EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 15 2009
Luca Pignataro
Abstract A small library of chiral BINOL-derived monodentate phosphites containing either a carboxylic acid (-COOH, A1,A3) or a tertiary amine (-NMe2, B1,B4) was synthesized. The ligand combinations were screened in the enantioselective rhodium-catalyzed hydrogenation of methyl 2-acetamidoacrylate. The heterocombinations of the amine-derived phosphite (R)- B2 and of a carboxylic-phosphite [(R)- A1, (R)- A2, (R)- A3] displayed a slightly higher level of enantioselectivity compared to the corresponding homocombinations [up to 90,% ee using (R)- A1/(R)- B2]. The nature and extent of the interaction between the acidic and basic ligands in the rhodium complexes were studied by IR and 31P-NMR spectroscopy. The formation of an intramolecular salt in the Rh-heterocomplex, between the carboxylic acid and the tertiary amine, was suggested by the IR spectra. The selective formation of the Rh-heterocomplex was quantitatively assessed by 31P-NMR spectroscopy, using a modified acidic ligand (R)- A1-Me. In this way, a moderate (ca. 70:30) heterocomplex/homocomplexes ratio was determined.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009) [source]

Concave Pyridines for Bifunctional Acid,Base Catalysis,

EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 6 2007
Timo Liebig
Abstract Two bifunctional concave acid,base catalysts, 1 and 2, have been synthesized starting from 2,6-dibromopyridine (9) and 2,6-bis(,-alkenyloxy)phenylboronic acids 8 and 10 which end up as bridgeheads in final bimacrocycles 1 and 2. One bridgehead contained an additional substituent in the 4-position. The respective protected 4-hydroxymethyl-substituted phenylboronic acids 8 were synthesized from 4-bromo-3,5-dihydroxybenzoic acid (3) in five steps. 4-Unsubstituted boronic acid 10 and 4-substituted boronic acid 8 were then attached to 9 by subsequent Suzuki couplings to give tetra-,-alkenes 12. By ring-closing metathesis of 12, bimacrocyclic dienes 13 and 17 were formed. After deprotection of the 4-hydroxymethyl group of one bridgehead, a 3-hydroxybenzoate was coupled to 14 to give ester 15 which gave bifunctional acid,base catalyst 1 upon hydrolysis. Analogously, homologue 2 was synthesized, but before coupling the bimacrocycle to the benzoate, tetraene 14 was hydrogenated to 18. Acidic and basic centers in 1 (49,% from 9) and 2 (19,% from 9) are at least 5 Å apart.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007) [source]

Reinvestigation of the Mechanism of gem -Diacylation: Chemoselective Conversion of Aldehydes to Various gem -Diacylates and Their Cleavage under Acidic and Basic Conditions

EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 2 2005
Veerababurao Kavala
Abstract The mechanism of gem -diacylate formation has been studied extensively using tetrabutylammonium tribromide (TBATB) as the catalyst. The reaction proceeds by a nucleophilic attack of an anhydride on an aldehydic carbonyl group, nucleophilic attack of the hemiacylate intermediate on a second molecule of the anhydride, followed by an intermolecular attack of a second acetate group to regenerate the anhydride. gem -Diacylates of various aliphatic and aromatic aldehydes were obtained directly from the reaction of a variety of aliphatic and aromatic acid anhydrides in the presence of a catalytic quantity of tetrabutylammonium tribromide (TBATB) under solvent-free conditions. A significant electronic effect was observed during its formation as well as deprotection to the corresponding aldehyde. Chemoselective gem -diacylation of the aromatic aldehyde containing an electron-donating group has been achieved in the presence of an aldehyde containing an electron-withdrawing group. Deprotection of the gem -diacylate to the parent carbonyl compound can be accomplished in methanol in presence of the same catalyst. Here again, chemoselective deprotection of the gem -diacylate of a substrate containing an electrondonating group has been achieved in the presence of a substrate containing an electron-withdrawing group. Both the acid and base stability order of the various gem -diacylates examined follow a similar order. The stability order determined from the present study is: gem -dibenzoate > gemdipivalate > gem -diisobutyrate > gem -diacetate > gem -dipropionate. All the gem -diacylals are more stable under basic conditions than acidic condition. No correlation was found between the stability order and the pKa's of the corresponding acids; rather, the stability order is directly related to the steric crowding around the carbonyl carbon. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005) [source]

Mineral-Coated Polymer Microspheres for Controlled Protein Binding and Release

ADVANCED MATERIALS, Issue 19 2009
Leenaporn Jongpaiboonkit
Polymer microspheres with a bone-like mineral coatings are generated via a biomimetic process, and this biodegradable coating is used as a carrier for delivery of biological molecules. Acidic and basic proteins are controllably bound and released from these microspheres, suggesting that this approach can be used for binding and delivery of a broad range of biologically active molecules. [source]

Direct B -Alkyl Suzuki,Miyaura Cross-Coupling of Trialkyl- boranes with Aryl Bromides in the Presence of Unmasked Acidic or Basic Functions and Base-Labile Protections under Mild Non-Aqueous Conditions

ADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 3 2009
Bing Wang
Abstract An efficient and chemoselective palladium-catalyzed direct B -alkyl Suzuki,Miyaura cross-coupling of trialkylboranes with diversely functionalized aryl bromides is described. A wide variety of unmasked acidic or basic functions are tolerated. The mild non-aqueous conditions are compatible with aldehydes, ketones, nitriles, chloro substitution as well as base-labile phenolic Piv and TBS protecting groups. The anhydrous conditions were found to be advantageous for aryl bromide substrates. A potent CEPT inhibitor was efficiently synthesised using this protocol. [source]

An initial assessment of the use of gradient elution in microemulsion and micellar liquid chromatography

JOURNAL OF SEPARATION SCIENCE, JSS, Issue 17-18 2004
Simon M. Bryant
Abstract Novel microemulsion and micellar HPLC separations have been achieved using gradient elution and columns packed with reverse phase material. Initial attempts at gradient microemulsion liquid chromatography proved impossible on use of a microemulsion successfully used in capillary electrophoresis. Optimisation of the microemulsion composition allowed the generation of stable microemulsions to achieve separations in HPLC. The novel use of organic-solvent micellar chromatography in gradient elution mode was shown to give efficient separations. A range of efficient separations of pharmaceuticals and related impurities were obtained. Acidic, basic, and neutral solutes were resolved covering a wide range of water solubilities and polarities. Elution times were in the order of 4,15 minutes. Separations were briefly compared to those accomplished with a micellar HPLC system. It is proposed that gradient elution in both microemulsion and micellar HPLC can be regarded as a highly successful means of achieving resolution of complex mixtures and should be considered for routine analysis and further investigation. [source]

Synthesis of Polycaprolactone Using Free/Supported Enzymatic and Non-Enzymatic Catalysts

MACROMOLECULAR RAPID COMMUNICATIONS, Issue 24 2004
María Laura Foresti
Abstract Summary: Polymerization of caprolactone using lipases from Candida antarctica B, Rhizomucor meihei, Candida rugosa, and Pseudomonas fluorescens is highly effective, with 97% conversion into polycaprolactone. Poly(propylene)-supported Candida rugosa lipase achieves higher conversion values (85,92%) than free lipase (75%). Acidic and basic non-conventional catalysis with butanol yields 50,85% conversion. Simple UV/visible techniques gave the same results for measuring conversion than other studies. Applications are opened for the non-conventional catalysts. Mechanism of the polymerization of caprolactone polymerization using a basic catalyst. [source]

Pyruvate Preserves Neutrophilic Superoxide Production in Acidic, High Glucose-Enriched Peritoneal Dialysis Solutions

ARTIFICIAL ORGANS, Issue 3 2003
Yi Tai Wu
Abstract: Aim: To investigate effects of pyruvate (Py)-based peritoneal dialysis solutions (P-PDS) on neutrophilic superoxide (O2,) production against high glucose (HG) concentrations at acidic or physiologic pH value, and explore potential mechanisms. Methods: Human neutrophils were incubated with both dl -lactate (La, 40 mM)-based PDS (L-PDS) and equimolar P-PDS at various pH and HG levels, respectively. Hanks' balanced salt solution (HBSS) served as controls. O2, generation was determined by the reduction of ferricytochrome c. Results: Acidic pH and high La induced acute and substantial inhibitions of O2, production. HG in both PDS and HBSS resulted in a suppression of O2, in a dose-dependent manner. P-PDS generated near twofold O2, formation of L-PDS counterparts at various pH and HG levels. P-PDS with HG produced significantly more O2, than Py-free HBSS counterparts. Conclusions: Py in PDS effectively protected neutrophils from HG-induced inhibition of O2, production, even at a physiological pH. The Py cytoprotection may be associated with the preservation of carbohydrate metabolic pathways in addition to its alkalization. [source]

The effect of low intensity ultraviolet-C light on monoclonal antibodies

BIOTECHNOLOGY PROGRESS, Issue 2 2009
Christopher M. Lorenz
Abstract As part of an investigation to identify potential new viral reduction strategies, ultraviolet-C (UV-C) light was examined. Although this technology has been known for decades to possess excellent virus inactivation capabilities, UV-C light can also introduce significant unwanted damage to proteins. To study the effect on monoclonal antibodies, three different antibodies were subjected to varying levels of UV-C light using a novel dosing device from Bayer Technology Services GmbH. The range of fluencies (or doses) covered was between 0 and 300 J/m2 at a wavelength of 254 nm. Product quality data generated from the processed pools showed only minimal damage done to the antibodies. Aggregate formation was low for two of the three antibodies tested. Acidic and basic variants increased for all three antibodies, with the basic species increasing more than the acidic species. Peptide maps made for the three sets of pools showed no damage to two of the three antibody backbones, whereas the third antibody had very low levels of methionine oxidation evident. Samples held at 2,8°C for 33 days showed no increase in aggregates or charge variants, indicating that the proteins did not degrade and were not damaged further by reactive or catalytic species that may have been created on exposure to UV-C light. Overall, UV-C light was shown to induce very little damage to monoclonal antibodies at lower fluencies and appears to be a viable option for viral inactivation in biotechnology applications. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source]

Parallel Solution-Phase Synthesis of Acrylonitrile Scaffolds Carrying L-,-Amino Acidic or D-Glycosyl Residues.

