Steroid 5alpha-reductase activity of Treponema denticola.

INTRODUCTION: Previously we have shown that reference and freshly isolated Treponema denticola cultures possess 5alpha-reductase (5alpha-R) and 3beta- and 17beta-hydroxysteroid dehydrogenase activity. A gene matching the 3-oxo-5alpha-steroid 4-dehydrogenase family protein (gene ID: 2739284; locus tag: TDE2697) has been identified in T. denticola ATCC 35405. The aim of the work presented here was to optimize assay conditions and determine steroid substrate specificities for the 5alpha-R activity of T. denticola ATCC 33520. METHODS: 5alpha-R activity of cell-free preparations was assayed with radioactive steroid substrates. 5alpha-R-reduced products were identified using thin-layer chromatography and a radioisotope scanner. Assay conditions were optimized for co-factor, buffer and pH requirements. Apparent substrate specificities were determined for progesterone, 4-androstenedione, testosterone and corticosterone. The time-course for metabolism of radiolabelled progesterone and cholesterol substrates was investigated with anaerobic cultures. RESULTS: The optimum pH for 5alpha-R was 5.5 and the preferred co-factor was NADPH. The order of the steroids with respect to their 5alpha-R substrate specificities was (in descending order): progesterone, 4-androstenedione, testosterone and corticosterone. There are at least two intermediates in the synthesis of 5alpha-dihydrocholesterol from cholesterol. CONCLUSION: These results suggest that the 3-oxo-5alpha-steroid 4-dehydrogenase family protein gene of T. denticola codes for a functional protein that resembles mammalian 5alpha-R isoenzyme 2 with regard to co-factor requirement and pH optimum.

Effect of withdrawal of fluoride-containing toothpaste on the interproximal plaque microflora.

To compare the effects of fluoride-containing and fluoride-free toothpaste on plaque microflora, 15 subjects were enrolled in a double-blind crossover trial. All subjects used a fluoride toothpaste for 7 days before the trial started. Then, 4 interproximal sites per subject were professionally cleaned and subjects used one of the toothpastes for 5 days. On the 5th day plaque was collected from 2 sites, 12 and 6 h after toothpaste use. There was no difference between the groups in the numbers or proportions of aciduric bacteria (recovered at pH 4.8 or 5.2), or of yeasts, neisseriae, lactobacilli or streptococci (total or individual species, including Streptococcus mutans). However, the numbers and proportions of Gram-positive pleomorphic rods, primarily Actinomyces naeslundii, increased in 6-hour samples from subjects using fluoride toothpaste. The data suggest that the anti-caries effect of fluoride toothpaste is not mediated primarily through effects on the plaque microflora, although effects on plaque physiology could be important.

The microflora of the erupting first permanent molar.

The relationship between microflora, eruption status and caries status in the first permanent molar of young children was investigated in 177 children aged 6-7 years. A significantly greater proportion of fully erupted teeth were classified as sound and plaque-free compared to partially erupted teeth. Fully erupted teeth yielded greater numbers and proportions of mutans streptococci compared with significantly greater numbers and proportions of Actinomyces israelii in partially erupted teeth. Logistical regression analysis showed significant associations between white spot lesions in partially erupted teeth and increased numbers of Streptococcus oralis, mutans streptococci and Streptococcus salivarius whereas the presence of Actinomyces naeslundii was associated with health. Significantly greater numbers and proportions of S. oralis and S. salivarius were isolated from partially erupted teeth with white spot lesions whereas Streptococcus mutans was isolated in significantly greater numbers and proportions from fully erupted molars with white spots. This study suggests that organisms other than mutans streptococci are associated with caries development in erupting permanent molar teeth.

Intra-oral acid production associated with eating whole or pulped raw fruits.

The hypotheses that raw fruits, whether whole or pulped, were cleared rapidly from the mouth and that the sugars in the whole and pulped fruits are fermented with equal efficiency to acids by the oral microflora were tested in this study. Groups of 7-9 adult subjects chewed 10 g of raw, whole or pulped fruit (apple, banana, orange, pear and pineapple) for 1 min and whole, unstimulated saliva samples were collected during the following 60-min interval. Each saliva sample was assayed for the concentrations of fruit-derived sugars (glucose, fructose and sucrose), fruit-derived acids (malic and citric) and acids which may be produced as a result of bacterial fermentation (acetic, lactic, formic and succinic). We found the fruit-derived sugars were rapidly cleared from the mouth (within 5 min). The major bacterially produced acids were lactic and succinic, which reached maximum concentrations in the 5-min sample. There was no significant difference, within a fruit, in the salivary levels of any of the sugars or acids between the raw whole or raw pulped forms. In light of these findings it seems unwise to assume that fruits may be consumed without consideration of their acidogenic potential.

