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Physiological Temperature (physiological + temperature)
Selected AbstractsThe Drosophila cacts2 mutation reduces presynaptic Ca2+ entry and defines an important element in Cav2.1 channel inactivationEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2006G. T. Macleod Abstract Voltage-gated Ca2+ channels in nerve terminals open in response to action potentials and admit Ca2+, the trigger for neurotransmitter release. The cacophony gene encodes the primary presynaptic voltage-gated Ca2+ channel in Drosophila motor-nerve terminals. The cacts2 mutant allele of cacophony is associated with paralysis and reduced neurotransmission at non-permissive temperatures but the basis for the neurotransmission deficit has not been established. The cacts2 mutation occurs in the cytoplasmic carboxyl tail of the ,1 -subunit, not within the pore-forming trans-membrane domains, making it difficult to predict the mutation's impact. We applied a Ca2+ -imaging technique at motor-nerve terminals of mutant larvae to test the hypothesis that the neurotransmission deficit is a result of impaired Ca2+ entry. Presynaptic Ca2+ signals evoked by single and multiple action potentials showed a temperature-dependent reduction. The amplitude of the reduction was sufficient to account for the neurotransmission deficit, indicating that the site of the cacts2 mutation plays a role in Ca2+ channel activity. As the mutation occurs in a motif conserved in mammalian high-voltage-activated Ca2+ channels, we used a heterologous expression system to probe the effect of this mutation on channel function. The mutation was introduced into rat Cav2.1 channels expressed in human embryonic kidney cells. Patch-clamp analysis of mutant channels at the physiological temperature of 37 °C showed much faster inactivation rates than for wild-type channels, demonstrating that the integrity of this motif is critical for normal Cav2.1 channel inactivation. [source] Development of Biomimetic Chitosan-Based Hydrogels Using an Elastin-Like Polymer,ADVANCED ENGINEERING MATERIALS, Issue 1-2 2010Joaquim S. Barbosa Chitosan and an elastin-like polymer, containing a specific osteoconductive sequence in the primary structure, have been combined to obtain bioactive injectable systems with enhanced mechanical properties and hydrogels. Obtained results indicate that the combination of such polymers may be very promising in the development of biomaterials for minimal invasive orthopaedic reconstructive applications or in bone tissue engineering. The figure shows a thermo-sensitive hydrogel, with a gelation point under physiological temperature. [source] Control of thermo reversible gelation of methylcellulose using polyethylene glycol and sodium chloride for sustained delivery of ophthalmic drugJOURNAL OF APPLIED POLYMER SCIENCE, Issue 2 2010Mrinal Kanti Bain Abstract The effect of molecular weight of polyethyleneglycol (PEG) and sodium chloride (NaCl) on the gelation temperature of methylcellulose (MC) was studied with the objective to develop a MC based formulation for sustained delivery of ophthalmic drug. The gelation temperature of 1% MC was 60 ± 0.40°C. It was found that the gelation temperature of MC was reduced with the addition of 10% PEG and extent of reduction of gelation temperature was depended on the molecular weight of PEG at same PEG concentration of 10%. The gelation temperature of MC was reduced by 10.4 to 5.9°C with the increasing molecular weight of PEG starting from 400 to 20,000 (Mn) depending on the method of determination of gelation temperature. To reduce the gelation temperature of MC close to physiological temperature (37°C), 6% NaCl was added in the different MC-PEG combinations containing different molecular weight of PEG. It was observed that the drug release time increased from 5 to 8 h with the increase in molecular weight of PEG from 400 to 20,000 (Mn) and this was due to the maximum viscosity and gel strength of MC-PEG20000-NaCl ternary combination. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010 [source] Synthesis and characterization of injectable bioadhesive hydrogels for nucleus pulposus replacement and repair of the damaged intervertebral discJOURNAL OF BIOMEDICAL MATERIALS RESEARCH, Issue 2 2010J. Vernengo Abstract Bioadhesive polymers are natural or synthetic materials that can be used for soft tissue repair. The aim of this investigation was to develop an injectable, bioadhesive hydrogel with the potential to serve as a synthetic replacement for the nucleus pulposus of the intervertebral disc or as an annulus closure material. Branched copolymers of poly(N -isopropylacrylamide) (PNIPAAm) and poly(ethylene glycol) (PEG) were blended with poly(ethylene imine) (PEI). This three component injectable system can form a precipitated gel at physiological temperature due to the phase transition of PNIPAAm. The injection of glutaraldehyde into the gel core will adhere the implant to the surrounding tissues. 