Suspension Assay Research Articles

Antimicrobial activities of pomegranate rind extracts: enhancement by addition of metal salts and vitamin C

BackgroundPunica granatum L. or pomegranates, have been reported to have antimicrobial activity against a range of Gram positive and negative bacteria. Pomegranate formulations containing ferrous salts have enhanced although short-term, antibacteriophage activities which are rapidly diminished owing to instability of the ferrous combination. The aim of this study was to determine the antimicrobial activities of combinations of pomegranate rind extracts (PRE) with a range of metals salts with the added stabiliser vitamin C.MethodsPRE solutions, prepared by blending rind sections with distilled water prior to sterilisation by autoclaving or filtration, were screened with a disc diffusion assay using penicillin G as a control. Suspension assays were used to determine the antimicrobial activities of PRE alone and in combination with salts of the following metals; Fe (II), Cu (II), Mn (II) or Zn (II), and vitamin C, against a panel of microbes following exposure for 30 mins. The test organisms included Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and Proteus mirabilis.ResultsThe screening assay demonstrated that PRE exhibited activity against the Gram positive organisms at 24 h with no observable effect on any of the Gram negative bacteria. However, after 12 h, zones of inhibition were only observed for Ps. aeruginosa. In contrast, using the suspension assay, addition of Cu (II) salts to PRE solutions extended the activities resulting in no detectable growth being observed for the PRE/Cu (II) combination against E. coli, Ps. aeruginosa and P. mirabilis. Minimal antimicrobial activity was observed following incubation with Fe (II), Mn (II) or Zn (II) salts alone or in combination with PRE against any of the organisms in the test panel. The addition of vitamin C markedly enhanced the activities of both PRE/Fe (II) and PRE/Cu (II) combinations against S. aureus.ConclusionThis is the first report demonstrating the enhanced efficacy of PRE/metal salt combinations in the presence of the stabilising agent vitamin C, to which all isolates were sensitive with the exception of B. subtilis. This study has validated the exploration of PRE along with additives such as metal salts and vitamin C as novel antimicrobial combinations.

Bioavailability of Cd, Zn and Hg in Soil to Nine Recombinant Luminescent Metal Sensor Bacteria.

A set of nine recombinant heavy metal-specific luminescent bacterial sensors belonging to Gram-negative (Escherichia and Pseudomonas) and Gram-positive (Staphylococcus and Bacillus) genera and containing various types of recombinant metal-response genetic elements was characterized for heavy metal bioavailability studies. All nine strains were induced by Hg and Cd and five strains also by Zn. As a lowest limit, the sensors were detecting 0.03 μg·L-1 of Hg, 2 μg·L-1 of Cd and 400 μg·L-1 of Zn. Limit of determination of the sensors depended mostly on metal-response element, whereas the toxicity of those metals towards the sensor bacteria was mostly dependent on the type of the host bacterium, with Gram-positive strains being more sensitive than Gram-negative ones. The set of sensors was used to evaluate bioavailability of Hg, Cd and Zn in spiked soils. The bioavailable fraction of Cd and Zn in soil suspension assay (2.6 – 5.1% and 0.32 – 0.61%, of the total Cd and Zn, respectively) was almost comparable for all the sensors, whereas the bioavailability of Hg was about 10-fold higher for Gram-negative sensor cells (30.5% of total Hg), compared to Gram-positive ones (3.2% of the total Hg). For Zn, the bioavailable fraction in soil-water suspensions and respective extracts was comparable (0.37 versus 0.33% of the total Zn). However, in the case of Cd, for all the sensors used and for Hg concerning only Gram-negative sensor strains, the bioavailable fraction in soil-water suspensions exceeded the water-extracted fraction about 14-fold, indicating that upon direct contact, an additional fraction of Cd and Hg was mobilized by those sensor bacteria. Thus, for robust bioavailability studies of heavy metals in soils any type of genetic metal-response elements could be used for the construction of the sensor strains. However, Gram-positive and Gram-negative senor strains should be used in parallel as the bioavailability of heavy metals to those bacterial groups may be different.

