Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “HEPES”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 397 records · Page 22Linked to original sources
PubMedDate not supplied

Validation of analytical capillary electrophoresis methods for use in a regulated environment.

Since its introduction into the analytical laboratory, CE has had to prove that it was capable of generating results comparable to HPLC or GC techniques in the six areas (specificity, precision, accuracy, linearity, ruggedness, and range) typically required of validated methods intended for submission to governmental agencies. This paper will showcase the development and validation of two analytical methods: one for specific identification of HEPES (N-[2-hydroxyethyl]piperazine-N'-[2-ethane sulfonic acid]) and the other to quantitate millimolar concentrations of tris (tris[hydroxymethyl]aminomethane) in high electrolyte solutions. Utilizing neutral markers and internal standards, results for HEPES demonstrate that migration time reproducibility, expressed as %RSD of 2% or less on a variety of capillaries, is obtainable. Additionally, for quantitation of tris, the values obtained for accuracy (%relative mean bias), precision (%RSD), and linearity (r2) over multiple days and capillaries meet the rigorous standards we require of HPLC or GC methods.

Electrophoresis, Capillary↗
PubMed1997

Chiral assay of atenolol present in microdialysis and plasma samples of rats using chiral CBH as stationary phase.

Two different enantioselective chiral chromatographic methods were developed and validated to investigate the disposition of the beta 1-receptor antagonist atenolol in blood and in brain extracellular fluid of rats (tissue dialysates). System A for the plasma samples was a one-column chromatographic system with a chiral CBH column with an aqueous buffer as mobile phase into which cellobiose was added for selective regulation of the retention of the internal standard, (S)-metoprolol. The plasma samples were analysed after a simple extraction procedure. The limit of quantitation was 0.2 micrograms/ml for the atenolol enantiomers. The repeatability of the medium concentration quality control plasma sample (6.0 micrograms rac-atenolol/ml) was 11-18% for the enantiomers. The dynamic linear range of the plasma samples was 0.5-20 micrograms/ml. For system B, since atenolol is an extremely hydrophilic drug, the tissue dialysate sample required a much more sensitive system as compared to the plasma samples. A coupled column system was used for peak compression of the enantiomers in the eluate after the separation on the Chiral CBH column, hence increasing the detection sensitivity. The limit of quantification was 0.045 micrograms/ml for the atenolol enantiomers in artificial CSF. The repeatability of the medium concentration quality control samples (0.1 and 4.0 micrograms rac-atenolol/ml in artificial CSF and Hepes Ringer, respectively) was 2.8-9.3% for the two enantiomers. The dynamic linear range of the brain samples was 0.05-1.0 and 0.5-20 micrograms/ml in artificial CSF and Hepes Ringer, respectively.

Adrenergic beta-Antagonists↗
PubMed2000

In vitro and in vivo studies for modified ethyl cyanoacrylate regimens for sclerotherapy.

Cyanoacrylates have known for their ability to polymerize rapidly in the presence of traces of weakly basic moieties such as water. The tissue adhesive, Histoacryl(R) (N-butyl 2-cyanoacrylate), has been reported to control bleeding through endoscopic sclerotherapy. But the commercially available Histoacryl(R) is expensive, and it has the problem like other cyanoacrylates that the glue tends to flow/run away from the point of application, which is inherent to the low viscosity, making precise application difficult. In this study, ethyl cyanoacrylate (ECA), the main constituent of "super glue," was employed instead of Histoacryl(R) due to its lower cost. The aim of the research is to modify the compositions of ECA regimen and evaluate its feasibility for sclerosant application through both in vitro flow circuit model and in vivo animal tests. It was noted that the difference in the relative hardening rate between the in vitro Hepes-Tyrodes buffer flowing model and the in vivo rat model for the ECA and Histoacryl(R) was related to the existence of the blood protein, such as albumin, in the physiological milieu. It was also noticed that the ECA setting rate was greatly increased either in Hepes-Tyrodes buffer or in blood (to a comparable rate as Histoacryl(R) in vivo) by adding a few doses of caffeine, which acts as a polymerization initiator. This would lead to far better injection precision during sclerotherapy. Furthermore, in vivo histological examination for the occluded lumen of the rat's inferior vena cava and a clinical piglet portal vein occlusion experiment have suggested this new sclerosant regimen, caffeine/ECA, is of great promise in endoscopic sclerotherapy.

Animals↗
PubMedDate not supplied

In vivo gene delivery to the lung using polyethylenimine and fractured polyamidoamine dendrimers.