1H NMR results indicated the successful physical incorporation of PEI into the PNIPAAm-PEG network by blending. In addition, the covalent crosslinking between the amine functionalities on the PEI and the aldehyde functionalities on the glutaraldehyde was verified using FTIR difference spectroscopy. Mechanical characterization of these blends showed a significant increase (p < 0.05) in compressive modulus following glutaraldehyde injection. The in vitro bioadhesive force studies with porcine skin showed a significant increase (p < 0.05) in the mean maximum force of detachment for PNIPAAm-PEG/PEI gels when glutaraldehyde was injected into the gel core. The results of this study indicate that the reactivity between amines and aldehyde functionalities can be exploited to impart bioadhesive properties to PNIPAAm-PEG/PEI copolymers. © 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2010 [source] Temperature-dependent localization of GPI-anchored intestinal alkaline phosphatase in model rafts,JOURNAL OF MOLECULAR RECOGNITION, Issue 6 2007Marie-Cécile Giocondi Abstract In plasma membranes, most of glycosylphosphatidylinositol (GPI)-anchored proteins would be associated with rafts, a category of ordered microdomains enriched in sphingolipids and cholesterol (Ch). They would be also concentrated in the detergent resistant membranes (DRMs), a plasma membrane fraction extracted at low temperature. Preferential localization of GPI-anchored proteins in these membrane domains is essentially governed by their high lipid order, as compared to their environment. Changes in the temperature are expected to modify the membrane lipid order, suggesting that they could affect the distribution of GPI-anchored proteins between membrane domains. Validity of this hypothesis was examined by investigating the temperature-dependent localization of the GPI-anchored bovine intestinal alkaline phophatase (BIAP) into model raft made of palmitoyloleoylphosphatidylcholine/sphingomyelin/cholesterol (POPC/SM/Chl) supported membranes. Atomic force microscopy (AFM) shows that the inserted BIAP is localized in the SM/Chl enriched ordered domains at low temperature. Above 30°C, BIAP redistributes and is present in both the ,fluid' POPC enriched and the ordered SM/Chl domains. These data strongly suggest that in cells the composition of plasma membrane domains at low temperature differs from that at physiological temperature. Copyright © 2007 John Wiley & Sons, Ltd. [source] Echogenic liposome compositions for increased retention of ultrasound reflectivity at physiologic temperatureJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 6 2008Kyle D. Buchanan Abstract Targetable echogenic liposomes (ELIP) for ultrasound enhancement of atheroma have recently been developed; however, their retention of echogenicity at physiological temperature is less than desirable. The purpose of this study was to improve ELIP stability and increase clinical potential. The approach utilized the original procedures but involved manipulation of the lipid composition by reducing the level of unsaturation of the phospholipids components to minimize the rate of loss of echogenicity. Echogenicity was measured using a 20 MHz intravascular ultrasound (IVUS) catheter and quantified (as mean gray scale values) using computer-assisted videodensitometry. The optimal preparation for retention of echogenicity stability at physiologic temperature was egg phosphatidylcholine/dipalmitoylphosphatidylcholine/dipalmitoylphos-phatidylethanolamine/dipalmitoylphosphatidylglycerol/cholesterol (27:42:8:8:15, molar percent). This preparation retained 51,±,3.5% of its echogenicity after 1 h at 37°C, more than 5× that retained by the previously descried preparation. In this composition nearly 2/3 of the phosphosphatidylcholine is fully saturated. Such an increase in saturation is anticipated to stiffen the lipid acyl chains. The air pockets that are responsible for reflection of ultrasound waves can be assumed to be stabilized by a lipid monolayer at the interface between the air and bulk water. The increased rigidity of that monolayer is presumed to be responsible for reducing the loss of air and extending the duration of echogenic activity. The stability of this improved formulation now appears to be more than adequate for clinical applications. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:2241,2248, 2008 [source] Determination of the binding constant between alprostadil and alpha-cyclodextrin by capillary electrophoresis: Implications for a freeze-dried formulationJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 7 2005Benjamin R. Schipper Abstract The binding constant between alprostadil (PGE1) and ,-cyclodextrin (,-CD) was determined at three temperatures by capillary electrophoresis. ,-CD is an excipient material in Caverject Dual Chamber Syringe (DCS), added to enhance stability. The binding constant was used to calculate the amount of PGE1 free upon reconstitution and injection, the latter of which is critical to product performance. Measurement was made in a pH 7.2 separation buffer to ensure a negative charge on PGE1. The concentration of PGE1 was fixed while the concentration of ,-CD was varied over a suitable range. As the amount of PGE1 bound to ,-CD increases, the weighted average of the resultant mobility decreases, thereby allowing a binding isotherm to be generated from which the stability constant was extracted via nonlinear regression analysis. A value of 708,±,64 M,1 was obtained at 27°C, while at physiological temperature (37°C) the value was 537,±,27 M,1. These results compare favorably to values previously obtained by NMR. Calculation of the percent PGE1 free upon reconstitution and injection show it to be near the desired outcome of 100%. Hence, we were able to conclude that the amount of free drug delivered by Caverject DCS is nominally the same as for Caverject