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R J Genco

Publications and source records attributed to R J Genco.

At least 91 records · Page 5Linked to original sources
PubMed1994

Salivary receptors for recombinant fimbrillin of Porphyromonas gingivalis.

Fimbriae are considered important in the adherence and colonization of Porphyromonas gingivalis in the oral cavity. It has been demonstrated that purified fimbriae bind to whole human saliva adsorbed to hydroxyapatite (HAP) beads, and the binding appears to be mediated by specific protein-protein interactions. Recently, we expressed the recombinant fimbrillin protein (r-Fim) of P. gingivalis corresponding to amino acid residues 10 to 337 of the native fimbrillin (A. Sharma, H.T. Sojar, J.-Y. Lee, and R.J. Genco, Infect. Immun. 61:3570-3573, 1993). We examined the ability of individual salivary components to promote the direct attachment of r-Fim to HAP beads. Purified r-Fim was radiolabeled with 125I and incubated with HAP beads which were coated with saliva or purified individual salivary components. Whole, parotid, and submandibular-sublingual salivas increased the binding of 125I-r-Fim to HAP beads. Submandibular-sublingual saliva was most effective in increasing the binding of 125I-r-Fim to HAP beads (1.8 times greater than that to uncoated HAP beads). The binding of 125I-r-Fim to HAP beads coated with acidic proline-rich protein 1 (PRP1) or statherin was four and two times greater, respectively, than that to uncoated HAP beads. PRP1 and statherin molecules were also found to bind 125I-r-Fim in an overlay assay. The binding of intact P. gingivalis cells to HAP beads coated with PRP1 or statherin was also enhanced, by 5.4 and 4.3 times, respectively, over that to uncoated HAP beads. The interactions of PRP1 and statherin with 125I-r-Fim were not inhibited by the addition of carbohydrates or amino acids. PRP1 and statherin in solution did not show inhibitory activity on 125I-r-Fim binding to HAP beads coated with PRP1 or statherin. These results suggest that P. gingivalis fimbriae bind strongly through protein-protein interactions to acidic proline-rich protein and statherin molecules which coat surfaces.

Adult↗
PubMed1994

Inactivation of the Porphyromonas gingivalis fimA gene blocks periodontal damage in gnotobiotic rats.

Fimbrial production by Porphyromonas gingivalis was inactivated by insertion-duplication mutagenesis, using the cloned gene for the P. gingivalis major fimbrial subunit protein, fimA. by several criteria, this insertion mutation rendered P. gingivalis unable to produce fimbrilin or an intact fimbrial structure. A nonfimbriated mutant, DPG3, hemagglutinated sheep erythrocytes normally and was unimpaired in the ability to coaggregate with Streptococcus gordonii G9B. The cell surface hydrophobicity of DPG3 was also unaffected by the loss of fimbriae. However, DPG3 was significantly less able to bind to saliva-coated hydroxyapatite than wild-type P. gingivalis 381. This suggested that P. gingivalis fimbriae are important for adherence of the organism to saliva-coated oral surfaces. Further, DPG3 was significantly less able to cause periodontal bone loss in a gnotobiotic rat model of periodontal disease. These observations are consistent with other data suggesting that P. gingivalis fimbriae play an important role in the pathogenesis of human periodontal disease.

Alveolar Bone Loss↗
PubMed1994

The distribution and transmission of Actinobacillus actinomycetemcomitans in families with established adult periodontitis.