CHEMINFORM, Issue 12 2007
Stefania Fioravanti
Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF. [source]

One-Pot Synthesis of Benzo[b]thiophenes and Naphtho[2,1-b]thiophenes in the Presence of Acidic and Basic Supported Reagents.

CHEMINFORM, Issue 14 2006
Tadashi Aoyama
Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF. [source]

SPARC is expressed by macroglia and microglia in the developing and mature nervous system

DEVELOPMENTAL DYNAMICS, Issue 5 2008
Adele J. Vincent
Abstract SPARC (secreted protein, acidic and rich in cysteine) is a matricellular protein that is highly expressed during development, tissue remodeling, and repair. SPARC produced by olfactory ensheathing cells (OECs) can promote axon sprouting in vitro and in vivo. Here, we show that in the developing nervous system of the mouse, SPARC is expressed by radial glia, blood vessels, and other pial-derived structures during embryogenesis and postnatal development. The rostral migratory stream contains SPARC that becomes progressively restricted to the SVZ in adulthood. In the adult CNS, SPARC is enriched in specialized radial glial derivatives (Müller and Bergmann glia), microglia, and brainstem astrocytes. The peripheral glia, Schwann cells, and OECs express SPARC throughout development and in maturity, although it appears to be down-regulated with maturation. These data suggest that SPARC may be expressed by glia in a spatiotemporal manner consistent with a role in cell migration, neurogenesis, synaptic plasticity, and angiogenesis. Developmental Dynamics 237:1449-1462, 2008. © 2008 Wiley-Liss, Inc. [source]

Identification of some human genes oppositely regulated during esophageal squamous cell carcinoma formation and human embryonic esophagus development

DISEASES OF THE ESOPHAGUS, Issue 3 2010
M. V. Zinovyeva
SUMMARY Here we directly compared gene expression profiles in human esophageal squamous cell carcinomas and in human fetal esophagus development. We used the suppression subtractive hybridization technique to subtract cDNAs prepared from tumor and normal human esophageal samples. cDNA sequencing and reverse transcription polymerase chain reaction (RT-PCR) analysis of RNAs from human tumor and the normal esophagus revealed 10 differentially transcribed genes: CSTA, CRNN, CEACAM1, MAL, EMP1, ECRG2, and SPRR downregulated, and PLAUR, SFRP4, and secreted protein that is acidic and rich in cysteine upregulated in tumor tissue as compared with surrounding normal tissue. In turn, genes up- and downregulated in tumor tissue were down- and upregulated, respectively, during development from the fetal to adult esophagus. Thus, we demonstrated that, as reported for other tumors, gene transcriptional activation and/or suppression events in esophageal tumor progression were opposite to those observed during development from the fetal to adult esophagus. This tumor ,embryonization' supports the idea that stem or progenitor cells are implicated in esophageal cancer emergence. [source]

A quantitative approach to probe the dependence and correlation of food-effect with aqueous solubility, dose/solubility ratio, and partition coefficient (Log P) for orally active drugs administered as immediate-release formulations

DRUG DEVELOPMENT RESEARCH, Issue 2 2005
Brahma N. Singh
Abstract The purpose of the present review was to systematically evaluate if aqueous solubility, dose/solubility ratio, and partition coefficient (Log P) could be used as useful parameters to quantitatively probe the dependence and correlation of in vivo food effects with these physicochemical properties of orally active drugs administered as immediate-release (IR) formulations. Mean AUC data obtained under fasted and fed states of over 100 structurally diverse orally active drugs with different physicochemical properties were obtained from the primary literature. Correlations of AUC ratio (Fed/Fasted) with aqueous solubility, dose/solubility ratio, and Log P were derived and statistically evaluated by Pearson's correlation test (two-tailed). A negative correlation was obtained between the logarithm of the aqueous solubility and the AUC ratio (r=,0.5982, N=93), whereas a positive correlation existed between AUC ratio and Log P (r=0.5147, N=110) and between AUC ratio and dose/solubility ratio (r=0.5511, N=87). All these correlations were significant (P<0.0001). Based on this study, the estimated range within which a drug is not expected to be significantly affected by food falls between 0.148,89.39 mg/ml for aqueous solubility and between 0.23,624 ml for the dose:solubility ratio. The corresponding range of Log P for expecting a lack of food-effect lies between ,1.13 and 2.98. Quantitatively, the effect of food was most pronounced for lipophilic, poorly water-soluble drugs (with only a few exceptions), irrespective of whether the drug is acidic, basic, or neutral. It is concluded that aqueous solubility, dose/solubility ratio, and partition coefficient can be used as useful parameters to probe the dependence and correlation of food-effect with these physicochemical parameters for immediate-release formulations. Drug Dev. Res. 65:55,75, 2005. © 2005 Wiley-Liss, Inc. [source]

