p = 0.01;) and 3 weeks (3.0% vs -1.1%; p < 0.02, respectively) following exposure. Haematocrit was greater in LHTH than CONTROL at 2 (p = 0.01) and 3 weeks (p = 0.04) following exposure. No significant interaction effect was observed for haemoglobin concentration (p = 0.06), serum ferritin (p = 0.43), transferrin (p = 0.52) or reticulocyte percentage (p = 0.16). The results of this study indicate that three week of natural classic (i.e. LHTH) low altitude exposure (1800 m) results in a significant increase in Hbmass of elite distance runners, which is likely due to the continuous exposure to hypoxia. Key points Two and three weeks of LHTH altitude exposure (1800 m) results in a significant increase in Hbmass LHTH altitude exposure increased Hbmass by 3.1% after 2 weeks, and 3.0% after 3 weeks of exposure LHTH altitude exposure may be a practical method to increase Hbmass in well-trained athletes. PMID:25983592

  3. Haem degradation in human haemoglobin in vitro. Separation of the contribution of the alpha- and beta-subunits.

    PubMed Central

    Docherty, J C; Brown, S B

    1984-01-01

    Human haemoglobin was prepared containing [14C]haem in either the alpha- or the beta-subunits. Coupled oxidation of such hybrid haemoglobins with ascorbate and O2 showed that the biliverdin produced by the alpha-subunits contained approx. 55% alpha-isomer and 45% beta-isomer, whereas that produced by the beta-subunits contained approx. 75% alpha-isomer and 25% beta-isomer. Coupled oxidation of isolated alpha- and beta-subunits gave approx. 70% alpha-isomer, 30% beta-isomer and 78% alpha-isomer, 22% beta-isomer respectively. These results are consistent with calculations of differences in the haem environment in the two subunit types. PMID:6477522

  4. NOTE: A haemodynamic model for the physiological interpretation of in vivo measurements of the concentration and oxygen saturation of haemoglobin

    NASA Astrophysics Data System (ADS)

    Fantini, Sergio

    2002-09-01

    We present a model that describes the effect of physiological parameters such as the speed of blood flow, local oxygen consumption, capillary recruitment, and vascular dilation/constriction on the concentration and oxygen saturation of haemoglobin in tissue. This model can be used to guide the physiological interpretation of haemodynamic and oximetric data collected in vivo with techniques such as optical imaging, near-infrared spectroscopy and functional magnetic resonance imaging. In addition to providing a formal description of well-established results (exercise-induced hyperemia, reperfusion hyperoxia, decrease in the concentration of deoxyhaemoglobin induced by brain activity, measurement of arterial saturation by pulse oximetry, etc.), this model suggests that the superposition of asynchronous contributions from the arterial, capillary and venous haemoglobin compartments may be at the origin of observed out-of-phase oscillations of the oxyhaemoglobin and deoxyhaemoglobin concentrations in tissue.

  5. Effect of late vs early clamping of the umbilical cord (on haemoglobin level) in full-term neonates.

    PubMed

    Nesheli, Hassan Mahmoodi; Esmailzadeh, Seddigheh; Haghshenas, Mohsen; Bijani, Ali; Moghaddams, Tahereh Galini

    2014-11-01

    Sixty term infants delivered vaginally were assigned randomly to one of the two management groups; early cord clamping (ECC) or delayed cord clamping (DCC). Six months after delivery, the children in both groups were called back for follow-up. Blood samples were obtained for measuring haemoglobin (Hb), haematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), serum iron (SI), transferrin saturation (TS) and serum ferritin (SF) levels. The mean Hb, HCT, SI and TS at 6 months were significantly higher in the DCC group (95% confidence interval (CI); p<0.001, p<0.001, p<0.024 and p<0.009). The mean SF at 6 months was also higher in the DCC group but it was not significant (p<0.071). Polycythaemia, jaundice and other undesirable side-effects of DCC were not seen.

  6. Diagnosis of iron deficiency anaemia in hospital patients: Use of the reticulocyte haemoglobin content to differentiate iron deficiency anaemia from anaemia of chronic disease.

    PubMed

    Schapkaitz, Elise; Buldeo, Suvarna; Mahlangu, Johnny Ndoni

    2015-11-20

    The diagnosis of iron deficiency anaemia in hospital patients with chronic infections and inflammation presents a challenge. Recently laboratory tests such as the reticulocyte haemoglobin content, which are independent of infection and inflammation, have become available for routine diagnostic use.

