Prevention of artificial dental plaque formation in vitro by plant extracts.

AIMS: A number of previous studies have shown that plant extracts can inhibit formation of dental plaque. The ability of extracts of Rosmarinus officianalis L., Salvia officianalis L., unfermented cocoa, red grape seed and green tea to inhibit plaque bacteria, glucosyltransferase activity, glucan and plaque formation in an in vitro model using bovine teeth was examined. METHODS AND RESULTS: The antimicrobial activity of the plant extracts against oral bacteria was determined using a standard susceptibility agar dilution technique. Inhibition of growth and acid production from glucose and sucrose by Streptococcus mutans in liquid culture was investigated. Prevention of plaque formation on bovine teeth initiated by Strep. mutans was studied using an artificial mouth. The plant extracts inhibited the growth of oral bacteria and prevented acid production by Strep. mutans. Extracts inhibited glucosyltransferase activity and glucan production and inhibited adhesion to glass. Extracts of R. officianalis L. and S. officianalis L. at 0·25 mg ml(-1) reduced plaque growth by >80%. Green tea extract completely inhibited plaque formation but resulted in a greenish discolouration of the teeth which could not be removed by scrubbing. CONCLUSIONS: The plant extracts, particularly those from R. officianalis L. and S. officianalis L., inhibited glucosyltranferase activity, glucan production and plaque formation in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that the extracts of R. officianalis L. and S. officianalis L. may be useful as antiplaque agents in foods and dental preparations. Bovine teeth can be used as an alternative to hydroxyapatite for studies of plaque formation, but they need to be carefully sterilized before use.

Regulation of IL-2 expression by transcription factor BACH2 in umbilical cord blood CD4+ T cells.

On activation, umbilical cord blood (UCB) CD4(+) T cells demonstrate reduced expression of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), whereas maintaining equivalent interleukin-2 (IL-2) levels, as compared with adult peripheral blood (PB) CD4(+) T cells. Nuclear factor of activated T cells (NFAT1) protein, a transcription factor known to regulate the expression of IL-2, TNF-alpha and IFN-gamma, is reduced in resting and activated UCB CD4(+) T cells. In contrast, expression of Broad-complex-Tramtrack-Bric-a-Brac and Cap'n'collar homology 1 bZip transcription factor 2 (BACH2) was shown by gene array analyses to be increased in UCB CD4(+) T cells and was validated by qRT-PCR. Using chromatin immunoprecipitation, BACH2 was shown binding to the human IL-2 proximal promoter. Knockdown experiments of BACH2 by transient transfection of UCB CD4(+) T cells with BACH2 siRNA resulted in significant reductions in stimulated IL-2 production. Decreased IL-2 gene transcription in UCB CD4(+) T cells transfected with BACH2 siRNA was confirmed by a human IL-2 luciferase assay. In summary, BACH2 maintains IL-2 expression in UCB CD4(+) T cells at levels equivalent to adult PB CD4(+) T cells despite reduced NFAT1 protein expression. Thus, BACH2 expression is necessary to maintain IL-2 production when NFAT1 protein is reduced, potentially impacting UCB graft CD4(+) T-cell allogeneic responses.

MethylScreen: DNA methylation density monitoring using quantitative PCR.

Aberrant gene silencing of genes through cytosine methylation has been demonstrated during the development of many types of cancers including prostate cancer Several genes including GSTP1 have been shown to be methylated in prostate cancer leading to the suggestion and demonstration that methylation status of such genes could be used as cancer diagnosis markers alone or in support of histology. We developed a bisulfite-free alternative, MethylScreen technology, an assay for DNA methylation detection utilizing combined restriction from both methylation-sensitive restriction enzymes (MSRE) and methylation-dependent restriction enzymes (MDRE). MethylScreen was used to analyze the 5' region of GSTP1 in cell lines, in vitro methylated DNA populations, and flash-frozen tissue samples in an effort to characterize the output and analytical performance characteristics of the assay. The output from the quantitative PCR assay suggested that it could not only detect fully methylated molecules in a mixed population below the 1% level, but it could also quantify the abundance of intermediately methylated molecules. Interestingly, the interpreted output from the four quantitative PCRs closely resembled the molecular population as described by clone-based bisulfite genomic sequencing.

Comparison of the efficacies of disinfectants to control microbial contamination in dental unit water systems in general dental practices across the European Union.