Novel 3,3-disubstituted pyrrolidines as selective triple serotonin/norepinephrine/dopamine reuptake inhibitors

A series of 3,3-disubstituted pyrrolidine monoamine triple reuptake inhibitors were discovered. Analogues with low nanomolar potency, good human in vitro microsomal stability and in vitro permeability, and low drug–drug interaction potential are described. One example showed in vivo anti-depressant-like effects in the mouse tail suspension assay with a minimum effective dose of 30mg/kg ip.

Analysis of the Chemical Constituents of Agaricus brasiliensis

This study examined the chemical composition of A. blasiliensis and the chemical structural properties of an immuno-stimulating polysaccharide. The amino acids, free sugars, and organic acids by HPLC and fatty acids by GC were analyzed. The immuno-stimulating substance from A. blasiliensis was extracted with hot water and purified by ethanol precipitation. It underwent ion exchange chromatography on DEAE-cellulose and gel filtration on Toyopearl HW 65 F. Through GP-HPLC, the substance was found to be homogeneous. Its chemical structure was determined by 13 C-NMR. Fatty acids, organic acids, and sugar alcohol composition consisted exclusively of linoleic acid, fumaric acid and mannitol, respectively. The amino acids were mainly glutamic acid, glycine, and arginine. By 13 C-NMR analysis, the immuno-stimulating substance was identified as β-(1→3) (1→6)-glucan, composed of a backbone with (1→3)-linked D-glucopyranosyl residues branching a (1→6)-linked D-glucopyranosyl residue. The β-glucan from A. blasiliensis showed pronounced immuno-stimulating activity on the antibody-production ability of B-lymphocytes by the hemolytic suspension assay. In these results, A. blasiliensis was estimated to have potent pharmacological properties and potential nutritional values.

Synthesis and characterization of polyoxometalates loaded starch nanocomplex and its antitumoral activity

According to a water-in-oil emulsion technique using POCl 3 as a cross-linking agent, starch nanoparticles have been prepared. Using this starch nanoparticles, loaded polyoxometalates (POMs) α-K 8H 6 [Si 2W 18Ti 6O 77] (Si 2W 18Ti 6) have been prepared and structurally characterized by elemental analyses, IR spectroscopy and NMR spectroscopy. The result shows that the Si 2W 18Ti 6 molecule retains its parent structure after encapsulated by starch nanoparticles and Si 2W 18Ti 6 incorporates into starch matrix to form the nanoparticles. This new complex exhibits higher stability, antitumoral activity and can extend the survival days of the mice with tumor owing to the encapsulation by starch nanoparticles. The Si 2W 18Ti 6 has tested negative for mutagenicity in the standard Ames assay with TA97, TA98, TA100 and TA102, indicating that Si 2W 18Ti 6 has no mutagenic effect in the microsome suspension assay.

Distinct Roles for Two CYP226 Family Cytochromes P450 in Abietane Diterpenoid Catabolism by Burkholderia xenovorans LB400

The 80-kb dit cluster of Burkholderia xenovorans LB400 encodes the catabolism of abietane diterpenoids. This cluster includes ditQ and ditU, predicted to encode cytochromes P450 (P450s) belonging to the poorly characterized CYP226A subfamily. Using proteomics, we identified 16 dit-encoded proteins that were significantly more abundant in LB400 cells grown on dehydroabietic acid (DhA) or abietic acid (AbA) than in succinate-grown cells. A key difference in the catabolism of DhA and AbA lies in the differential expression of the P450s; DitU was detected only in the AbA-grown cells, whereas DitQ was expressed both during growth on DhA and during growth on AbA. Analyses of insertion mutants showed that ditQ was required for growth on DhA, ditU was required for growth on AbA, and neither gene was required for growth on the central intermediate, 7-oxo-DhA. In cell suspension assays, patterns of substrate removal and metabolite accumulation confirmed the role of DitU in AbA transformation and the role of DitQ in DhA transformation. Spectral assays revealed that DitQ binds both DhA (dissociation constant, 0.98 +/- 0.01 microM) and palustric acid. Finally, DitQ transformed DhA to 7-hydroxy-DhA in vitro. These results demonstrate the distinct roles of the P450s DitQ and DitU in the transformation of DhA and AbA, respectively, to 7-oxo-DhA in a convergent degradation pathway.