S. Po., an earlier-developed product that contains no ,-CD. © 2005 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:1528,1537, 2005 [source] Effect of UV irradiation on type I collagen fibril formation in neutral collagen solutionsPHOTODERMATOLOGY, PHOTOIMMUNOLOGY & PHOTOMEDICINE, Issue 3 2001Julian M. Menter Background: Collagens have the well-known ability to spontaneously self-associate to form fibrils at physiological temperature and neutral pH in vitro and in vivo. Because solar UV may photochemically alter collagen, the kinetics of fibril formation may be modified. Thus, we have begun a systematic study of the effect of various UV wavebands on fibril formation. Methods: Citrate-soluble calf skin collagen (Elastin Products) was dissolved at 0.05% in 0.5 M HOAc, dialyzed over 2 days into two changes of 0.0327 M phosphate buffer, pH 7.0 at 4 °C, and centrifuged at 48 000×g. Photolysis was carried out at 4 °C with either (a) UVC (UVG,11 lamp), (b) filtered solar-simulating radiation (SSR) or UVA (SSR or UVL,21 lamp filtered with a 2.0 mm Schott WG 345 filter). Gelation was commenced by rapidly raising the temperature from 8 °C to 33 °C. Nucleation and growth were followed by turbidimetric measurements at 400 nm. Results: UVC radiation (0,17.3 J/cm2) resulted in a dose-dependent decrease in the rate of fibril growth. Under these conditions, concomitant collagen cross-linking and degradation occurred. Fibril nucleation, a prerequisite for growth, was rapid (threshold , 2 min) and was not affected by UVC, UVA or SSR. SSR (0,1320 J/cm2) caused a small decrease in growth rate and in the degree of fibril formation. UVA radiation (0,1080 J/cm2) had a similar effect. "Direct" photochemical damage thus paralleled absorption via various collagen chromophores, with UVC>SSR,UVA. The presence of riboflavin (RF) resulted in ground-state interactions that markedly altered both nucleation and growth kinetics. Irradiation with 29.6 J/cm2 UVA in the presence of RF photosensitizer caused relatively minor additional changes in fibrillation kinetics. Conclusions: These results collectively indicate that fibril formation is markedly dependent on specific ground state interactions and relatively insensitive to nonspecific UV damage. On the other hand, fibrils thus formed from photochemically altered collagen may have altered structural properties that could have subtle but unfavorable effects on the local dermal milieu in vivo. Notwithstanding, the relative insensitivity of fibrillogenesis to non-specific photochemical damage probably represents a favorable adaptation, overall, which tends to conserve the mechanical integrity of the skin. [source] In situ crosslinked hydrogels formed using Cu(I)-free Huisgen cycloaddition reactionPOLYMER INTERNATIONAL, Issue 10 2009Meredith Clark Abstract BACKGROUND: ,Click' chemistry, or the 1,3-dipolar cycloaddition of organic azides with alkynes, has been evaluated for many biomedical purposes; however, its utility in crosslinking hydrogels in situ is limited by the toxicity of the requisite copper(I) catalyst. We report the first use of catalyst-free Huisgen cycloaddition to generate crosslinked hydrogels under physiological conditions using multivalent azide-functionalized polymers and an electron-deficient dialkyne crosslinker. RESULTS: Water-soluble azide-functionalized polymers were crosslinked with an electron-deficient dialkyne crosslinker to form hydrogels at physiological temperature without the addition of copper(I) catalyst. Crosslinking was confirmed using scanning electron microscopy, Fourier transform infrared and 1H NMR analyses. Flow by vial inversion and dynamic rheological methodologies were implemented to evaluate gelation kinetics at 37 °C of variable polymer compositions, concentrations and stoichiometric ratios. Kinetic studies revealed gelation in as little as 12 h at 37 °C, although strong gels that withstand inversion were observed by 1,8 days. CONCLUSION: The ability to form hydrogel networks under mild conditions demonstrates the potential viability of the catalyst-free ,click' crosslinking chemistry for in situ gelling and other biological applications. Further chemical modifications in the crosslinking moieties, as well as polymer and crosslinker conformations, are expected to enhance gelation kinetics to a more biomedically practical rate. Copyright © 2009 Society of Chemical Industry [source] Folding and misfolding mechanisms of the p53 DNA binding domain at physiological temperaturePROTEIN SCIENCE, Issue 11 2006James S. Butler Abstract p53 modulates a large number of cellular response pathways and is critical for the prevention of cancer. Wild-type p53, as well as tumorigenic mutants, exhibits the singular property of spontaneously losing DNA binding activity at 37°C. To understand the molecular basis for this effect, we examine the folding mechanism of the p53 DNA binding domain (DBD) at elevated temperatures. Folding kinetics do not change appreciably from 5°C to 35°C. DBD therefore folds by the same two-channel mechanism at physiological temperature as it does at 10°C. Unfolding rates, however, accelerate by 10,000-fold. Elevated temperatures thus dramatically increase the frequency of cycling between folded and unfolded states. The results suggest that function is lost because a fraction of molecules become trapped in misfolded conformations with each folding-unfolding cycle. In addition, at 37°C, the equilibrium stabilities