On the fit of statistical and the k-C* models to projecting treatment performance in a constructed wetland system.

The objective of this study was to assess the suitability of statistical and the k-C* models to projecting treatment performance of constructed wetlands by applying the models to predict the final effluent concentrations of a pilot field-scale constructed wetlands system (CWs) treating animal farm wastewater. The CWs achieved removal rates (in g/m(2).d) ranging from 7.1-149.8 for BOD(5), 49.8-253.8 for COD and 7.1-47.0 for NH(4)-N. Generally, it was found that the statistical models developed from multiple regression analyses (MRA) were stronger in predicting final effluent concentrations than the k-C* model. However, both models were inadequate in predicting the final effluent concentrations of NO(3)-N. The first-order area-based removal rate constants (k, m/yr) determined from the experimental data were 200.5 for BOD(5), 80.1 for TP and 173.8 for NH(4)-N and these indicate a high rate of pollutant removal within the CWs.

Multiplexed PCR genotyping of HPVs from plantaris verrucae.

BACKGROUND: Plantaris verrucae are a common diagnosis in childhood, consume a significant amount of health-care resources, have many painful treatment options and many recurrences. OBJECTIVES: The objective of this study was to design and test a single site-anchored, multiplexed and expandable PCR assay for common types of cutaneous HPVs. STUDY DESIGN: Common forward and unique reverse primers were selected from the E2 open reading frames of five cutaneous HPV genotypes. These were analyzed for sensitivity and selectivity using pHPV plasmids and several control DNAs in an optimized multiplexed assay. This standardized assay was used to analyze human verruca plantaris tissue for genome type and to evaluate the effect of a commonly used treatment protocol. RESULTS: A sensitive, multiplexed PCR assay for human cutaneous HPV genotypes 1a, 2a and 4 was developed. Specific-unique primers and a consensus anchor primer were selected within the HPV E2 region to produce amplicons varying by greater than 100bp. In analytical sensitivity studies, fewer than 100 genome copies of HPV1a and 2a were detected, and fewer than 1000 copies of HPV4 were detected. The multiplexed assay did not amplify regions of human placenta, calf thymus, CaSki or SiHa DNA and E. coli, pBR322 or non-HPV virus DNAs. In combination with a forensic DNA extraction procedure, the multiplexed HPV assay detected and identified HPV types in 23 of 51 (45%) deep plantaris verrucae. Two patients were found with two different genotypes in single deep plantaris verruca. Detection of the HPV genome was followed as a function of tissue ablation and Mediplast treatment in one patient. In healing tissue, the genome content was reduced but had not totally disappeared. CONCLUSIONS: The multiplexed HPV assay can be used to determine genotype prevalence that may correlate with treatment efficacy.

Elastic, flexible peptidoglycan and bacterial cell wall properties.

The peptidoglycan sacculus serves as a mechanical framework for the cell walls of most eubacteria and largely determines cell shape. The notion that the structure is a rigid shell is contradicted by findings that peptidoglycan can expand and contract. Thus, the sacculus functions as an elastic, flexible, polyionic, amphoteric, restraining network.

Glycine prevents the phenotypic expression of streptococcal glucan-binding lectin.

Glycine has been used extensively in bacterial cell surface research. Some researchers employ glycine in growth media so as to increase the transformability of streptococci during electroporation. Others have found that glycine, similar to wall antibiotics, 'weakens' peptidoglycan. It is now shown that when glycine is incorporated into the growth medium, Streptococcus sobrinus exhibits a diminished ability to aggregate with high molecular weight alpha-1,6-glucan. Growth of the bacteria in either a rich or a chemically defined medium results in a cell population with full lectin (glucan-binding) fidelity. Incorporation of glycine, but not serine or other amino acids, at concentrations of 100-200 mM gives rise to bacteria with lowered lectin activities. Bacteriolytic enzymes were able to lyse bacteria from glycine-grown cultures more readily than from cultures without the glycine supplement. The bacteria produce glucan-binding proteins, including glucosyltransferases, but they do not readily aggregate with added dextran. Furthermore, SDS-PAGE gels of supernatants of growth media (+/-glycine) are similar, suggesting the bacteria do not produce a unique set of proteins. Western blotting with a fluorescein isothiocyanate-labeled dextran probe reveals normal amounts of glucan-binding proteins in glycine-grown streptococci. Glycine may be acting as a type of antibiotic, reducing wall integrity upon which glucan promoted cellular aggregation depends.

Modulation of glucan-binding protein activity in streptococci by fluoride.

