Serum regulates Na+/H+ exchange in Caco-2 cells by a mechanism which is dependent on F-actin.

Regulation of Na+/H+ exchange by fetal bovine serum was studied in Caco-2 cells, an established cell line derived from a human colon carcinoma. Cells were grown as polarized monolayers on collagen-coated filters and intracellular pH measured fluorometrically with 2',7'-bis(2-carboxymethyl)-5,6-carboxyfluorescein. Na+/H+ exchange was reduced 64% when cells were deprived of serum for 4 h. In contrast to other cell types, readdition of serum for 10 min did not activate Na+/H+ exchange; however, readdition of serum for 4 h restored Na+/H+ exchange to control values. This long-term effect of serum on Na+/H+ exchange activity could not be explained by changes in intracellular buffering capacity or intracellular [Na+]. 4-h serum deprivation reduced the K(t) of the exchanger for external Na+ from 21 to 6 mM, and reduced the V(max) by 57%, but did not alter the IC50 for amiloride in the presence of 140 mM Na+. Inhibition of protein synthesis with cycloheximide (5 microM) did not alter the effect of serum removal or readdition on Na+/H+ exchange. Low temperature (13 degrees C) completely prevented the inhibition of Na+/H+ exchange caused by the removal of serum. In addition, once Na+/H+ exchange was inhibited by serum removal at 37 degrees C, maintaining cells at 13 degrees C also blocked the recovery of Na+/H+ exchange caused by serum readdition. Conversely, cytochalasin D (0.1-20 microM) blocked the reduction of Na+/H+ exchange which occurred due to 4-h serum deprivation, but did not block the restoration of Na+/H+ exchange when the cells were re-exposed to serum for a further 4 h. Colchicine (20 microM) did not alter the effect of serum removal or readdition. These data suggest that serum regulates Na+/H+ exchange activity by a posttranslational mechanism which is dependent on F-actin.     

Membrane potentials, electrolyte contents, cell pH, and some enzyme activities of fibroblasts

The resting membrane potential of the cultured fibroblasts derived from rabbit subcutaneous tissues was -10.2 +/- 0.20 mV (n = 390). This potential was affected by the potassium concentration in the culture medium, but not by other chemical or hormonal preparations, such as dibutyryladenosine 3',5'-cyclic monophosphate (0.5 to 5.0 mmol/l), sodium fluoride (10(-5) to 10(-4) M), hydrocortisone (10(-7) to 10(-6) M), parathyroid extract (0.5 to 1.0 U/ml), or thyrotrophin (5 to 10 mU/ml). The Na+, K+, and Cl- concentrations of the cultured fibroblasts were 35.4, 85.7, and 22.6 mmol/l cell water, respectively. The water and protein contents of these cells were 82.1 and 9.18 g/100-g cells, respectively. The intracellular pH of fibroblasts as determined by [14C] dimethyloxazolidine-2, 4-dione, and 3H2O ranged between 6.9 and 7.1 when the pH of the culture medium was maintained at 7.4. The activities of Na+, K+-, HCO3(-)-, and Ca++, Mg++-ATPases in these cultured cells were 19.0 +/- 2.1, 13.6 +/- 2.1, and 6.6 +/- 1.2 nmol pi/mg protein per minute, respectively, and the carbonic anhydrase activity was 0.054 U/mg protein. Calculations based on the values for the membrane potential and the electrolyte concentrations observed in this study indicate that Na+, K+, Cl-, and H+ are not distributed according to their electrochemical gradients across the cell membrane. Na+, Cl-, and H+ are actively transported out of the cells and K+ into the cells.     

Proton dissociation from nigericin at the membrane-water interface, the rate-limiting step of K+/H+ exchange on the bilayer lipid membrane.