of the off-pathway species are predicted to rival that of the native state, particularly in the case of destabilized mutants. We propose that it is the presence of these misfolded species, which can aggregate in vitro and may be degraded in the cell, that leads to p53 inactivation. [source] Is the efficiency of mammalian (mouse) skeletal muscle temperature dependent?THE JOURNAL OF PHYSIOLOGY, Issue 19 2010C. J. Barclay Myosin crossbridges in muscle convert chemical energy into mechanical energy. Reported values for crossbridge efficiency in human muscles are high compared to values measured in vitro using muscles of other mammalian species. Most in vitro muscle experiments have been performed at temperatures lower than mammalian physiological temperature, raising the possibility that human efficiency values are higher than those of isolated preparations because efficiency is temperature dependent. The aim of this study was to determine the effect of temperature on the efficiency of isolated mammalian (mouse) muscle. Measurements were made of the power output and heat production of bundles of muscle fibres from the fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus muscles during isovelocity shortening. Mechanical efficiency was defined as the ratio of power output to rate of enthalpy output, where rate of enthalpy output was the sum of the power output and rate of heat output. Experiments were performed at 20, 25 and 30°C. Maximum efficiency of EDL muscles was independent of temperature; the highest value was 0.31 ± 0.01 (n= 5) at 30°C. Maximum efficiency of soleus preparations was slightly but significantly higher at 25 and 30°C than at 20°C; the maximum mean value was 0.48 ± 0.02 (n= 7) at 25°C. It was concluded that maximum mechanical efficiency of isolated mouse muscle was little affected by temperature between 20 and 30°C and that it is unlikely that differences in temperature account for the relatively high efficiency of human muscle in vivo compared to isolated mammalian muscles. [source] The kinetics of competitive antagonism of nicotinic acetylcholine receptors at physiological temperatureTHE JOURNAL OF PHYSIOLOGY, Issue 4 2008Deeptankar Demazumder Detailed information about the ligand-binding site of nicotinic acetylcholine receptors has emerged from structural and mutagenesis experiments. However, these approaches provide only static images of ligand,receptor interactions. Kinetic measurements of changes in protein function are needed to develop a more dynamic picture. Previously, we measured association and dissociation rate constants for competitive inhibition of current through embryonic muscle acetylcholine receptor channels at 25°C. Little is known about competitive antagonism at physiological temperatures. Here, we performed measurements at 37°C and used thermodynamics to estimate the energetics of antagonism. We used rapid solution exchange protocols to determine equilibrium and kinetics of inhibition of acetylcholine-activated currents in outside-out patches by (+)-tubocurarine, pancuronium and cisatracurium. Kinetic rates as high as 600 s,1 were resolved by this technique. Binding was primarily enthalpy driven. The 12°C increase in temperature decreased equilibrium antagonist binding by 1.7- to 1.9-fold. In contrast, association and dissociation rate constants increased 1.9- to 6.0-fold. Activation energies for dissociation were 90 ± 6, 106 ± 8 and 116 ± 10 kJ mol,1 for cisatracurium, (+)-tubocurarine and pancuronium, respectively. The corresponding apparent activation energies for association were 38 ± 6, 85 ± 6 and 107 ± 13 kJ mol,1. The higher activation energy for association of (+)-tubocurarine and pancuronium compared with cisatracurium is notable. This may arise from either a more superficial binding site for the large antagonist cisatracurium compared to the other ligands, or from a change in receptor conformation upon binding of (+)-tubocurarine and pancuronium but not cisatracurium. Differences in ligand desolvation and ligand conformation are not likely to be important. [source] Influence of ionic strength on the time course of force development and phosphate release by dogfish muscle fibresTHE JOURNAL OF PHYSIOLOGY, Issue 3 2005Timothy G. West We measured the effects of ionic strength (IS), 200 (standard) and 400 mmol l,1 (high), on force and ATP hydrolysis during isometric contractions of permeabilized white fibres from dogfish myotomal muscle at their physiological temperature, 12°C. One goal was to test the validity of our kinetic scheme that accounts for energy release, work production and ATP hydrolysis. Fibres were activated by flash photolysis of the P3 -1-(2 nitrophenyl) ethyl ester of ATP (NPE-caged ATP), and time-resolved phosphate (Pi) release was detected with the fluorescent protein MDCC-PBP, N -(2[1-maleimidyl]ethyl)-7-diethylamino-coumarin-3-carboxamide phosphate binding protein. High IS slowed the transition from rest to contraction, but as the fibres approached the isometric force plateau they showed little IS sensitivity. By 0.5 s of contraction, the force and the rate of Pi release at standard and high IS values were not significantly different. A five-step reaction mechanism was used to account for the observed time courses of force and Pi release in all conditions explored here. Only the rate constants for reactions of ATP, ADP and Pi with the contractile proteins varied with IS, thus suggesting that the actin,myosin