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Free Solution (free + solution)
Selected AbstractsReview modeling the free solution and gel electrophoresis of biopolymers: The bead array-effective medium modelBIOPOLYMERS, Issue 2-3 2007Stuart A. Allison Abstract Free solution and gel electrophoresis is an extremely useful tool in the separation of biopolymers. The complex nature of biopolymers, coupled with the usefulness of electrophoretic methods, has stimulated the development of theoretical modeling over the last 30 years. In this work, these developments are first reviewed with emphasis on Boundary Element and bead methodologies that enable the investigator to design realistic models of biopolymers. In the present work, the bead methodology is generalized to include the presence of a gel through the Effective Medium model. The biopolymer is represented as a bead array. A peptide, for example, made up of N amino acids is modeled as 2N beads. Duplex DNA is modeled as a discrete wormlike chain consisting of touching beads. The technical details of the method are placed in three Appendices. To illustrate the accuracy and effectiveness of the approach, two applications are considered. Model studies on both the free solution mobility of 73 peptides ranging in size from 2 to 42 amino acids, and the mobility of short duplex DNA in dilute agarose gels are discussed. © 2007 Wiley Periodicals, Inc. Biopolymers 87: 102,114, 2007. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] Regulation of early response genes in pancreatic acinar cells: external calcium and nuclear calcium signalling aspectsACTA PHYSIOLOGICA, Issue 1 2009N. Fedirko Abstract Nuclear calcium signalling has been an important topic of investigation for many years and some aspects have been the subject of debate. Our data from isolated nuclei suggest that the nuclear pore complexes (NPCs) are open even after depletion of the Ca2+ store in the nuclear envelope (NE). The NE contains ryanodine receptors (RyRs) and Ins(1,4,5)P3 receptors [Ins(1,4,5)P3Rs], most likely on both sides of the NE and these can be activated separately and independently: the RyRs by either NAADP or cADPR, and the Ins(1,4,5)P3Rs by Ins(1,4,5)P3. We have also investigated the possible consequences of nuclear calcium signals: the role of Ca2+ in the regulation of immediate early genes (IEG): c-fos, c-myc and c-jun in pancreatic acinar cells. Stimulation with Ca2+ -mobilizing agonists induced significant increases in levels of expression. Cholecystokinin (CCK) (10 nm) evoked a substantial rise in the expression levels, highly dependent on external Ca2+: the IEG expression level was lowest in Ca2+ -free solution, increased at the physiological level of 1 mm [Ca2+]o and was maximal at 10 mm [Ca2+]o, i.e.: 102 ± 22% and 163 ± 15% for c-fos; c-myc ,73 ± 13% and 106 ± 24%; c-jun ,49 ± 8% and 59 ± 9% at 1 and 10 mm of extracellular Ca2+ respectively. A low CCK concentration (10 pm) induced a small increase in expression. We conclude that extracellular Ca2+ together with nuclear Ca2+ signals induced by CCK play important roles in the induction of IEG expression. [source] High-sensitive determination of human ,-thrombin by its 29-mer aptamer in affinity probe capillary electrophoresisELECTROPHORESIS, Issue 12 2008Yilin Li Abstract ACE technique provides an effective tool for the separation and identification of disease-related biomarkers in clinical analysis. In recent years, a couple of synthetic DNA or RNA oligonucleotides, known as aptamers, rival the specificity and affinity for targets to antibodies and are employed as one kind of powerful affinity probe in ACE. In this work, based on high affinity between antithrombin aptamer and thrombin (their dissociation constant is 0.5,nM), a carboxyfluorescein-labeled 29-nucleotide (nt) aptamer (F29-mer) was used and an aptamer-based affinity probe CE (aptamer-based APCE) method was successfully established for high-sensitive detection and quantitative analysis of thrombin. Experimental conditions including incubation temperature and time, buffer composition, and concentration of cations were investigated and optimized. Under the optimized condition, the linear range was from 0 to 400,nM and the LOD was 2,nM (74,ng/mL, S/N,=,3), i.e., 40,amol, both in running buffer and in 5% v/v human serum. This LOD is the lowest one than those achieved by the previous APCE methods but based on a 15-mer aptamer. This approach offers a promising method for the rapid, selective, and sensitive detection of thrombin in practical utility. Further binding experiments using one carboxyfluorescein-labeled aptamer and the other nonlabeled aptamer or vice versa were carried out to deduce the formation of ternary complex when these two aptamers coexisted in the free solution with thrombin. [source] Glutamate-induced elevations in intracellular chloride concentration in hippocampal cell cultures derived from EYFP-expressing miceEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2004Jennifer E. Slemmer Abstract The homeostasis of intracellular Cl, concentration ([Cl,]i) is critical for neuronal function, including ,-aminobutyric acid (GABA)ergic synaptic transmission. Here, we investigated activity-dependent changes in [Cl,]i using a transgenetically expressed Cl, -sensitive enhanced yellow-fluorescent protein (EYFP) in cultures of mouse hippocampal neurons. Application of glutamate (100 µm for 3 min) in a bath perfusion to cell cultures of various days in vitro (DIV) revealed a decrease in EYFP fluorescence. The EYFP signal increased in amplitude with increasing DIV, reaching