Implementation of an HPLC polystyrene divinylbenzene column for separation of activated sludge exopolymers

A high-pressure size-exclusion chromatography procedure for separation of activated sludge exopolymers was investigated and implemented in order to achieve a documented and faster separation procedure than the conventional low-pressure size-exclusion chromatography methods previously suggested in studies of activated sludges from a traditional and an advanced from activated sludges from a traditional and an advanced activated sludge treatment plant performing biological nitrogen and phosphorus removal were used. For both types of exopolymers the separation was largely dependent on the mobile-phase. Using NaCl and ortho-phosphate in the molar proportion 10:1 it was shown that for a mobile-phase ionic strength of 0.011 and pH in the range 7.0–10.0 no irreversible column adsorption occurred. For a standard procedure a mobile-phase pH of 7.0 was selected in order to separate the exopolymers into the maximal number of peaks. Alterations in the mobile-phase, i.e. using a pH below 7.0 or a mobile-phase ionic strength above 0.011, changed the separation for both types of exopolymers and caused irreversible column adsorption. Similarly, using deionized water as the mobile-phase irreversible column adsorption was introduced and the separation was strongly affected. The method applicability for qualitative characterization of exopolymers was demonstrated. The method was found to be successful in showing differences and similarities between exopolymers from two different activated sludge treatment plants, showing degradation of exopolymer compounds due to exoenzymes in the exopolymers and showing that snow melting and subsequent high conductivity in the inlet to the waste-water treatment plant had an impact on the chromatographic fingerprint of the extracted exopolymers.     

Determination of proton dissociation constants by ion-exchange high-performance liquid chromatography

The common methods to determine dissociation constants of solutes, e.g., uv spectrophotometry, potentiometry, and conductimetry, are accurate but require at least 1 nmol of compound. High-performance liquid chromatography (HPLC) allows 1 pmol of a uv-absorbing compound to be detected. By adjusting the polarity of the mobile phase, reverse and normalphase properties of an ion-exchanger can be minimized, resulting in a high correlation between charge and retardation of the solute. Thus, the degree of ionization of several compounds was monitored in mobile-phase compositions of different pH values using cation exchange. The pK values of several pterin derivatives corresponded to those obtained by other methods. In addition, pK values of two unidentified pterin derivatives were determined, using only 20 pmol of each.     

A radiochemical study of irreversible adsorption of proteins on reversed-phase chromatographic packing materials

A radiochemical study of the irreversible adsorption of proteins on commercial reversed-phase HPLC packing materials is reported. The conditions of study are similar to those used in HPLC separation of protein. The effects of the amount and contact time of two proteins, ovalbumin and cytochrome c, are reported. Additional results include the effect of column pretreatment with protein, silanophilic mobile-phase blocking agent, and type of packing material on the extent of irreversible adsorption. The loss process is shown to be at least biphasic and the mechanisms of loss distinct for different proteins.     

High-performance liquid chromatographic determination of modafinil and its two metabolites in human plasma using solid-phase extraction

A simple procedure for the simultaneous determination of modafinil, its acid and sulfone metabolites in plasma is described. The assay involved an extraction of the drug, metabolites and internal standard from plasma with a solid-phase extraction using C₁₈ cartridges. These compounds were eluted by methanol. The extract was evaporated to dryness at 40°C under a gentle stream of nitrogen. The residue was redissolved in 250 μl of mobile-phase and a 30 μl aliquot was injected via an automatic sampler into the liquid chromatograph and eluted with the mobile-phase (26%, v/v acetonitrile in 0.05 M orthophosphoric acid buffer adjusted to pH 2.6) at a flow-rate of 1.1 ml/min on a C₈ Symmetry cartridge column (5 μm, 150 mm×3.9 mm, Waters) at 25°C. The eluate was detected at 225 nm. Intra-day coefficients of variation ranged from 1.0 to 2.9% and inter-day coefficients from 0.9 to 6.1%. The limits of detection and quantitation of the assay were 0.01 μg/ml and 0.10 μg/ml respectively.     