BACKGROUND: Gene transfer into the airways could be of importance for the treatment of chronic lung diseases such as cystic fibrosis. In the past few years several attempts have been made to effectively deliver DNA to the lung using different viral and non-viral vector systems. Viral vectors and cationic lipids have been tested intensively but the properties of cationic polymers such as polyethylenimine (PEI) 25 kDa and fractured polyamidoamine dendrimers to deliver DNA to the airways have not been studied. Surfactant preparations have been shown to influence pulmonary adenoviral and naked plasmid DNA mediated gene transfer in vivo. We investigated the gene delivery efficiency of branched PEI 25 kDa and fractured dendrimers to the murine lung in vivo and also examined the effect of surfactant on PEI 25 kDa mediated gene transfer to the lung. METHODS: Cationic polymer/DNA complexes were prepared in 25 mM HEPES buffer (pH = 7.4) or double distilled water and administered to the lungs of BALB/c mice via cannula intubation. The trachea, left and right lung, heart, liver and esophagus were examined for luciferase activity. Inflammation was assessed by performing standard histology. RESULTS: PEI/DNA complexes showed a high level of luciferase gene expression in the lung. Complexes formed in double distilled water exhibited higher gene expression than complexes formed in 25 mM HEPES buffer (pH 7.4). The optimal N/P ratio was found to be N/P = 10 in double distilled water. Luciferase activity was only detected in the lung and decreased rapidly in a time-dependent manner. The addition of a natural surfactant preparation, Alveofact, slightly reduced gene transfer of branched PEI 25 kDa. Luciferase gene expression obtained by using fractured dendrimers was very low. CONCLUSION: The present study demonstrates that PEI 25 kDa, but not polyamidoamine dendrimers, effectively mediates transient gene transfer to the murine lung after intratracheal intubation. In conclusion, branched PEI 25 kDa was found to be an effective vector for pulmonary gene delivery in vivo, being superior to fractured dendrimers.

Animals↗
PubMed2003

Fabrication of nickel and chromium nanoparticles using the protein cage of apoferritin.

The iron storage protein, apoferritin, has a cavity in which iron is oxidized and stored as a hydrated oxide core. The size of the core is about 7 nm in diameter and is regulated by the cavity size. The cavity can be utilized as a nanoreactor to grow inorganic crystals. We incubated apoferritin in nickel or chromium salt solutions to fabricate hydroxide nanoparticles in the cavity. By using a solution containing dissolved carbon dioxide and by precisely controlling the pH, we succeeded in fabricating nickel and chromium cores. During the hydroxylation process of nickel ions a large portion of the apoferritin precipitated through bulk precipitation of nickel hydroxide. Bulk precipitation was suppressed by adding ammonium ions. However, even in the presence of ammonium ions the core did not form using a degassed solution. We concluded that carbonate ions were indispensable for core formation and that the ammonium ions prevented precipitation in the bulk solution. The optimized condition for nickel core formation was 0.3 mg/mL horse spleen apoferritin and 5 mM ammonium nickel sulfate in water containing dissolved carbon dioxide. The pH was maintained at 8.65 using two buffer solutions: 150 mM HEPES (pH 7.5) and 195 mM CAPSO (pH 9.5) with 20 mM ammonium at 23 degrees C. The pH had not changed after 48 h. After 24 h of incubation, all apoferritins remained in the supernatant and all of them had cores. Recombinant L-ferritin showed less precipitation even above a pH of 8.65. A chromium core was formed under the following conditions: 0.1 mg/mL apoferritin, 1 mM ammonium chromium sulfate, 100 mM HEPES (pH 7.5) with a solution containing dissolved carbon dioxide. About 80% of the supernatant apoferritin (0.07 mg/mL) formed a core. In nickel and chromium core formation, carbonate ions would play an important role in accelerating the hydroxylation in the apoferritin cavity compared to the bulk solution outside.

Alkanesulfonic Acids↗
PubMed1988

Mass spectrometry of underivatized 15-hydroxyeicosatetraenoic acid and 15-hydroxyeicosapentaenoic acid.