a maximal response after 7 DIV. Glutamate application resulted in a slight neuronal acidification. Although EYFP fluorescence is sensitive to pH, EYFP signals were virtually abolished in Cl, -free solution, demonstrating that the EYFP signal represented an increase in [Cl,]i. Similar to glutamate, a rise in [Cl,]i was also induced by specific ionotropic glutamate receptor agonists and by increasing extracellular [K+], indicating that an increase in driving force for Cl, suffices to increase [Cl,]i. To elucidate the membrane mechanisms mediating the Cl, influx, a series of blockers of ion channels and transporters were tested. The glutamate-induced increase in [Cl,]i was resistant to furosemide, bumetanide and 4,4,-diisothiocyanato-stilbene-2,2,-disulphonic acid (DIDS), was reduced by bicuculline to about 80% of control responses, and was antagonized by niflumic acid (NFA) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). We conclude that membrane depolarization increases [Cl,]i via several pathways involving NFA- and NPPB-sensitive anion channels and GABAA receptors, but not through furosemide-, bumetanide- or DIDS-sensitive Cl, transporters. The present study highlights the vulnerability of [Cl,]i homeostasis after membrane depolarization in neurons. [source] No evidence for calcium electrogenic exchanger in frog semicircular canal hair cellsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 9 2002M. Martini Abstract We investigated the possibility that, in hair cells mechanically isolated from frog semicircular canals, Ca2+ extrusion occurs via a Na+ : Ca2+ (cardiac type) or a Na+ : Ca2+,K+ (retinal type) exchanger. Cells concurrently imaged during whole-cell patch-clamp recordings using the Ca2+ sensitive fluorescent dye Oregon Green 488 BAPTA-1 (100 µm) showed no voltage dependence of Ca2+ clearance dynamics following a Ca2+ load through voltage-gated Ca2+ channels. Reverse exchange was probed in hair cells dialyzed with a Ca2+ - and K+ -free solution, containing a Na+ concentration that saturates the exchanger, after zeroing the contribution to the whole-cell current from Ca2+ and K+ conductances. In these conditions, no reverse exchange current was detected upon switching from a Ca2+ -free external solution to a solution containing concentrations of Ca2+ alone, or Ca2+ + K+ that saturated the exchanger. By contrast, the same experimental protocol elicited peak exchange currents exceeding 100 pA in gecko rod photoreceptors, used as positive controls. In both cell types, we also probed the forward mode of the exchanger by rapidly increasing the intracellular Ca2+ concentration using flash photolysis of two novel caged Ca2+ complexes, calcium 2,2,-{[1-(2-nitrophenyl)ethane-1,2-diyl]bis(oxy)}bis(acetate) and calcium 2,2,-{[1-(4,5-dimethoxy-2-nitrophenyl)ethane-1,2-diyl]bis(oxy)} bis(acetate), in the presence of internal K+ and external Na+. No currents were evoked by UV-triggered Ca2+ jumps in hair cells, whereas exchanger conformational currents up to 400 pA, followed by saturating forward exchange currents up to 40 pA, were recorded in rod photoreceptors subjected to the same experimental conditions. We conclude that no functional electrogenic exchanger is present in this hair cell population, which leaves the abundant plasma membrane Ca2+ -ATPases as the primary contributors to Ca2+ extrusion. [source] Noxious heat-induced CGRP release from rat sciatic nerve axons in vitroEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2001S. K. Sauer Abstract Noxious heat may act as an endogenous activator of the ionotropic capsaicin receptor (VR1) and of its recently found homologue VRL1, expressed in rat dorsal root ganglion cells and present along their nerve fibres. We have previously reported that capsaicin induces receptor-mediated and Ca++ -dependent calcitonin gene-related peptide (CGRP) release from axons of the isolated rat sciatic nerve. Here we extended the investigation to noxious heat stimulation and the transduction mechanisms involved. Heat stimulation augmented the CGRP release from desheathed sciatic nerves in a log,linear manner with a Q10 of ,,15 and a threshold between 40 and 42 °C. The increases were 1.75-fold at 42 °C, 3.8-fold at 45 °C and 29.1-fold at 52 °C; in Ca++ -free solution these heat responses were abolished or reduced by 71 and 92%, respectively. Capsazepine (10 µm) and Ruthenium Red (1 µm) used as capsaicin receptor/channel antagonists did not significantly inhibit the heat-induced release. Pretreatment of the nerves with capsaicin (100 µm for 30 min) caused complete desensitization to 1 µm capsaicin, but a significant heat response remained, indicating that heat sensitivity is not restricted to capsaicin-sensitive fibres. The sciatic nerve axons responded to heat, potassium and capsaicin stimulation with a Ca++ -dependent CGRP release. Blockade of the capsaicin receptor/channels had little effect on the heat-induced neuropeptide release. We conclude therefore that other heat-activated ion channels than VR1 and VRL1 in capsaicin-sensitive and -insensitive nerve fibres may cause excitation, axonal Ca++ influx and subsequent CGRP release. [source] Cell shrinkage evoked by Ca2+ -free solution in rat alveolar type II cells: Ca2+ regulation of Na+,H+ exchangeEXPERIMENTAL PHYSIOLOGY, Issue 2 2005Hitoshi Murao The effects of intracellular Ca2+ concentration, [Ca2+]i, on the volume of rat alveolar type II cells (AT-II cells) were examined. Perfusion with a Ca2+ -free solution induced shrinkage of the AT-II cell volume in the absence or presence of amiloride (1 ,m, an inhibitor of Na+ channels); however, it did not in the presence of 5-(N -methyl- N -isobutyl)-amiloride (MIA, an inhibitor of Na+,H+ exchange). MIA decreased the volume of AT-II cells. Inhibitors of Cl,,HCO3, exchange, 4,4,-diisothiocyanostilbene-2,2,-disulfonic acid (DIDS) and 4-acetamido-4,-isothiocyanatostilbene-2,2,-disulfonic acid (SITS) also decreased the volume of AT-II cells. This indicates that the cell shrinkage induced by a Ca2+ -free solution is caused by a decrease in NaCl influx via Na+,H+ exchange and Cl,,HCO3, exchange. Addition of ionomycin (1 ,m), in contrast, induced cell swelling when AT-II cells were pretreated with quinine and amiloride. This swelling of the AT-II cells is not detected in the presence of MIA. Intracellular pH (pHi) measurements demonstrated that the Ca2+ -free solution or MIA decreases pHi, and that ionomycin increases it. Ionomycin stimulated the pHi recovery after an acid loading (NH4+ pulse method), which was not noted in MIA-treated AT-II cells. Ionomycin increased [Ca2+]i in fura-2-loaded AT-II cells. In conclusion, the Na+,H+ exchange activities of AT-II cells, which maintain the volume and pHi, are regulated by [Ca2+]i. [source] The Secretory Response of the Rat Colon to the Flavonol Quercetin is Dependent on Ca2+ -CalmodulinEXPERIMENTAL PHYSIOLOGY, Issue 3 2000R. Cermak The dietary flavonol quercetin induces chloride secretion in rat intestine. To clarify the underlying mechanisms, experiments were performed in Ussing chambers with tissue from rat proximal and distal colon. Quercetin induced an increase in short-circuit current (Isc), which was largely independent of submucosal neurons, as it was not affected by the neurotoxin tetrodotoxin. The effect of quercetin was blocked by the calmodulin antagonists trifluoperazine and ophiobolin A and was diminished by a blocker of Ca2+ release from intracellular stores (TMB-8), whereas the muscarinic receptor antagonist atropine was ineffective. The quercetin-induced Isc was abolished in Ca2+ -free solution. The flavonol was able to further increase Isc after maximal stimulation of the cAMP pathway by forskolin. The Isc increase by the flavonol was differently affected by two analogous phosphodiesterase inhibitors. Whereas 3-isobutyl-1-methylxanthine (IBMX) antagonized the effect of quercetin, 8-methoxymethyl-IBMX had no effect. Both phosphodiesterase inhibitors similarly influenced the Isc increase induced by forskolin. These results indicate that the chloride secretion induced by quercetin in rat colon depends on Ca2+ and calmodulin. The cAMP pathway and inhibition of phosphodiesterase appear not to be responsible for the secretory activity of the flavonol. [source] The substituent effects on the structure and surface morphology of polyanilineJOURNAL OF APPLIED POLYMER SCIENCE, Issue 5 2010Mutlu Sahin Abstract In this work, poly(2-fluoroaniline), poly(2-chloroaniline), poly(2-methylaniline), and poly(N -ethylaniline) were prepared by a self-assembly method using an oxidizing system consisting of a dopant anion, p-toluene sulfonate with ammonium peroxydisulfate. The effects of substituents on the surface morphology, conductivity, molecular weight, spectral and thermal properties of the polymers were studied. SEM results revealed that the surface morphology of the resulting polymers changed from nanofiber to spherical structure by changing the substituent on the aniline monomers. The structure and properties of these conducting films were characterized by FTIR, UV-vis, elemental analysis, TGA, conductivity, and cyclic voltammetry. The polymer films show electroactivity in monomer free solution. Molecular weight of the polymers was determined by gel permeation chromatography. The dry electrical conductivity values of the substituted-polyanilines were found to be lower than that of PANI. The results revealed that the molecular structures of the polymers were similar to those of the emeraldine form of polyaniline. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2010 [source] Effects on the removal of Pb2+ from aqueous solution by crab shellJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 11 2001Dong Seog Kim Abstract The effects of temperature, pH, chitin and chitosan on Pb2+ removal by crab shell were investigated. Pb2+ removal by crab shell was greater than that of chitin and chitosan, indicating that chitin did not contribute to Pb2+ removal by crab shell. The quantity and rate of Pb2+ removal increased as the pH value increased. The rate of Pb2+ removal increased with increased temperature, but the maximum amount of Pb2+ removal was constant irrespective of temperature. Metal ions (K+, Na+, Mg2+, Ca2+) were released from crab shell concomitant with Pb2+ removal by ion exchange. The amount of Ca2+ released was greater than any for other metal ions in both Pb2+ and Pb2+ -free solutions. The amount of Ca2+ released in Pb2+ solution was greater than that in Pb2+ -free solution, whereas CO32, release in Pb2+ solution was less than in Pb2+ -free solution. Pb2+ removal was mainly a consequence of dissolution of CaCO3(s) with consequence precipitation of Pb3(CO3)2(OH)2(s) and PbCO3(s). Pb2+ accelerated the dissolution of CaCO3(s) by ion exchange and the precipitation occurred both at the surface and in the inner part of the crab shell. © 2001 Society of Chemical Industry [source] Protein ion-exchange adsorption kineticsAICHE JOURNAL, Issue 7 2001J. A. Wesselingh The kinetics of the adsorption of the protein BSA on the ion exchanger Q-Sepharose FF were measured for several values of the pH and ionic strength, using several techniques. The measurements were