A Low Gastric pH Mouse Model to Evaluate Live Attenuated Bacterial Vaccines

The low pH of the stomach serves as a barrier to ingested microbes and must be overcome or bypassed when delivering live bacteria for vaccine or probiotic applications. Typically, the impact of stomach acidity on bacterial survival is evaluated in vitro, as there are no small animal models to evaluate these effects in vivo. To better understand the effect of this low pH barrier to live attenuated Salmonella vaccines, which are often very sensitive to low pH, we investigated the value of the histamine mouse model for this application. A low pH gastric compartment was transiently induced in mice by the injection of histamine. This resulted in a gastric compartment of approximately pH 1.5 that was capable of distinguishing between acid-sensitive and acid-resistant microbes. Survival of enteric microbes during gastric transit in this model directly correlated with their in vitro acid resistance. Because many Salmonella enterica serotype Typhi vaccine strains are sensitive to acid, we have been investigating systems to enhance the acid resistance of these bacteria. Using the histamine mouse model, we demonstrate that the in vivo survival of S. Typhi vaccine strains increased approximately 10-fold when they carried a sugar-inducible arginine decarboxylase system. We conclude that this model will be a useful for evaluating live bacterial preparations prior to clinical trials.     

Glycosylated liposomes against Helicobacter pylori: Behavior in acidic conditions

Helicobacter pylori was isolated in 1982 and confirmed as a gastric pathogenic agent at the end of the 1980s. The present work deals with liposomes formulations in which are incorporated cholesteryl tetraethylene glycol oside as model ligands for H. pylori adhesins. This study is devoted to the behavior of liposomes in gastric conditions. The glycosylated vesicles are stable and the pH of the internal aqueous compartment remains close to 4 even through more acidic conditions are imposed to the external phase (pH 1.2-2). Such a pH gradient depends essentially on the nature of phospholipids used and is not extensively affected by the incorporation of the targeting agent. These aspects are particularly important to the development of liposome formulations against H. pylori, bacteria sensitive to antibiotics which are unstable in very acidic conditions.     

Use of models in understanding the mechanism of action of haemoglobin

Models with three, four and eight salt-bridges have been used to study the mechanism of action of haemoglobin. Both side chains forming a salt-bridge, i.e. the proton acceptor and the proton donor, are postulated to change pK on ligation of oxygen. The eight salt-bridge model is able to predict, as a unified theory, both the degree of oxygenation and the Bohr effect at any PH and p_O2 value; this has not been done by any other published model. The predicted pK values for the Borh groups corresponde well with those measured experiemntally. This model predicts the pK values of those side chains responsible for the acid Bohr effect, suggesting that these correspond to the proton acceptors of the salt-bridges. The model also fulfils the condition of linearity between the fractional degree of oxygenation and fractional number of protons released. It is postulated that there is a gradual change in structure on going from deoxy to oxyhaemoglobin, due to the rupture of salt-bridges. The path folowed during this process will be both pH and p_O2 dependent. A formula describing the number of intact or broken salt-bridges as a function of pH and p_O2 was developed. This formula shows that the fractional number of broken salt-bridges reaches a minimum value of 0.2 at around pH 6.3 in the absence of oxygen. However, if oxygen is added, this fractional number approaches 1.0 soon after the partial pressure of oxygen goes above 40 mm Hg.     

Identification of pH-sensing Sites in the Light Harvesting Complex Stress-related 3 Protein Essential for Triggering Non-photochemical Quenching in Chlamydomonas reinhardtii

Light harvesting complex stress-related 3 (LHCSR3) is the protein essential for photoprotective excess energy dissipation (non-photochemical quenching, NPQ) in the model green alga Chlamydomonas reinhardtii. Activation of NPQ requires low pH in the thylakoid lumen, which is induced in excess light conditions and sensed by lumen-exposed acidic residues. In this work we have used site-specific mutagenesis in vivo and in vitro for identification of the residues in LHCSR3 that are responsible for sensing lumen pH. Lumen-exposed protonatable residues, aspartate and glutamate, were mutated to asparagine and glutamine, respectively. By expression in a mutant lacking all LHCSR isoforms, residues Asp¹¹⁷, Glu²²¹, and Glu²²⁴ were shown to be essential for LHCSR3-dependent NPQ induction in C. reinhardtii. Analysis of recombinant proteins carrying the same mutations refolded in vitro with pigments showed that the capacity of responding to low pH by decreasing the fluorescence lifetime, present in the wild-type protein, was lost. Consistent with a role in pH sensing, the mutations led to a substantial reduction in binding the NPQ inhibitor dicyclohexylcarbodiimide.     

