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pH Gradient (ph + gradient)
Kinds of pH Gradient Selected AbstractsMicrochip isoelectric focusing with monolithic immobilized pH gradient materials for proteins separationELECTROPHORESIS, Issue 23 2009Yu Liang Abstract Monolithic immobilized pH gradient (M-IPG) materials were prepared in microchannles by photoinitiated polymerization of acrylamide, glycidylmethacrylate and Bis, followed by the attachment of focused Ampholine onto the surface of porous monoliths via epoxide groups. With M-IPG materials as matrix, FITC-labeled ribonuclease B, myoglobin and ,-casein were well separated by microchip isoelectric focusing (,CIEF) without carrier amphocytes (CAs) added in the buffer. Both chemical and pressure mobilization were applied to drive focused zones for LIF detection. Our experimental results showed that pressure mobilization was preferable with neglectable band broadening, and good peak shape and high detection sensitivity were obtained. All these results demonstrate that ,CIEF with M-IPG materials is not only an efficient mode for protein enrichment and separation but also attractive to couple with other CE modes to achieve multi-dimensional separation or MS for further identification, without the interference of mobile CAs. [source] Simulations of IEF in microchannel with variable cross-sectional areaELECTROPHORESIS, Issue 5 2009Yin Chou Abstract This study develops a 1-D mass transport model to describe the electrophoresis transport behavior within a microchannel with a variable cross-sectional area. Utilizing three different numerical schemes, simulations are performed to investigate the IEF of proteins in ampholyte-based pH gradients within both a planar microchannel and a contraction,expansion microchannel, respectively. The simulation results obtained using the modified 1-D mass transport model and the finite-volume method (FVM) for the IEF separation of a single protein sample in a ten-ampholyte-based pH gradient within a planar microchannel are consistent with those presented by Shim et al. [Electrophoresis 2007, 28, 572,586] using a 2-D FVM scheme. In addition, the Courant,Friedrichs,Lewy number insensitive conservation element and solution element (CNI-CESE) method is found to be both more robust and more computationally efficient than the conventional CESE scheme when modeling IEF phenomena within a contraction,expansion microchannel. In modeling the IEF separation of four sample ampholytes in a 20-ampholtye-based pH gradient within a contraction,expansion microchannel, the results obtained using the CNI-CESE scheme are in good agreement with those published in literature. Moreover, the simulations can be performed significantly faster with the new 1-D model and the CNI-CESE scheme. Finally, the results obtained using the modified 1-D mass transport model and the CNI-CESE scheme demonstrate that the concentration of the focused test sample and the resolution of the pH gradient within the microchannel increase as the number of ampholytes used to accomplish the IEF separation process is increased. [source] Study of Joule heating effects on temperature gradient in diverging microchannels for isoelectric focusing applicationsELECTROPHORESIS, Issue 10 2006Brian Kates Abstract IEF is a high-resolution separation method taking place in a medium with continuous pH gradients, which can be set up by applying electrical field to the liquid in a diverging microchannel. The axial variation of the channel cross-sectional area will induce nonuniform Joule heating and set up temperature gradient, which will generate pH gradient when proper medium is used. In order to operationally control the thermally generated pH gradients, fundamental understanding of heat transfer phenomena in microfluidic chips with diverging microchannels must be improved. In this paper, two 3-D numerical models are presented to study heat transfer in diverging microchannels, with static and moving liquid, respectively. Through simulation, the temperature distribution for the entire chip has been revealed, including both liquid and solid regions. The model for the static liquid scenario has been compared with published results for validation. Parametric studies have showed that the channel geometry has significant effects on the peak temperature location, and the electrical conductivity of the medium and the wall boundary convection have effects on the generated temperature gradients and thus the generated pH gradients. The solution to the continuous flow model, where the medium convection is considered, shows that liquid convection has significant effects on temperature distribution and the peak temperature location. [source] Dynamic analyte introduction and focusing in plastic microfluidic devices for proteomic analysisELECTROPHORESIS, Issue 1-2 2003Yan Li Abstract Isoelectric focusing (IEF) separations, in general, involve the use of the entire channel filled with a solution mixture containing protein/peptide analytes and carrier ampholytes for the creation of a pH gradient. Thus, the preparative capabilities of IEF are inherently greater than most microfluidics-based electrokinetic separation techniques. To further increase sample loading and therefore the concentrations of focused analytes, a dynamic approach, which is based on electrokinetic injection of proteins/peptides from solution reservoirs, is demonstrated in this study. The proteins/peptides continuously migrate into the plastic microchannel and encounter a pH gradient established by carrier ampholytes originally present in the channel for focusing and separation. Dynamic sample introduction and analyte focusing in plastic microfluidic devices can be directly controlled by various electrokinetic conditions, including the injection time and the applied electric field strength. Differences in the sample loading are contributed by electrokinetic injection bias and are affected by the individual analyte's electrophoretic mobility. Under the influence of 30 min electrokinetic injection at constant electric field strength of 500 V/cm, the sample loading is enhanced by approximately 10,100 fold in comparison with conventional IEF. [source] Proteome analysis of human liver tumor tissue by two-dimensional gel electrophoresis and matrixassisted laser desorption/ionization-mass spectrometry for identification of disease-related proteinsELECTROPHORESIS, Issue 24 2002Jina Kim Abstract Hepatocellular carcinoma (HCC) is a common malignancy worldwide and is a leading cause of death. To contribute to