In vitrobiofilm model for studying tongue flora and malodour

To develop a perfusion biofilm system to model tongue biofilm microflora and their physiological response to sulfur-containing substrates (S-substrates) in terms of volatile sulfide compound (VSC) production. Tongue-scrape inocula were used to establish in vitro perfusion biofilms which were examined in terms of ecological composition using culture-dependent and independent (PCR-DGGE) approaches. VSC-specific activity of cells was measured by a cell suspension assay, using a portable industrial sulfide monitor which was also used to monitor VSC production from biofilms in situ. Quasi steady states were achieved by 48 h and continued to 96 h. The mean (+/-SEM) growth rate for 72-h biofilms (n=4) was micro=0.014 h(-1) (+/-0.005 h(-1)). Comparison of biofilms, perfusate and original inoculum showed their ecological composition to be similar (Pearson coefficient>0.64). Perfusate and biofilm cells derived from the same condition (co-sampled) were equivalent with regard to VSC-specific activities which were up-regulated in the presence of S-substrates. The model maintained a stable tongue microcosm suitable for studying VSC production; biofilm growth in the presence of S-substrates up-regulated VSC activity. The method is apt for studying ecological and physiological aspects of oral biofilms and could be useful for screening inhibitory agents.

Effect of dietary zinc on post‐absorptive activation of insulin‐ and stress‐stimulated cellular signaling using multiplex anaylsis

Supplemental dietary zinc (Zn) has been shown to participate in regulatory processes of cellular growth; however the cellular regulatory steps are not fully understood. We investigated the effects of Zn on insulin- (Akt, p70s6k and GSK-3) and stress- activated (p38 MAPK, ERK, p90RSK, and JNK) signaling pathways in mouse tissues utilizing a multiplex suspension assay, which allows for simultaneous detection of multiple signaling events. Briefly, mice were fed Zn-marginal (ZM, 5 ppm) diets for 4 wks. Thereafter, mice were fasted for 12 h (F) and/or re-fed with ZM or Zn-supplemental (ZS, 300 ppm) diet for 3 or 6 h. Gastrocnemius (GA) muscle and liver tissues were collected at each time point and protein content and phosphorylation of the aforementioned kinases were determined. In ZS tissue, a significant (188 %, P=0.03) increase in phosphorylation of p70s6k was seen in liver at 6 h with a similar non-significant increase (311 %; P = 0.09) observed in GA at 3 h as compared to F. Thus, Zn may potentiate the feeding induction of translation initiation. GSK-3 phosphorylation was elevated at 3 h (~40 %; P<0.05) in liver tissue independent of Zn status and persisted (P=0.02) at 6 h in the ZS group. Increased p38 MAPK phosphorylation was also seen (153 %; P<0.03) at 6 h in ZS liver tissue. In conclusion, multiplex analysis has proven to be a powerful tool for investigating multiple signaling events simultaneously by demonstrating that Zn may potentiate cellular regulation of signaling pathways involving p70s6k in GA and liver and GSK-3 and p38 MAPK in liver.

Cryopreserved human hepatocytes in suspension are a convenient high throughput tool for the prediction of metabolic clearance

Hepatocyte assays, routinely used to assess the metabolic stability of new chemical entities, were recently improved by using hepatocytes in suspension instead of primary cultures [N. Blanchard, L. Richert, B. Notter, F. Delobel, P. David, P. Coassolo, T. Lavé, Impact of serum on clearance predictions obtained from suspensions and primary cultures of rat hepatocytes, Eur. J. Pharm. Sci. 23 (2004) 189–199]. The aim of the present study was to investigate miniaturising the suspension assay by using cryopreserved human hepatocytes, i.e., 150,000 cells/well in 96-well plates, to predict hepatic clearance (CL H) in order to increase compound throughput and decrease cost and tissue requirements. For this, an evaluation was first carried out with rat hepatocytes. Then, human hepatocytes from various donors were used under these predetermined conditions, either immediately after isolation, either after a 20-h-cold storage period in UW or after cryopreservation. The values of CL int and CL H determined using human hepatocytes in suspension in 96-well plates, immediately after isolation, after cold storage or after cryopreservation, were comparable to those obtained with hepatocytes in primary culture. In particular, the use of cryopreserved human hepatocytes in suspension in a 96-well format appeared to be largely satisfactory as a tool for screening and ranking of compounds in the early phase of the drug discovery process.