Theoretical considerations and modeling of chemical inactivation of microorganisms: Inactivation of Giardia Cysts by free chlorine

Chemical inactivation of microorganisms is a common process widely employed in many fields such as in treatment of water, preservation in food industry and antimicrobial treatments in healthcare. For economy of applications and efficiency of treatment establishment the minimum dosage of breakpoint in the chemical application becomes essential. Even though experimental investigations have been extensive, theoretical understanding of such processes are demanding. Commonly employed theoretical analyses for the inactivation of microorganisms and depletion of chemicals include kinetics expressing the rates of depletion of chemical and microorganisms. The terms chemical demand (x) and specific disinfectant demand (α) are often used in theoretical modeling of inactivation. The value of specific disinfectant demand (α) has always been assumed to be a constant in these models. Intracellular concentration built up within the cells of the microorganisms during inactivation could lead to possible weakening effects of microorganisms thereby requiring lower doses as disinfection proceeds makes the assumption of constant α inaccurate. Model equations are formulated based on these observations co-relating the parameters α and x with a progressive inactivation (N/N₀). The chemical concentration (C) is also presented in terms of the inactivation time (t) and the survival ratio (N/N₀) for given pH and temperature conditions. The model is examined using experimentally verified Ct data of Giardia Cysts/chlorine system. The respective values of x for different survival ratios were evaluated from the data using MatLab software. Proposed model correlating for the disinfectant demand (x) with the survival ratio (N/N₀) fits satisfactorily with those evaluated from data. The rate constants for different pH and temperature conditions are evaluated which showed compatibility with the Arrhenius model. The dependence of frequency factors with pH indicated compatibility with accepted models. The Ct values regenerated with the kinetic data shows a very accurate fit with published data.     

The Fab1/PIKfyve Phosphoinositide Phosphate Kinase Is Not Necessary to Maintain the pH of Lysosomes and of the Yeast Vacuole

Lysosomes and the yeast vacuole are degradative and acidic organelles. Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P₂), a master architect of endolysosome and vacuole identity, is thought to be necessary for vacuolar acidification in yeast. There is also evidence that PtdIns(3,5)P₂ may play a role in lysosomal acidification in higher eukaryotes. Nevertheless, these conclusions rely on qualitative assays of lysosome/vacuole pH. For example, quinacrine, an acidotropic fluorescent base, does not accumulate in the vacuoles of fab1Δ yeast. Fab1, along with its mammalian ortholog PIKfyve, is the lipid kinase responsible for synthesizing PtdIns(3,5)P₂. In this study, we employed several assays that quantitatively assessed the lysosomal and vacuolar pH in PtdIns(3,5)P₂-depleted cells. Using ratiometric imaging, we conclude that lysosomes retain a pH < 5 in PIKfyve-inhibited mammalian cells. In addition, quantitative fluorescence microscopy of vacuole-targeted pHluorin, a pH-sensitive GFP variant, indicates that fab1Δ vacuoles are as acidic as wild-type yeast. Importantly, we also employed fluorimetry of vacuoles loaded with cDCFDA, a pH-sensitive dye, to show that both wild-type and fab1Δ vacuoles have a pH < 5.0. In comparison, the vacuolar pH of the V-ATPase mutant vph1Δ or vph1Δ fab1Δ double mutant was 6.1. Although the steady-state vacuolar pH is not affected by PtdIns(3,5)P₂ depletion, it may have a role in stabilizing the vacuolar pH during salt shock. Overall, we propose a model in which PtdIns(3,5)P₂ does not govern the steady-state pH of vacuoles or lysosomes.     

Modeling alcoholic fermentation of glucose/xylose mixtures by ethanologenic Escherichia coli as a function of pH

In order to understand the effect of pH on growth and ethanol production in ethanologenic Escherichia coli, we investigated the kinetic behavior of ethanologenic E. coli during alcoholic fermentation of glucose or xylose in a controlled pH environment and the fermentation of glucose, xylose, or their mixtures without pH control. Based on the Monod equation, an unstructured and unsegregated kinetic model was proposed as a function of the pH of the fermentation medium. The pH effects on cell growth, sugar consumption, and ethanol production were taken into account in the proposed model. Both cell growth and ethanol production were found to be significantly influenced by the pH of the fermentation medium. The optimal pH range for ethanol production by ethanologenic E. coli on either glucose or xylose was 6.0–6.5. The highest value of the maximum specific growth rate (μ _m) was obtained at pH 7.0. In the kinetic model of the fermentations of the sugar mixture, two inhibition terms related to glucose concentrations were included in both the cell growth and ethanol production equations because of the strong inhibitions of glucose and glucose metabolites on xylose metabolism. A good fit was found between model predictions and experimental data for both single-sugar and mixed-sugar fermentations without pH control within the experimental domain.     