The rate of K+/H+ exchange through bilayer lipid membranes (BLM) induced by nigericin was measured by the method of pH gradient offset according to Antonenko, Yu.N. and Yaguzhinsky L.S. [(1990) Biochim. Biophys. Acta 1026, 236-240]. It was shown that under the conditions of high potassium ion concentration the rate of nigericin-mediated K+/H+ exchange increased with an increase in the concentrations of such buffer compounds as citric acid and MES. The concentration dependence was different for citrate and MES. The buffer concentration effect was absent at low potassium ion concentrations. Citrate increased the rate of K+/H+ exchange being added to the side of BLM where the K+ concentration was higher and had no effect at the opposite side. At high KCl and citrate concentrations, the rate of K+/H+ exchange was about 6 times lower in D2O when compared to H2O solutions. It is concluded that under certain experimental conditions the overall rate of the K+/H+ exchange induced by nigericin is determined by the rate of proton dissociation from nigericin at the membrane-water interface.     

Adsorption of Methylene Blue Dye on Pure and Carbonized Water Weeds

The influence of process variables in batch adsorption has been used to assess the removal of methylene blue dye from aqueous solution using pure and carbonized biomasses of water hyacinth and water spinach. Dried leaves of the water weeds were carbonized at temperature up to 750°C. The optimum removal of dye was achieved at pH 10, 30°C, and 55 min at a dye concentration of 10 mg/L. In an attempt to describe the adsorption process, the equilibrium isotherm for each adsorbent was determined using Langmuir and Freundlich adsorption isotherm models. Maximum adsorption capacities based on the Langmuir model for pure and carbonized water hyacinth were (mg/g) 7.05 and 2.07, respectively, whereas those of pure and carbonized water spinach were 1.25 and 5.32, respectively. It was observed that the equilibrium data were well fit by both the Freundlich and Langmuir isotherms as R ² > .97. This study demonstrates that the two waterweeds are effective, environmentally friendly, and inexpensive biomaterials for the removal of color from industrial effluents.     

Thermodynamics of the hydrolysis of sucrose

A thermodynamic investigation of the hydrolysis of sucrose to fructose and glucose has been performed using microcalorimetry and high-pressure liquid chromatography. The calorimetric measurements were carried out over the temperature range 298-316 K and in sodium acetate buffer (0.1 M, pH 5.65). Enthalpy and heat capacity changes were obtained for the hydrolysis of aqueous sucrose (process A): sucrose(aq) + H2O(liq) = glucose(aq) + fructose (aq). The determination of the equilibrium constant required the use of a thermochemical cycle calculation involving the following processes: (B) glucose 1-phosphate2-(aq) = glucose 6-phosphate2-(aq); (C) sucrose(aq) + HPO4(2-)(aq) = glucose 1-phosphate2-(aq) + fructose(aq); and (D) glucose 6-phosphate2-(aq) + H2O(liq) = glucose(aq) + HPO4(2-)(aq). The equilibrium constants determined at 298.15 K for processes B and C are 17.1 +/- 1.0 and 32.4 +/- 3.0, respectively. Equilibrium data for process D was obtained from the literature, and in conjunction with the data for processes B and C, used to calculate a value of the equilibrium constant for the hydrolysis of aqueous sucrose. Thus, for process A, delta G0 = -26.53 +/- 0.30 kJ mol-1, K0 = (4.44 +/- 0.54) x 10(4), delta H0 = -14.93 +/- 0.16 kJ mol-1, delta So = 38.9 +/- 1.2 J mol-1 K-1, and delta CoP = 57 +/- 14 J mol-1 K-1 at 298.15 K. Additional thermochemical cycles that bear upon the accuracy of these results are examined.     