The prevalence and genotype distribution of Actinobacillus actinomycetemcomitans strains in families where at least one adult family member (proband) suffered from periodontal disease was investigated to better understand how this periodontal organism is acquired or transmitted. Fifteen probands with severe (established) periodontal disease (EPD) and their 46 immediate family members were sampled for A. actinomycetemcomitans. Among the 15 families, 10 contained at least one additional family member colonized with oral A. actinomycetemcomitans. Genomic DNA from 3 subgingival A. actinomycetemcomitans strains from each of the 10 probands and their 17 family members were amplified and characterized by the polymerase chain reaction (PCR) using a single arbitrary primer known to distinguish A. actinomycetemcomitans strains. The PCR products from each strain were separated by electrophoresis on a 1% submarine agarose gel containing ethidium bromide and visualized by UV light transillumination. The amplification products migrated to form readily distinguishable bands and, since the banding patterns were characteristic of strains of A. actinomycetemcomitans, these patterns were called "amplitypes." The culture studies showed that 51% of all patients suffering from EPD carried oral A. actinomycetemcomitans. Moreover, 50% of their spouses and 30% of their children harbored the bacterium. Comparison of the PCR-generated amplitypes showed that 26 out of 27 individuals had strains exhibiting a single amplitype of A. actinomycetemcomitans, the 27th being colonized by 2 different amplitypes. They also showed that in 6 out of 7 families, the husband and wife did not harbor the same A. actinomycetemcomitans amplitype. Furthermore, most often children carried an an amplitype identical to one of the parents.(ABSTRACT TRUNCATED AT 250 WORDS)

Actinobacillus Infections↗
PubMed1994

Clinical, microbiological, and histological factors which influence the success of regenerative periodontal therapy.

The primary objectives of this double-blind, controlled clinical trial were to assess factor(s) which affect the success of guided tissue regeneration (GTR) procedures in mandibular Class II buccal furcation defects. Thirty subjects, with mandibular Class II furcation defects, were randomly assigned to one of two treatment groups; patients in Group A received oral hygiene instructions with scaling and root planing, while subjects in Group B received similar treatment but without subgingival scaling and root planing at the affected site. After initial oral hygiene instructions and scaling and root planing, GTR surgery was performed using ePTFE barrier membranes. Membranes were retrieved at 6 weeks and subjected to histological examination. Twelve months after regenerative therapy, clinical measurements and re-entry surgical measurements were repeated. Probing reduction (2.61 mm), horizontal probing attachment gain (2.59 mm), and vertical probing attachment gain (0.95 mm) were all significantly better compared to baseline. Likewise, significant improvements in furcation volume (8.0 microliters) and in bone measurements were observed at re-entry. There was no discernible difference between subjects for whom complete anti-infective therapy was deferred to the time of the surgery (Group B) compared to subjects in whom complete anti-infective therapy was performed as part of the hygienic phase of therapy (Group A). Pre-operative pocket depth was directly correlated with the magnitude of attachment gain as well as the amount of new bone formation in the furcation area. Subjects who maintained good oral hygiene and who had minimal gingival inflammation throughout the study demonstrated consistently better regenerative response.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Assessment of risk for periodontal disease. I. Risk indicators for attachment loss.

Specific risk indicators associated with either susceptibility or resistance to severe forms of periodontal disease were evaluated in a cross-section of 1,426 subjects, 25 to 74 years of age, mostly metropolitan dwellers, residing in Erie County, New York, and surrounding areas. The study sample exhibited a wide range of periodontal disease experience defined by different levels of attachment loss. Therefore, it was possible to accurately assess associations between the extent of periodontal disease and patient characteristics including age, smoking, systemic diseases, exposure to occupational hazards, and subgingival microbial flora. Age was the factor most strongly associated with attachment loss, with odds ratios for subjects 35 to 44 years old ranging from 1.72 (95% CI: 1.18 to 2.49) to 9.01 (5.86 to 13.89) for subjects 65 to 74 years old. Diabetes mellitus was the only systemic disease positively associated with attachment loss with an odds ratio of 2.32 (95% CI: 1.17-4.60). Smoking had relative risks ranging from 2.05 (95% CI: 1.47-2.87) for light smokers increasing to 4.75 (95% CI: 3.28-6.91) for heavy smokers. The presence of two bacteria, Porphyromonas gingivalis and Bacteroides forsythus, in the subgingival flora represented risks of 1.59 (95% CI: 1.11-2.25) and 2.45 (95% CI: 1.87-3.24), respectively. Our results show that age, smoking, diabetes mellitus, and the presence of subgingival P. gingivalis and B. forsythus are risk indicators for attachment loss. These associations remain valid after controlling for gender, socioeconomic status, income, education, and oral hygiene status expressed in terms of supragingival plaque accumulation and subgingival calculus. Longitudinal, intervention, and etiology-focused studies will establish whether these indicators are true risk factors.