Immobilization and Electrochemistry of Negatively Charged Proteins on Modified Nanocrystalline Metal Oxide Electrodes

ELECTROANALYSIS, Issue 12 2005
Emmanuel Topoglidis
Abstract The immobilization of two acidic, low isoelectric point proteins, green fluorescence protein and ferredoxin (FRD) is investigated on nanocrystalline, mesoporous TiO2 and SnO2 electrodes. Modification of these electrodes with a cationic polypeptide (poly- L -lysine) or an aminosilane prior to protein immobilization is found to enhance protein binding at least ten fold, attributed to more favorable protein/electrode electrostatic interactions. Cyclic voltammetry studies of FRD-modified SnO2 electrodes indicate reversible protein electrochemistry with a midpoint potential of ,0.59,V (vs. Ag/AgCl) and an interfacial electron transfer rate constant of 0.45,s,1. [source]

Evaluation of CE methods for global metabolic profiling of urine

ELECTROPHORESIS, Issue 14 2010
Rawi Ramautar
Abstract In this study, the usefulness of noncovalently coated capillaries with layers of charged polymers is investigated to obtain global electrophoretic profiles of urinary metabolites covering a broad range of different compound classes in a highly repeatable way. Capillaries were coated with a bilayer of polybrene (PB) and poly(vinyl sulfonate) (PVS), or with a triple layer of PB, dextran sulfate (DS) and PB. The bilayer and triple layer coatings were evaluated at acidic (pH 2.0) and alkaline (pH 9.0) separation conditions, thereby providing separation conditions for basic and acidic compounds. A representative metabolite mixture and spiked urine samples were used for the evaluation of the four CE methods. Migration time repeatability (RSD<2%) and plate numbers (N, 100,000,400,000) were similar for the test compounds in all CE methods, except for some multivalent ions that may exhibit adsorption to oppositely charged coatings. The analysis of cationic compounds with the PB-DS-PB CE method at low pH (i.e. after the EOF time) provided a larger separation window and number of separated peaks in urine compared to the analysis with the PB-PVS CE method at low pH (i.e. before the EOF time). Approximately, 600 molecular features were detected in rat urine by the PB-DS-PB CE-MS method whereas about 300 features were found with the PB-PVS CE-MS method. This difference can be attributed to reduced comigration of compounds with the PB-DS-PB CE-MS method and a related decrease of ion suppression. With regard to the analysis of anionic compounds by CE-MS, in general analyte responses were significantly lower than that for cationic compounds, most probably due to less efficient ionization and to ion suppression effects caused by the background electrolyte. Hence, further optimization is required for the sensitive CE-MS analysis of anionic compounds in body fluids. It is concluded that the selection of a CE method for profiling of cationic metabolites in urine depends on the purpose of the study. For high-throughput analyses, the PB-PVS CE-MS method is favored whereas the PB-DS-PB CE-MS method provides a more information-rich metabolic profile, but at the cost of prolonged analysis time. [source]

Electrophoretic behaviors of human hepatoma HepG2 cells

ELECTROPHORESIS, Issue 9 2009
Jyh-Ping Hsu
Abstract The electrophoretic mobility of HepG2 cells was measured and a charge-regulated model was proposed to simulate the results obtained. Here, a cell was simulated by a rigid core and an ion-penetrable membrane layer containing both acidic and basic functional groups. The influences of the key parameters, including the pH, the ionic strength, the thickness of the membrane layer of a cell, the density and the dissociation constant of the dissociable functional groups in the membrane layer, and the binding constant of divalent cations on the electrophoretic mobility of a cell were investigated. In particular, the role of the buffer used in the experiment was discussed; this effect was neglected in almost all the relevant theoretical analyses in the literature. We showed that the binding ability of divalent cations to the dissociated functional groups in the membrane layer of a cell ranks as Ca2+>Mg2+>hexamethonium. [source]

Electrophoresis of a charge-regulated toroid normal to a large disk

ELECTROPHORESIS, Issue 2 2008
Jyh-Ping Hsu
Abstract The electrophoresis of a charge-regulated toroid (doughnut-shaped entity) normal to a large disk is investigated under the conditions of low surface potential and weak applied electric field. The system considered is capable of modeling the electrophoretic behavior of various types of biocolloids such as bacterial DNA, plasmid DNA, and anabaenopsis near a perfectly conducting planar wall. The influences of the size of the toroid, the separation distance between the toroid and the disk, the charged conditions on the surfaces of the toroid and the disk, and the thickness of electric double layer on the electrophoretic mobility of the toroid are discussed. The results of numerical simulation reveal that under typical conditions the electrophoretic behavior of the toroid can be different from that of an integrated entity. For instance, if the surface of the toroid carries both acidic and basic functional groups, its mobility may have a local maximum as the thickness of double layer varies. We show that the electrophoretic behavior of the toroid is different, both qualitatively and quantitatively, from that of the corresponding integrated particle (particle without hole). [source]