the development and improvement of molecular markers for diagnostics and prognostics and of therapeutic targets for the disease, we have largely expanded the currently available human liver tissue maps and studied the differential expression of proteins in normal and cancer tissues. Reference two-dimensional electrophoresis (2-DE) maps of human liver tumor tissue include labeled 2-DE images for total homogenate and soluble fraction separated on pH 3,10 gels, and also images for soluble fraction separated on pH 4,7 and pH 6,9 gels for a more detailed map. Proteins were separated in the first dimension by isoelectric focusing on immobilized pH gradient (IPG) strips, and by 7.5,17.5% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels in the second dimension. Protein identification was done by peptide mass fingerprinting with delayed extraction-matrix assisted laser desorption/ionization-time of flight-mass spectrometry (DE-MALDI-TOF-MS). In total, 212 protein spots (117 spots in pH 4,7 map and 95 spots in pH 6,9) corresponding to 127 different polypeptide chains were identified. In the next step, we analyzed the differential protein expression of liver tumor samples, to find out candidates for liver cancer-associated proteins. Matched pairs of tissues from 11 liver cancer patients were analyzed for their 2-DE profiles. Protein expression was comparatively analyzed by use of image analysis software. Proteins whose expression levels were different by more than three-fold in at least 30% (four) of the patients were further analyzed. Numbers of protein spots overexpressed or underexpressed in tumor tissues as compared with nontumorous regions were 9 and 28, respectively. Among these 37 spots, 1 overexpressed and 15 underexpressed spots, corresponding to 11 proteins, were identified. The physiological significance of the differential expressions is discussed. [source] Digestive peptidases in Tenebrio molitor and possibility of use to treat celiac diseaseENTOMOLOGICAL RESEARCH, Issue 3 2007Elena 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] Carbohydrate expression and modification during keratinocyte differentiation in normal human and reconstructed epidermisEXPERIMENTAL DERMATOLOGY, Issue 5 2003Bruno Méhul Abstract:, Using fluorescein isothiocyanate (FITC)-labeled lectins we were able to demonstrate the presence of specific carbohydrate moieties in normal human and reconstructed epidermis. Evidence is provided that in both cases the strongly reduced lectin staining at the level of the stratum corneum is the result of a hindered accessibility of the lectins in this lipid-rich hydrophobic environment. Isolated corneocytes and purified cornified envelopes (CEs) exhibited clearly glycosylated structures reacting with distinct lectins. The presence of glycosidase activity, particularly in the upper layers of the epidermis characterized by an acidic environment (pH 5.5), indicates that modifications of the sugar residues might be important in epidermal homeostasis, barrier behavior and desquamation. Absent or strongly reduced glycosidase activity in the stratum corneum of reconstructed epidermis with an impaired pH gradient could be in part responsible for the reduced barrier function and the lack of desquamation in this model. [source] Ambient pH controls the expression of endopolygalacturonase genes in the necrotrophic fungus Sclerotinia sclerotiorumFEMS MICROBIOLOGY LETTERS, Issue 2 2003Pascale Cotton Abstract In the necrotrophic fungus Sclerotinia sclerotiorum, secretion of polygalacturonases (PGs) and decrease of the environmental pH via oxalic acid production are considered as the main pathogenicity determinants. In order to evaluate the relationship between these two aspects of the infection process, we analyzed the expression of the endoPG-encoding genes pg1,3. Transcription of pg1,3 was not carbon regulated but was strictly controlled by pH and highly favored in a narrow range of acidic pH. During plant infection, a pH gradient was established in relation to oxalic acid secretion. Transcripts of pg1,3 were localized to the zone of colonization of healthy tissues while transcripts of genes encoding other lytic enzymes were restricted to the more acidic zones of the infected tissues. Our results show that progressive acidification of the ambient medium by the fungus is a major strategy for the sequential expression of pathogenicity factors. [source] Consumer body composition and community structure in a stream is altered by pHFRESHWATER BIOLOGY, Issue 3 2010A. LARRAÑAGA Summary 1.,Low pH inhibits microbial conditioning of leaf-litter, which forms the principal energy input to many headwater streams. This reduces food quality and availability for the shredder assemblage, thereby creating a potential bottleneck in the flux of energy and biomass through acidified food webs. 2.,We explored the consequences of acidity on the well-characterised community of Broadstone Stream in southeast England, by quantifying the physiological condition (protein and lipid content) of three dominant shredder species (Leuctra nigra, L. hippopus and Nemurella pictetii) and relating this to changes in the numerical abundance and biomass of invertebrates across a longitudinal pH gradient (5.3,6.5). 3.,Total taxon richness increased with pH, as did shredder diversity. The acid-tolerant stonefly, L. nigra, exhibited a positive correlation between pH and protein content, but its abundance was suppressed in the less acid reaches. These results suggest that the impacts of environmental stressors might be manifested differently at the population (i.e. numerical and biomass abundance) versus the physiological (i.e. protein content of individuals) levels of organisation. Body composition of L. hippopus and N. pictetii did not exhibit any significant relationship with stream pH in the field. 4.,The survey data were corroborated with a laboratory rearing experiment using N. pictetii, in which survival rate, growth rate, and protein and lipid content of individuals were measured in stream water of differing pH and acid versus circumneutral microbial conditioning regimes. Acid-conditioned leaves were associated with increased mortality and reduced protein content in consumers' tissues, with acid water also having the latter effect. 