Adult↗
PubMed1994

Assessment of risk of periodontal disease.

The Periodontal Disease Research Center at the State University of New York at Buffalo has studied two sets of subjects--those with a high prevalence of diabetes (the Pima Indians) and an urban population in Erie County, New York--to determine factors that put a patient at risk for periodontal disease. These and other studies have established that smoking and diabetes are important risk indicators for periodontal disease. Local etiologic factors associated with periodontal disease in adults include the presence of the putative periodontal pathogens Porphyromonas gingivalis and Bacteroides forsythus. Longitudinal studies and intervention studies are needed to fully establish the role of risk indicators. However, information already available from present studies suggests that modulation of risk factors, such as smoking and diabetes mellitus, as well as suppression of the pathogenic flora, will be effective in the management of periodontal disease.

Adult↗
PubMed1993

Recombinant expression and partial characterization of the human formyl peptide receptor.

FMLP-receptor DNA was expressed in Escherichia coli. The expressed product could specifically bind FMLP. This is the first-reported expression of a functional FMLP receptor in Escherichia coli. We confirm that receptor glycosylation is not essential for ligand binding. A deletion mutant did not bind FMLP, suggesting that the deleted portion plays a role in ligand binding.

Amino Acid Sequence↗
PubMed1993

Evidence for up-regulation of epidermal growth-factor receptors on rat periodontal ligament fibroblastic cells associated with stabilization of phenotype in vitro.

This study sought to understand the role of epidermal growth factor receptor (EGF-R) in periodontal ligament (PDL) fibroblasts. Rat PDL fibroblastic cells and ROS 17/2.8 cells (highly differentiated osteoblastic osteosarcoma cells) were cultured and treated with transforming growth factor-alpha (TGF-alpha), EGF, dexamethasone (Dex) or a combination of EGF and Dex. Alkaline phosphatase (ALP) activity, an early differentiation marker for mineralized tissue-forming cells, was measured using p-nitrophenylphosphate as a substrate. For Scatchard analysis of [125I]-EGF binding, cells were incubated in Dulbecco's modified Eagle's medium containing 0.2% bovine serum albumin and 0-64 ng/ml of [125I]-EGF for 4 h at 4 degrees C. Also, the synthesis of EGF-R protein and the expression of mRNA for EGF-R were measured by immunoprecipitation and Northern blot analysis, respectively. Untreated PDL fibroblastic cells showed a gradual increase in spontaneous ALP activity from 32.4 U/10(6) cells at 2 days to 49.6 U/10(6) cells at 7 days of culture. ALP activity was further increased to 70.8 U/10(6) cells at 7 days after treatment with Dex, whereas EGF treatment reduced it to 19.4 U/10(6) cells. Culture of PDL fibroblastic cells in the presence of a combination of Dex and EGF decreased the Dex-induced ALP activity from 70.8 U to 41.8 U/10(6) cells at 7 days. A similar inhibitory effect on ALP activity was found after treatment with TGF-alpha. In contrast, ROS cells maintained a high ALP activity (1748 U/10(6) cells) throughout culture, unaffected by EGF. Scatchard analysis demonstrated that PDL fibroblastic cells have both high- and low-affinity forms of EGF-R, while ROS cells did not have any detectable EGF-R. Treatment of PDL cells with Dex for 2 days decreased the synthesis of EGF-R protein, the expression of EGF-R mRNA and the number of EGF-R. In contrast, EGF treatment increased the expression of EGF-R mRNA. These data suggest that PDL fibroblastic cells express numerous EGF-R, but the number decreases during their differentiation into mineralized tissue-forming cells under the influence of Dex. Thus, EGF-R may function in the stabilization of phenotype in PDL fibroblastic cells.