best described with a model incorporating both surface and pore diffusion and with the chemical potential gradient as the driving force for diffusion. The surface-diffusion coefficients from this model show an inverse exponential dependency on the binding strength. This dependency can be explained by an activated jump mechanism. The pore-diffusion coefficient is much lower than that in free solution, which is probably caused by a combination of steric and electric exclusion. [source] Genetically engineered normal flora for oral polypeptide delivery: Dose,absorption responseJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 8 2009Gagan Kaushal Abstract Genetically modified Lactococcus lactis (L. lactis), a probiotic bacterium, able to secrete ,-lactamase (29 kDa), was used as a vector for the oral delivery of ,-lactamase to the rats. Three different doses of L. lactis were administered to the rats, and the resulted ,-lactamase oral bioavailability was studied, and compared to the solution form. The oral administration of 1.2,×,107, 3,×,107, and 8,×,107 colony-forming units of L. lactis led to 145, 209, and 364 mU of ,-lactamase absorbed, and the corresponding bioavailability was 8.7%, 15.5%, and 20.8% based on the in vitro production of ,-lactamase by L. lactis. The oral administration of 504 mU and 1008 mU ,-lactamase free solution resulted in 30 and 47 mU absorbed, a bioavailability of 5.9% and 4.7%, respectively. L. lactis significantly (p,<,0.01) increased the oral bioavailability compared to the free solution form. A significant (p,<,0.01) increase in the MAT value as compared to the solution, demonstrated that L. lactis can be used as a sustained delivery system. In conclusion, there is a linear relationship between L. lactis dose and these absorption PK parameters within L. lactis dose range of the current study. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:2573,2580, 2009 [source] Carrier proteins determine local pharmacokinetics and arterial distribution of paclitaxelJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 9 2001Mark A. Lovich Abstract The growing use of local drug delivery to vascular tissues has increased interest in hydrophobic compounds. The binding of these drugs to serum proteins raises their levels in solution, but hinders their distribution through tissues. Inside the arterial interstitium, viscous and steric forces and binding interactions impede drug motion. As such, this might be the ideal scenario for increasing the amount of drug delivered to, and residence time within, arterial tissues. We quantified carrier-mediated transport for paclitaxel, a model hydrophobic agent with potential use in proliferative vascular diseases, by determining, in the presence or absence of carrier proteins, the maximum concentration of drug in aqueous solution, the diffusivity in free solution, and the diffusivity in arterial tissues. Whereas solubility of paclitaxel was raised 8.1-, 21-, and 57-fold by physiologic levels of ,1 -acid glycoproteins, bovine serum albumin, and calf serum over that in protein-free solution, diffusivity of paclitaxel in free solution was reduced by 41, 49, and 74%, respectively. When paclitaxel mixed in these solutions was applied to arteries both in vitro and in vivo, drug was more abundant at the tissue interface, but protein carriers tended to retain drug in the lumen. Once within the tissue, these proteins did not affect the rate at which drug traverses the tissue because this hydrophobic drug interacted with the abundant fixed proteins and binding sites. The protein binding properties of hydrophobic compounds allow for beneficial effects on transvascular transport, deposition, and distribution, and may enable prolonged effect and rationally guide local and systemic strategies for their administration. © 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:1324,1335, 2001 [source] Vasorelaxant effect of Valeriana edulis ssp. procera (Valerianaceae) and its mode of action as calcium channel blockerJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 9 2010Samuel Estrada-Soto Abstract Objectives, The aim was to evaluate the relaxant effect of extracts from Valeriana edulis and determine the possible mechanism of action of the hexanic extract as vasorelaxant agent. Methods, Extracts from rhizomes obtained by maceration (hexanic (HEVe), dichloromethanic (DEVe), methanolic (MEVe) and hydroalcoholic (HAEVe) (3.03,500 µg/ml)) were evaluated on aortic rat rings with and without endothelium. Key findings, Extracts induced a significant concentration-dependent and endothelium-independent relaxation on isolated rat aorta pre-contracted with noradrenaline (0.1 µm). HEVe, the most potent extract (0.15,50 µg/ml), induced relaxation in aortic rings pre-contracted with KCl (80 mm), with an IC50 value of 34.61 ± 1.41 µg/ml and Emax value of 85.0 ± 4.38%. Pretreatment with HEVe (30 µg/ml) also inhibited contractile responses to noradrenaline and CaCl2. HEVe (9.98 ± 2.0 µg/ml) reduced noradrenaline-induced transient contraction in Ca2+ -free solution, and inhibited contraction induced by KCl (80 mm). In endothelium-denuded rings, the vasorelaxant effect of HEVe was not modified by 1- H -[1,2,4]-oxadiazolo-[4,3a]-quinoxalin-1-one (1 µm), tetraethylammonium (5 mm), glibenclamide (10 µm) or 2-aminopyridine (100 µm). Conclusions, Our results suggest that HEVe induces relaxation through an endothelium-independent pathway, involving blockade of Ca2+ channels, and this effect could be related to the presence of valepotriates. [source] Endothelium-independent Vasorelaxant Effect of