Water delivered by dental unit water systems (DUWS) in general dental practices can harbor high numbers of bacteria, including opportunistic pathogens. Biofilms on tubing within DUWS provide a reservoir for microorganisms and should be controlled. This study compared disinfection products for their ability to meet the American Dental Association's guideline of <200 CFU x ml(-1) for DUWS water. Alpron, BioBlue, Dentosept, Oxygenal, Sanosil, Sterilex Ultra, and Ster4Spray were tested in DUWS (n = 134) in Denmark, Germany, Greece, Ireland, The Netherlands, Spain, and the United Kingdom. Weekly water samples were tested for total viable counts (TVCs) on yeast extract agar, and, where possible, the effects of products on established biofilm (TVCs) were measured. A 4- to 5-week baseline measurement period was followed by 6 to 8 weeks of disinfection (intermittent or continuous product application). DUWS water TVCs before disinfection ranged from 0 to 5.41 log CFU x ml(-1). Disinfectants achieved reductions in the median water TVC ranging from 0.69 (Ster4Spray) to 3.11 (Dentosept) log CFU x ml(-1), although occasional high values (up to 4.88 log CFU x ml(-1)) occurred with all products. Before treatment, 64% of all baseline samples exceeded American Dental Association guidelines, compared to only 17% following commencement of treatment; where tested, biofilm TVCs were reduced to below detectable levels. The antimicrobial efficacies of products varied (e.g., 91% of water samples from DUWS treated with Dentosept or Oxygenal met American Dental Association guidelines, compared to 60% of those treated with Ster4Spray). Overall, the continuously applied products performed better than those applied intermittently. The most effective products were Dentosept and Oxygenal, although Dentosept gave the most consistent and sustained antimicrobial effect over time.

Gas chromatographic method for the analysis of allelopathic natural products in rye (Secale cereale L.).

Accurate and reproducible methods for the analysis of plant allelochemicals are a requirement for the study of chemical interactions between plants. This paper describes a method for sample preparation and quantitative analysis of the allelopathic chemical content of rye (Secale cereale L.) using gas chromatography (GC). Sample preparation consists of extraction of freeze-dried rye vegetative tissue with aqueous ethanol followed by partitioning of the allelochemicals into ethyl acetate, evaporation, and derivatization using the trimethylsilylating reagent N-methyl-N-trimethylsilyltrifluoroacetamide. GC analysis of the silylated mixture was performed using flame ionization detection. This method permits analysis of all known rye allelopathic agents including 2,4-dihydroxy-1,4-benzoxazin-3-one, its corresponding glucoside, 2-benzoxazolinone, beta-hydroxybutyric acid, and beta-phenyllactic acid. Identities of all compounds were confirmed by GC/MS analysis.

Microbiological evaluation of dental unit water systems in general dental practice in Europe.

A range of opportunistic pathogens have been associated with dental unit water systems (DUWS), particularly in the biofilms that can line the tubing. This study therefore aimed to assess the microbiology of DUWS and biofilms in general dental practices across seven European countries, including the United Kingdom (UK), Ireland (IRL), Greece (GR), Spain (ES), Germany (D), Denmark (DK) and the Netherlands (NL). Water supplied by 51% of 237 dental unit water lines exceeded current American Dental Association recommendations of < or = 200 colony-forming units (CFU) ml(-1). Microbiological loading of the source waters was between 0 (Denmark, the Netherlands and Spain) and 4.67 (IRL) log CFU ml(-1); water line samples from the DUWS ranged from 1.52 (ES) to 2.79 (GR) log CFU ml(-1); and biofilm counts ranged from 1.49 (GR) to 3.22 (DK) log CFU.cm(-2). Opportunistic pathogens such as legionellae (DK and ES), including Legionella pneumophila SG1 (DK and GR), and Mycobacterium spp. (DK, NL, GR, D and ES) were recovered occasionally. Presumptive oral streptococci (ES and NL), oral anaerobes (GR), Candida spp. (UK, NL and ES) and blood (GR and IRL) were detected at relatively low frequencies, but their presence indicated a failure of the 3-in-1 antiretraction valve, leading to back siphonage of oral fluids into the water and biofilm phase. These findings confirm that a substantial proportion of DUWS have high levels of microbial contamination, irrespective of country, type of equipment and source water. The study emphasizes the need for effective mechanisms to reduce the microbial burden within DUWS, and highlights the risk of occupational exposure and cross-infection in general dental practice.

Evaluation of Chromocult coliform agar for the detection and enumeration of Enterobacteriaceae from faecal samples from healthy subjects.