Alkaline Phosphatase↗
PubMed1993

Modification of experimental Porphyromonas gingivalis murine infection by immunization with a polysaccharide-protein conjugate.

To better understand the role of the capsular polysaccharide in the virulence of Porphyromonas gingivalis, the effect of immunization with a polysaccharide-protein conjugate on experimental murine infection was evaluated. The conjugate was prepared using polysaccharide isolated from P. gingivalis strain ATCC 53977 and bovine serum albumin. One group of 22 mice was immunized by intraperitoneal injection with the conjugate and a control group of 25 mice was similarly immunized with bovine serum albumin. Serum antibody reactive to the polysaccharide, as determined by enzyme-linked immunosorbent assay, was elevated in the group of mice immunized with the polysaccharide-protein conjugate but not in the mice immunized with bovine serum albumin. Both groups of mice were challenged with P. gingivalis strain ATCC 53977 (10(10) cells) administered subcutaneously on the dorsal surface. Following challenge, the mice immunized with the polysaccharide-protein conjugate appeared healthier and demonstrated less weight loss than did the control group of mice. Ulcerative lesions at secondary locations were smaller in mice immunized with the polysaccharide-protein conjugate. Thus, immunization of mice with a conjugate containing P. gingivalis polysaccharide could reduce the severity of but not prevent an invasive infection with P. gingivalis.

Animals↗
PubMed1993

Alternative methods for screening periodontal disease in adults.

Clinical measurements of periodontal attachment loss, while not always representing the histological changes, are used to establish the diagnosis of adult periodontitis. Such measurements are difficult to perform accurately and are labor intensive. To counter these problems, index teeth and index sites have been employed in an attempt to estimate the severity of the periodontal condition without the need to resort to elaborate attachment measurements. Unfortunately, such indices usually tend to underestimate prevalence while often overestimating severity. The purpose of the present study was to examine the correlation of alternative clinical, radiographic and microbiological parameters, with periodontal disease using the diagnostic criteria of established periodontitis. 508 adults included in this study received thorough periodontal examination which included probing pocket depth, clinical attachment level, plaque, gingival and calculus scores, together with radiographic analysis and assays of subgingival periopathogenic microorganisms. Radiographic alveolar bone loss and probing pocket depth had the highest correlation with clinical attachment loss (phi = 0.72 and phi = 0.75 respectively). Plaque scores (phi = 0.17), like gingival scores (phi = 0.06) and calculus scores (phi = 0.42) had poor correlation with established periodontitis. Periodontopathogenic species demonstrated high specificity and negative predictive values; but low sensitivity and positive predictive values make for an overall low correlation of these species with established periodontitis. However, when used in a logistic regression model, the presence of P. gingivalis (odds ratio = 6.25) has shown to contribute significantly to the estimate of probability for established periodontitis. The use of these various alternative parameters for screening of periodontal disease is discussed in light of their sensitivity, specificity and predictive value.

Adult↗
PubMed1993

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

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.

Antigens, Bacterial↗
PubMed1993

Purification and characterization of a protease from Porphyromonas gingivalis capable of degrading salt-solubilized collagen.