interactions are largely non-ionic. Our reaction scheme also fits previous results for intact fibres. [source] The Rewarming Index Formula for Pediatric Cardiopulmonary PerfusionARTIFICIAL ORGANS, Issue 11 2009Hideshi Itoh Abstract Careful rewarming of perfusion blood following cardiopulmonary bypass surgery is critical to a successful outcome, but the optimal rewarming strategy is not clear. The purpose of this study was to derive a formula for a rewarming index (defined as [rewarming time × perfusion flow]/[body weight × body surface area]) that would enable the calculation of the ideal rewarming conditions for pediatric cardiopulmonary perfusion. We retrospectively investigated 220 pediatric cardiopulmonary bypass operations conducted from July 2005 to June 2008 in Okayama University Hospital, Japan. We determined the formula as , = (T × Q)/(R × S) = |0.9127P , 0.0152|, where , = rewarming index, T = rewarming time (min), Q = perfusion volume (L), R = body weight (kg), S = body surface area (m2), and P = temperature gap (). The formula will help those who perform pediatric cardiopulmonary bypass surgery to establish ideal perfusion flow conditions and to control physiological temperature during rewarming. [source] Antithrombogenic Polynitrosated Polyester/Poly(methyl methacrylate) Blend for the Coating of Blood-Contacting SurfacesARTIFICIAL ORGANS, Issue 4 2008Amedea B. Seabra Abstract:, A nitric oxide (NO) donor polyester containing multiple S-nitrosothiol (S-NO) groups covalently attached to the polymer backbone was synthesized through the esterification of poly(ethylene glycol) with mercaptosuccinic acid, followed by the nitrosation of the ,SH moieties. The polynitrosated polyester (PNPE) obtained was blended with poly(methyl methacrylate) (PMMA), yielding solid films capable of releasing NO. Scanning electron microscopy analysis showed that acrylic plates and stainless steel intracoronary stents can be coated with continuous and adherent PNPE/PMMA films. After an initial NO burst, these films release NO spontaneously in dry condition or immersed in aqueous solution at constant rates of 1.8 and 180 nmol/g/h, respectively, for more than 24 h at physiological temperature. PNPE/PMMA coated surfaces were shown to inhibit platelet adhesion when in contact with whole blood. These results show that PNPE/PMMA blend can be used for the coating of blood-contacting surfaces, with potential to inhibit thrombosis and restenosis after stenting. [source] Surface Physiochemistry Affects Protein Adsorption to Stoichiometric and Silicate-Substituted Microporous Hydroxyapatites,ADVANCED ENGINEERING MATERIALS, Issue 4 2010Katharina Guth An important factor in the bioactivity and success of a bone-graft substitute is the nature of the adsorbed protein layer, which plays a vital role in orchestrating cell attachment and development through the presence of adhesion proteins such as fibronectin (Fn) and vitronectin (Vn). In this study, microporous hydroxyapatite (HA) and silicate-substituted hydroxyapatite (SA) discs with matched porosity and surface morphology are developed to mimic the topography found in commercial bone-graft substitutes in order to identify whether the introduction of microporosity and associated surface roughness eliminates the beneficial effect that silicate substitution has on protein adsorption. The introduction of microporosity does not abolish the relative enrichment of the protein layer that is adsorbed to the microporous SA discs, as opposed to HA, but appears to accelerate it. Fibronectin and Vn adsorption in a range of competitive environments at physiological temperatures confirm that the microporous SA discs have a greater affinity for Fn and Vn compared with HA, suggesting differences in the mechanisms behind the surface affinity to SA. Thus, development of a surface protein layer on SA and HA is likely to be dependent on the nature of the local protein environment and a combination of factors that are associated with the addition of silicate: the surface charge, the nature of the ionic species at the interface and the resultant hydrophilicity of the surface. Total protein adsorption is not found to be a good indicator of potential implant performance, particularly at early time points. [source] Large-scale expression and thermodynamic characterization of a glutamate receptor agonist-binding domainFEBS JOURNAL, Issue 13 2000Dean R. Madden The ionotropic glutamate receptors (GluR) are the primary mediators of excitatory synaptic transmission in the brain. GluR agonist binding has been localized to an extracellular domain whose core is homologous to the bacterial periplasmic binding proteins (PBP). We have established routine, baculovirus-mediated expression of a complete ligand-binding domain construct at the 10-L scale, yielding 10,40 milligrams of purified protein. This construct contains peptides that lie outside the PBP-homologous core and that connect the domain core to the transmembrane domains of the channel and to the N-terminal ,X'-domain. These linker peptides have been implicated in modulating channel physiology. Such extended constructs have proven difficult to express in bacteria, but the protein described here is stable and monomeric. Isothermal titration calorimetry reveals that glutamate binding to the domain involves a substantial heat capacity change and that at physiological temperatures, the reaction is both entropically and enthalpically