Thermodynamics of hydrolysis of oligosaccharides

Microcalorimetry has been used to determine enthalpy changes for the hydrolysis of a series of oligosaccharides. High-pressure liquid chromatography was used to determine the extents of reaction and to check for any possible side reactions. The enzyme glucan 1,4-alpha-glucosidase was used to bring about the following hydrolysis reactions: (A) maltose(aq) + H2O(liq) = 2D-glucose(aq); (B) maltotriose(aq) + 2H2O(liq) = 3D-glucose(aq); (C) maltotetraose(aq) + 3H2O(liq) = 4D-glucose(aq); (D) maltopentaose(aq) + 4H2O(liq) = 5D-glucose(aq); (E) maltohexaose(aq) + 5H2O(liq) = 6D-glucose(aq); (F) maltoheptaose(aq) + 6H2O(liq) = 7D-glucose(aq); (G) amylose(aq) + nH2O(liq) = (n + 1) D-glucose(aq); and (H) panose(aq) + 2H2O(liq) = 3D-glucose(aq); (J) isomaltotriose(aq) + 2H2O(liq) = 3D-glucose(aq). The enzyme beta-fructofuranosidase was used for the reactions: (K) raffinose(aq) + H2O(liq) = alpha-D-melibiose(aq) + D-fructose(aq); and (L) stachyose(aq) + H2O(liq) = o-alpha-D-galactopyranosyl-(1----6)- alpha-o-D-galactopyranosyl-(1----6)-alpha-D-glucopyranose + D-fructose(aq). The results of the calorimetric measurements (298.15 K, 0.1 M sodium acetate buffer, pH 4.44-6.00) are: delta H0A = -4.55 +/- 0.10, delta H0B = -9.03 +/- 0.10, delta H0C = -13.79 +/- 0.15, delta H0D = -18.12 +/- 0.10, delta H0E = -22.40 +/- 0.15, delta H0F = -26.81 +/- 0.20, delta H0H = 1.46 +/- 0.40, delta H0J = 11.4 +/- 2.0, delta H0K = -15.25 +/- 0.20, and delta H0L = -14.93 +/- 0.20 kJ mol-1. The enthalpies of hydrolysis of two different samples of amylose were 1062 +/- 20 and 2719 +/- 100 kJ mol-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biphasic effect of hydrogen peroxide on skeletal muscle arteriolar tone via activation of endothelial and smooth muscle signaling pathways.

We hypothesized that hydrogen peroxide (H2O2) has a role in the local regulation of skeletal muscle blood flow, thus significantly affecting the myogenic tone of arterioles. In our study, we investigated the effects of exogenous H2O2 on the diameter of isolated, pressurized (at 80 mmHg) rat gracilis skeletal muscle arterioles (diameter of approximately 150 microm). Lower concentrations of H2O2 (10(-6)-3 x 10(-5) M) elicited constrictions, whereas higher concentrations of H2O2 (6 x 10(-5)-3 x 10(-4) M), after initial constrictions, caused dilations of arterioles (at 10(-4) M H2O2, -19 +/- 1% constriction and 66 +/- 4% dilation). Endothelium removal reduced both constrictions (to -10 +/- 1%) and dilations (to 33 +/- 3%) due to H2O2. Constrictions due to H2O2 were completely abolished by indomethacin and the prostaglandin H2/thromboxane A2 (PGH2/TxA2) receptor antagonist SQ-29548. Dilations due to H2O2 were significantly reduced by inhibition of nitric oxide synthase (to 38 +/- 7%) but were unaffected by clotrimazole or sulfaphenazole (inhibitors of cytochrome P-450 enzymes), indomethacin, or SQ-29548. In endothelium-denuded arterioles, clotrimazole had no effect, whereas H2O2-induced dilations were significantly reduced by charybdotoxin plus apamin, inhibitors of Ca(2+)-activated K+ channels (to 24 +/- 3%), the selective blocker of ATP-sensitive K+ channels glybenclamide (to 14 +/- 2%), and the nonselective K(+)-channel inhibitor tetrabutylammonium (to -1 +/- 1%). Thus exogenous administration of H2O2 elicits 1) release of PGH2/TxA2 from both endothelium and smooth muscle, 2) release of nitric oxide from the endothelium, and 3) activation of K+ channels, such as Ca(2+)-activated and ATP-sensitive K+ channels in the smooth muscle resulting in biphasic changes of arteriolar diameter. Because H2O2 at low micromolar concentrations activates several intrinsic mechanisms, we suggest that H2O2 contributes to the local regulation of skeletal muscle blood flow in various physiological and pathophysiological conditions.     