Dioclein, a New Flavonoid Isolated from Dioclea grandiflora, in the Rat AortaJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 11 2000F. TRIGUEIRO We have investigated the endothelium-independent vasorelaxant effect of the new Flavonoid dioclein (5,2,,5,-trihydroxy-6,7-dimethoxyflavanone) in the rat aorta. In endothelium-denuded vessels, dioclein induced a concentration-dependent relaxation of aortic rings precontracted with noradrenaline (IC50 = 3.5 ± 0.89 times 10,4 M and KCl (IC50 = 5.2 ± 1.2 times 10,4 M). In the absence of extracellular calcium, dioclein reduced the contraction induced by noradrenaline (maximal reduction approximately 33%) but not that induced by caffeine. Dioclein also produced a concentration-dependent inhibition of the sustained contractions induced by the phorbol ester 12- O -tetradecanoyl phorbol-13-acetate in normal (IC50 = 4.0 ± 0.2 times 10,4 M) and Ca2+ -free solution (IC50 = 4.0 ± 0.3 times 10,4 M). The results indicate that the endothelium-independent vasorelaxant effect of dioclein may be explained by inhibition of contractions dependent on activation of protein kinase C, voltage-dependent Ca2+ influx and on the release of intracellular Ca2+ stores sensitive to noradrenaline. [source] Dissociation of intermolecular disulfide bonds in P22 tailspike protein intermediates in the presence of SDSPROTEIN SCIENCE, Issue 7 2006Junghwa Kim Abstract Each chain of the native trimeric P22 tailspike protein has eight cysteines that are reduced and buried in its hydrophobic core. However, disulfide bonds have been observed in the folding pathway and they are believed to play a critical role in the registration of the three chains. Interestingly, in the presence of sodium dodecyl sulfate (SDS) only monomeric chains, rather than disulfide-linked oligomers, have been observed from a mixture of folding intermediates. Here we show that when the oligomeric folding intermediates were separated from the monomer by native gel electrophoresis, the reduction of intermolecular disulfide bonds did not occur in the subsequent second-dimension SDS,gel electrophoresis. This result suggests that when tailspike monomer is present in free solution with SDS, the partially unfolded tailspike monomer can facilitate the reduction of disulfide bonds in the tailspike oligomers. [source] Review modeling the free solution and gel electrophoresis of biopolymers: The bead array-effective medium modelBIOPOLYMERS, Issue 2-3 2007Stuart A. Allison Abstract Free solution and gel electrophoresis is an extremely useful tool in the separation of biopolymers. The complex nature of biopolymers, coupled with the usefulness of electrophoretic methods, has stimulated the development of theoretical modeling over the last 30 years. In this work, these developments are first reviewed with emphasis on Boundary Element and bead methodologies that enable the investigator to design realistic models of biopolymers. In the present work, the bead methodology is generalized to include the presence of a gel through the Effective Medium model. The biopolymer is represented as a bead array. A peptide, for example, made up of N amino acids is modeled as 2N beads. Duplex DNA is modeled as a discrete wormlike chain consisting of touching beads. The technical details of the method are placed in three Appendices. To illustrate the accuracy and effectiveness of the approach, two applications are considered. Model studies on both the free solution mobility of 73 peptides ranging in size from 2 to 42 amino acids, and the mobility of short duplex DNA in dilute agarose gels are discussed. © 2007 Wiley Periodicals, Inc. Biopolymers 87: 102,114, 2007. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] Design and characterization of a prototype enzyme microreactor: Quantification of immobilized transketolase kineticsBIOTECHNOLOGY PROGRESS, Issue 1 2010S. Matosevic Abstract In this work, we describe the design of an immobilized enzyme microreactor (IEMR) for use in transketolase (TK) bioconversion process characterization. The prototype microreactor is based on a 200-,m ID fused silica capillary for quantitative kinetic analysis. The concept is based on the reversible immobilization of His6 -tagged enzymes via Ni-NTA linkage to surface derivatized silica. For the initial microreactor design, the mode of operation is a stop-flow analysis which promotes higher degrees of conversion. Kinetics for the immobilized TK-catalysed synthesis of L -erythrulose from substrates glycolaldehyde (GA) and hydroxypyruvate (HPA) were evaluated based on a Michaelis,Menten model. Results show that the TK kinetic parameters in the IEMR (Vmax(app) = 0.1 ± 0.02 mmol min,1, Km(app) = 26 ± 4 mM) are comparable with those measured in free solution. Furthermore, the kcat for the microreactor of 4.1 × 105 s,1 was close to the value for the bioconversion in free solution. This is attributed to the controlled orientation and monolayer surface coverage of the His6 -immobilized TK. Furthermore, we show quantitative elution of the immobilized TK and the regeneration and reuse of the derivatized capillary over five cycles. The ability to quantify kinetic parameters of engineered enzymes at this scale has benefits for the rapid and parallel evaluation of evolved enzyme libraries for synthetic biology applications and for the generation of kinetic models to aid bioconversion process design and bioreactor selection as a more efficient alternative to previously established microwell-based systems for TK bioprocess characterization. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Tamoxifen dilates porcine coronary arteries: roles for nitric oxide and ouabain-sensitive mechanismsBRITISH JOURNAL OF PHARMACOLOGY, Issue 6 2006H S Leung Background and purpose: Experiments were designed to determine the mechanism of the relaxation induced by tamoxifen in porcine coronary arteries at the tissue, cellular and molecular levels. Experimental approach: Porcine left circumflex coronary arteries were isolated and isometric tension was measured. [Ca2+]i in native endothelial cells of intact arteries was determined by a calcium fluorescence imaging technique and eNOS ser1177 phosphorylation was assayed by Western blotting. Key results: Tamoxifen induced an endothelium-dependent relaxation that was antagonized by ICI 182,780 and abolished by NG -nitro-L-arginine methyl ester (L-NAME) or 1H-[1,2,4]oxadizolo[4,3-a]quinoxalin-1-one (ODQ). L-Arginine reversed the effect of L-NAME while indomethacin was without effect. Tamoxifen-induced relaxation was attenuated by charybdotoxin (CTX) plus apamin, ouabain or by incubation in a K+ -free solution. Moreover, tamoxifen triggered extracellular Ca2+ -dependent increases in endothelial [Ca2+]i and this effect was abolished by ICI 182,780. Endothelium-independent relaxation to sodium nitroprusside was also inhibited by ouabain or in a K+ -free solution. Furthermore, tamoxifen increased endothelial nitric oxide synthase (eNOS) phosphorylation at Ser-1177 and ICI 182,780 prevented this effect. Conclusions and Implications: The present results suggest that tamoxifen mainly induces endothelium-dependent relaxation and that endothelial nitric oxide (NO) is the primary mediator of this effect. NO-dependent responses may result from elevated [Ca2+]i in endothelial cells; an effect abolished by ICI 182,780. NO activates Na+/K+ -ATPase in vascular smooth muscle, leading to relaxation. These results suggest that tamoxifen is able to modulate eNOS phosphorylation directly. British Journal of Pharmacology (2006) 149, 703,711. doi:10.1038/sj.bjp.0706921 [source] The mechanism for the contraction induced by leukotriene C4 in guinea-pig taenia coliBRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2001Satoshi Ieiri The mechanism underlying the LTC4 -induced contraction of guinea-pig taenia coli was determined using the simultaneous measurements of [Ca2+]i and force in whole muscle preparations. Additional experiments were performed in receptor coupled permeabilized preparation. For comparison purposes, the contraction which was induced by a typical G-protein mediated agonist, carbachol was also characterized. LTC4 induced a contraction in the guinea-pig taenia coli in a concentration-dependent manner. The maximal response was obtained at 100 nM and the EC50 value was 5.4±1.9 nM. Both LTC4 and carbachol induced increases in [Ca2+]i and force. The maximum force induced by 100 nM LTC4 was significantly smaller than that induced by 10 ,M carbachol, although an increase in [Ca2+]i produced by both agonists was similar. In the permeabilized preparations, carbachol, but not LTC4, induced an additional force development at a fixed Ca2+ concentration. LTC4 induced no increase in [Ca2+]i and force in the Ca2+ -free solution, while carbachol induced transient increases in both [Ca2+]i and force in a Ca2+ -free solution. Both diltiazem and SK&F 96365 significantly inhibited the LTC4, and carbachol-induced increases in [Ca2+]i and force in normal PSS. The inhibitory pattern of [Ca2+]i by these drugs was also similar. We thus conclude that LTC4 induces the contraction of the guinea-pig taenia coli mainly through Ca2+ influx via both the diltiazem-sensitive and SK&F 96365-sensitive Ca2+ channels, without affecting either the Ca2+ -sensitivity or the intracellular Ca2+ release. These results indicated that the mechanism underlying the LTC4 -induced contraction differs greatly from that for conventional G-protein mediated agonists, such as carbachol. British Journal of Pharmacology (2001) 133, 529,538; doi:10.1038/sj.bjp.0704122 [source] Selective phenylalkylamine block of IKr over other K+ currents in guinea-pig ventricular myocytesBRITISH JOURNAL OF PHARMACOLOGY, Issue 8 2000Stephen E Jones Previous studies on verapamil and D600 have established that the Ca2+ -channel blockers also inhibit delayed-rectifier K+ currents in cardiac tissues and myocytes. However, estimated IC50 values range over two to three orders of concentration, and it is unclear whether this reflects a high selectivity by one or both of the phenylalkylamines for particular K+ channels. The purpose of the present study was to determine the concentration-dependent actions of verapamil and D600 on three defined cardiac K+ currents. Guinea-pig ventricular myocytes in the conventional whole-cell configuration were bathed with normal Tyrode's or K+ -free solution, and pulsed from ,80 mV for measurement of the effects of 0.01 ,M to 3 mM verapamil and D600 on the inwardly-rectifying K+ current (IKl) and the two delayed-rectifier K+ currents, rapidly-activating IKr and slowly-activating IKs. The phenylalkylamines inhibited both inward- and outward-directed IKl. The IC50 values for outward IKl were approximately 220 ,M. Verapamil and D600 were approximately equipotent inhibitors of the delayed-rectifier K+ currents. They inhibited IKr with IC50 near 3 ,M, and IKs with IC50280 ,M. These results are discussed in relation to previous findings on K+ currents and to the clinical actions of the drugs. British Journal of Pharmacology (2000) 131, 1809,1816; doi:10.1038/sj.bjp.0703758 [source] One-Armed Artificial Receptors for the Binding of Polar Tetrapeptides in Water: Probing the Substrate Selectivity of a Combinatorial Receptor LibraryCHEMISTRY - A EUROPEAN JOURNAL, Issue 5 2006Carsten Schmuck Prof. Dr. Abstract We have recently developed a new class of one-armed artificial receptors 1 for the binding of the polar tetrapeptide N -Ac- D -Glu- L -Lys- D -Ala- D -Ala-OH (EKAA) 2 in water using a combined combinatorial and statistical approach. We have now further probed the substrate selectivity of this receptor library 1 by screening a second tetrapeptide substrate (3) with the inverse sequence N -Ac- D -Ala- D -Ala- L -Lys- D -Glu-OH (AAKE). This "inverse" substrate is also efficiently bound by our receptors, with Kass ,6000,m,1 for the best receptors, as determined both by a quantitative on-bead binding assay and by UV and fluorescence titration studies in free solution. Hence, the inverse tetrapeptide 3 is in general bound two to three times less efficiently than the "normal" peptide 2 (Kass ,17,000,m,1), even though the complexation mainly involves long-range electrostatic interactions and both the receptor and substrate are rather flexible. Molecular modeling and ab initio calculations have been used to rationalize the observed substrate selectivity and to analyze the various binding interactions within the complex. [source] A ROLE FOR RHO-KINASE IN Ca2+ -INDEPENDENT CONTRACTIONS INDUCED BY PHORBOL-12,13-DIBUTYRATECLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 3 2009Inji Baek SUMMARY 1Phorbol-12,13-dibutyrate (PDBu) is an activator of protein kinase C (PKC) that causes contractions in both physiological salt solutions and Ca2+ -depleted solutions. In the present study, we tested the hypothesis that Rho-kinase plays a role in Ca2+ -independent contractions induced by PDBu in vascular smooth muscles. 2In Ca2+ -free solution, 0.1 and 1 µmol/L PDBu induced contraction and myosin light chain (MLC20) phosphorylation, both of which were approximately 40% of responses obtained in normal Krebs' solution. Hydroxyfasudil (H1152; 1 µmol/L), an inhibitor of Rho-kinase, but not ML7 (10 µmol/L), an inhibitor of myosin light chain kinase, inhibited Ca2+ -independent contractions induced by PDBu. 3In Ca2+ -free solution, PDBu increased phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and CPI-17 (PKC-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa). This action was inhibited by H1152, with the phosphorylation of CPI-17 almost completely abolished by 1 µmol/L Ro31-8220, an inhibitor of PKC. 4In Ca2+ -free solution, PDBu increased the amount of GTP-RhoA (an activated form of RhoA). This increase was blocked by the PKC inhibitor Ro31-8220, but not by the Rho kinase inhibitor H1152. 5In conclusion, RhoA/Rho-kinase plays an important role in Ca2+ -independent contractions induced by PDBu in vascular smooth muscles. The results of the present study suggest that PDBu induces Ca2+ -independent contractions by inhibiting myosin light chain phospatase (MLCP) through activation of GTP-RhoA and subsequent phosphorylation of MYPT1 and CPI-17. [source] NANOMOLAR LEVEL OF OUABAIN INCREASES INTRACELLULAR CALCIUM TO PRODUCE NITRIC OXIDE IN RAT AORTIC ENDOTHELIAL CELLSCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 5-6 2004Xian Hui Dong Summary 1.,Changes in [Ca2+]i across the cell membrane and/or the sarcoplasmic reticulum regulate endothelial nitric oxide (NO) synthase activity. 2.,In the present study, we investigated the effect of ouabain, a specific inhibitor of Na+/K+ -ATPase, on NO release and [Ca2+]i movements in cultured rat aortic endothelial cells (RAEC) by monitoring NO production continuously using an NO-specific real-time sensor and by measuring the change in [Ca2+]i using a fluorescence microscopic imaging technique with high-speed wavelength switching. The t½ (half-time of the decline of [Ca2+]i to basal levels after stimulation with 10 µmol/L bradykinin) was used as an index of [Ca2+]i extrusion. 3.,A very low concentration of ouabain (10 nmol/L) did not increase the peak of NO production, but decreased the decay of NO release and, accordingly, increased integral NO production by the maximal dose,response concentration induced by bradykinin. The same dose of ouabain affected [Ca2+]i movements across the cell membrane and/or sarcoplasmic reticulum induced by bradykinin with a time-course similar to that of NO release. Moreover, the t½ was significantly increased. 4.,Pretreatment of RAEC with Na+ -free solution, an inhibitor of the Na+/Ca2+ exchanger, and nickel chloride hexahydrate prevented the effects induced by bradykinin and ouabain. 5.,These observations using real-time recording indicate that a small amount of ouabain contributes to the bradykinin-stimulated increase of NO production through inhibition of plasma membrane Na+/K+ -ATPase activity and an increase in intracellular Na+ concentrations. The membrane was then depolarized, leading to a decline in the bradykinin-stimulated increase in [Ca2+]i by forward mode Na+/Ca2+ exchange to prolong the Ca2+ signal time. 6.,From these results, we suggest that nanomolar levels of ouabain modulate [Ca2+]i movements and NO production in RAEC. [source] Motility-induced but not vasoactive intestinal peptide-induced increase in luminal alkalinization in rat duodenum is dependent on luminal Cl,ACTA PHYSIOLOGICA, Issue 2 2010L. Pihl Abstract Aim:, To investigate whether the motility- and the vasoactive intestinal peptide (VIP)-induced increase in luminal alkalinization in the duodenum is dependent on luminal Cl,. Methods:, Experiments were performed in anaesthetized rats in vivo. The proximal duodenum was perfused luminally with an