favorable. [source] Luminescent Europium(III) Nanoparticles for Sensing and Imaging of Temperature in the Physiological RangeADVANCED MATERIALS, Issue 6 2010Hongshang Peng Europium(III) Nanoparticles are fabricated for sensing and imaging of physiological temperatures (see image). The material shows visible-light excitation, line-like emission, inertness to external perturbers (such as oxygen in air), and a dynamic range that covers temperatures encountered in medicine and (cellular) biology. The resolution is ±0.3,°C. The nanoparticles may also be incorporated into a (conceivably sprayable) sensor film. [source] Stabilized Copper(I) Oxide Nanoparticles Catalyze Azide-Alkyne Click Reactions in WaterADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 10 2010Zhenfang Zhang Abstract A novel form of polyvinylpyrrolidone (PVP) coated copper(I) oxide nanoparticle (Cu2O-NP) was prepared and used to catalyze azide-alkyne click reactions in water under aerobic conditions. The nanoparticles were well dispersed in aqueous solutions and have a size of 20±10,nm, as determined by transmission electron microscope (TEM). Inductively coupled plasma (ICP), X-ray powder diffraction (XRD), and X-ray photoelectron spectroscopy (XPS) analyses demonstrated that the main content of Cu2O-NP is copper(I). The cytotoxicity of it was evaluated by an in vitro 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and its catalytic efficiency for azide-alkyne click reactions was studied in water and organic solvents at physiological temperatures. Our results indicate that Cu2O-NP is more efficient in catalytic reactions in water for both aliphatic and aromatic azides and alkynes and less toxic than the commonly used CuSO4/reductant catalyst systems. [source] Anthrax vaccine powder formulations for nasal mucosal deliveryJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 1 2006Ge Jiang Abstract Anthrax remains a serious threat worldwide as a bioterror agent. A second-generation anthrax vaccine currently under clinical evaluation consists of a recombinant Protective Antigen (rPA) of Bacillus anthracis. We have previously demonstrated that complete protection against inhalational anthrax can be achieved in a rabbit model, by intranasal delivery of a powder rPA formulation. Here we describe the preformulation and formulation development of such powder formulations. The physical stability of rPA was studied in solution as a function of pH and temperature using circular dichroism (CD), and UV-visible absorption and fluorescence spectroscopies. Extensive aggregation of rPA was observed at physiological temperatures. An empirical phase diagram, constructed using a combination of CD and fluorescence data, suggests that rPA is most thermally stable within the pH range of 6,8. To identify potential stabilizers, a library of GRAS excipients was screened using an aggregation sensitive turbidity assay, CD, and fluorescence. Based on these stability profiles, spray freeze-dried (SFD) formulations were prepared at pH 7,8 using trehalose as stabilizer and a CpG-containing oligonucleotide adjuvant. SFD formulations displayed substantial improvement in storage stability over liquid formulations. In combination with noninvasive intranasal delivery, such powder formulations may offer an attractive approach for mass biodefense immunization. © 2005 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 95:80,96, 2006 [source] Skeletal tissue engineering using silk biomaterialsJOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 2-3 2008Ana C. MacIntosh Abstract Silks have been proposed as potential scaffold materials for tissue engineering, mainly because of their physical properties. They are stable at physiological temperatures, flexible and resist tensile and compressive forces. Bombyx mori (silkworm) cocoon silk has been used as a suture material for over a century, and has proved to be biocompatible once the immunogenic sericin coating is removed. Spider silks have a similar structure to silkworm silk but do not have a sericin coating. This paper provides a general overview on the use of silk protein in biomaterials, with a focus on skeletal tissue engineering. Copyright © 2008 John Wiley & Sons, Ltd. [source] Maintenance of integrity and function of isolated hepatocytes during extended suspension culture at 25°CLIVER INTERNATIONAL, Issue 3 2003Alan J. Wigg Abstract: Isolated hepatocytes in suspension provide a number of advantages for use in bioartificial liver device, however, poor stability of this cell preparation at physiological temperatures is an apparent barrier preventing their use. We therefore investigated the integrity and differentiated function of isolated rat hepatocytes under conditions of mild hypothermia. Isolated hepatocytes were suspended in a bicarbonate buffered saline medium, supplemented with glucose and bovine serum albumin (BSA), and maintained for 48 h at 25 °C on a rotary shaker under an atmosphere of 95% O2 and 5% CO2. Under these conditions there was no significant decline in cell viability and good preservation of cellular morphology on transmission electron microscopy for at least 24 h. Isolated hepatocytes in suspension at 25 °C were also able to maintain normal Na + and K + ion gradients. The cellular energy status ([ATP], ATP/ADP ratio, cytoplasmic and mitochondrial redox potentials), metabolic function (urea synthesis and ammonia removal), albumin synthesis and phase I and