5.,Our results suggest that biochemical constraints within key taxa might create energy flux bottlenecks in detrital-based food webs, and that this could ultimately determine the productivity of the entire system. Hence assays of the body composition of macroinvertebrates could be an effective new tool that complements population level studies of the impacts of stressors in fresh waters. [source] The evolutionary species pool hypothesis and patterns of freshwater diatom diversity along a pH gradientJOURNAL OF BIOGEOGRAPHY, Issue 3 2005Jason Pither Abstract Aim, To interpret the unimodal relationship between diatom species richness and lake pH within the context of the evolutionary species pool hypothesis (SPH). We test the following primary prediction arising from the SPH: the size of the potential species pool (PSP) will increase along a gradient representing the historical commonness of different pH environments (pH commonness). To do this we assume that the present-day spatial dominance of near-neutral pH conditions compared with acidic and alkaline conditions reliably mimics the relative spatial availabilities of historical pH conditions among freshwater lakes. We also determine whether local richness represents a constant proportion of PSP size along the pH commonness gradient. Location, Two hundred and thirty-four lakes distributed over a 405,000 km2 region of the north-eastern United States of America. Methods, Sediment diatom morphospecies lists and pH data were acquired from the US Environmental Protection Agency's Environmental Monitoring and Assessment Program (EMAP) website. Using 248 morphospecies that occurred in at least 10 of the 234 lakes, four different measures of PSPs were calculated along the pH gradient. Local species richness was equated with the number of species occurring within the lake. Alpha diversity was equated with the average species richness of lakes with similar pH values. A combination of statistical methods were employed, including correlations, quadratic regression and piecewise regression. Results, PSP size increased significantly with pH commonness for all four measures of PSP size, thus supporting the primary prediction of the evolutionary SPH. Local richness comprised a larger proportion of the PSP within acidic lakes than within circumneutral lakes. Alpha diversity and lake species richness both increased significantly with pH commonness, but the former did so in a two-step fashion. We test and reject several alternative contemporary time-scale explanations for our findings. Main Conclusions, Our findings are consistent with the hypothesis that diatom taxonomic richness is presently lower within acidic and highly alkaline lakes than in circumneutral lakes owing to the limited opportunity in space and/or time for the evolution of suitably adapted species. Whereas ecological processes can explain why certain species are excluded from particular habitats, e.g. acidic lakes, they cannot account for why so few species are adapted to those habitats in the first place. [source] Regulatory Mechanisms and Physiological Relevance of a Voltage-Gated H+ Channel in Murine Osteoclasts: Phorbol Myristate Acetate Induces Cell Acidosis and the Channel Activation,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 11 2003Hiroyuki Mori Abstract The voltage-gated H+ channel is a powerful H+ extruding mechanism of osteoclasts, but its functional roles and regulatory mechanisms remain unclear. Electrophysiological recordings revealed that the H+ channel operated on activation of protein kinase C together with cell acidosis. Introduction: H+ is a key signaling ion in bone resorption. In addition to H+ pumps and exchangers, osteoclasts are equipped with H+ conductive pathways to compensate rapidly for pH imbalance. The H+ channel is distinct in its strong H+ extrusion ability and voltage-dependent gatings. Methods: To investigate how and when the H+ channel is available in functional osteoclasts, the effects of phorbol 12-myristate 13-acetate (PMA), an activator for protein kinase C, on the H+ channel were examined in murine osteoclasts generated in the presence of soluble RANKL (sRANKL) and macrophage-colony stimulating factor (M-CSF). Results and Conclusions: Whole cell recordings clearly showed that the H+ current was enhanced by increasing the pH gradient across the plasma membrane (,pH), indicating that the H+ channel changed its activity by sensing ,pH. The reversal potential (Vrev) was a valuable tool for the real-time monitoring of ,pH in clamped cells. In the permeabilized patch, PMA (10 nM-1.6 ,M) increased the current density and the activation rate, slowed decay of tail currents, and shifted the threshold toward more negative voltages. In addition, PMA caused a negative shift of Vrev, suggesting that intracellular acidification occurred. The PMA-induced cell acidosis was confirmed using a fluorescent pH indicator (BCECF), which recovered quickly in a K+ -rich alkaline solution, probably through the activated H+ channel. Both cell acidosis and activation of the H+ channel by PMA were inhibited by staurosporine. In ,80% of cells, the PMA-induced augmentation in the current activity remained after compensating for the ,pH changes, implying that both ,pH-dependent and -independent mechanisms mediated the channel activation. Activation of the H+ channel shifted the membrane potential toward Vrev. These data suggest that the H+ channel may contribute to regulation of the pH environments and the membrane potential in osteoclasts activated by protein kinase C. [source] Altered distribution of mitochondria impairs calcium homeostasis in rat hippocampal neurons in cultureJOURNAL OF NEUROCHEMISTRY, Issue 1 2003Guang Jian Wang Abstract The specificity of Ca2+ signals is conferred in part by limiting changes in cytosolic Ca2+ to subcellular domains. Mitochondria play a major role in regulating Ca2+ in neurons and may participate in its spatial localization. We examined the effects of changes in the distribution of mitochondria on NMDA-induced Ca2+ increases. Hippocampal cultures were treated with the microtubule-destabilizing agent vinblastine, which caused the mitochondria to aggregate and migrate towards one side of the neuron. This treatment did not appear to decrease the energy status of mitochondria, as indicated by a normal membrane potential and pH gradient across the inner membrane. Moreover, electron microscopy showed that vinblastine treatment altered the distribution but not the ultrastructure of mitochondria. NMDA (200 µm, 1 min) evoked a greater increase in cytosolic Ca2+ in vinblastine-treated cells than in untreated cells. This increase did not result from impaired Ca2+ efflux, enhanced Ca2+ influx, opening of the mitochondrial permeability transition pore or altered function of endoplasmic reticulum Ca2+ stores. Ca2+ uptake into mitochondria was reduced by 53% in vinblastine-treated cells, as reported by mitochondrially targeted aequorin. Thus, the distribution of mitochondria maintained by microtubules is critical for buffering Ca2+ influx. A subset of mitochondria close to a Ca2+ source may preferentially regulate Ca2+ microdomains, set the threshold for Ca2+ -induced toxicity and participate in local ATP production. [source] Uptake of lamivudine by rat renal brush border membrane vesiclesJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 1 2002Takatoshi Takubo Uptake of lamivudine, a nucleoside analogue antiviral agent, by brush border membrane vesicles (BBMV) prepared from rat renal cortex was investigated. Initial uptake of lamivudine by BBMV was stimulated in the presence of an outward pH gradient. Determination of the kinetic parameters of the initial uptake yielded apparent Km and Vmax values of 2.28 mM and 1.56 nmol (mg protein),1 (20 s),1, respectively. The pH-driven uptake of lamivudine was inhibited by organic cations such as trimethoprim and cimetidine. The inhibitory effect of trimethoprim on lamivudine uptake was competitive, with an apparent Ki of 27.6 ,M. The uptake of lamivudine was also inhibited by nitrobenzylthioinosine, a representative inhibitor of nucleoside transport, and by other nucleoside analogues, such as azidothymidine and dideoxycytidine, that are excreted by renal tubular secretion. These findings suggest that efflux of lamivudine at the brush border membrane of renal tubular epithelium is mediated by an H+/lamivudine antiport system, which may correspond to the H+/organic cation antiport system, and that this system is also involved in the renal secretion of other nucleoside analogues. [source] Does H+ pumping by plasmalemma ATPase limit leaf growth of maize (Zea mays) during the first phase of salt stress?JOURNAL OF PLANT NUTRITION AND SOIL SCIENCE, Issue 4 2005Christian Zörb Abstract In the first phase of salt stress, growth of plants is impaired mainly by osmotic stress. To elucidate the effect of NaCl salinity on elongation growth of maize leaves in the first phase of salt stress, we investigated the effect of NaCl on gene expression and activity of the plasmalemma H+ ATPase of elongating leaves of maize (Zea mays L.). Treatment of maize plants with 125 mM NaCl for 3 d decreased leaf growth relative to control plants (1 mM NaCl). Whereas H+ ATPase hydrolytic activity was unaffected, the ability of the H+ ATPase to establish a pH gradient was strongly reduced. Total mRNA of plasmalemma H+ ATPase was slightly increased. However, mRNA of the ATPase isoform MHA1 was significantly reduced and ATPase isoform MHA4 was strongly increased at the mRNA level. Synthesis of total H+ ATPase protein was unchanged as revealed by western blot. The results indicate that reduced pumping of H+ ATPase in leaf plasmalemma under salt stress may be caused by a switch to gene expression of the specific isoform MHA4, which shows inferior H+ -pumping efficiency in comparison to isoforms expressed under control conditions. We propose that reduced H+ pumping of plasmalemma H+ ATPase is involved in the reduction of leaf growth of maize during the first phase of salt stress. Limitiert die H+ -Pumpaktivität der Plasmalemma-ATPase das Blattwachstum von Mais (Zea mays) während der ersten Phase eines Salzstresses? In der ersten Phase eines Salzstresses wird das Pflanzenwachstum hauptsächlich durch osmotischen Stress beeinträchtigt. Zur Klärung des Einflusses der NaCl-Salinität auf das Streckungswachstum von Maisblättern in der ersten Phase eines Salzstresses wurde der Einfluss von NaCl auf die Genexpression und die Aktivität der H+ -ATPase im Plasmalemma von unter Salzstress wachsenden Maisblättern (Zea mays L.) untersucht. Dreitägige Behandlung von Maispflanzen mit 125 mM NaCl verminderte im Vergleich zu Kontrollpflanzen (1 mM NaCl) das Blattwachstum. Während die hydrolytische H+ -ATPase-Aktivität unbeeinflusst blieb, wurde die Fähigkeit, einen pH-Gradienten aufzubauen, stark reduziert. Die Gesamt-mRNA der H+ -ATPase war unverändert. Während jedoch die ATPase-Isoform MHA1 signifikant vermindert war, wurde die Isoform MHA4 auf dem mRNA-Niveau stark erhöht. Die Western-Blot-Analyse zeigte keine Veränderung der Menge des Gesamtproteins der H+ -ATPase. Unsere Ergebnisse sprechen dafür, dass die reduzierte Fähigkeit der H+ -ATPase im Plasmalemma von unter Salzstress gewachsenen Blättern einen pH-Gradienten aufzubauen, durch Genexpression der spezifischen Isoform MHA4 verursacht wird. Diese Isoform weist eine geringere H+ -Pumpeffizienz im Vergleich zu Isoformen auf, die unter Kontrollbedingungen gebildet werden. Wir schlagen daher vor, dass die reduzierte Fähigkeit der H+ -ATPase einen pH-Gradienten aufzubauen, an der Verminderung des Blattwachstums von Mais in der ersten Phase des Salzstresses beteiligt ist. [source] Patterns of species richness and turnover along the pH gradient in deciduous forests: testing the continuum hypothesisJOURNAL OF VEGETATION SCIENCE, Issue 6 2009Cord Peppler-Lisbach Abstract Question: (i) How do species richness and species turnover change along a pH gradient? (ii) What are possible driving factors behind these patterns? (iii) Can the observed patterns be explained by an individualistic continuum concept that postulates independence of species responses and constant turnover rates? Location: Semi-natural, deciduous hardwood forests in NW Germany (558 plots). Methods: The instantaneous rate of compositional turnover is measured by the sum of slope angles of modelled response curves (119 understorey species) at any point along the pH gradient. Total turnover rate, positive turnover rate (species increasing in probability of occurrence) and negative turnover rate (species decreasing in probability of occurrence) are calculated separately. Species richness is modelled using Poisson regression and by calculating the sum of predicted probabilities at any gradient point. Turnover rates are compared with those calculated from a null model based on a Gaussian community model. Soil chemical analyses of 49 plots are used to interpret biodiversity patterns. Results: Species richness shows a hump-shaped relation to pH(CaCl2) with a minor decline at approximately pH>5.0. The decline is possibly due to the confounding influence of water regime and local species pool effects. Increasing richness from pH 2.5 to 4.7 can be traced back to positive turnover exceeding negative turnover. Peaks in turnover rates, dominated by positive turnover, are located