isotonic solution, containing zero or low Cl, and the effects on luminal alkalinization, motility, fluid flux and epithelial permeability were determined. Parecoxib, a COX-2 inhibitor, was used to induce duodenal contractions. Results:, Control rats lacked duodenal wall contractions while parecoxib-treated ones exhibited contractions throughout the experiment. Most animals had a net fluid absorption during the perfusion with isotonic NaCl. Luminal alkalinization was about 100% higher in parecoxib-treated rats than in controls. Cl, -free solutions did not affect epithelial permeability or motility but decreased luminal alkalinization by ,50% and decreased net fluid absorption in both control and parecoxib-treated animals. Reduction in luminal Cl, decreased alkalinization in a concentration-dependent manner. The parecoxib-induced increase in alkalinization was markedly reduced in the absence of luminal Cl,. VIP increased luminal alkalinization and induced fluid secretion. The lack of luminal Cl, did not affect the VIP-induced increase in alkalinization but reduced fluid secretion. Conclusions:, The parecoxib-induced increase in luminal alkalinization is highly dependent on luminal Cl, and it is proposed that COX-2 inhibition, via induction of duodenal motility, enhances HCO3, efflux through stimulation of apical Cl,/HCO3, exchange in duodenal epithelial cells. Although the VIP-induced stimulation of fluid secretion is partly dependent on luminal Cl,, the VIP-induced increase in luminal alkalinization is not. [source] Tetanic stimulation of Schaffer collaterals induces rhythmic bursts via NMDA receptor activation in rat CA1 pyramidal neuronsHIPPOCAMPUS, Issue 4 2002Christian Bonansco Abstract Exploring the principles that regulate rhythmic membrane potential (Vm) oscillations and bursts in hippocampal CA1 pyramidal neurons is essential to understanding the , rhythm (,). Recordings were performed in vitro in hippocampal slices from young rats, and a group of the recorded CA1 pyramidal cells were dye-filled with carboxifluorescein and immunolabeled for the R1 subunit of the NMDA receptor. Tetanic stimulation of Schaffer collaterals (SCs) and iontophoresis of glutamate evoked rhythmic Vm oscillations and bursts (,10 mV, ,7 Hz, 2,5 spikes per burst) in cells (31%) placed close to the midline ("medial cells"). Rhythmic bursts remained under picrotoxin (10 ,M) and Vm oscillations persisted with tetrodotoxin (1.5 ,M), but bursts were blocked by AP5 (25 ,M) and Mg2+ -free solutions. Depolarization and AMPA never induced rhythmic bursts. The rest of the neurons (69%), recorded closer to the CA3 region ("lateral cells"), discharged rhythmically single repetitive spikes under SC stimulation and glutamate in control conditions, but fired rhythmic bursts under similar stimulation, both when NMDA was applied and when non-NMDA receptors were blocked with CNQX (20 ,M). Medial cells exhibited a larger NMDA current component and a higher NMDAR1 density at the apical dendritic shafts than lateral cells, suggesting that these differences underlie the dissimilar responses of both cell groups. We conclude that the ",-like" rhythmic oscillations and bursts induced by glutamate and SC stimulation relied on the activation of NMDA receptors at the apical dendrites of medial cells. These results suggest a role of CA3 pyramidal neurons in the generation of CA1 , via the activation of NMDA receptors of CA1 pyramidal neurons. Hippocampus 2002;12:434,446. © 2002 Wiley-Liss, Inc. [source] Food applications of trans fatty acid substitutesINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 5 2007Paul Wassell Summary The review outlines the increasing need to reduce trans fatty acids, and addresses the functionality issues of various trans free solutions through discussion of hydrogenation, interesterification, and fractionation, and their influence on fat crystallisation and solid fat content. Caution is urged not to focus solely on physio-chemical aspects, but to approach trans free designing for specific food applications from a multidisciplinary angle. Examples of specific applications; margarines, shortenings and frying oils are given. The review also offers a glimpse into what the future trans free trends may hold. [source] Effects on the removal of Pb2+ from aqueous solution by crab shellJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 11 2001Dong Seog Kim Abstract The effects of temperature, pH, chitin and chitosan on Pb2+ removal by crab shell were investigated. Pb2+ removal by crab shell was greater than that of chitin and chitosan, indicating that chitin did not contribute to Pb2+ removal by crab shell. The quantity and rate of Pb2+ removal increased as the pH value increased. The rate of Pb2+ removal increased with increased temperature, but the maximum amount of Pb2+ removal was constant irrespective of temperature. Metal ions (K+, Na+, Mg2+, Ca2+) were released from crab shell concomitant with Pb2+ removal by ion exchange. The amount of Ca2+ released was greater than any for other metal ions in both Pb2+ and Pb2+ -free solutions. The amount of Ca2+ released in Pb2+ solution was greater than that in Pb2+ -free solution, whereas CO32, release in Pb2+ solution was less than in Pb2+ -free solution. Pb2+ removal was mainly a consequence of dissolution of CaCO3(s) with consequence precipitation of Pb3(CO3)2(OH)2(s) and PbCO3(s). Pb2+ accelerated the dissolution of CaCO3(s) by ion exchange and the precipitation occurred both at the surface and in the inner part of the crab shell. © 2001 Society of Chemical Industry [source] | ||||||||||||||||