CE analysis of the acidic organelles of a single cell

ELECTROPHORESIS, Issue 14 2007
Yun Chen
Abstract The properties of organelles within a cell have been shown to be highly heterogeneous. Until now, it has been unclear just how much of this heterogeneity is endemic to the organelle subpopulations themselves and how much is actually due to stochastic cellular noise. An attractive approach for investigating the origins of heterogeneity among the organelles of a single cell is CE with LIF detection (CE-LIF). As a proof of principle, in this report we optimize and use a single cell CE-LIF method to investigate the properties of endocytic (acidic) organelles. Our results show that the properties of individual acidic organelles containing Alexa Fluor® 488 Dextran suggest that there are two groups of CCRF-CEM cells: a group with a high dextran content per cell, and a group with a low dextran content per cell. Furthermore, the individual organelle measurements of the single cells allow us to compare in each group the distributions of doxorubicin content per acidic organelle and electrophoretic mobilities of these organelles. [source]

pH transients during salt removal in isoelectric trapping separations: A curse revisited

ELECTROPHORESIS, Issue 4 2007
Evan Shave
Abstract The pH transients that occur during isoelectric trapping separations as a result of the removal of nonampholytic ionic components have been re-examined. Salts containing strong electrolyte anions and cations, both with equal and dissimilar mobilities, have been studied using anodic and cathodic buffering membranes whose pH values were both equidistant and nonequidistant from pH,7. The direction and magnitude of the pH transient (acidic or basic) was found to depend on both the mobilities of the anion and cation (,anion/,cation) and the pH difference between pH,7 and the pH of the buffering membranes (|pHmembanodic,,,7|/|7,,,pHmembcathodic|). When |pHmembanodic,,,7|/|7,,,pHmembcathodic|,=,1, ,anion/,cation<1 leads to an acidic pH transient, ,anion/,cation,=,1 eliminates the pH transient and ,anion/,cation>1 leads to a basic pH transient. When ,anion/,cation,=,1, |pHmembanodic ,,7|/|7,,,pHmembcathodic|<1 leads to a basic pH transient, |pHmembanodic,,,7|/|7,,,pHmembcathodic|,=,1 eliminates the pH transient and |pHmembanodic,,,7|/|7,,,pHmembcathodic|>1 leads to an acidic pH transient. By selecting appropriate anodic and cathodic buffering membranes to adjust the |pHmembanodic,,,7|/|7,,,pHmembcathodic| value, pH transients caused by dissimilar anion and cation mobilities can be avoided. [source]

High-efficiency peptide analysis on monolithic multimode capillary columns: Pressure-assisted capillary electrochromatography/capillary electrophoresis coupled to UV and electrospray ionization-mass spectrometry

ELECTROPHORESIS, Issue 21 2003
Alexander R. Ivanov
Abstract High-efficiency peptide analysis using multimode pressure-assisted capillary electrochromatography/capillary electrophoresis (pCEC/pCE) monolithic polymeric columns and the separation of model peptide mixtures and protein digests by isocratic and gradient elution under an applied electric field with UV and electrospray ionization-mass spectrometry (ESI-MS) detection is demonstrated. Capillary multipurpose columns were prepared in silanized fused-silica capillaries of 50, 75, and 100 ,m inner diameters by thermally induced in situ copolymerization of methacrylic monomers in the presence of n -propanol and formamide as porogens and azobisisobutyronitrile as initiator. N -Ethylbutylamine was used to modify the chromatographic surface of the monolith from neutral to cationic. Monolithic columns were termed as multipurpose or multimode columns because they showed mixed modes of separation mechanisms under different conditions. Anion-exchange separation ability in the liquid chromatography (LC) mode can be determined by the cationic chromatographic surface of the monolith. At acidic pH and high voltage across the column, the monolithic stationary phase provided conditions for predominantly capillary electrophoretic migration of peptides. At basic pH and electric field across the column, enhanced chromatographic retention of peptides on monolithic capillary column made CEC mechanisms of migration responsible for separation. The role of pressure, ionic strength, pH, and organic content of the mobile phase on chromatographic performance was investigated. High efficiencies (exceeding 300,000 plates/m) of the monolithic columns for peptide separations are shown using volatile and nonvolatile, acidic and basic buffers. Good reproducibility and robustness of isocratic and gradient elution pressure-assisted CEC/CE separations were achieved for both UV and ESI-MS detection. Manipulation of the electric field and gradient conditions allowed high-throughput analysis of complex peptide mixtures. A simple design of sheathless electrospray emitter provided effective and robust low dead volume interfacing of monolithic multimode columns with ESI-MS. Gradient elution pressure-assisted mixed-mode separation CE/CEC-ESI-MS mass fingerprinting and data-dependent pCE/pCEC-ESI-MS/MS analysis of a bovine serum albumin (BSA) tryptic digest in less than 5 min yielding high sequence coverage (73%) demonstrated the potential of the method. [source]