Novel 5-HT7 Ligands as Antidepressants: Automated Synthesis of NSubstituted- N-[1-Methyl-3-(4-Methylpiperidin-1-yl)propyl]-Arylsulfonamides

The 5-HT7 receptor is linked with various CNS disorders. Using an automated solution phase synthesis a combinatorial library of 384 N-substituted N-[1-methyl-3-(4-methylpiperidin-1-yl)propyl]- arylsulfonamides was prepared with 24 chemically diverse amines 1-24 and 16 sulfonyl chlorides A-P. The chemical library of alkylated sulfonamides was evaluated in a receptor binding assay with [3]H-5-CT as ligand. The key synthetic step was the alkylation of a sulfonamide with iodide E, which was prepared from butanediol in 4 synthetic steps. The target compounds 1A, 1B.....24A ... 24P were purified by solvent extraction on a Teacan liquid handling system. Sulfonamide J20, B23, D23, G23, G23, J23 , I24 and O24 displayed a binding affinity IC50 between 100 nM and 10 nM. The crystalline J20 (IC50=39 nM) and O24 (IC50=83 nM) were evaluated further in the despair swimming test and the tail suspension assay. A significant antidepressant activity was found in mice of a greater magnitude than imipramine and fluoxetine at low doses. Keywords: 5-HT7 ligands, sulfonamides, automated synthesis, depression

Comparison of osteoblast spreading on microstructured dental implant surfaces and cell behaviour in an explant model of osseointegration

To compare interactions between rat calvarial osteoblasts and titanium dental implants with different microstructured surfaces. Seven commercially available implants were used. Surfaces included plasma-sprayed, grit-blasted and/or acid-etched, smooth-machined and anodised titanium. Two methods were used to compare cell behaviour: (1) A cell-spreading assay in which percentages of cells at four different stages of attachment were identified by scanning electron microscopy and quantified within a 30 min attachment period. (2) Implants were placed in 'pocket culture' within nylon mesh sacs in contact with explanted calvarial bone fragments for 2 and 4 weeks. Surfaces combining grit blasting and acid etching, of microporous topography, showed significantly enhanced rates of cell spreading in comparison with the others. Differential cell morphology was observed in both suspension assays and pocket cultures. In the latter, cells migrated onto all surfaces. Multicellular layers with extracellular matrix (ECM) were present between the layers and on the material surfaces after 2 weeks. After 4 weeks, cell layers were more consolidated, and microstructures were obscured by layers of cells and ECM. Mineralised tissue was seen in association with ECM on grit-blasted surfaces of rough and smooth microtopography. The two methods provided complementary information: a rough surface of porous microstructure may enhance the rate of cell spreading. Differentiation and calcification occurred on surfaces of both rough and smooth microstructure.

Biotests and biosensors in ecotoxicological risk assessment of field soils polluted with zinc, lead, and cadmium

The combined chemical and ecotoxicological hazard evaluation study was conducted on 60 smelter-influenced soils containing 1 to 13, 50 to 653, and 100 to 1,198 mg/kg of Cd, Pb, and Zn, respectively. For these soils (liquid-to-soil ratio = 10), water extractability of Zn, Cd, and Pb was less than 0.19% (median values). Acetic acid (0.11 M) extracted 23, 9.7, and 0.7% of Cd, Zn, and Pb, respectively. Although heavy metal concentrations in the studied soils were high, the toxic effects of water extracts were observed only in few samples and in few biotests (algae Selenastrum capricornutum and metal detector assay). For most of the aquatic test organisms (e.g., crustaceans, photobacteria), the bioavailable concentrations of metals in soil-water extracts were either subtoxic, or the adverse effects were compensated by soil nutrients, etc. However, analysis of the soils with recombinant Cd sensor Bacillus subtilis (pTOO24) showed that about 65% of these apparently subtoxic samples contained bioavailable Cd when analyzed in the suspension assay (detection limit 1.5 mg Cd/kg soil), indicating the desorption of Cd induced by direct contact of bacteria with soil particles. The median bioavailable fraction of Cd (1%) was 23-fold lower than the fraction extracted by acetic acid. The Pb-Cd sensor Staphylococcus aureus (pT0024) detected bioavailable Pb only in the suspensions of five of the most lead-polluted soils (>417 mg Pb/kg): the median bioavailability of Pb was 0.42%. Consequently, the hazard assessment relying on total metal levels in soils should be revised by critical comparison with data obtained from bioassays. Development and use of biosensors (excellent tools for mechanistic studies and signaling hazard already at subtoxic level) should be encouraged.