at pH 3.7 and 2.8, where turnover rates considerably exceed rates derived from the null model. The turnover pattern can be related to soil chemical conditions, e.g. decreasing base saturation, Al and H+ toxicity and the occurrence of mor. Conclusions: The high turnover rates and the massive imbalance in positive and negative turnover rates found in deciduous forests cannot be explained by the individualistic continuum concept. Physiological constraints at the gradient limits and species pool effects could be responsible for this. Their role should be considered more explicitly in vegetation concepts dealing with the continuum-discontinuum controversy. The presented approach can be regarded as a comprehensive analytical tool for a better understanding of biodiversity patterns along environmental gradients by linking species richness, turnover and response curve types. [source] ,Smart' delivery systems for biomolecular therapeuticsORTHODONTICS & CRANIOFACIAL RESEARCH, Issue 3 2005PS Stayton Structured Abstract Authors ,, Stayton PS, El-Sayed MEH, Murthy N, Bulmus V, Lackey C, Cheung C, Hoffman AS Objective ,, There is a strong need for drug delivery systems that can deliver biological signals from biomaterials and tissue engineering scaffolds, and a particular need for new delivery systems that can efficiently deliver biomolecules to intracellular targets. Viruses and pathogens have evolved potent molecular machinery that sense the lowered pH gradient of the endosomal compartment and become activated to destabilize the endosomal membrane, thereby enhancing protein or DNA transport to the cytoplasmic compartment. A key feature of many of these biological delivery systems is that they are reversible, so that the delivery systems are not directly toxic. These delivery systems have the ability to change their structural and functional properties and thus display remarkable ,smart' material properties. The objective of this presentation is to review the initial development of smart polymeric carriers that mimic these biological delivery systems and combine similar pH-sensitive, membrane-destabilizing activity for the delivery of therapeutic biomolecules. Design ,, We have developed new ,smart' polymeric carriers to more effectively deliver and broaden the available types of biomolecular therapeutics. The polymers are hydrophilic and stealth-like at physiological pH, but become membrane-destabilizing after uptake into the endosomal compartment where they enhance the release of therapeutic cargo into the cytoplasm. They can be designed to provide a range of pH profiles and membrane-destabilizing activities, allowing their molecular properties to be matched to specific drugs and loading ranges. A versatile set of linker chemistries is available to provide degradable conjugation sites for proteins, nucleic acids, and/or targeting moieties. Results ,, The physical properties of several pH-responsive polymers were examined. The activity and pH profile can be manipulated by controlling the length of hydrophobic alkyl segments. The delivery of poly(propyl acrylic acid) (PPAA)-containing lipoplexes significantly enhanced wound healing through the interconnected effects of altered extracellular matrix organization and greater vascularization. PPAA has also been shown to enhance cytoplasmic delivery of a model protein therapeutic. Polymeric carriers displaying pH-sensitive, membrane-destabilizing activity were also examined. The pH profile is controlled by the choice of the alkylacrylic acid monomer and by the ratio of the carboxylate-containing alkylacrylic acid monomer to alkylacrylate monomer. The membrane destabilizing activity is controlled by the lengths of the alkyl segment on the alkylacrylic acid monomer and the alkylacrylate monomer, as well as by their ratio in the final polymer chains. Conclusion ,, The molecular mechanisms that proteins use to sense and destabilize provide interesting paradigms for the development of new polymeric delivery systems that mimic biological strategies for promoting the intracellular delivery of biomolecular drugs. The key feature of these polymers is their ability to directly enhance the intracellular delivery of proteins and DNA, by destabilizing biological membranes in response to vesicular compartment pH changes. The ability to deliver a wide variety of protein and nucleic acid drugs to intracellular compartments from tissue engineering and regenerative scaffolds could greatly enhance control of important processes such as inflammation, angiogenesis, and biomineralization. [source] Allelic variants of granule-bound starch synthase proteins in European bread wheat varietiesPLANT BREEDING, Issue 4 2000C. Marcoz-Ragot Abstract The composition of 324 European wheat cultivars were analysed at the three granule-bound starch synthase (GBSS I) loci. Protein separation was first made by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE. A specific two-dimensional (2D) electrophoresis (immobilized pH gradient × SDS-PAGE) using an Immobiline dry strip in the first dimension was developed to resolve the GBSS I proteins more clearly and to confirm some results. Very low polymorphism was found. Among the 324 cultivars analysed, only one carried a Wx-A1 null allele (Wx-A1b) and none was found to have the Wx-2D null allele. As described in the literature the Wx-B1 locus was more polymorphic and the null allele was encountered in 11 cultivars. The use of 2D electrophoresis allowed us to find another type of variant which presented as having thicker band with same mobility as the Wx-D1 protein in SDS-PAGE. Twelve per cent of the cultivars analysed presented this band and could have been previously mistaken for cultivars carrying the Wx-B1 null allele. Indeed this band probably corresponded to the Wx-B1, or Wx-B1e allele overlapping with the Wx-D1a allele in SDS-PAGE. [source] Adaptation of plasma membrane H+ -ATPase of rice roots to low pH as related to ammonium nutritionPLANT CELL & ENVIRONMENT, Issue 10 2009YIYONG ZHU ABSTRACT The preference of paddy rice for NH4+ rather than NO3 - is associated with its tolerance to low pH since a rhizosphere acidification occurs during NH4+ absorption. However, the adaptation of rice root to low pH has not been fully elucidated. This study investigated the acclimation of plasma membrane H+ -ATPase of rice root to low pH. Rice seedlings were grown either with NH4+ or NO3 - . For both nitrogen forms, the pH value of nutrient solutions was gradually adjusted to pH 6.5 or 3.0. After 4 d cultivation, hydrolytic H+ -ATPase