Digestive peptidases in Tenebrio molitor and possibility of use to treat celiac disease

ENTOMOLOGICAL RESEARCH, Issue 3 2007
Elena N. ELPIDINA
Abstract Digestion in Tenebrio molitor larvae occurs in the midgut, where there is a sharp pH gradient from 5.6 in the anterior midgut (AM) to 7.9 in the posterior midgut (PM). Accordingly, digestive enzymes are compartmentalized to the AM or PM. Enzymes in the AM are soluble and have acidic or neutral pH optima, while PM enzymes have alkaline pH optima. The main peptidases in the AM are cysteine endopeptidases presented by two to six subfractions of anionic proteins. The major activity belongs to cathepsin L, which has been purified and characterized. Serine post-proline cleaving peptidase with pH optimum 5.3 was also found in the AM. Typical serine digestive endopeptidases, trypsin-like and chymotrypsin-like, are compartmentalized to the PM. Trypsin-like activity is due to one cationic and three anionic proteinases. Chymotrypsin-like activity consists of one cationic and four anionic proteinases, four with an extended binding site. The major cationic trypsin and chymotrypsin have been purified and thoroughly characterized. The predicted amino acid sequences are available for purified cathepsin L, trypsin and chymotrypsin. Additional sequences for putative digestive cathepsins L, trypsins and chymotrypsins are available, implying multigene families for these enzymes. Exopeptidases are found in the PM and are presented by a single membrane aminopeptidase N-like peptidase and carboxypeptidase A, although multiple cDNAs for carboxypeptidase A were found in the AM, but not in the PM. The possibility of the use of two endopeptidases from the AM , cathepsin L and post-proline cleaving peptidase , in the treatment of celiac disease is discussed. [source]

Autoinducers extracted from microbial mats reveal a surprising diversity of N -acylhomoserine lactones (AHLs) and abundance changes that may relate to diel pH

ENVIRONMENTAL MICROBIOLOGY, Issue 2 2009
Alan W. Decho
Summary Microbial mats are highly structured and diverse communities, and one of the earliest-known life assemblages. Mat bacteria interact within an environment marked by strong geochemical gradients and fluctuations. We examined natural mat systems for the presence of autoinducers involved in quorum sensing, a form of cell,cell communication. Our results revealed that a diverse array of N -acylhomoserine lactones (AHLs) including C4 - to C14 -AHLs, were identified from mat extracts using mass spectrometry (MS), with further confirmation by MS/MS-collision-induced dissociation (CID), and additions of external standards. Microelectrode measurements showed that mats exhibited diel pH fluctuations, ranging from alkaline (pH 9.4) during daytime (net photosynthesis) to acidic (pH 6.8) during darkness (net respiration/fermentation). Under laboratory conditions, AHLs having shorter acyl-chains were degraded within the time frame that daily alkaline pH (> 8.2) conditions exist in mats. Intensive sampling of mats after full day- or night-time incubations revealed that accumulations of extractable shorter-chain AHLs (e.g. C8 - and C10 -AHLs) were significantly (P < 0.001) diminished during daytime. Our study offers evidence that stabilities of AHLs under natural conditions may be influenced by the proximal extracellular environment. We further propose that the ancient periodicity of photosynthesis/respiration in mats may potentially drive a mechanism for diel differences in activities of certain autoinducers, and hence bacterial activities mediated through quorum sensing. [source]

Linking microbial oxidation of arsenic with detection and phylogenetic analysis of arsenite oxidase genes in diverse geothermal environments

ENVIRONMENTAL MICROBIOLOGY, Issue 2 2009
N. Hamamura
Summary The identification and characterization of genes involved in the microbial oxidation of arsenite will contribute to our understanding of factors controlling As cycling in natural systems. Towards this goal, we recently characterized the widespread occurrence of aerobic arsenite oxidase genes (aroA -like) from pure-culture bacterial isolates, soils, sediments and geothermal mats, but were unable to detect these genes in all geothermal systems where we have observed microbial arsenite oxidation. Consequently, the objectives of the current study were to measure arsenite-oxidation rates in geochemically diverse thermal habitats in Yellowstone National Park (YNP) ranging in pH from 2.6 to 8, and to identify corresponding 16S rRNA and aroA genotypes associated with these arsenite-oxidizing environments. Geochemical analyses, including measurement of arsenite-oxidation rates within geothermal outflow channels, were combined with 16S rRNA gene and aroA functional gene analysis using newly designed primers to capture previously undescribed aroA -like arsenite oxidase gene diversity. The majority of bacterial 16S rRNA gene sequences found in acidic (pH 2.6,3.6) Fe-oxyhydroxide microbial mats were closely related to Hydrogenobaculum spp. (members of the bacterial order Aquificales), while the predominant sequences from near-neutral (pH 6.2,8) springs were affiliated with other Aquificales including Sulfurihydrogenibium spp., Thermocrinis spp. and Hydrogenobacter spp., as well as members of the Deinococci, Thermodesulfobacteria and ,- Proteobacteria. Modified primers designed around previously characterized and newly identified aroA -like genes successfully amplified new lineages of aroA- like genes associated with members of the Aquificales across all geothermal systems examined. The expression of Aquificales aroA- like genes was also confirmed in situ, and the resultant cDNA sequences were consistent with aroA genotypes identified in the same environments. The aroA sequences identified in the current study expand the phylogenetic distribution of known Mo-pterin arsenite oxidase genes, and suggest the importance of three prominent genera of the order Aquificales in arsenite oxidation across geochemically distinct geothermal habitats ranging in pH from 2.6 to 8. [source]