Antialgal, antibacterial and antifungal activity of two metabolites produced and excreted by cyanobacteria during growth

The antimicrobial activity of two cyanobacterial exometabolites, norharmane (9H-pyrido(3,4-b)indole) and 4,4'-dihydroxybiphenyl, was determined in suspension assays. Good anticyanobacterial activities (concentrations of 8-80 microg ml(-1)) and moderate antibacterial (16-160 microg ml(-1)) and antifungal (32-40 microg ml(-1)) activities were found for both compounds. The natural function as allelopathic chemicals and a possible applicability as antifouling agents or leads for the development of new antifouling chemicals are discussed.

Effiziente Abtötung von Milzbrandsporen durch wässrige und alkoholische Peressigsäure–Lösungen

We analysed the sporicidal effect of different concentrations of aqueous and alcoholic peracetic acid (PAA) solutions on anthrax spores in suspension and germ carrier tests. In activation of anthrax spores in suspension assays was achieved in less than 2 min using 1% PAA solution and in less than 3 min using 0.5% PAA solution, respectively. In contrast, in germ carrier as says, a test under practical conditions, spores on 38% of the germ carriers survived treatment with 1% PAA solution for 15 min. The use of PAA in 80% ethyl alcohol outclassed the sporicidal effect of aqueous PAA solutions in both suspension and germ carrier assays. Anthrax spores on 14% of germ carriers tested survived 30 min of treatment with a 1% aqueous PAA solution. In contrast anthrax spores were reliably inactivated under the same test procedure using a 1% alcoholic PAA solution for 30 min. The proven enhancement of the sporicidal effect of alcoholic PAA solutions should be kept in mind when using disinfectants in practice. In further surveys we will optimise the test conditions.

Isolation and Characterization of a Genetically Tractable Photoautotrophic Fe(II)-Oxidizing Bacterium, Rhodopseudomonas palustris Strain TIE-1

We report the isolation and characterization of a phototrophic ferrous iron [Fe(II)]-oxidizing bacterium named TIE-1 that differs from other Fe(II)-oxidizing phototrophs in that it is genetically tractable. Under anaerobic conditions, TIE-1 grows photoautotrophically with Fe(II), H2, or thiosulfate as the electron donor and photoheterotrophically with a variety of organic carbon sources. TIE-1 also grows chemoheterotrophically in the dark. This isolate appears to be a new strain of the purple nonsulfur bacterial species Rhodopseudomonas palustris, based on physiological and phylogenetic analysis. Fe(II) oxidation is optimal at pH 6.5 to 6.9. The mineral products of Fe(II) oxidation are pH dependent: below pH 7.0 goethite (alpha-FeOOH) forms, and above pH 7.2 magnetite (Fe3O4) forms. TIE-1 forms colonies on agar plates and is sensitive to a variety of antibiotics. A hyperactive mariner transposon is capable of random insertion into the chromosome with a transposition frequency of approximately 10(-5). To identify components involved in phototrophic Fe(II) oxidation, mutants of TIE-1 were generated by transposon mutagenesis and screened for defects in Fe(II) oxidation in a cell suspension assay. Among approximately 12,000 mutants screened, 6 were identified that are specifically impaired in Fe(II) oxidation. Five of these mutants have independent disruptions in a gene that is predicted to encode an integral membrane protein that appears to be part of an ABC transport system; the sixth mutant has an insertion in a gene that is a homolog of CobS, an enzyme involved in cobalamin (vitamin B12) biosynthesis.