  7. Preferred temperature of juvenile Atlantic cod Gadus morhua with different haemoglobin genotypes at normoxia and moderate hypoxia.

    PubMed

    Petersen, Maria Faldborg; Steffensen, John Fleng

    2003-01-01

    Atlantic cod Gadus morhua has polymorphic haemoglobin, which can be separated into two homozygous types, HbI-1 and HbI-2, and one heterozygous type HbI-1/2. The geographical distribution of Atlantic cod with the different haemoglobin types varies, with the HbI(2) allele occurring at high frequency in northern regions, and the HbI(1) allele dominant in warmer areas. To determine if temperature is a selective parameter in the distribution of the haemoglobin types, the preferred temperature of the homozygous genotypes HbI-1 and HbI-2 was measured. We found that HbI-2 cod preferred a temperature of 8.2+/-1.5 degrees C while HbI-1 cod preferred 15.4+/-1.1 degrees C, and this preference was significant. The effect of hypoxia (35% oxygen saturation) on the preferred temperature was also measured. Previous studies showed that the preferred temperature of fish decreases during hypoxia, and this was the case for HbI-1 cod, which preferred 9.8+/-1.8 degrees C during hypoxia, whereas HbI-2 cod did not show this effect. The results indicate that environmental temperature changes will lead to a distributional change in the different haemoglobin types of Atlantic cod, global warming providing an advantage for HbI-1 cod. However, since HbI-1 cod prefer a low temperature under hypoxic conditions, a combination of increased water temperature and hypoxia could be unfavourable for Atlantic cod stocks.

  8. Total haemoglobin mass and spleen contraction: a study on competitive apnea divers, non-diving athletes and untrained control subjects.

    PubMed

    Prommer, Nicole; Ehrmann, Ulrich; Schmidt, Walter; Steinacker, Jürgen M; Radermacher, Peter; Muth, Claus-Martin

    2007-12-01

    In diving mammals splenic contraction increases circulating red cell volume, whereas in humans increased haemoglobin concentrations have been reported. It is unknown, however, whether repetitive apnea diving also comprises an adaptive increase in total red cell volume as reported in endurance athletes. The first aim of the study therefore was to investigate the effect of repeated apnea dives on splenic size and putative red cell release in trained apnea divers (n = 10) and control subjects (SCUBA divers performing apneas without long-term apnea training, n = 7). Long-term effects of repetitive apnea diving may elevate the oxygen transport capacity by an adaptive increase in total haemoglobin mass as reported in endurance athletes. The second goal, therefore, was to compare the trained apnea divers' and the control divers' total haemoglobin mass (tHb-mass) with that of endurance-trained (n = 9) and untrained (n = 10) non-divers. Before and immediately after a series of five dives to a depth of 4 m in a heated pool, spleen volume was assessed with ultrasound tomography. tHb-mass and plasma volume were measured using the CO-rebreathing method. In the trained apnea divers, repeated apnea dives resulted in a 25% reduction of spleen size (P < 0.001), whereas no significant effect was observed in the control subjects. While tHb-mass did not differ between trained apnea divers, untrained SCUBA divers performing apneas and untrained non-divers, it was 30% lower than in endurance-trained non-divers. We conclude that prolonged apnea training causes marked apnea-induced splenic contraction. In contrast to athletes in endurance sports, the trained apnea divers did not present with increased total haemoglobin mass and, hence, no increase in blood oxygen stores.

  9. Heating of food and haemoglobin adducts from carcinogens: possible precursor role of glycidol.

    PubMed

    Hindsø Landin, H; Tareke, E; Rydberg, P; Olsson, U; Törnqvist, M

    2000-11-01

    Studies of adducts from reactive compounds to haemoglobin (Hb) by gas chromatography-tandem mass spectrometry according to the N-alkyl Edman method reveals the occurrence of N-(2,3-dihydroxypropyl)valine (diHOPrVal) at levels of 1-2 pmol/g Hb, in persons without known exposure. The hypothesis that this background originates from glycidol or related compounds during heating of food was tested in experiments with rats. Animals fed fried animal feed for 30 or 72 days showed an increase of the diHOPrVal level by about 50% compared with controls. Several arguments, such as the formation of reactive oxiranes by heat-induced dehydration of glycol configurations in glycerol and sugars, support the idea that glycidol (or e.g. glycidyl esters) are precursors of the adduct. In Hb samples, reduced for stabilisation of aldehyde adducts, relatively high levels of adducts determined as diHOPrVal were found, although without significant relation to frying of the feed. There is thus no indication that reduction in vivo of, for example, the Schiff base from glyceraldehyde, is a pathway for formation of the diHOPrVal. The background level of diHOPrVal in humans Hb is low, and the cancer risk associated with exposure to the specific alkylator-probably glycidol-formed in cooking, is therefore presumably low. The result implies, however, that low-molecular mass mutagenic oxiranes formed during the heating of food should be studied further.