A direct mass spectrometric method for 15-hydroxy metabolites of arachidonic acid and of eicosapentaenoic acid is described. 15-Hydroxyeicosanoids have the capacity to inhibit the formation of leukotrienes, potent mediators of inflammation formed by the 5-lipoxygenase pathway of arachidonic acid metabolism and may therefore have anti-inflammatory properties. 15-Hydroxyeicosatetraenoic acid (15-HETE) and 15-hydroxyeicosapentaenoic acid (15-HEPE) were synthesized using soybean lipoxygenase and arachidonic acid and eicosapentaenoic acid as substrates. These hydroxy fatty acids were then purified by reversed-phase high-performance liquid chromatography. This modified procedure provides rapid synthesis of multimilligram quantities of 15-hydroxyeicosanoids for in vitro and in vivo studies. Electron impact mass spectra of 15-HETE and 15-HEPE could be obtained directly without derivatizations.

Chromatography, High Pressure Liquid↗
PubMed1994

A physiological measure of carbonic anhydrase in Müller cells.

Carbonic anhydrase activity was characterized in freshly dissociated Müller cells of the salamander retina. Intracellular pH was monitored using ratio imaging of the indicator dye BCECF as extracellular PCO2 was varied. The extracellular solution was switched rapidly (141 ms rise time) from a HEPES buffered to a CO2-HCO3- buffered solution (both pH 7.4). Introduction of CO2-HCO3- produced a rapid cell acidification. Cell pH dropped from a steady-state pH of 7.02 in HEPES solution to pH 6.81 in CO2-HCO3-. Methazolamide, a carbonic anhydrase inhibitor, dramatically reduced the initial rate of acidification, demonstrating that the acidification was produced by the carbonic anhydrase-catalyzed hydration of CO2. The initial rate of acidification, 52.6 pH units per min (0.88 pH units per s), was reduced approximately 150-fold to 0.36 pH units per min by 10(-3) M methazolamide. Half-maximal inhibition occurred at a methazolamide concentration of 5.6.10(-7) M. The carbonic anhydrase inhibitor acetazolamide (10(-3) M) also greatly reduced the rate of cell acidification. The latency to the onset of carbonic anhydrase inhibition was 660 ms for methazolamide and 7.5 s for acetazolamide. The carbonic anhydrase inhibitor benzolamide (10(-4) M, 4 min exposure), which is poorly membrane permeant, had little effect on the rate of cell acidification, indicating that the site of carbonic anhydrase action was intracellular. The activity of Müller cell carbonic anhydrase may help to buffer extracellular CO2 variations in the retina.

Acetazolamide↗
PubMed2005

Eicosanoid generation in the intertidal barnacle, Balanus perforatus--effect of season and reproductive status.

The nature and quantity of the principal lipoxygenase (LO) products generated by the intertidal barnacle Balanus perforatus were examined at monthly intervals and their potential involvement in reproductive events was investigated. The main mono-hydroxy products generated were found to be formed through the action of an 8-lipoxygenase (8-LO) activity and were the mono-hydroxy fatty acids, 8-hydroxyeicosapentaenoic acid (HEPE) and 8-hydroxyeicosatetraenoic acid. Generation of these products was found to be correlated with the environmental seawater temperature, although no change in either 8-LO activity or the precursor fatty acid levels in total phospholipids was found with the time of the year. Changes in fatty acid composition measured in animals collected from summer and winter conditions were found to follow the pattern expected by homeoviscous adaptation of a cold-acclimated animal. Oogenesis was found to occur in August and was linked to a significant reduction in 8-HEPE generation. Spermatozoa were found to be present year-round in the seminal vesicles although the testes became atrophic during the winter months.

Animals↗
PubMed1987

Arachidonic and eicosapentaenoic acid metabolism in bovine neutrophils and platelets: effect of calcium ionophore.

Substitution of dietary fatty acids has potential for altering the inflammatory response. The purpose of the present study was to define the metabolites of arachidonic acid (AA) and eicosapentaenoic acid (EPA) secreted by bovine peripheral blood neutrophils and platelets. High performance liquid chromatography was used to characterize cyclooxygenase and lipoxygenase metabolites secreted in response to the calcium ionophore A23187. Cells were prelabelled with 3H-AA or 3H-EPA prior to challenge with the calcium ionophore. Bovine neutrophils secreted leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) as the major metabolites of AA, as well as the corresponding leukotriene B5 (LTB5) and 5-hydroxyeicosapentaenoic acid (5-HEPE) metabolites of EPA. Peptidoleukotrienes derived from 3H-AA or 3H-EPA were not detected under these conditions. The major tritiated metabolites secreted from bovine platelets were: thromboxane A2, measured as the stable metabolite thromboxane B2 (TXB2); hydroxyheptadecatrienoic acid (HHT) and 12-HETE derived from 3H-AA; and the omega-3 analogs TXB3 and 12-HEPE, derived from 3H-EPA. Preferred substrate specificities existed amongst the AA- and EPA-derived metabolites for the intermediary enzymes involved in the arachidonic acid cascade. These findings support the hypothesis that substitution of membrane-bound AA by EPA has potential for modulation of the host inflammatory response following cellular phospholipid mobilization.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1988

Prevention of calcification of bioprosthetic heart valve leaflets by Ca2+ diphosphonate pretreatment.