Thermodynamic redox behavior of the heme centers in A-type heme-copper oxygen reductases: comparison between the two subfamilies

The study of the thermodynamic redox behavior of the hemes from two members of the A family of heme-copper oxygen reductases, Paracoccus denitrificans aa₃ (A1 subfamily) and Rhodothermus marinus caa₃ (A2 subfamily) enzymes, is presented. At different pH values, midpoint reduction potentials and interaction potentials were obtained in the framework of a pairwise model for two interacting redox centers. In both enzymes, the hemes have different reduction potentials. For the A1-type enzyme, it was shown that heme a has a pH-dependent midpoint reduction potential, whereas that of heme a₃ is pH independent. For the A2-type enzyme the opposite was observed. The midpoint reduction potential of heme c from subunit II of the caa₃ enzyme was determined by fitting the data with a single-electron Nernst curve, and it was shown to be pH dependent. The results presented here for these A-type enzymes are compared with those previously obtained for representative members of the B and C families.     

Cellulase–lignin interactions—The role of carbohydrate-binding module and pH in non-productive binding

Non-productive cellulase adsorption onto lignin is a major inhibitory mechanism preventing enzymatic hydrolysis of lignocellulosic feedstocks. Therefore, understanding of enzyme–lignin interactions is essential for the development of enzyme mixtures and processes for lignocellulose hydrolysis. We have studied cellulase–lignin interactions using model enzymes, Melanocarpus albomyces Cel45A endoglucanase (MaCel45A) and its fusions with native and mutated carbohydrate-binding modules (CBMs) from Trichoderma reesei Cel7A. Binding of MaCel45A to lignin was dependent on pH in the presence and absence of the CBM; at high pH, less enzyme bound to isolated lignins. Potentiometric titration of the lignin preparations showed that negatively charged groups were present in the lignin samples and that negative charge in the samples was increased with increasing pH. The results suggest that electrostatic interactions contributed to non-productive enzyme adsorption: Reduced enzyme binding at high pH was presumably due to repulsive electrostatic interactions between the enzymes and lignin. The CBM increased binding of MaCel45A to the isolated lignins only at high pH. Hydrophobic interactions are probably involved in CBM binding to lignin, because the same aromatic amino acids that are essential in CBM–cellulose interaction were also shown to contribute to lignin-binding.     

Ozone alteration of transport of cations and the Na+/K+-ATPase in human erythrocytes.

We have purified glutaminase 65-fold from cow brain; the final specific activity is 24 μmol/min/mg. The enzyme is stable between pH 7.5 and 9.0 and has maximal activity at pH 8.8. It requires P_i for activity. The dependence of activity on P_i concentration is sigmoidal; 50 mmP_i gives half-maximal velocity at pH 8.8. At 0.2 mP_i, pH 8.8, the dependence of activity on glutamine concentration is hyperbolic; the observed K_Gln was 30 mm. Increasing P_i concentrations increase the apparent V_m and decrease the apparent K_Gln. NH₄⁺ does not inhibit at concentrations up to 0.1 m. Glutamic acid inhibits competitively with respect to glutamine; at 0.2 mP_i pH 8.8, K_Gln was 30 mm and K_Glu was 19 mm. The results are consistent with a model in which NH₄⁺ is released irreversibly from the enzyme-substrate complex and is the first product released. The activity of glutaminase appears to be independent of the nature of the buffer with which it is equilibrated before being assayed.     