An enzyme capable of hydrolyzing the substrate 4-phenylazobenzyloxycarbonyl-L-prolyl-leucyl-glycyl-prolyl-D-ar gin ine (pZ-peptide), pZ-peptidase, was purified from the oral bacterium Porphyromonas gingivalis. pZ-peptidase hydrolyzed salt-solubilized type I collagen from rat skin, rat plasma low-molecular-weight kininogen, and transferrin at room temperature in the presence of calcium and dithiothreitol. pZ-peptidase did not cleave acid-soluble type I calf skin collagen, type V placental collagen, lysozyme, albumin, or human plasma fibrinogen. Furthermore, the purified enzyme did not hydrolyze N-alpha-benzoyl-DL-Arg-p-nitroanilide, Gly-Pro-p-nitroanilide, N-p-tosyl-Gly-Pro-Arg-p-nitroanilide, N-p-tosyl-Gly-Pro-Lys-p-nitroanilide, azoalbumin, or azocasein. Under reducing conditions, the native enzyme migrated as a single band at 120 kDa on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. However, when heated to 100 degrees C for 10 min in SDS under reducing conditions, the enzyme migrated as a major band at 50 kDa and a minor band at 60 kDa on SDS-polyacrylamide gel electrophoresis. Zymography using calf skin gelatin revealed the gelatin-cleaving activity of the enzyme as evidenced by a diffuse band in the range of 120 to 300 kDa under reducing conditions at room temperature, suggesting that this is the native form of the enzyme. However, incubation at 50 degrees C for 10 min under reducing conditions showed gelatin-cleaving activity at a distinct band of 60 kDa. A minimum temperature of 50 degrees C was required to dissociate the 60-kDa chain from the native complex in active form on gelatin zymography. The ability of the enzyme to cleave other proteins, including kininogen and transferrin, suggests that it has specificity for the Pro-X-Gly sequence found in several proteins, including collagen.

Amino Acid Sequence↗
PubMed1993

Expression of a functional Porphyromonas gingivalis fimbrillin polypeptide in Escherichia coli: purification, physicochemical and immunochemical characterization, and binding characteristics.

Fimbriae have been reported to play an important role in the adherence of Porphyromonas gingivalis to oral surfaces and possibly in triggering host responses. A structural subunit of the fimbriae, fimbrillin, has been shown to be important in binding of the bacterium to saliva-coated oral surfaces. In the present study, a coding region of the fimbrillin gene from P. gingivalis 2561 was amplified by the polymerase chain reaction and cloned into the pET-11d vector. The recombinant plasmid was transformed into Escherichia coli BL21, and protein expression was induced with isopropyl-beta-D-thiogalactopyranoside. The expressed protein was purified from insoluble inclusion bodies after solubilization with urea and gel filtration chromatography. The purified recombinant fimbrillin polypeptide, r-fim 10-337, corresponded to amino acid residues 10 to 337 of the deduced amino acid sequence of fimbrillin. In immunoblot analysis, r-fim 10-337 reacted with antibodies to fimbrillin purified from P. gingivalis, as well as with antibodies to synthetic peptides corresponding to the amino acid sequence of fimbrillin. The apparent molecular mass of r-fim 10-337 was estimated to be 41 kDa on sodium dodecyl sulfate-polyacrylamide gels. The r-fim 10-337 polypeptide was capable of inhibiting the binding of P. gingivalis 2561 to saliva-coated hydroxyapatite beads. These results suggest that the fimbrillin subunit polypeptide plays an important role in binding of P. gingivalis cells to saliva-coated surfaces. We describe here the successful expression and purification of a functionally and immunologically reactive recombinant P. gingivalis fimbrillin subunit from E. coli.

Bacterial Adhesion↗
PubMed1993

Differentiation of strains of Actinobacillus actinomycetemcomitans by arbitrarily primed polymerase chain reaction.

The present study describes a method for amplifying DNA in Actinobacillus actinomycetemcomitans by using short, synthetic oligonucleotides of random sequence as primers in the polymerase chain reaction. Genomic DNA from each of 20 human isolates of A. actinomycetemcomitans was successfully amplified in a thermal cycler with a single synthetic primer (GGGTAACGCC) and reproducibly produced 14 different DNA amplification profiles (amplitypes). A. actinomycetemcomitans isolates from the same subject revealed the same amplitype. The arbitrarily primed polymerase chain reaction appears to be useful in characterizing human isolates of A. actinomycetemcomitans for studies of epidemiology and bacterial transmission.

Aggregatibacter actinomycetemcomitans↗