The purpose of this study was to examine the use of Chromocult agar medium for isolation and enumeration of Enterobacteriaceae from human faecal samples, to compare it to MacConkey agar and to evaluate its usefulness as a possible alternative selective medium in human faecal studies. The medium was shown to be effective in identifying Escherichia coli and coliforms in faeces without the need for extensive accompanying biochemical tests for confirmation of identity. A positive correlation (r=0.86) was found between the recovery of Enterobacteriaceae on the two media, and no significant difference (P>0.05) between overall mean bacterial counts for the whole study group or at different intervals of faecal collection were observed. Chromocult agar is an effective replacement for MacConkey agar in human faecal studies and has the advantage of differentiating E. coli from other coliforms.

Molecular cloning of PCR products.

It is often desirable to clone PCR products to establish a permanent source of cloned DNA for hybridization studies, to obtain high-quality DNA sequencing results, or to separate products when PCR amplification yields a complex mixture. The efficiency of direct cloning of PCR products can be improved by generating suitable ends on the amplified fragments. This unit describes the strategies for generating and manipulating suitable ends on the PCR fragments.

Pulsed-field gel electrophoresis.

DNA molecules longer than 25 kb are poorly resolved by standard agarose gel electrophoresis. These longer molecules can be resolved using several techniques that periodically change the direction of the electric field in the gel. This unit describes the simplest and most generally useful of the pulsed-field techniques, field inversion electrophoresis, which can be tuned to resolve molecules from 10 to 2000 kb (or more with specialized equipment). To resolve molecules beyond the range of field inversion, it is necessary to use some sort of field-angle alternation electrophoresis such as CHEF (contour-clamped homogeneous electric field; described in an Alternate Protocol). A method is also provided for preparing high-molecular-weight DNA samples and size markers embedded in agarose blocks.

Using wound fluid analyses to identify trace element requirements for efficient healing.

A series of wound fluid and blood plasma samples from 20 patients with breast cancer were analysed by Potentiometric Stripping Analysis and computer-aided chemical speciation to quantify the concentrations of the trace elements of copper and zinc in the samples and to investigate the individual species of copper and zinc present. Comparisons were made between total concentrations of copper and zinc in wound fluid, pre-operative blood plasma levels and reference values. A wound fluid model constructed using JESS identified the main copper and zinc species present. It was also used to investigate the effects of a change in pH and changes in the total concentrations of certain components on their predominance. The clinical significance of the research is discussed, together with suggestions for a continuation in the research.

What the mouth has to say about diabetes. Careful examinations can avert serious complications.

Periodontal disease is a major but preventable complication of diabetes mellitus. Patient education, good glycemic control, regular dental care, appropriate diet, and a team approach that involves physicians, dietitians, dentists, and other health professionals offer the best chance for optimum care for these patients. Other oral complications of diabetes include tooth decay, xerostomia, candidiasis, and oral peripheral neuropathy. The mouth may also reflect secondary causes of diabetes, and oral examination may provide clues to diseases that coexist with type 1 diabetes. Truly, the mouth has much to say about diabetes.

Self-seal reagent: evaporation control for molecular histology procedures without chambers, clips or fingernail polish.

Sensitive nucleic acid based detection methods such as in situ PCR, in situ RT-PCR and PRINS have great potential in the areas of developmental biology, pathogenesis and diagnostics. However, control of evaporation from in situ reactions is critical to ensure reliable data. Self-Seal Reagent, a component added directly to the in situ reaction mixture, effectively controls evaporation during in situ procedures by creating an evaporation-limiting barrier around the periphery of a standard cover glass as the reaction proceeds. At the end of the procedure, the cover glass is easily removed by soaking in an aqueous solution. A model is presented for how Self-Seal Reagent controls evaporation while maintaining reagent concentrations. Self-Seal Reagent is shown to be effective in the detection of HIV sequences in cells by in situ PCR.

Engagement of CD20 suppresses apoptosis in germinal center B cells.

In a screen of 67 monoclonal antibodies (mAb) included in the Blind Panel of B cell antibodies for the 5th International Workshop on Human Leukocyte Differentiation Antigens, only the CD20 mAb--included as a positive control for immunophenotyping studies--was found to suppress the spontaneous apoptosis which occurs in human germinal center (GC) B cells when placed in tissue culture at 37 degrees C. Further detailed study using the 1F5 mAb confirmed this observation, showing that rescue from apoptosis via CD20, while not as efficient as that obtained on ligating CD40, was of similar magnitude to that achieved on engagement of surface immunoglobulin (sIg) by immobilized antibody. Also similar to anti-Ig, the CD20 mAb rescued from apoptosis without priming for the proliferation of GC B cells: this was quite different to its action on resting, non-GC B cells, where it provides a potent priming signal for cell cycle progression in response to IL-4 or anti-CD40. Unlike the survival signal engendered via sIg, CD20 engagement neither mobilized Ca2+ from intracellular stores or opening of a Ca2+ channel with 1F5, nor did it affect the ability of anti-Ig to open a Ca2+ gate in GC B cells. An unexpected feature of CD20-mediated rescue of GC B cells from apoptosis was a failure to turn on Bcl-2 expression.