Reductive damage in directly ionized DNA: saturation of the C5=C6 bond of cytosine in d(CGCG)(2) crystals

Electron paramagnetic resonance (EPR) was used to study an oligodeoxynucleotide duplex of d(CGCG)(2) that is known to crystallize in Z-form. After X irradiation at 4 K, EPR data were collected on single crystals and polycrystalline samples as a function of annealing temperature and dose. A radical produced by the net gain of a hydrogen atom at C6 and a proton at N3, Cyt(C6+H, N3+H(+))(+*), is identified. This radical had not been positively identified in polymeric DNA previously. The Cyt(C6+H, N3+H(+))(+*) makes up about 4% of the total radical population at 4 K, increasing to about 10-15% after the DNA is annealed to 240 K. There appears to be neither an increase nor a decrease in the absolute concentration of Cyt(C6+H, N3+H(+))(+*) upon annealing from 4 K to 240 K. Additionally, the presence of another radical, one due to the net gain of hydrogen at C5 of cytosine, the Cyt(C5+H)(*), is implicated. Together, these two radicals appear to account for 60-80% of the reduced species in DNA that has been irradiated at 4 K and annealed to 240 K.     

红曲菌利用生物柴油废液生产红曲色素

对生物柴油废液作简单处理,利用红曲茵发酵生物柴油废液中副产物甘油生产红曲色素。通过响应面方法确定最佳发酵培养基为：甘油48．49g/L,蛋白胨3．12g／L,K2HPO4·3H202．01g/L,MgSO4 0．48g／L,ZnSO4·7H2O 0．04g／L,MnSO4·H2O 0．03g／L,玉米浆13mL／L,植物油10mL／L,起始pH为6。发酵结果表明：在接种量6％（v／v）,转速140r／min,35℃的条件下发酵培养6d,红曲色素最高产量到达204U／mL。说明用生物柴油废液中的粗甘油为原料生产红曲色素是基本可行的。可望为生物柴油废液的资源化提供一条环境友好型的途径。     

Biosorption of Pb(II) from contaminated water onto Moringa oleifera biomass: kinetics and equilibrium studies

Abstract

The present study aims at evaluating a batch scale biosorption potential of Moringa oleifera leaves (MOL) for the removal of Pb(II) from aqueous solutions. The MOL biomass was characterized by FTIR, SEM, EDX, and BET. The impact of initial concentrations of Pb (II), adsorbent dosage, pH, contact time, coexisting inorganic ions (Ca²⁺, Na⁺, K⁺, Mg²⁺, CO₃^2?, HCO₃^?, Cl^?), electrical conductivity (EC) and total dissolved salts (TDS) in water was investigated. The results revealed that maximum biosorption (45.83?mg/g) was achieved with adsorbent dosage 0.15?g/100?mL while highest removal (98.6%) was obtained at adsorbent biomass 1.0?g/100?mL and pH 6. The presence of coexisting inorganic ions in water showed a decline in Pb(II) removal (8.5% and 5%) depending on the concentrations of ions. The removal of Pb(II) by MOL decreased from 97% to 89% after five biosorption/desorption cycles with 0.3?M HCl solution. Freundlich model yielded a better fit for equilibrium data and the pseudo-second-order well described the kinetics of Pb(II) biosorption. FTIR spectra showed that –OH, C–H, –C–O, –C?=?O, and –O–C functional groups were involved in the biosorption of Pb(II). The change in Gibbs free energy (ΔG = ?28.10?kJ/mol) revealed that the biosorption process was favorable and thermodynamically driven. The results suggest MOL as a low cost, environment-friendly alternative biosorbent for the remediation of Pb(II) contaminated water.     