phase II drug detoxification activity of these cells were also maintained for at least 24 h post isolation. These observations demonstrate the robust nature of mildly hypothermic isolated hepatocytes in suspension and encourage further studies re-examining the feasibility of using this cell preparation in bioartificial livers. [source] The effect of elevated dietary cholesterol on pulmonary surfactant function in adolescent micePEDIATRIC PULMONOLOGY, Issue 5 2008K.C. McCrae PhD Abstract It has been established that phospholipids and cholesterol interact in films of pulmonary surfactant (PS). Generally it is thought that phospholipids increase film stability whereas cholesterol increases film fluidity. To study this further, we modified dietary cholesterol in mice which received either standard rodent lacking cholesterol (sd), or high cholesterol (2%) diet (hc) for 1 month. Phospholipid stability was investigated by a capillary surfactometer (CS), which measures airflow resistance and patency. PS was collected by bronchiolar lavage and centrifuged to obtain the surface-active film (SAF). Results showed that the hc-SAF had significantly more cholesterol than sd-SAF. CS analyses at 37°C showed no significance differences in airflow resistance between hc-SAF and sd-SAF. However, at 37°C, sd-SAF showed greater ability to maintain patency compared to hc-SAF, whereas at 42°C hc-SAF showed patency ability similar to sd-SAF. The results suggested that increased cholesterol in hc-SAF induced less stability in the SAF possibly due to cholesterol's fluidizing effect on phospholipids at physiological temperatures. Pediatr Pulmonol. 2008; 43:426,434. © 2008 Wiley-Liss, Inc. [source] Variations in Photosystem I Properties in the Primordial Cyanobacterium Gloeobacter violaceus PCC 7421PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 1 2010Mamoru Mimuro We compared the optical properties of the trimeric photosystem (PS) I complexes of the primordial cyanobacterium Gloeobacter violaceus PCC 7421 with those of Synechocystis sp. PCC 6803. Gloeobacter violaceus PS I showed (1) a shorter difference maximum of P700 by approximately 2 nm, (2) a smaller antenna size by approximately 10 chlorophyll (Chl) a molecules and (3) an absence of Red Chls. The energy transfer kinetics in the antennae at physiological temperatures were very similar between the two species due to the thermal equilibrium within the antenna; however, they differed at 77 K where energy transfer to Red Chls was clearly observed in Synechocystis sp. PCC 6803. Taken together with the lower P700 redox potential in G. violaceus by approximately 60 mV, we discuss differences in the optical properties of the PS I complexes with respect to the amino acid sequences of core proteins and further to evolution of cyanobacteria. [source] Coexistence of Domains with Distinct Order and Polarity in Fluid Bacterial Membranes,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 1 2002Sharon Vanounou ABSTRACT In this study we sought the detection and characterization of bacterial membrane domains. Fluorescence generalized polarization (GP) spectra of laurdan-labeled Escherichia coli and temperature dependencies of both laurdan's GP and fluorescence anisotropy of 1,3-diphenyl-1,3,5-hexatriene (DPH) (rDPH) affirmed that at physiological temperatures, the E. coli membrane is in a liquid-crystalline phase. However, the strong excitation wavelength dependence of rlaurdan at 37°C reflects membrane heterogeneity. Time-resolved fluorescence emission spectra, which display distinct biphasic redshift kinetics, verified the coexistence of two subpopulations of laurdan. In the initial phase, <50 ps, the redshift in the spectral mass center is much faster for laurdan excited at the blue edge (350 nm), whereas at longer time intervals, similar kinetics is observed upon excitation at either blue or red edge (400 nm). Excitation in the blue region selects laurdan molecules presumably located in a lipid domain in which fast intramolecular relaxation and low anisotropy characterize laurdan's emission. In the proteo-lipid domain, laurdan motion and conformation are restricted as exhibited by a slower relaxation rate, higher anisotropy and a lower GP value. Triple-Gaussian decomposition of laurdan emission spectra showed a sharp phase transition in the temperature dependence of individual components when excited in the blue but not in the red region. At least two kinds of domains of distinct polarity and order are suggested to coexist in the liquid-crystalline bacterial membrane: a lipid-enriched and a proteo-lipid domain. In bacteria with chloramphenicol (Cam),inhibited protein synthesis, laurdan showed reduced polarity and restoration of an isoemissive point in the temperature-dependent spectra. These results suggest a decrease in membrane heterogeneity caused by Cam-induced domain dissipation. [source] The kinetics of competitive antagonism of nicotinic acetylcholine receptors at physiological temperatureTHE JOURNAL OF PHYSIOLOGY, Issue 4 2008Deeptankar Demazumder Detailed information about the ligand-binding site of nicotinic acetylcholine receptors has emerged from structural and mutagenesis experiments. However, these approaches provide only static images of ligand,receptor interactions. Kinetic measurements of changes in protein function are needed to develop a more dynamic picture. Previously, we measured association and dissociation rate constants for