Dental caries, oral hygiene, and oral clearance in children with craniofacial disorders.

The reason that children with cleft palates tend to have a greater prevalence of tooth decay than normal children is unclear. We hypothesized that children with cleft palates would have increased oral clearance times for foods and, consequently, higher levels of caries and caries-associated micro-organisms than control children. Children aged 6-16 yrs, with (n = 81) or without (n = 61) cleft palates, were studied. Children with cleft palates had DMFT and dmft scores greater (p < 0.01) than those of the control group. The number of caries-associated organisms was greater in the saliva of the cleft palate children (all p < 0.001). The oral hygiene, plaque and gingival index scores were greater (p < 0.0001), oral clearance was longer (p < 0.01), and levels of sucrose and starch-derived saccharides higher (p < 0.01) in the cleft palate group. However, salivary concentrations of organic acids were lower in the children with craniofacial disorders, probably reflecting the altered physiology of the more mature dental biofilm. The longer oral clearance times of foods and the consequent generation of fermentable sugars from starches may contribute to the higher caries prevalence observed in children with cleft palates.

The influence of specific foods and oral hygiene on the microflora of fissures and smooth surfaces of molar teeth: A 5-day study.

A group of 20 students, harbouring >10(4) mutans streptococci per millilitre of saliva, was enrolled into the study. Models for sampling, reproducibly, the dental plaque present in specific sites (fissure and smooth surface) on the dentition were developed and validated. Withdrawal of normal oral hygiene procedures for only 1 day resulted in approximately 10-fold increases in the number of micro-organisms recovered from both sites. The effect of supplementing the subjects' diets with particular food items given 5 times per day [lemonade (5.8% w/v sugars, 250 ml), biscuits (digestive biscuits, 67.6% w/w carbohydrate of which 22% w/w was sugars and 45.6% w/w was starch), caramel toffees and sugar lumps] on the number of micro-organisms recovered and on the composition of the flora at both sites was determined. Dental plaque samples were taken after 5 days and it was found that supplementation of the diet with toffee and sugar lumps resulted in significantly more micro-organisms at both sampling sites. The supplementation of the diets with lemonade or biscuits did not significantly alter the numbers of micro-organisms recovered from either site. The percentage composition of the plaque samples from both dental sites remained relatively unaffected by oral hygiene although there were lower levels of mutans streptococci which might be related to the use of an antimicrobial toothpaste containing fluoride, triclosan and zinc citrate. This study suggests that the cariogenicity of certain sucrose-containing foods may, in part, be due to the enhancement of plaque accumulation in addition to other effects on the percentage composition of the plaque which may become manifest on prolonged usage of these dietary foodstuffs. These observations are consistent with dietary survey findings which often find consumption of confectionery related to caries experience or incidence.

Characterization of protease activities in Capnocytophaga spp., Porphyromonas gingivalis, Prevotella spp., Treponema denticola and Actinobacillus actinomycetemcomitans.

Protease activities in cell sonicates of defined bacterial strains were examined using peptide substrates and class-specific inhibitors. Capnocytophaga spp. all produced serine dipeptidyl peptidase activity and arginine/lysine, elastase- and chymotrypsin-like enzymes with some metalloprotease characteristics. The elastase-like activity was strongest in Capnocytophaga sputigena, but the others were greatest in Capnocytophaga gingivalis. The latter also had a separate arginine-specific enzyme which appeared not to be present in the other two species. Porphyromonas gingivalis showed serine dipeptidyl peptidase activity and very strong arginine and lysine cysteine protease activities. Prevotella spp. had inhibitor-resistant dipeptidyl peptidase activity and arginine cysteine protease activity that was much weaker but biochemically similar to P. gingivalis. Treponema denticola possessed a strong trypsin-like serine protease activity as well as very weak dipeptidyl peptidase and chymotrypsin-like activities that were sensitive to some cysteine protease reagents. Actinobacillus actinomycetemcomitans showed a novel alanine- and lysine-specific activity, but its nature was unclear.