Optimization of pretreatment and saccharification for the production of bioethanol from water hyacinth by Saccharomyces cerevisiae

Alkaline-oxidative (A/O) pretreatment and enzymatic saccharification were optimized for bioethanol fermentation from water hyacinth by Saccharomyces cerevisiae. Water hyacinth was subjected to A/O pretreatment at various NaOH and H(2)O(2) concentrations and reaction temperatures for the optimization of bioethanol fermentation by S. cerevisiae. The most effective condition for A/O pretreatment was 7% (w/v) NaOH at 100 °C and 2% (w/v) H(2)O(2). The carbohydrate content was analyzed after reaction at various enzyme concentrations and enzyme ratios using Celluclast 1.5 L and Viscozyme L to determine the effective conditions for enzymatic saccharification. After ethanol fermentation using S. cerevisiae KCTC 7928, the concentration of glucose, ethanol and glycerol was analyzed by HPLC using a RI detector. The yield of ethanol in batch fermentation was 0.35 g ethanol/g biomass. Continuous fermentation was carried out at a dilution rate of 0.11 (per h) and the ethanol productivity was 0.77 [g/(l h)].     

Applications of ammonium formate-catalytic transfer hydrogenation. Part V (1). Transfer hydrogenolysis of peptides containing p-chlorophenylalanine as a convenient method for preparation of deuterium labeled peptides

Dehalogenation of chlorine bound to aromatic nuclei has been achieved using ammonium formate-catalytic transfer hydrogenation (AF-CTH) techniques. The use of deuterated ammonium formate as the transfer agent in the CTH process when performed in a deuterated solvent medium results in the predominant formation of the labeled product. Thus, ND+4 DCOO(-)-CTH of [D-Ala2, p-ClPhe4]-Leu-enkephalin in 80% CD3COOD/D2O yielded [D-Ala2, Phe (4D)4]-Leu-enkephalin. Levels of D-incorporation were measured by fast atom bombardment-mass spectrometry (FAB-MS) and/or by 13C n.m.r. spectroscopy. Using D-labeled and unlabeled ammonium formate in stoichiometrically similar reactions, it was concluded that the hydrogenation exhibited a primary kinetic isotope effect. Reactant and product concentrations were determined by amino acid analysis and reversed phase HPLC.     

Identification of oxygen nucleophiles in tetrahedral intermediates: 2H and 18O induced isotope shifts in 13C NMR spectra of pepsin-bound peptide ketone pseudosubstrates

The synthetic ketone peptide analogue of pepstatin, isovaleryl-L-valyl-[3-13C]-(3-oxo-4S)-amino-6-methylheptanoyl-L-al anyl-isoamylamide is a strong inhibitor of aspartyl proteases. When the peptide is added to porcine pepsin in H2O at pH 5.1, the 13C NMR chemical shift of the ketone carbon moves from 208 ppm for the inhibitor in solution to 99.07 ppm when bound to the enzyme active site. In 2H2O the bound shift is 98.71 ppm, 0.36 ppm upfield. For the analogous experiment contrasting H216O and H218O, the 13C chemical shift was 0.05 ppm to higher field for the heavier isotope. These data show that water, and not an enzyme nucleophile, adds to the peptide carbonyl to yield a tetrahedral diol adduct in the enzyme-catalyzed reaction, and provide a method for differentiating between covalent and non-covalent mechanisms.     

The efficient role of aquatic plant (water hyacinth) in treating domestic wastewater in continuous system

In this study, water hyacinth (Eichhornia crassipes) was used to treat domestic wastewater. Ten organic and inorganic parameters were monitored in three weeks for water purification. The six chemical, biological and physical parameters included Dissolved Oxygen (DO), Biological Oxygen Demand (BOD), Chemical Oxygen Demand (COD), Ammoniacal Nitrogen (NH₃-N), Total Suspended Solids (TSS), and pH were compared with the Interim National Water Quality Standards, Malaysia River classification (INWQS) and Water Quality Index (WQI). Between 38% to 96% of reduction was observed and water quality has been improved from class III and IV to class II. Analyses for Electricity Conductivity (EC), Salinity, Total Dissolved Solids (TDS) and Ammonium (NH₄) were also investigated. In all parameters, removal efficiency was in range of 13–17th day (optimum 14th day) which was higher than 3 weeks except DO. It reveals the optimum growth rate of water hyacinth has great effect on waste water purification efficiency in continuous system and nutrient removal was successfully achieved.     