activity, Vmax, Km, H+ -pumping activity, H+ permeability and pH gradient across the plasma membrane were significantly higher in rice roots grown at pH 3.0 than at 6.5, irrespective of the nitrogen forms supplied. The higher activity of plasma membrane H+ -ATPase of adapted rice roots was attributed to the increase in expression of OSA1, OSA3, OSA7, OSA8 and OSA9 genes, which resulted in an increase of H+ -ATPase protein concentration. In conclusion, a high regulation of various plasma membrane H+ -ATPase genes is responsible for the adaptation of rice roots to low pH. This mechanism may be partly responsible for the preference of rice plants to NH4+ nutrition. [source] Voltage-activated proton currents in human lymphocytesTHE JOURNAL OF PHYSIOLOGY, Issue 1 2002Tom Schilling Voltage-activated proton currents are reported for the first time in human peripheral blood T and B lymphocytes and in the human leukaemic T cell line Jurkat E6-1. The properties of H+ currents studied using tight-seal voltage-clamp recording techniques were similar in all cells. Changing the pH gradient by one unit caused a 47 mV shift in the reversal potential, demonstrating high selectivity of the channels for protons. H+ current activation upon membrane depolarisation had a sigmoidal time course that could be fitted by a single exponential function after a brief delay. Increasing pHo shifted the activation threshold to more negative potentials, and increased both the H+ current amplitude and the rate of activation. In lymphocytes studied at pHi 6.0, the activation threshold was more negative and the H+ current density was three times larger than at pHi 7.0. Increasing the intracellular Ca2+ concentration to 1 ,m did not change H+ current amplitude or kinetics detectably. Extracellularly applied Zn2+ and Cd2+ inhibited proton currents, slowing activation and shifting the voltage-activation curve to more positive potentials. The H+ current amplitude was 100 times larger in CD19+ B lymphocytes and in Jurkat E6-1 cells than in CD3+ T lymphocytes. Following stimulation with the phorbol ester PMA, the H+ current density in peripheral blood T lymphocytes and Jurkat T cells increased. In contrast, the H+ current density of phorbol ester (PMA)-stimulated B lymphocytes was reduced and activation became slower. The pattern of expression of H+ channels in lymphocytes appears well suited to their proposed role of charge compensation during the respiratory burst. [source] Antibiotic therapy , rationale and evidence for optimal drug concentrations in prostatic and seminal fluid and in prostatic tissueANDROLOGIA, Issue 5 2003Kurt G. Naber Summary. The theoretical background of drug penetration into the prostate is outlined, emphasizing the phenomenon of ion-trapping and the role of nonionic diffusion of weak acids, bases and amphoteric drugs across biological membranes with a pH gradient. Determination of drug concentrations in human prostatic secretion are problematic because of possible urinary contamination. Studies have been carried out mainly in healthy volunteers. The results have to be interpreted with caution, if not care was taken to rule out or at least identify urinary contamination. Analysing the concentrations of various fluoroquinolones in prostatic and seminal fluid as well as in prostatic tissue, it becomes obvious that the fluoroquinolones differ not only in plasma concentrations but also in their penetration ability to these sites. In spite of intensive investigations, our knowledge is still limited concerning the mechanisms that govern the transport of antibiotic drugs into and their activity in the various prostatic compartments and how the findings can be applied clinically. Nevertheless, overall the concentrations at the site of infection of most of the fluoroquinolones should be sufficient for the treatment of chronic bacterial prostatitis and vesiculitis caused by susceptible pathogens. [source] Uptake/Efflux Transport of Tramadol Enantiomers and O -Desmethyl-Tramadol: Focus on P -GlycoproteinBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 3 2009Mouna Kanaan P -glycoprotein (P -gp) might be of importance in the analgesic and tolerability profile variability of TMD. Our study investigated the involvement of P -gp in the transepithelial transport of (+)-TMD, (,)-TMD and M1, using a Caco-2 cell monolayer model. The bidirectional transport of racemic TMD and M1 (1,100 µM) across the monolayers was investigated at two pH conditions (pH 6.8/7.4 and 7.4/7.4) in the presence and absence of P -gp inhibitor cyclosporine A (10 µM) and assessed with the more potent and specific P -gp inhibitor GF120918 (4 µM). Analytical quantification was performed by liquid chromatography coupled to the fluorescence detector. A net secretion of (+)-TMD, (,)-TMD and M1 was observed when a pH gradient was applied (TR: Papp(B , A)/Papp(A , B): 1.8,2.7; P < 0.05). However, the bidirectional transport of all compounds was equal in the non-gradient system. In the presence of P -gp inhibitors, a slight but significant increase of secretory flux was observed (up to 26%; P < 0.05) at both pH conditions. In conclusion, (+)-TMD, (,)-TMD and M1 are not P -gp substrates. However, proton-based efflux pumps may be involved in limiting the gastrointestinal absorption of TMD enantiomers as well as enhancing TMD enantiomers and M1 renal excretion. A possible involvement of uptake carriers in the transepithelial transport of TMD enantiomers and M1 is suggested. [source] Transporters involved in apical and basolateral uptake of ceftibuten into Caco-2 cellsBIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 8 2002Rajeev M. Menon Abstract Ceftibuten uptake from the apical and basolateral side of Caco-2 cells grown on transwells was studied. Uptake into the cells showed concentration dependent saturation. The apical transporter(s) showed a higher capacity and lower affinity for ceftibuten than the basolateral transporter(s). Uptake was inhibited in the presence of higher pH and in the presence of 2,4-dinitro phenol (DNP). A proton gradient had a greater effect on the apical than on the basolateral transporter. Glycyl proline, a dipeptide transport system (PEPT1) substrate, inhibited ceftibuten uptake into Caco-2 cells. Benzoic acid, a monocarboxylic acid (MCT) transporter substrate also exhibited a strong inhibition of ceftibuten uptake, but acetic acid had no effect. Adipic acid inhibited apical uptake of ceftibuten but had no effect on the basolateral uptake. None of the inhibitors had a significant effect on ceftibuten uptake