Glucan-binding lectin (GBL) activity of Streptococcus sobrinus was significantly reduced by fluoride in the growth medium. Approximately 1.5 mM fluoride was required for a 50% reduction in GBL activity. In addition to the GBL, several other glucan-binding proteins were reduced when the bacteria were grown in subinhibitory fluoride. Fluoride had no effect on glucosyltransferases (GTFs), enzymes capable of converting sucrose into alpha-1,6-glucans. All the proteins were detected by use of enhanced chemiluminescence (ECL of fluorescein-labeled dextran) and Western blotting of renatured SDS-PAGE gels. The effects of fluoride on the bacteria were abrogated when the manganous ion was included in the growth medium. It thus appears that one mechanism of action of fluoridated water is its effects on glucan-binding proteins. The fluoride may be reducing metabolism of the mangano aquo ion, essential for expression of the glucan-binding proteins.

Polycarboxylates inhibit the glucan-binding lectin of Streptococcus sobrinus.

Polycarboxylates, such as carboxymethylcellulose and hyaluronan, were found to be reversible inhibitors of the glucan-binding lectin of Streptococcus sobrinus. When the carboxylate groups were coupled to ethylenediamine, or reduced with carbodiimide-borohydride, inhibitory powers were lost. Similarly, N-deacetylated hyaluronan had poor inhibitory powers, probably due to the introduction of positive charges into the polymer. Other polymers, such as chondroitin sulfates, dextran sulfate, fetuin, heparin were not inhibitors. It appears that inhibition is based on repeating carboxylates, free of influence from ammonium groups. Such polymers have the property of complexing with metals. Earlier studies had concluded that the streptococcal lectin depended on manganese for activity. It is likely the carboxymethylcellulose and hyaluronan perturb essential metal coordination centers in the lectin. Polycarboxylates may have value in oral health care by acting on glucan-dependent microbial adhesion and biofilm formation.

On the specificity of the D-galactose-binding lectin (PA-I) of Pseudomonas aeruginosa and its strong binding to hydrophobic derivatives of D-galactose and thiogalactose.

The D-galactose-binding lectin (PA-I) from the bacterium Pseudomonas aeruginosa, isolated by affinity chromatography on Sepharose, was examined for its relative affinities for simple sugars and their derivatives using equilibrium dialysis and hemagglutination inhibition tests. The lectin, which was found to bind 0.68 mol of D-galactose per subunit of 12.8 kDa, exhibited an association constant (Ka) of 3.4 x 10(4) M-1 for D-galactose and higher affinities for hydrophobic and thio derivatives of D-galactose (with highest affinity for the hydrophobic thio derivatives). alpha-Methyl-galactoside was a stronger inhibitor than the beta-methyl derivative and alpha-lactose was a weak inhibitor but the hydrophobic phenylated derivatives of the beta-configuration of D-galactose were more potent inhibitors than the respective alpha-galactosides.

Anomalies in cell wall turnover associated with the growth temperature of Bacillus subtilis.

Cell wall turnover appeared to be anomalously fast in Bacillus subtilis when the cells were grown at temperatures below 29 degrees C. Turnover rates k(generation-1), of exponential cultures at 25 degrees were approximately double those of cells grown at 37 degrees C. When autolysin levels were assayed in cell walls, it was found that the enzyme activities were constant between 25 degrees C and 40 degrees C, suggesting that there was no greater synthesis of autolysin at the lower temperature. Analyses of walls for individual components, extent of aminosugar substitution and extent of crosslinking, did not reveal significant differences between samples obtained from 25 degrees C or 37 degrees C cultures. The N-acetylmuramoyl-L-alanine amidase was stable over the temperature range studied. Lysis of cells, induced by carbonylcyanide-m-chlorophenylhydrazone, occurred at a faster rate for cells obtained at 25 degrees C than for cells obtained at 37 degrees C. In addition, the lysis of cells by hen egg white lysozyme was slightly faster when the cells were obtained from 25 degrees C cultures than from 37 degrees C cultures. It is possible the autolysin(s) responsible for cell wall turnover are cold-activated.

A new mechanism of action of fluoride on streptococci.

Addition of fluoride to the growth medium of Streptococcus sobrinus resulted in a loss of glucan-binding lectin activity. Upon removal of fluoride, the bacteria regained their ability to bind glucan in about one generation. Chloramphenicol prevented recovery of ability to produce the lectin, showing the requirement for protein synthesis. Fluoride also caused a significant reduction in the tendency of the streptococci to form chains of cells, although the spent medium from fluoride-containing growth media did not dechain control cells. The fluoride thus does not activate autolytic enzymes. Importantly, 2-D electrophoresis and SDS-PAGE revealed several proteins were synthesized in the presence of fluoride that were not synthesized in its absence. It seems possible that fluoride places a stress on the bacteria, causing the synthesis of proteins that may play a role in protecting the cells against the stress. Numerous stress proteins are known for bacteria, including those resulting from heat, enzymes and osmotic shocks. The ability of fluoride to cause loss of glucan-binding may be related to its reported beneficial effects on oral health.