Tetrahydromethanopterin, a carbon carrier in methanogenesis

Evidence obtained by 13C NMR spectroscopy indicates that tetrahydromethanopterin (H4MPT) serves as a carbon carrier for C1 units at the methine, methylene, and methyl levels of oxidation. All three derivatives of H4MPT served as substrates for methanogenesis by cell extracts under a hydrogen atmosphere; in each instance, methane evolved at a rate comparable to that obtained when 2-(methylthio)ethanesulfonic acid was used as the substrate. Each C1 derivative of H4MPT stimulated the reduction of CO2 as efficiently as 2-(methylthio)ethanesulfonic acid. High resolution fast atom bombardment mass spectrometry indicated that the product of the spontaneous reaction of formaldehyde with H4MPT was methylene-H4MPT, with the molecular formula C31H45N6O16P. 13C NMR spectroscopy of hexamethylenetetramine, a model compound, suggested that the methylene group in methylene-H4MPT was bound to two nitrogen atoms. Molecular formulas of C31H44N6O16P and C31H47N6O16P were assigned to methenyl-H4MPT+, and methyl-H4MPT, by high resolution fast atom bombardment mass spectrometry. 1H NMR spectroscopy of methyl-H4MPT indicated that the methyl group was bound to a nitrogen atom. Sensitivity of each derivative to oxygen was noted. Apparent extinction coefficients of H4MPT and its derivatives were recorded. Evidence for the enzymatic synthesis of methylene-H4MPT from methenyl-H4MPT+ is presented.     

Voltage and temperature dependence of single K+ channels isolated from canine cardiac sarcoplasmic reticulum.

The temperature and voltage dependence of gating and conductance of sarcoplasmic reticulum K+ channels (S-R K+) isolated from adult canine hearts were studied using the reconstituted bilayer technique. Fusion of vesicles from this preparation frequently resulted in the incorporation of a single channel. Only bilayers into which a single S-R K+ channel had fused were studied. The three conductance states of the channel, fully open (O2), substate conductance (O1), and closed (C) were studied as a function of voltage (-50 to +50 mV) and temperature (16 to 37 degrees C). Permeation through the O1 state showed the same temperature dependence as the O2 state corresponding to an enthalpy of permeation of 4.1-4.2 kcal/mol, which is similar to that for K+ diffusion through water. As expected, increased temperature increased the frequency of gating transitions and shortened the average dwell time spent in any conductance state. Over the range of 25 to 37 degrees C, the average dwell time spent in the O1, O2, and C states decreased by 44 +/- 11, 36 +/- 13, and 78 +/- 7% (n = 3 to 4 channels), respectively. The ratio of probabilities between the various conductance states was not strongly temperature sensitive. Analysis of the voltage dependence of this channel was carried out at 37 degrees C and revealed that the dwell times of the O1 and O2 states were voltage insensitive and the probability ratio (PO2:PO1) was approximately 7 and was voltage insensitive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pex, analytical tools for PDB files. II. H-Pex: noncanonical H-bonds in alpha-helices

We use the H-Pex (Thomas et al., this issue) to analyze the main chain interactions in 131 proteins. In antiparallel beta-sheets, the geometry of the N...O bond is: median N...O distances, 2.9 SA, C==O...N angles at 154 degrees and the C alpha--C==O...H angles are dispersed around 3 degrees. In some instances, the other side of the C==O axis is occupied by a HC alpha. As recently supported by Vargas et al. (J Am Chem Soc 2000;122:4750-4755) C alpha H...O and NH...O could cooperate to sheet stability. In alpha-helices, the main chain C==O interact with the NH of their n + 4 neighbor on one side, and with a C beta H or C gamma H on the other side. The median O...N distance (3.0 A) and C==N angle (147 degrees) suggest a canonical H-bond, but the C alpha--C==O...H dihedral angle invalidates this option, since the hydrogen attacks the oxygen at 122 degrees, i.e., between the sp(2) and pi orbitals. This supports that the H-bond is noncanonical. In many instances, the C gamma H or the C beta H of the n + 4 residue stands opposite to the NH with respect to the oxygen. Therefore, we propose that, in alpha-helices, the C gamma H or C beta H and the NH of the n + 4 residue hold the oxygen like an electrostatic pincher. Proteins 2001;43:37-44.     