Regulation of neurite outgrowth from differentiated human neuroepithelial cells: a comparison of the activities of prothrombin and thrombin.

The mechanism by which thrombin and prothrombin control neurite retraction was studied in Ad12E1HER10 human neuroepithelial cells. Morphological changes in differentiated cells were apparent within minutes of the addition of very low concentrations of thrombin (3 pM). Higher concentrations (2 nM) of prothrombin were required to elicit a similar response. Doses of thrombin and prothrombin sufficient to cause neurite retraction stimulated protein tyrosine kinase activity. Protein tyrosine kinase activation also correlated positively with thrombin- and prothrombin-induced phosphoinositide 3-kinase activation and InsP6 dephosphorylation. However, thrombin-stimulated Ins(1,4,5)P3 generation and intracellular Ca2+ mobilization only occurred at concentrations in excess of those needed to induce retraction. No fluctuations in Ins(1,4,5)P3 were detected after stimulation with prothrombin, and no rapid synchronized release of Ca2+ was observed, even at very high concentrations. Prothrombin did, however, cause small oscillations in the intracellular Ca2+ concentration, similar to those produced by low concentrations of thrombin, after approximately 30 min. We conclude that prothrombin- and thrombin-induced neurite retractions are not dependent on PtdIns(4,5)P2 and Ca2+ mobilization, but are more probably mediated through an effector mechanism involving protein tyrosine kinase activation. No intracellular Ca2+ mobilization, protein tyrosine kinase activity or neurite retraction was observed after treatment of cells with proteolytically inactive mutant thrombin (S205-->A). Prothrombin-mediated intracellular Ca2+ mobilization and neurite retraction were inhibited by hirudin, which was shown to interact with thrombin but not prothrombin. It is concluded that cleavage of prothrombin to thrombin is a necessary prerequisite for biological activity on differentiated Ad12E1HER10 cells and that differences in agonist concentration are capable of coupling the thrombin receptor to different pathways within the cell.

Targeted mutations in the Caenorhabditis elegans POU homeo box gene ceh-18 cause defects in oocyte cell cycle arrest, gonad migration, and epidermal differentiation.

We used targeted gene inactivation to analyze the function of a Caenorhabditis elegans POU gene, ceh-18, and to dissect its functional domains in vivo. In ceh-18 mutants, oocytes exhibit an incompletely penetrant failure to arrest in diakinesis of meiotic prophase I and instead undergo multiple rounds of DNA replication without cytokinesis. ceh-18 is expressed in the gonadal sheath cells that signal the oocyte, but not in the oocyte. This suggests that ceh-18 affects, directly or indirectly, a sheath cell signal that causes oocytes to maintain diakinesis arrest. ceh-18 also participates in directing gonad migration and in specifying the differentiated phenotypes of epidermal cells during postembryonic development. Analysis of targeted deletions that disrupt half of the POU domain selectively by deleting either the POUhd or the POUsp alone, indicates that each CEH-18 POU subdomain is sufficient for partial activity in vivo.

Quantification of nasal involvement in a guinea pig plethysmograph.

We have developed a technique for measuring lung function in conscious guinea pigs using a whole body plethysmograph. Because guinea pigs breathe through the nose, a technique was also developed to measure nasal and lower respiratory system conductance simultaneously in anesthetized animals. The upper and the lower airways could be challenged separately and studied in a manner similar to the conditions in the plethysmograph. Aerosols of histamine, carbachol, or ovalbumin delivered to the nose in sensitized animals had no effect on nasal conductance, even in doses 100 times higher than that required to reduce lower respiratory system conductance. However, intravenous histamine increased nasal conductance. Thus, although nasal resistance constitutes the majority of the total respiratory system resistance measured in the plethysmograph, nasal resistance is unaffected by the aerosol drugs studied. We therefore consider changes in resistance measured in the plethysmograph to originate at or below the larynx. The plethysmographic technique described here is a reliable, reproducible, and rapid technique that enables repeated measurement in animals and minimizes animal trauma.