in absence of a pH gradient. Addition of inhibitors in presence of DNP led to a greater decrease in ceftibuten uptake, when compared to the effect of DNP alone, indicating a facilitated diffusion process. These results indicate that ceftibuten uptake in Caco-2 cells involve multiple transport pathways. Apical uptake is mediated by an energy dependent carrier-mediated process and an energy independent facilitated diffusion process. The apical transport system is different from the basolateral transporter. Copyright © 2002 John Wiley & Sons, Ltd. [source] Engineering Propionibacterium acidipropionici for enhanced propionic acid tolerance and fermentationBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2009An Zhang Abstract Propionibacterium acidipropionici, a Gram-positive, anaerobic bacterium, has been the most used species for propionic acid production from sugars. In this study, the metabolically engineered mutant ACK-Tet, which has its acetate kinase gene knocked out from the chromosome, was immobilized and adapted in a fibrous bed bioreactor (FBB) to increase its acid tolerance and ability to produce propionic acid at a high final concentration in fed-batch fermentation. After about 3 months adaptation in the FBB, the propionic acid concentration in the fermentation broth reached ,100,g/L, which was much higher than the highest concentration of ,71,g/L previously attained with the wild-type in the FBB. To understand the mechanism and factors contributing to the enhanced acid tolerance, adapted mutant cells were harvested from the FBB and characterized for their morphology, growth inhibition by propionic acid, protein expression profiles as observed in SDS,PAGE, and H+ -ATPase activity, which is related to the proton pumping and cell's ability to control its intracellular pH gradient. The adapted mutant obtained from the FBB showed significantly reduced growth sensitivity to propionic acid inhibition, increased H+ -ATPase expression and activity, and significantly elongated rod morphology. Biotechnol. Bioeng. 2009; 104: 766,773 © 2009 Wiley Periodicals, Inc. [source] Purification of homogeneous forms of fungal peroxygenaseBIOTECHNOLOGY JOURNAL, Issue 11 2009René Ullrich Abstract Extracellular peroxygenase from the agaric fungus Agrocybe aegerita is a versatile biocatalyst that oxygenates various substrates by means of hydrogen peroxide. The enzyme is routinely produced in suspensions of soybean meal and has until now been purified by several steps of fast protein liquid chromatography (FPLC) using different ion exchangers. The final protein fraction had a molecular mass of 46 kDa but still consisted of several incompletely separated proteins with slightly differing isoelectric points (pI 5.2, 5.6, 6.1), probably representing differently glycosylated isoforms. This made it difficult to further purify the individual protein forms. Since homogeneous protein fractions are a pre-requisite for X-ray crystallography and specific structure-function studies, an appropriate FPLC procedure was developed starting with pre-purification of crude peroxygenase on SP Sepharose followed by chromatofocusing on a Mono P column and elution with a pH gradient. Three sufficiently separated main protein peaks were eluted from the Mono P column and confirmed to be distinct forms of aromatic peroxygenase with different pIs. All A. aegerita peroxygenase forms oxygenated toluene and naphthalene and no catalytic differences were observed between them. We tested also two devices for preparative isoelectric focusing (Rotofor, IsoPrime systems) for peroxygenase separation but resolution and protein recovery were not sufficient. [source] Simultaneous extraction and concentration of penicillin G by hollow fiber renewal liquid membraneBIOTECHNOLOGY PROGRESS, Issue 2 2009Zhongqi Ren Abstract In this article, hollow fiber renewal liquid membrane (HFRLM) technique was used for recovery of penicillin G from aqueous solution. The organic solution of 7 vol % di-n-octylamine (DOA) + 30 vol % iso-octanol + kerosene was used as liquid membrane phase, and Na2CO3 aqueous solution was used as stripping phase. Experiments were performed as a function of carrier concentration in the organic phase, organic/aqueous volume ratio, pH, and initial penicillin G concentration in the feed phase, pH in the stripping phase, flow rates, etc. The results showed that the HFRLM process was stable and could carry out simultaneous extraction and concentration of penicillin G from aqueous solutions. As a carrier facilitated transport process, the addition of DOA in organic phase could greatly enhance the mass transfer rate; and there was a favorable organic/aqueous volume ratio of 1:20 to 1:30 for this system. The mass transfer flux and overall mass transfer coefficient increased with decreasing pH in the feed phase and increasing pH in the stripping phase, because of variation of the mass transfer driving force caused by pH gradient and distribution equilibrium. The flow rate of the shell side had significant influence on the mass transfer performance, whereas the effect of flow rate of lumen side on the mass transfer performance was slight because of the mass transfer intensification of renewal effect in the lumen side. The results indicated that the HFRLM process was a promising method for the recovery of penicillin G from aqueous solutions. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] pH and nitric oxide synthase activity and expression in bovine aortic endothelial cellsACTA PAEDIATRICA, Issue 7 2006Sandor Nagy Abstract Aim: Nitric oxide (NO) plays an important role in the transition from intrauterine to extrauterine life. If this transition fails, a condition called persistent pulmonary hypertension of the neonate (PPHN) may develop. The current treatment modalities for this disease include induction of alkalosis by hyperventilation or alkali infusion, inhaled nitric oxide (iNO) and extracorporeal membrane oxygenation. There is evidence from animal studies that the elevated pH, not the low pCO2 is responsible for the resultant pulmonary vasodilatation. In this study, we examined the effect of pH on the activity and expression of endothelial nitric oxide synthase (eNOS) in cultured bovine aortic endothelial cells (BAEC) as a possible explanation for the pH dependent drop in pulmonary vascular resistance. Methods: BAEC were exposed to a pH gradient of 7.1,7.6 for 4 h (short-term) and 16 h (long-term). Standard Western blotting technique was used to detect expression of eNOS. Activity was measured by an indirect assay