Fusarium sp. growth inhibition by wheat germ agglutinin.

The antifungal role of wheat germ agglutinin (WGA) isolated from a Romanian dihaploid variety of wheat against two pathogenic fungal species of Fusarium, F. graminearum and F. oxysporum, is demonstrated. WGA was prepared from unprocessed wheat germs by a new purification procedure using chitin and fetuin-Sepharose as affinity chromatography supports. SDS-PAGE and chitinase assay showed that the WGA preparation migrated as a single protein band and was devoid of any contaminating enzyme chitinase, well known for its antifungal effects. Based on its affinity for N-acetylglucosamine residues, WGA binding to the chitin-containing walls of the fungi was detected by fluorescence microscopy using WGA coupled with fluorescein isothiocyanate (FITC). In vitro testing of WGA action on early developmental stages of both fungal strains resulted in various modifications of the germ tubes, visualised by light microscopy: swelling, vacuolation of the cellular content and lysis of cell walls. Viability tests performed on potato tuber slices showed that the microbial infection was prevented from spreading by pretreatment of the fungal suspension with WGA.

Binding of hydrophobic ligands by Pseudomonas aeruginosa PA-I lectin.

The ability of Pseudomonas aeruginosa PA-I lectin to bind the fluorescent hydrophobic probe, 2-(p-toluidinyl) naphthalene sulfonic acid (TNS), and adenine was examined by spectrofluorametry and equilibrium dialysis. Interaction of TNS with PA-I caused significant enhancement of TNS fluorescence. The Hill coefficient (3.8+/-0.3) and the dissociation constant (8.7+/-0.16 microM) showed that TNS probably bound to four high affinity hydrophobic sites per PA-I tetramer. Interactions between PA-I and adenine were examined by equilibrium dialysis using [3H] adenine. The results indicated the presence of at least two classes of binding sites--one high and four lower affinity sites per tetramer with dissociation constants of 3.7+/-1.5 and 42.6+/-1.2 microM, respectively. These were distinct from the TNS sites as titration of TNS-equilibrated PA-I with adenine caused TNS fluorescence enhancement. The titration curve confirmed the existence of two classes of adenine-binding sites. Conversely, when PA-I was first equilibrated with adenine and then titrated with TNS, no TNS-binding was registered. This may indicate that conformational rearrangements of the lectin molecule caused by adenine prevent allosterically TNS binding.

Specificity and stability of guinea pig anti-progesterone antibodies.

Antibodies against progesterone were induced in guinea pigs of both sexes by injection of progesterone-y beta-hemisuccinate conjugated to bovine serum albumin (BSA) in a ratio of 16 moles of steroid per mole of protein. The concentration of antibody binding sites for progesterone of the animals studied ranged from 5 to 20 microM. The expected heterogeneity of binding affinity for progesterone was observed with two major populations apparently predominating. On bound progesterone with an average affinity greater than 2 X 10(9) M-1 and the other showed an average affinity less than or equal to 6 X 10(6) M-1. The antibodies were fond to be stable to extremes of pH and temperature in serum as well as in solutions of ammonium sulfate precipitates. The antibodies were not stable, however, in a more highly purified form. Attempts to obtain active preparations in high yield by purification beyond the ammonium sulfate step were unsuccessful. Competition studies and direct analysis with radiolabeled steroids showed the high-affinity population to be relatively specific for progesterone binding, whereas other steroids were bound according to the polarity rule indicating that the binding forces are predominantly hydrophobic.

Contribution of the hydrophobic effect to microbial infection.

The hydrophobic effect has been known for decades. Numerous researchers have invoked the hydrophobic effect to explain how pathogens adhere to tissues. In some cases, inhibition of adhesion can be brought about by low concentrations of aromatic compounds, such as p-nitrophenol or tryptophan. Because the hydrophobic effect has been considered to be nonspecific, the molecular biology of adhesive hydrophobins has not been studied in as much detail as lectin adhesins. The literature provides compelling evidence that a large number of bacterial and fungal pathogens depend on hydrophobic interactions for successful colonization of a host. Several laboratories are now developing effective antiadhesins, based on inhibition of hydrophobic interactions between the host and the pathogen.

Tetranitromethane as a surface antiviral disinfectant.