  10. Crystal structure of truncated haemoglobin from an extremely thermophilic and acidophilic bacterium.

    PubMed

    Jamil, Farrukh; Teh, Aik-Hong; Schadich, Ermin; Saito, Jennifer A; Najimudin, Nazalan; Alam, Maqsudul

    2014-08-01

    A truncated haemoglobin (tHb) has been identified in an acidophilic and thermophilic methanotroph Methylacidiphilium infernorum. Hell's Gate Globin IV (HGbIV) and its related tHbs differ from all other bacterial tHbs due to their distinctively large sequence and polar distal haem pocket residues. Here we report the crystal structure of HGbIV determined at 1.96 Å resolution. The HGbIV structure has the distinctive 2/2 α-helical structure with extensions at both termini. It has a large distal site cavity in the haem pocket surrounded by four polar residues: His70(B9), His71(B10), Ser97(E11) and Trp137(G8). This cavity can bind bulky ligands such as a phosphate ion. Conformational shifts of His71(B10), Leu90(E4) and Leu93(E7) can also provide more space to accommodate larger ligands than the phosphate ion. The entrance/exit of such bulky ligands might be facilitated by positional flexibility in the CD1 loop, E helix and haem-propionate A. Therefore, the large cavity in HGbIV with polar His70(B9) and His71(B10), in contrast to the distal sites of other bacterial tHbs surrounded by non-polar residues, suggests its distinct physiological functions.

  11. Exploring the Nitric Oxide Detoxification Mechanism of Mycobacterium tuberculosis Truncated Haemoglobin N

    NASA Astrophysics Data System (ADS)

    Bidon-Chanal, A.; Martí, M. A.; Estrín, D. A.; Luque, F. J.

    Mycobacterium tuberculosis, the causative agent of human tuberculosis, encodes a haemoprotein named Truncated Haemoglobin N (trHbN), which in its active site transforms nitric oxide (NO) to nitrate anion.left( {{text{NO}}_3^ - } right). The NO-dioxygenase activity of trHbN seems to be crucial for the bacillus, which can survive under the nitrosative stress conditions that occur upon infection of the host. As a defense mechanism against the copious amounts of NO produced by macrophages upon infection, the protein must achieve a high level of NO-dioxygenase activity to eliminate NO, but this is modulated by its efficiency in capturing O2 and NO. Migration of small diatomic ligands through the protein matrix is related to the presence of a doubly branched tunnel system connecting the surface and the haem cavity site. In this work, we have studied the mechanism that controls ligand diffusion and product egression with state-of-the-art molecular dynamics simulations. The results support a dual path mechanism for migration of O2 and NO through distinct branches of the tunnel, where migration of NO is facilitated upon binding of O2 to the haem group. Finally, egression of left( {{text{NO}}_3^ - } right) is preceded by the entrance of water to the haem cavity and occurs through a different pathway. Overall, the results highlight the intimate relationship between structure, dynamical behavior and biological function of trHbN.

  12. New insights into the proton-dependent oxygen affinity of Root effect haemoglobins.

    PubMed

    Bonaventura, C; Crumbliss, A L; Weber, R E

    2004-11-01

    A long-standing puzzle with regard to protein structure/function relationships is the proton-dependent modification of haemoglobin (Hb) structure that causes oxygen to be unloaded from Root effect Hbs into the swim bladders and eyes of fish even against high oxygen pressure gradients. Although oxygen unloading in Root effect Hbs has generally been attributed to proton-dependent stabilization of the T-state, protonation of Root effect Hbs can alter their ligand affinities in both R- and T-state conformations and either stabilize the T-state or destabilize the R-state. The C-terminal residues that are so important in the Bohr effect of human Hb appear to be involved in the Root effects of some fish Hbs and not in others, indicating that several evolutionary pathways have resulted in expression of highly pH-dependent Hbs. New data are presented that show surprising similarities in the pH- and anion-dependence of sulfhydryl group reactivity and anaerobic oxidation of human and fish Hbs. The available evidence supports the concept that in both Bohr effect and Root effect Hbs a large steric component acts in addition to quaternary shifts between R and T conformations to regulate ligand affinity. Allosteric effectors moderate these steric effects within both R- and T-state conformations and allow for an elegant match between Hb function and the wide-ranging physiological needs of diverse organisms.