Ultrastructural and electron energy-loss spectroscopic analysis of an extracellular filamentous matrix of an environmental bacterial isolate

ENVIRONMENTAL MICROBIOLOGY, Issue 9 2007
Uta Böckelmann
Summary Strain F8, a bacterial isolate from ,river snow', was found to produce extracellular fibres in the form of a filamentous network. These extracellular filaments, which were previously shown to be composed of DNA, have been studied for the first time by ultrastructural and electron energy-loss spectroscopy in the present work. ,Whole mount' preparations of strain F8 indicate these polymers are ultrastructurally homogeneous and form a network of elemental filaments, which have a width of 1.8,2.0 nm. When incubated at pH 3.5 with colloidal cationic ThO2 tracers they become intensely stained (electron dense), affording direct evidence that the fibres are negatively charged and thus acidic chemically. Elemental analysis of the extracellular filaments by Energy-filtered Transmission Electron Microscopy revealed phosphorus to be the main element present and, because pretreatment of F8 cells with DNase prevented thorium labelling, the fibres must be composed of extracellular DNA (eDNA). Neither ultrathin sections nor ,whole mount negative stain' caused DNA release by general cell lysis. Additionally, cells infected with phages were never observed in ultrathin sections and phage particles were never detected in whole mount samples, which rules out the possibility of phages being directly involved in eDNA release. [source]

Vertical profiles of methanogenesis and methanogens in two contrasting acidic peatlands in central New York State, USA

ENVIRONMENTAL MICROBIOLOGY, Issue 8 2006
Hinsby Cadillo-Quiroz
Summary Northern acidic peatlands are important sources of atmospheric methane, yet the methanogens in them are poorly characterized. We examined methanogenic activities and methanogen populations at different depths in two peatlands, McLean bog (MB) and Chicago bog (CB). Both have acidic (pH 3.5,4.5) peat soils, but the pH of the deeper layers of CB is near-neutral, reflecting its previous existence as a neutral-pH fen. Acetotrophic and hydrogenotrophic methanogenesis could be stimulated in upper samples from both bogs, and phylotypes of methanogens using H2/CO2 (Methanomicrobiales) or acetate (Methanosarcinales) were identified in 16S rRNA gene clone libraries and by terminal restriction fragment length polymorphism (T-RFLP) analyses using a novel primer/restriction enzyme set that we developed. Particularly dominant in the upper layers was a clade in the Methanomicrobiales, called E2 here and the R10 or fen group elsewhere, estimated by quantitative polymerase chain reaction to be present at ,108 cells per gram of dry peat. Methanogenic activity was considerably lower in deeper samples from both bogs. The methanogen populations detected by T-RFLP in deeper portions of MB were mainly E2 and the uncultured euryarchaeal rice cluster (RC)-II group, whereas populations in the less acidic CB deep layers were considerably different, and included a Methanomicrobiales clade we call E1-E1,, as well as RC-I, RC-II, marine benthic group D, and a new cluster that we call the subaqueous cluster. E2 was barely detectable in the deeper samples from CB, further evidence for the associations of most organisms in this group with acidic habitats. [source]

Effects of estimates from different geochemical models on metal fate predicted by coupled speciation-fate models,

ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 5 2008
Satyendra P. Bhavsar
Abstract Coupled metal speciation-fate models are an improvement over stand-alone fate-transport models for accurately assessing metal fate and transport. These coupled models estimate fate-controlling partition coefficients using geochemical speciation/complexation models. Commercially available geochemical models are practical options for a two-step, loose coupling with fate-transport models. These models differ in their partitioning estimates because of differences in assumptions, databases, and so on. The present study examines the effects of differences in estimates from geochemical models on estimates of cationic metal fate using two geochemical models: the Windermere humic aqueous model (WHAM) and the minicomputer equilibrium+ model (MINEQL+). The results from each geochemical model were used as input to the fate module of TRANSPEC (a general, coupled metal transport and speciation model). The two versions of the TRANSPEC model were then used to assess the fate of five cationic metals (Cd, Cu, Ni, Pb, and Zn) in Ross Lake (Flin Flon, MB, Canada; alkaline, eutrophic, mine impacted), Kelly Lake (Sudbury, ON, Canada; circumneutral, mesotrophic, mine influenced), and Lake Tantaré (Quebec City, QC, Canada; acidic, oligotrophic, pristine). For relatively soluble metals (Cd, Ni, and Zn), the WHAM and MINEQL+ estimates of speciation/complexation were similar for Ross and Kelly lakes but differed for Lake Tantaré. These differences, however, did not result in significant differences in overall fate estimates. Marked differences were observed between the WHAM and MINEQL+ estimates of partition coefficient, Kd, for more particle-reactive Cu and Pb that translated into the greatest impact on fate in mesotrophic Kelly Lake, in which particle movement is important for fate. [source]