A comparison of cysteine and serine proteinases in human gingival crevicular fluid with tissue, saliva and bacterial enzymes by analytical isoelectric focusing.

Gingival crevicular fluid (GCF) contains several different proteinase activities and the study sought to clarify their sources. Gingival tissue and GCF were collected from chronic periodontitis patients. Gel-filtration chromatography of crude tissue extracts yielded cathepsin B and tryptase fractions sensitive to cysteine and serine proteinase inhibitors, respectively. Cell sonicates of suspected periodontal pathogens were prepared from broth cultures of reference strains. Of these, Porphyromonas gingivalis showed much the strongest activity and this had an effector response consistent with the metal-dependent cysteine proteinase described by others. Banding patterns in GCF, tissue and bacterial samples were compared on substrate-impregnated overlay membranes applied to isoelectric focusing gels. On Z-Val-Lys-Lys-Arg-AFC overlays, GCF had bands corresponding to tissue cathepsin B and the enzyme from P. gingivalis, though a contribution from Treponema denticola could not be ruled out. Use of D-Val-Leu-Arg-AFC overlays showed GCF activity similar to tissue tryptase. In GCF there were additional bands that did not correspond to any tissue or bacterial samples and on Z-Ala-Ala-Lys-AFC overlays these closely resembled activity in parotid saliva. The results confirmed that GCF contains tissue cathepsin B and tryptase, while the apparent presence of enzymes from P. gingivalis and possibly T. denticola is consistent with previous reports linking activity to these organisms. The saliva bands demonstrated that contamination of GCF may occur despite rigorous collection procedures.

Use of CHROMagar Candida medium for isolation of yeasts from dental samples.

A new differential medium, CHROMagar Candida, for the isolation of clinically important yeasts was investigated to determine its usefulness in facilitating the study of oral yeasts. The recovery of yeasts on the medium was not significantly different from the recovery on Sabouraud dextrose agar. The identities of 450 green colonies on CHROMagar Candida, presumptively identified as Candida albicans on the basis of the manufacturer's instructions, were confirmed by testing for beta-N-acetylgalactosaminidase. Candida tropicalis also formed distinctive colonies, and other yeasts including Candida (Torulopsis) glabrata, Candida Parapsilosis, Candida Magnoliae, Candida lusitaniae, Candida Famata, Candida kefir, and Saccharomyces cerevisiae were readily distinguished from C. albicans and C. tropicalis isolates. CHROMagar Candida is a very useful medium, and its use will facilitate the study of yeasts associated with dental diseases.

Comparison of host tissue and bacterial dipeptidyl peptidases in human gingival crevicular fluid by analytical isoelectric focusing.

Earlier work has shown that gingival crevicular fluid (GCF) contains dipeptidyl peptidase (DPP) activities that resemble those in host tissue. Here, further comparisons were made with enzymes from suspected periodontal pathogens. Gingival tissue and GCF were collected from patients with chronic periodontitis. DPP II and DPP IV fractions with acid and alkaline pH optima, respectively, were separated from crude tissue extracts by gel-filtration chromatography. Bacterial cell sonicates were prepared from broth cultures of reference strains. There was moderate to strong DPP activity with Capnocytophaga spp., Porphyromonas gingivalis and Prevotella spp., very weak activity with Treponema denticola and no detectable activity with Actinobacillus actinomycetemcomitans or Fusobacterium nucleatum. Banding patterns in GCF, tissue and bacterial samples were compared on substrate-impregnated overlay membranes applied to isoelectric focusing gels. In gels washed with acid buffer, GCF had bands corresponding to tissue DPP II. Use of an alkaline washing buffer showed GCF activity which closely matched tissue DPP IV that had been pretreated with neuraminidase, an enzyme found by others in the gingival crevice. P. Gingivalis gave multiple bands and several of these had counterparts in GCF. The apparent presence in GCF of the DPP from P. gingivalis is consistent with the association of this organism with destructive periodontitis.

Prophylactic application of an intra-alveolar socket medicament to reduce postextraction complications in HIV-seropositive patients.