using bovine aortic smooth muscle cells (BASM) as reporter cells and measuring cGMP levels as a marker of NO production. The cells were exposed to the pH gradient for a total of 4 h and measurement were made at 30, 60 and 90 min, and 2, 3 and 4 hours. Results: eNOS activity and expression remained unchanged during the four and sixteen hours of exposure. Conclusion: In this in vitro experiment, we could not demonstrate an alkalosis-induced increase in eNOS activity and expression. The clinically observed pH dependent vasodilatation does not appear to be directly mediated through the induction of eNOS. [source] Simulations of IEF in microchannel with variable cross-sectional areaELECTROPHORESIS, Issue 5 2009Yin Chou Abstract This study develops a 1-D mass transport model to describe the electrophoresis transport behavior within a microchannel with a variable cross-sectional area. Utilizing three different numerical schemes, simulations are performed to investigate the IEF of proteins in ampholyte-based pH gradients within both a planar microchannel and a contraction,expansion microchannel, respectively. The simulation results obtained using the modified 1-D mass transport model and the finite-volume method (FVM) for the IEF separation of a single protein sample in a ten-ampholyte-based pH gradient within a planar microchannel are consistent with those presented by Shim et al. [Electrophoresis 2007, 28, 572,586] using a 2-D FVM scheme. In addition, the Courant,Friedrichs,Lewy number insensitive conservation element and solution element (CNI-CESE) method is found to be both more robust and more computationally efficient than the conventional CESE scheme when modeling IEF phenomena within a contraction,expansion microchannel. In modeling the IEF separation of four sample ampholytes in a 20-ampholtye-based pH gradient within a contraction,expansion microchannel, the results obtained using the CNI-CESE scheme are in good agreement with those published in literature. Moreover, the simulations can be performed significantly faster with the new 1-D model and the CNI-CESE scheme. Finally, the results obtained using the modified 1-D mass transport model and the CNI-CESE scheme demonstrate that the concentration of the focused test sample and the resolution of the pH gradient within the microchannel increase as the number of ampholytes used to accomplish the IEF separation process is increased. [source] Determination of the operational pH value of a buffering membrane by an isoelectric trapping separation of a carrier ampholyte mixtureELECTROPHORESIS, Issue 5 2008Robert Y. North Abstract The operational pH value of a buffering membrane used in an isoelectric trapping separation is determined by installing the membrane as the separation membrane into a multicompartmental electrolyzer operated in the two-separation compartment configuration. A 3 Study of Joule heating effects on temperature gradient in diverging microchannels for isoelectric focusing applicationsELECTROPHORESIS, Issue 10 2006Brian Kates Abstract IEF is a high-resolution separation method taking place in a medium with continuous pH gradients, which can be set up by applying electrical field to the liquid in a diverging microchannel. The axial variation of the channel cross-sectional area will induce nonuniform Joule heating and set up temperature gradient, which will generate pH gradient when proper medium is used. In order to operationally control the thermally generated pH gradients, fundamental understanding of heat transfer phenomena in microfluidic chips with diverging microchannels must be improved. In this paper, two 3-D numerical models are presented to study heat transfer in diverging microchannels, with static and moving liquid, respectively. Through simulation, the temperature distribution for the entire chip has been revealed, including both liquid and solid regions. The model for the static liquid scenario has been compared with published results for validation. Parametric studies have showed that the channel geometry has significant effects on the peak temperature location, and the electrical conductivity of the medium and the wall boundary convection have effects on the generated temperature gradients and thus the generated pH gradients. The solution to the continuous flow model, where the medium convection is considered, shows that liquid convection has significant effects on temperature distribution and the peak temperature location. [source] pH-dependent antitumor activity of proton pump inhibitors against human melanoma is mediated by inhibition of tumor acidityINTERNATIONAL JOURNAL OF CANCER, Issue 1 2010Angelo De Milito Abstract Metastatic melanoma is associated with poor prognosis and still limited therapeutic options. An innovative treatment approach for this disease is represented by targeting acidosis, a feature characterizing tumor microenvironment and playing an important role in cancer malignancy. Proton pump inhibitors (PPI), such as esomeprazole (ESOM) are prodrugs functionally activated by acidic environment, fostering pH neutralization by inhibiting proton extrusion. We used human melanoma cell lines and xeno-transplated SCID mice to provide preclinical evidence of ESOM antineoplastic activity. Human melanoma cell lines, characterized by different mutation and signaling profiles, were treated with ESOM in different pH conditions and evaluated for proliferation, viability and cell death. SCID mice engrafted with human melanoma were used to study ESOM administration effects on tumor growth and tumor pH by magnetic resonance spectroscopy (MRS). ESOM inhibited proliferation of melanoma cells in vitro and induced a cytotoxicity strongly boosted by low pH culture conditions. ESOM-induced tumor cell death occurred via rapid intracellular acidification and activation of several caspases. Inhibition of caspases activity by pan-caspase inhibitor z-vad-fmk completely abrogated the ESOM-induced cell death. ESOM administration (2.5 mg kg,1) to SCID mice engrafted with human melanoma reduced tumor growth, consistent with decrease of proliferating cells and clear reduction of pH gradients in tumor tissue. Moreover, systemic ESOM administration dramatically increased survival of human melanoma-bearing animals, in absence of any relevant toxicity. These data show preclinical evidence supporting the use of PPI as novel therapeutic strategy for melanoma, providing the proof of concept that PPI target human melanoma modifying tumor pH gradients. [source]
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