In vivo study of the influence of adenine and guanosine on the erythrocytes of ACD (pH 5.0 and 5.6) preserved blood

Data are presented for percentage of recovery, survival time (T1/2) and mode of sequestration of erythrocytes from ACD [disodium citrate 95 mmol/l and glucose (C6H12O6 X H2O)] 152 mmol/l or ACD--adenine or adenine + guanosine (pH ranging from 5.0 to 5.6) preserved blood for 35 days at 4-8 degrees C (277-281 K). With the availability of guanosine in 0.25 mmol/l or 0.5 mmol/l final concentration in ACD + 0.25 or 0.5 mmol/l adenine preserved blood a positive effect can be exerted on erythrocyte 24 hrs recovery and survival time (T1/2). This effect is particularly evident when pH of the preservative solution is raised to 5.6. Final concentrations of 0.25 mmol/l adenine and guanosine in ACD preserved blood (whole or packed erythrocytes, pH 5.6, Hct. 0.73 or 0.61) are sufficient to ensure 35 days of storage at 4-8 degrees C (277-281 K).     

Structure of the O-polysaccharide from the lipopolysaccharide of Providencia stuartii O43 containing an amide of D-galacturonic acid with L-serine

The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia stuartii O43:H28 and studied by sugar and methylation analyses, Smith degradation and 1H and 13C NMR spectroscopy, including 2D ROESY, and H-detected 1H, 13C HSQC and HMBC experiments, as well as a NOESY experiment in a 9:1 H2O/D2O mixture to reveal correlations for NH protons. It was found that the polysaccharide is built up of linear tetrasaccharide repeating units containing an amide of D-galacturonic acid with L-serine [D-GalA6(L-Ser)] and has the following structure:[3)-beta-D-GalpA6(L-Ser)-(1-->3)-beta-D-GlcpNAc-(1-->2)-alpha-D-Rhap4NAc-(1-->4)-beta-D-GlcpA-(1-->]n.     

Evaluation of parameters affecting quantitative detection of Escherichia coli O157 in enriched water samples using immunomagnetic electrochemiluminescence

We report here the use of immunomagnetic (IM) electrochemiluminescence (ECL) for quantitative detection of Esherichia coli O157:H7 in water samples following enrichment in minimal lactose broth (MLB). IM beads prepared in-house with four commercial anti-O157 monoclonal antibodies were compared for efficiency of cell capture. IM-ECL responses for E. coli O157:H7 (strain SEA13B88) were similar for all four commercial anti-O157 LPS monoclonal antibodies. The ECL signal was linearly correlated with E. coli O157:H7 cell concentration, indicating a constant ECL response per cell. Twenty-two strains of E. coli O157:H7 or O157:NM gave comparable ECL signals using IM beads prepared in-house. To assess the potential for interference from background bacteria in MLB-enriched water samples, 10(4) cells of E. coli O157:H7 (strain SEA13B88) were added to enriched samples prior to analysis. There was considerable variability in recovery of E. coli O157:H7 cells; net ECL signals ranged from 1% to 100% of expected values (i.e., percent inhibition from 0% to 99%). Cultures of Klebsiella pneumoniae, Klebsiella oxytoca, and Enterobacter cloacae, subsequently isolated from MLB-enriched water samples via IM separation (IMS), were observed to interfere with the binding of E. coli O157:H7 cells to IM beads. Recoveries of 10(4) E. coli O157:H7 cells were 相似文献