A prospective, controlled trial was performed to investigate the effect of a prophylactic socket medicament containing chlortetracycline, aspirin, and local anesthetics, in reducing the incidence of postextraction pain and infection in patients who are HIV-seropositive. Fifty patients were in the study; 25 received the experimental dressing and 25 had no dressing. Patients were asked to complete a questionnaire rating the pain at 24 hours and 48 hours after surgery. Seven days after extraction, socket healing was scored, and sockets with delayed healing sampled for culture. Four of 25 (16%) patients who received the experimental dressing and 8 of 25 (32%) controls experienced pain during the 48 hours after extraction (p = not significant). None of the group who received the experimental dressing and 7 of 25 (28%) in the control group had delayed healing, of these 4 had alveolar osteitis and three had infected sockets (p = 0.0096). We conclude that the experimental agent is useful as a prophylactic agent to reduce delayed healing in HIV-positive patients who undergo exodontia.

The effects of Acacia arabica gum on the in vitro growth and protease activities of periodontopathic bacteria.

The antibacterial activity of acacia gum was assessed using fresh isolates and reference strains of Actinobacillus actinomycetemcomitans, Capnocytophaga spp., Porphyromonas gingivalis, Prevotella intermedia and Treponema denticola. A fine aqueous suspension of gum was produced by sonication and then a soluble fraction isolated by centrifugation and membrane filtration. These preparations were incorporated into columbia agar at doubling concentrations. Growth of P. gingivalis and P. intermedia cultures on the agar was inhibited by whole gum sonicate at concentrations of 0.5-1.0% w/v. Both species showed reduced susceptibility when horse blood was present in the agar. The gum soluble fraction did not inhibit growth of any bacterial culture. The effect of acacia on bacterial proteases was examined with cell sonicates from log phase broth cultures. Enzyme activities were determined by fluorimetric assay with various synthetic peptide substrates. Most protease activities reduced in the presence of 0.5% w/v gum sonicate, with the trypsin-like activities of P. gingivalis and P. intermedia proving most sensitive. The gum soluble fraction was nearly always less inhibitory than the sonicate. The action of acacia gum against suspected periodontal pathogens and their enzymes suggests that it may be of clinical value.

Zinc sulfide in intestinal cell granules of Ancylostoma caninum adults.

A source of confusion has existed since the turn of the century about the reddish brown, weakly birefringent "sphaerocrystals" located in the intestines of strongyle nematodes, Strongylus and Ancylostoma. X-ray diffraction and energy dispersive spectrometric analyses were used for accurate determination of the crystalline order and elemental composition of the granules in the canine hookworm Ancylostoma caninum. The composition of the intestinal pigmented granules was identified unequivocally as zinc sulfide. It seems most probable that the granules serve to detoxify high levels of metallic ions (specifically zinc) present due to the large intake of host blood.

An experimental and theoretical investigation of the core level spectra of a series of amino acids, dipeptides and polypeptides.

A series of simple amino acids, dipeptides and polypeptides have been studied experimentally by electron spectroscopy for chemical analysis and the results theoretically quantified using the charge potential model and semi-empirically computed charge distributions, and non-empirical calculations of absolute and relative binding energies for model systems. Core binding energies are shown to be characteristic of given structural features and the experimental data taken in conjunction with the model theoretical calculations indicate a zwitterionic structure with extensive intermolecular hydrogen bonding for simple amino acids in the solid state.

Relationship of pasture rotation to acquisition of gastrointestinal nematodes by sheep.

In a study of the relationship between pasture rotation in Illinois and acquisition of nematodes (mostly Haemonchus contortus) and body weight gains by lambs grazing with their ewes, 2 pasture rotation systems were tested. (1) Lambs and ewes were rotated through a series of 12 alfalfa-bromegrass-lespedeza pastures, each pasture being grazed for 3 to 4 days and rested for 5.5 weeks; 4 complete rotations were done during a 168-day grazing season. (2) Lambs and ewes were moved every 2 days, and 3 complete rotations of 50, 42, and 54 days, respectively, were done during the 146-day grazing season. The lambs under rotation had more nematodes and gained less weight than nonrotated control lambs, although rotation increased the amount of pasturage. Rotation is not recommended to control nematode parasitism of sheep in Illinois.