  13. Structures of haemoglobin from woolly mammoth in liganded and unliganded states.

    PubMed

    Noguchi, Hiroki; Campbell, Kevin L; Ho, Chien; Unzai, Satoru; Park, Sam-Yong; Tame, Jeremy R H

    2012-11-01

    The haemoglobin (Hb) of the extinct woolly mammoth has been recreated using recombinant genes expressed in Escherichia coli. The globin gene sequences were previously determined using DNA recovered from frozen cadavers. Although highly similar to the Hb of existing elephants, the woolly mammoth protein shows rather different responses to chloride ions and temperature. In particular, the heat of oxygenation is found to be much lower in mammoth Hb, which appears to be an adaptation to the harsh high-latitude climates of the Pleistocene Ice Ages and has been linked to heightened sensitivity of the mammoth protein to protons, chloride ions and organic phosphates relative to that of Asian elephants. To elucidate the structural basis for the altered homotropic and heterotropic effects, the crystal structures of mammoth Hb have been determined in the deoxy, carbonmonoxy and aquo-met forms. These models, which are the first structures of Hb from an extinct species, show many features reminiscent of human Hb, but underline how the delicate control of oxygen affinity relies on much more than simple overall quaternary-structure changes.

  14. 'Fit to fly': overcoming barriers to preoperative haemoglobin optimization in surgical patients.

    PubMed

    Muñoz, M; Gómez-Ramírez, S; Kozek-Langeneker, S; Shander, A; Richards, T; Pavía, J; Kehlet, H; Acheson, A G; Evans, C; Raobaikady, R; Javidroozi, M; Auerbach, M

    2015-07-01

    In major surgery, the implementation of multidisciplinary, multimodal and individualized strategies, collectively termed Patient Blood Management, aims to identify modifiable risks and optimise patients' own physiology with the ultimate goal of improving outcomes. Among the various strategies utilized in Patient Blood Management, timely detection and management of preoperative anaemia is most important, as it is in itself a risk factor for worse clinical outcome, but also one of the strongest predisposing factors for perioperative allogeneic blood transfusion, which in turn increases postoperative morbidity, mortality and costs. However, preoperative anaemia is still frequently ignored, with indiscriminate allogeneic blood transfusion used as a 'quick fix'. Consistent with reported evidence from other medical specialties, this imprudent practice continues to be endorsed by non-evidence based misconceptions, which constitute serious barriers for a wider implementation of preoperative haemoglobin optimisation. We have reviewed a number of these misconceptions, which we unanimously consider should be promptly abandoned by health care providers and replaced by evidence-based strategies such as detection, diagnosis and proper treatment of preoperative anaemia. We believe that this approach to preoperative anaemia management may be a viable, cost-effective strategy that is beneficial both for patients, with improved clinical outcomes, and for health systems, with more efficient use of finite health care resources.

  15. Haemoglobin levels in autoimmune haemolytic anaemias at diagnosis: relationship with immunoproteins on red blood cells.

    PubMed

    Lai, Marco; De Stefano, Valerio; Landolfi, Raffaele

    2014-10-01

    Previous investigations of the relationship between characteristics of immunoproteins on red blood cells (RBCs) and the occurrence of autoimmune haemolysis yielded divergent results. Here, we studied these characteristics in autoimmune haemolytic anaemias (AIHAs) to determine their relationship with the degree of anaemia at diagnosis. We studied at diagnosis 52 cases of warm AIHA with positive direct antiglobulin test. Immunohaematological testing and determination of immunoglobulin class, complement, and immunoglobulin G (IgG) were performed using gel technology (GCT). Median haemoglobin (Hb) levels significantly differed between cases with IgG1 only or negative for IgG subclasses (7.4 g/dl), those with IgG3 or IgG1 + IgG3 (6.5 g/dl), and those with multiple immunoglobulins (5 g/dl). Logistic regression indicated that IgG3 detection was the only variable significantly related to the occurrence of RBC transfusion in AIHA (odds ratio 4.05, 95 % CI 1.1-14.7). In our study, the type of immunoprotein(s) on the RBC surface was associated with different Hb levels at AIHA diagnosis. IgG3 and multiple immunoglobulins were associated with lower Hb levels; IgG3 was also associated with a higher percentage of patient transfusions in the first week after diagnosis. Thus, qualitative differences in these immunoproteins may