Modeling the Growth of Listeria monocytogenes in Soft Blue-White Cheese

The aim of this study was to develop a predictive model simulating growth over time of the pathogenic bacterium Listeria monocytogenes in a soft blue-white cheese. The physicochemical properties in a matrix such as cheese are essential controlling factors influencing the growth of L. monocytogenes. We developed a predictive tertiary model of the bacterial growth of L. monocytogenes as a function of temperature, pH, NaCl, and lactic acid. We measured the variations over time of the physicochemical properties in the cheese. Our predictive model was developed based on broth data produced in previous studies. New growth data sets were produced to independently calibrate and validate the developed model. A characteristic of this tertiary model is that it handles dynamic growth conditions described in time series of temperature, pH, NaCl, and lactic acid. Supplying the model with realistic production and retail conditions showed that the number of L. monocytogenes cells increases 3 to 3.5 log within the shelf life of the cheese.     

Reactivity-selectivity properties of reactions of carcinogenic electrophiles and nucleosides: Influence of pH on site selectivity

6-Chloromethylbenzo[a]pyrene (6-CMBP) labeled with ¹³C in the chloromethyl group was used as a model for those carcinogens which form essentially free carbocations. Using ¹³C-NMR to identify products, the selectivity with which this electrophile modifies nucleosides was investigated. At pH 7, guanosine and deoxyguanosine are the most nucleophilic nucleosides toward the carbocation generated by solvolysis of 6-CMBP. Attack at N-7 predominates over attack at N-2. At higher pH, the nucleophilicity of guanosine and deoxyguanosines increases markedly. In addition, the site of modification changes to N-1 with secondary modification at O-6. The pH dependence of the rate of this reaction implicates a group with pK-value approx. 8.7 which was assigned to the hydrogen on N-1. The presence of a methyl group on the N-7 position of guanosine lowers this pK-value to approx. 7.2. Consequently, N⁷-methylguanosine shows the high nucleophilicity at physiological pH that guanosine has at high pH. These observations lead to the suggestion of a one base: two-site model for chemical carcinogenesis.     

Effects of low pH and Pb2+ stress on living cyanobacterium, Phormidium angustissimum West & G.S.West : A test of its feasibility as a living biosorbent

Pb²⁺ adsorption by the living cyanobacterium, Phormidium angustissimum followed the Langmuir adsorption model, with the maximum adsorption capacity (q _max ) of 295.4?±?13.8 mg g^?1. This result suggests that P. angustissimum is a promising living biosorbent to remove Pb²⁺ from wastewaters. Living biosorbents are better able to remove Pb²⁺ from wastewater than dead biosorbents, however there are practical limitations for their use are encountered in extreme conditions such as low pH and high Pb²⁺ concentration. The feasibility of using cyanobacterium, P. angustissimum, as a living biosorbent for the extraction of Pb²⁺ from wastewater was studied by investigating its photosynthestic performance and tolerance under Pb²⁺ (0–5 mg ?L^?1) contamination and low pH (pH?3–7). Decreased photosynthetic performance caused by Pb²⁺ contamination and low pH stress was detected in this study by means of a reduction of the maximum photochemical efficiency of PSII (F_v/F_m). Detoxification mechanisms of P. angustissimum on Pb²⁺ appeared to increase its intracellular polysaccharides (IPS), exocellular polysaccharides (EPS), and protein. Living P. angustissimum could increase the pH of the solution which resulted in Pb²⁺ precipitation. The unique ability of P. angustissimum to remove Pb²⁺ and to grow under toxic conditions, demonstrated herein, indicates that it is a promising living biosorbent for mildly acidic water contaminated with Pb²⁺ in bioremoval systems in the which pH is not lower than 5 and Pb²⁺ is not higher than 5 mg L^?1.     

A quantitative HPLC–MS method for the simultaneous determination of testosterone, 11-ketotestosterone and 11-β hydroxyandrostenedione in fish serum

A simple and novel HPLC–MS method for the simultaneous quantification of testosterone, 11-ketotestosterone, and 11β-hydroxyandrostenedione in fish serum was developed and validated. Separation was achieved on a C-18 column using a water–acetonitrile mobile-phase with a cycle time of 12 min. Ion detection was performed using ESI positive SIM at [M+H] (m/z 303, 303, 289). The linear ranges (0.2–50 ng/ml), limits of detection (0.1–0.2 ng/ml) and quantification (0.2–0.5 ng/ml) were established. The method was validated by measuring the three androgens in goldfish sera, displaying comparable values to those reported by other analytical techniques (RIA, EIA).