Seasonality effects on pharmaceuticals and s -triazine herbicides in wastewater effluent and surface water from the Canadian side of the upper Detroit River

ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 9 2006
Wen Yi Hua
Abstract The influence of seasonal changes in water conditions and parameters on several major pharmacologically active compounds (PhACs) and s -triazine herbicides was assessed in the wastewater and sewage treatment plant (WSTP) effluent as well as the downstream surface water from sites on the Canadian side of the upper Detroit River, between the Little River WSTP and near the water intake of a major drinking water treatment facility for the City of Windsor (ON, Canada). The assessed PhACs were of neutral (carbamazepine, cotinine, caffeine, cyclophosphamide, fluoxetine, norfluoxetine, pentoxifylline, and trimethoprim) and acidic (ibuprofen, bezafibrate, clofibric acid, diclofenac, fenoprofen, gemfibrozil, indomethacin, naproxen, and ketoprofen) varieties. The major assessed s -triazine herbicides were atrazine, simazine, propazine, prometon, ametryn, prometryn, and terbutryn. At sampling times from September 2002 to June 2003, 15 PhACs were detected in the WSTP effluent at concentrations ranging from 1.7 to 1,244 ng/L. The PhAC concentrations decreased by as much 92 to 100% at the Little River/Detroit River confluence because of the river dilution effect, with further continual decreases at sites downstream from the WSTP. The only quantifiable s -triazine in WSTP effluent, atrazine, ranged from 6.7 to 200 ng/L and was higher in Detroit River surface waters than in WSTP effluent. Only carbamazepine, cotinine, and atrazine were detectable at the low-nanogram and subnanogram levels in surface waters near a drinking water intake site. Unlike the PhACs, atrazine in the Detroit River is not attributable to point sources, and it is heavily influenced by seasonal agricultural usage and runoff. Detroit River surface water concentrations of carbamazepine, cotinine, and atrazine may present a health concern to aquatic wildlife and to humans via the consumption of drinking water. [source]

Stable strontium accumulation by earthworms: A paradigm for radiostrontium interactions with its cationic analogue, calcium

ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 6 2001
John E. Morgan
Abstract The accumulation of stable strontium and its chemical analogue calcium by four species of earthworm, representing three distinct ecophysiological groups inhabiting celestite (SrSO4)-rich natural soils, was investigated. An increase in soil strontium concentration over a four-orders-of-magnitude range was accompanied by an increase in earthworm tissue strontium concentration. In contrast, no relationship between soil and tissue calcium concentrations was apparent, indicating that this essential cation is strongly regulated within earthworm tissues but that nonessential strontium burdens are not physiologically regulated. Concentration factors for strontium ranged from 0.1 to 1.42, with an inverse relationship between this parameter and soil strontium concentration. The use of concentration factors and discrimination coefficients lead to the conclusion that earthworms discriminate against strontium accumulation at increased soil calcium concentrations, such that strontium would be predicted to be more efficiently accumulated by worms inhabiting acidic, calcium-poor soils. Although few consistent trends in strontium and calcium concentration were apparent between earthworm species, the species with the highest calcium turnover (i.e., the litter-inhabiting Lumbricus rubellus) typically accumulated higher concentrations of strontium than representatives of contrasting ecophysiological classes. These findings are considered in the context of the distribution, retention, and cycling of radiostrontium in soils. [source]

Reactions of Pendant Boryl Groups in Cp,Metal Complexes: Heterocyclic Ring Annelation in a CpIr System

EUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 14 2008
Christoph Herrmann
Abstract Treatment of (cod)IrCl dimer with Li(allyl)cyclopentadienide gave (C5H4,CH2,CH=CH2)Ir(cod) (11). At 100 °C, 11 isomerizes cleanly into its ring-conjugated (trans -CH3,CH=CH,C5H4)Ir(cod) isomer (13). Subsequent addition of HB(C6F5)2 results in the formation of a product (16) that contains an annelated five-membered borata heterocycle at the Cp ring. This is probably formed by means of a reaction sequence involving regioselective hydroboration, followed by an electrophilic substitution reaction at the Cp ring by the strongly Lewis acidic ,B(C6F5)2 group, in which the iridium metal base acts as the proton abstractor. Products 13 and 16 were characterized by X-ray diffraction. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008) [source]