competitive inhibition of current through embryonic muscle acetylcholine receptor channels at 25°C. Little is known about competitive antagonism at physiological temperatures. Here, we performed measurements at 37°C and used thermodynamics to estimate the energetics of antagonism. We used rapid solution exchange protocols to determine equilibrium and kinetics of inhibition of acetylcholine-activated currents in outside-out patches by (+)-tubocurarine, pancuronium and cisatracurium. Kinetic rates as high as 600 s,1 were resolved by this technique. Binding was primarily enthalpy driven. The 12°C increase in temperature decreased equilibrium antagonist binding by 1.7- to 1.9-fold. In contrast, association and dissociation rate constants increased 1.9- to 6.0-fold. Activation energies for dissociation were 90 ± 6, 106 ± 8 and 116 ± 10 kJ mol,1 for cisatracurium, (+)-tubocurarine and pancuronium, respectively. The corresponding apparent activation energies for association were 38 ± 6, 85 ± 6 and 107 ± 13 kJ mol,1. The higher activation energy for association of (+)-tubocurarine and pancuronium compared with cisatracurium is notable. This may arise from either a more superficial binding site for the large antagonist cisatracurium compared to the other ligands, or from a change in receptor conformation upon binding of (+)-tubocurarine and pancuronium but not cisatracurium. Differences in ligand desolvation and ligand conformation are not likely to be important. [source] Reversible changes in Ca2+ -activation properties of rat skeletal muscle exposed to elevated physiological temperaturesTHE JOURNAL OF PHYSIOLOGY, Issue 3 2002Chris van der Poel Exposure of relaxed rat extensor digitorum longus (EDL; predominantly fast-twitch) muscle to temperatures in the upper physiological range for mammalian skeletal muscle (43-46 °C) led to reversible alterations of the contractile activation properties. These properties were studied using the mechanically skinned fibre preparation activated in Ca2+ -buffered solutions. The maximum Ca2+ -activated force (maximum force per cross-sectional area) and the steepness of force-pCa (-log10[Ca2+]) curves as measured by the Hill coefficient (nH) reversibly decreased by factors of 8 and 2.5, respectively, when the EDL muscle was treated at 43 °C for 30 min and 5 and 2.8, respectively, with treatment at 46 °C for 5 min. Treatment at 47 °C for 5 min produced an even more marked depression in maximum specific force, which fully recovered after treatment, and in the Hill coefficient, which did not recover after treatment. After all temperature treatments there was no change in the level of [Ca2+] at which 50 % maximum force was generated. The temperature-induced depression in force production and steepness of the force-pCa curves were shown to be associated with superoxide (O2,) production in muscle (apparent rate of O2, production at room temperature, 0.055 ± 0.008 nmol min,1 (g wet weight),1; and following treatment to 46 °C for 5 min, 1.8 ± 0.2 nmol min,1 (g wet weight),1) because 20 mm Tiron, a membrane-permeant O2, scavenger, was able to markedly suppress the net rate of O2, production and prevent any temperature-induced depression of contractile parameters. The temperature-induced depression in force production of the contractile apparatus could be reversed either by allowing the intact muscle to recover for 3-4 h at room temperature or by treatment of the skinned fibre preparation with dithiothreitol (a potent reducing agent) in the relaxing solution. These results demonstrate that mammalian skeletal muscle has the ability to uncouple force production reversibly from the activator Ca2+ as the temperature increases in the upper physiological range through an increase in O2, production. [source] P2X7 receptors in rat parotid acinar cells: formation of large poresAUTONOMIC & AUTACOID PHARMACOLOGY, Issue 4 2001Simon J. Gibbons 1 Permeabilization of cells mediated by P2X7 receptors occurs to varied degrees in native and heterologous expression systems. Previous studies on P2X7 receptors in parotid acinar cells suggested that ATP does not permeabilize these cells. 2 Modification of the assay conditions showed that ATP permeabilizes freshly dissociated rat parotid acinar cells to the fluorescent dye YOPRO-1. 3 The pharmacological and physiological properties of this effect indicate that permeabilization is mediated by the P2X7 receptor. Adenosine 5,-triphosphate (ATP) and 3,- O -(4-benzoyl)benzoyl adenosine 5,-triphosphate (BzBzATP) were effective agonists with EC50 values of 49.3 and 0.6 ,M, respectively. 4 Permeabilization was best observed in low divalent cation concentrations and at physiological temperatures. Previous studies failed to detect permeabilization because of the sensitivity of this effect to temperature and divalent cations. 5 An important consideration in understanding the effect of divalent cations is that the fluorescence of YOPRO-1/nucleic acid complexes is directly quenched by addition of divalent cations. This must be considered if quantitative study of the interaction of divalent cations with P2X7 receptors is carried out using fluorescent DNA-binding dyes. 6 In summary, our data show that P2X7 receptors in parotid acinar cells can form large pores in the plasma membrane. This property likely contributes to signalling and may be cytotoxic and have particular significance in damaged or inflamed salivary glands. [source] | |||||||||||||||||||