Tetranitromethane, a protein nitrating agent, was tested for its ability to disinfect surfaces from viruses. Different surfaces on commercially available pocket calculators were pretreated with either the Indiana strain of vesicular stomatitis virus or the Herts' strain of Newcastle disease virus. The calculators surfaces were then sprayed with either tetranitromethane or control solutions. The calculators were incubated for 30 min at ambient temperature, and then the surfaces were wiped with sterile swabs. The swabs were placed into test tubes containing phosphate-buffered saline. Samples of the phosphate-buffered saline were then titered on appropriate cell lines by plaque assay. The results indicated that the amount of vesicular stomatitis virus and Newcastle disease virus recovered from the tetranitromethane-treated surfaces was dramatically decreased compared to the amount of virus recovered from control-treated surfaces. These data suggest that tetranitromethane may be useful to disinfect surfaces from both enveloped and non-enveloped RNA viruses.

Inhibition of the cooperative adhesion of Streptococcus sanguis to hydroxylapatite.

The adhesion of Streptococcus sanguis to hydroxylapatite is a process involving several adhesins and receptors. Binding isotherms and Scatchard plots of the adhesion suggest that cooperative interactions occur at low cell densities. It was found that sulfolane, a hydrophobic-bond diluent, was capable of inhibiting the cooperative adhesion of S. sanguis to saliva-coated hydroxylapatite beads. Sodium thiocyanate, a chaotropic agent, inhibited not only cooperative adhesion, but also the adhesion thought to result from noncooperative interactions. It is suggested that strong chaotropic agents may not only inhibit adhesin-receptor complexes, but also may influence the secondary/tertiary structures of interacting species.

DNA from Serratia marcescens confers a hydrophobic character in Escherichia coli.

In order to determine whether hydrophobic surface properties of Serratia marcescens can be transferred to Escherichia coli, E. coli DH5 alpha cells were transformed by DNA fragments from S. marcescens RZ. Fifteen-hundred E. coli transformants were screened for adhesion to hexadecane and polystyrene. One transformant exhibited increased adhesion to hexadecane droplets, as well as altered kinetics of aggregation in the presence of ammonium sulfate. Western colony blotting revealed that antibodies raised against S. marcescens RZ recognized component(s) on the transformant outer surface.

Acrylonitrile induces autolysis Bacillus subtilis.

Acrylonitrile (AN) is an industrial chemical used in the manufacture of plastics and other polymers. AN has been reported to be an acute toxin and is a known carcinogen in rodents. When AN was mixed with suspensions of Bacillus subtilis, the bacteria began autolysis. It was determined that AN is partially converted to cyanide, a strong protonophore in B. subtilis. Autolytic enzymes in B. subtilis become active when the protonmotive force is dissipated. The amount of cyanide produced from AN, however, was not enough to promote autolysis in exponential B. subtilis. This is the first report showing that AN may induce autolytic reactions in bacteria. It is suggested the autolysis of B. subtilis may be useful in the environmental monitoring of AN. In addition, the metabolism of AN by bacilli may be useful in bioremediation.

On the origin of membrane vesicles in gram-negative bacteria.

It is proposed that the genesis of extracellular membrane vesicles in Gram-negative bacteria is a result of cell wall turnover. Peptidoglycan turnover would cause a turgor on the outer membrane, causing the outer membrane to bulge and finally bleb. Mechanical motion would then shear the blebs into the culture medium.

Isolation and purification of cell wall polysaccharide of Bacillus anthracis (delta Sterne).

A polysaccharide fraction was isolated form sodium-dodecyl-sulfate (SDS) treated cell walls of Bacillus anthracis (delta Sterne) by hydrofluoric acid (HF) hydrolysis and ethanolic precipitation. The polysaccharide fraction was subsequently purified by several washings with absolute ethanol. Purity of the isolated polysaccharide was tested using the anthrone assay and amino acid analyzer. The molecular mass of the polysaccharide fraction as determined by gel filtration chromatography was about 12000 Da. Preliminary analyses of the polysaccharide was done using thin layer chromatography and amino acid analyzer, and results obtained from these analyses were further confirmed by gas liquid chromatography and 13C-NMR spectroscopy. Results showed that the polysaccharide moiety contained galactose, N-acetylglucosamine, and N-acetylmannosamine in an approximate molar ratio of 3:2:1. This moiety was devoid of muramic acid, alanine, diaminopimelic acid, glutamic acid, and lipid, thus indicating that the isolated polysaccharide was of pure quality.

Fluoride inhibits the glucan-binding lectin of Streptococcus sobrinus.

The glucan-binding lectins of Streptococcus cricetus AHT and Streptococcus sobrinus 6715 were reversibly inhibited by sodium fluoride. Fluoride was superior to chloride, bromide, iodide and thiocyanate in preventing glucan-mediated aggregation of the bacteria. Fluoride was also an effective inhibitor of the sucrose-dependent adhesion of S. sobrinus to glass surfaces. The inhibition of glucan-binding lectin activities may be one of the mechanisms of action of fluoride in preventing dental disease.