Analysis of sorption and bioavailability of different species of mercury on model soil components using XAS techniques and sensor bacteria

The present work studies the adsorption behaviour of mercury species on different soil components (montmorillonite, kaolinite and humic acid) spiked with CH3HgCl and CH3HgOH at different pH values, by using XAS techniques and bacterial mercury sensors in order to evaluate the availability of methyl mercury on soil components. The study details and discusses different aspects of the adsorption process, including sample preparation (with analysis of adsorbed methyl mercury by ICP-OES), the various adsorption conditions, and the characterization of spiked samples by XAS techniques performed at two synchrotron facilities (ESRF in Grenoble, France and HASYLAB in Hamburg, Germany), as well as bioavailability studies using mercury-specific sensor bacteria. Results show that XAS is a valuable qualitative technique that can be used to identify the bonding character of the Hg in mercury environment. The amount of methyl in mercury adsorbed to montmorillonite was pH-dependent while for all soil components studied, the bond character was not affected by pH. On the other hand, clays exhibited more ionic bonding character than humic acids did with methyl mercury. This interaction has a higher covalent character and so it is more stable for CH3HgOH than for CH3HgCl, due to the higher reactivity of the hydroxyl group arising from the possible formation of hydrogen bonds. The bioavailability of methyl mercury adsorbed to montmorillonite, kaolinite and humic acids was measured using recombinant luminescent sensor bacterium Escherichia coli MC1061 (pmerBR(BS)luc). In case of contact exposure (suspension assays), the results showed that the bioavailability was higher than it was for exposure to particle-free extracts prepared from these suspensions. The highest bioavailability of methyl mercury was found in suspensions of montmorillonite (about 50% of the total amount), while the bioavailabilities of kaolinite and humic acids were five times lower (about 10%). The behaviour of methyl mercury in the presence of montmorillonite could be explained by the more ionic bonding character of this system, in contrast to the more covalent bonding character observed for humic acids. Thus, XAS techniques seem to provide promising tools for investigating the mechanisms behind the observed bioavailabilities of metals in various environmental matrices, an important topic in environmental toxicology.

Antidepressant-like activity of corticotropin-releasing factor type-1 receptor antagonists in mice

The development of selective corticotropin-releasing factor type-1 (CRF 1) receptor antagonists represents a potential novel treatment for depression. These studies evaluated CRF 1 receptor antagonists for antidepressant-like activity in mice. Subchronic dosing of both R 121919 (3-[6-(dimethylamino)-4-methyl-pyrid-3-yl]-2,5-dimethyl- N, N-dipropyl-pyrazolo[2,3- a]pyrimidin-7-amine) and DMP 696 (4-(1,3-dimethoxyprop-2-ylamino)-2,7-dimethyl-8-(2,4-dichlorophenyl)-pyrazolo[1,5- a]-1,3,5-triazine) significantly decreased immobility time in the tail suspension test (at 30 and at 3 and 10 mg/kg, i.p., respectively). These antidepressant-like effects were observed at doses that did not impair general locomotor activity. Neither antalarmin ( N-butyl- N-ethyl-[2,5,6-trimethyl-7-(2,4,6)trimethylphenyl)-7 H-pyrrolo[2,3- d]pyrimidin-4-yl]amine) nor DMP 904 (4-(3-pentylamino)-2,7-dimethyl-8-(2-methyl-4-methoxyphenyl)-pyrazolo-[1,5- a]-pyrimidine) had an effect indicative of antidepressant-like activity. These results suggest that the tail suspension assay may have utility to identify CRF 1 receptor antagonists with antidepressant-like activity. Moreover, the results lend support to the theory that some nonpeptidic CRF 1 receptor antagonists may possess antidepressant-like activity and therefore represent a promising novel pharmacotherapeutic strategy in the treatment of depression.

Efficacy of Egg Cleaning Compounds on Eggshells Contaminated with Salmonella enterica Serovar Enteritidis

Salmonella Enteritidis infections of egg contents can be related to external contamination of the shell. In this study, the efficacy of three commercial cleaning and/or sanitizing compounds (sodium carbonate, sodium hypochlorite, and potassium hydroxide) was evaluated for bactericidal activity at pH values of 10, 11, and 12 against various concentrations (102, 104, or 106 CFU/ml) of Salmonella Enteritidis inoculated onto the eggshell surface. Efficacy of these chemical agents was also assessed against Salmonella Enteritidis in aqueous suspension. Our results indicated that none of the chemicals applied at the recommended manufacturer's concentrations (sodium carbonate, 36 ppm; other treatments, 200 ppm) could eliminate Salmonella Enteritidis from eggshells artificially contaminated with the highest bacterial concentrations (104 or 106 CFU/ml). Higher concentrations of each product, at least 5 to 20 times greater than recommended doses, were needed to destroy the bacteria on egg surfaces. However, at or slightly above the manufacturer's recommended use concentrations, all three formulations were effective against Salmonella Enteritidis in aqueous suspension (108 CFU/ml) or on eggshells contaminated with 102 CFU/ml. For both shell and suspension assays, inactivation of Salmonella Enteritidis occurred at lower concentrations at pH 12 than at pH 11 and pH 10. Contact time between chemicals and Salmonella apparently influenced bacterial inactivation. Extended contact times (2 to 10 min) reduced minimum chemical concentrations necessary to inactivate the bacteria. However, neither pH nor contact time influenced Salmonella Enteritidis inactivation when the initial bacterial numbers on eggshells were high.

Inactivation of feline calicivirus, a surrogate of norovirus (formerly Norwalk-like viruses), by different types of alcohol in vitro and in vivo

Hand disinfection is an important measure to prevent transmission of norovirus (formerly called Norwalk-like viruses) from hands or environmental surfaces to other objects. Therefore, three types of alcohol (ethanol, 1- and 2-propanol) were examined for their virus-inactivating properties against feline calicivirus (FCV) as a surrogate for norovirus. Tests were performed as quantitative suspension assays or as in vivo experiments with artificially contaminated fingertips. The in vitro experiments showed that 1-propanol was more effective than ethanol and 2-propanol for the inactivation of FCV: in tests with the 50 and 70% solutions of the different alcohols, a 10 4-fold reduction was observed with 1-propanol after 30 s, whereas the other alcohols were effective only after 3 min contact time. The greatest efficacy did not occur at the highest concentrations (80%). The following concentrations (extrapolated data) showed the greatest virus-inactivating properties in the suspension test: ethanol 67%, 2-propanol 58% (exposure times of 1 min) and 1-propanol 60% (exposure time of 30 s). The results from fingertips experiments with 70 and 90% solutions and an application time of 30 s confirmed these findings: the 70% alcoholic solutions were more effective than the 90% solutions. In contrast to the suspension tests, 70% ethanol showed the greatest efficacy in vivo with a log 10 reduction factor (RF) of 3.78 compared with 70% 1-propanol (RF 3.58), 70% 2-propanol (RF 2.15) and hard water (RF 1.23). Ethanol and 1-propanol-based solutions with a high alcohol content thus appear most effective.

Application of a Multivalent Glycoprobe: Characterization of Sugar-Binding Specificity of Siglec Family Proteins

This chapter describes how the glycoprobe was prepared and then applied to the characterization of the binding specificity of sialic acid-binding Ig-like lectins (Siglecs). Siglecs () are a family of lectins expressed mainly on hematopoietic cells. The binding of Siglecs to specific sialoglycoconjugates is associated with their specific functions, for example, cellular attachment or recognition. The interaction of Siglecs with sialoglycoconjugates on the cell surface may comprise an initial step in the process of cellular attachment or recognition. The interaction is believed to occur in a multivalent manner that is, an array of Siglecs on the cell surface binds multiple sialoglycoconjugates on the recognized cells. To characterize the multivalent interaction of Siglecs, a polyvalent “glycoprobe” that carries more than 100 oligosaccharides was developed. Glycochain structures of gangliosides and oligosaccharides and their recognition by Siglecs are tabulated. The chapter discusses the preparation and polymerization of oligosaccharyl streptavidin, the assay for adherent cells, and suspension assay of Siglecs.