Calcification frequently causes failure of bioprosthetic heart valves (BHV) fabricated from glutaraldehyde-pretreated porcine aortic valve or bovine pericardium. Systemic diphosphonate therapy inhibits this disease process, but with adverse effects on overall growth, bone development, and calcium metabolism. The present study was designed to examine the hypothesis that the immobilization of ethanehydroxydiphosphonate (EHDP) within BHV as the poorly soluble Ca2+ salt would inhibit calcification at drug levels insufficient to produce side effects. Glutaraldehyde-pretreated pericardial BHV tissue was exposed to a physiologic concentration of Na2EHDP (0.14 M in 0.05 M HEPES, pH 7.4) and subsequently washed in a NaCl or CaCl2 (0.14 M in 0.05 M HEPES, pH 7.4) solution to precipitate the Na or Ca2+ salts of EHDP on/within the tissue, respectively. Incorporation of CaEHDP into BHV ranged from 74.8 nM/mg (after 1 h, 37 degrees C) to 353 nM/mg (2 weeks, 22 degrees C). None of the Na2EHDP was incorporated into BHV without exposure to CaCl2. In vitro release of CaEHDP from BHV into a physiologic buffer not containing Ca2+ was rapid, with greater than 95% removed after 4 d, while release of CaEHDP into buffer containing a physiologic concentration of Ca2+ ion (1.5 mM) was markedly reduced, with 30% of the precipitated CaEHDP remaining immobilized on or within the tissue matrix following 21 d.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Artificial activation of porcine oocytes matured in vitro.

These studies were undertaken to understand the biological basis of artificially induced activation of meiotic metaphase II oocytes and to develop a source of oocytes as recipients for cloning by nuclear transfer. In vitro matured porcine oocytes were pulsed with various voltages of electricity and evaluated for pronuclear formation. The percentage of eggs that activated was significantly greater for the higher voltages. The effect on activation of the temperature of the ovaries returning from the abattoir was evaluated and it was found that oocytes derived from ovaries returning at 29 degrees C activated at lower rates (45.5%) than those returning at 36 degrees C (78.9%). An experiment was designed to evaluate the pH of electroporation medium (EM) and the duration of exposure to EM on activation. Oocytes were placed in EM at various pHs for 5 minutes, pulsed, and immediately removed to TL-Hepes or allowed an additional 2 minutes in EM prior to rinsing in TL-Hepes. The results indicate an optimum activation rate at a pH of 7.0 and allowing the additional 2 minutes in EM. Additional glucosamine (5 mM) had no affect on development of the oocyte to metaphase but reduced the percent pronuclear formation from 61% and 47%. A final experiment evaluated the developmental competence of oocytes subjected to a optimum combination of the above treatments and illustrated that a significant portion of the activated oocytes can show limited signs of cleavage. Thus in vitro matured pig oocytes can be induced to activate at high rates.

Animals↗
PubMed1995

Chloride-bicarbonate exchange through the human red cell ghost membrane monitored by the fluorescent probe 6-methoxy-N-(3-sulfopropyl)quinolinium.

The exchange of chloride and bicarbonate across the human red cell membrane has been characterized by quenching of an intracellular fluorophore, 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). In 20 mM Hepes, pH 7.4, and varying concentrations of chloride and bicarbonate with total anion concentration of 150 mM, SPQ is quenched dynamically by chloride with an apparent Stern-Volmer quenching constant 0.071 +/- 0.016 mM-1 at 25 degrees C. Hepes alone quenched SPQ fluorescence with a quenching constant of 0.030 +/- 0.003 mM-1. Stopped-flow kinetic experiments give fluorescence time courses that, when corrected for Stern-Volmer quenching, are first order. Disulfonic stilbenes (inhibitors of anion exchange) decrease the rate of fluorescence equilibration. Transport of bicarbonate via hydration-dehydration of CO2 does not contribute to the observed kinetics. The chloride-bicarbonate exchange rate is independent of the anion concentration gradient, but increases at 25 degrees C from 1 to 4 s-1 as equilibrium chloride concentration increases from 20 to 130 mM (with concomitant decrease in bicarbonate concentration from 130 to 20 mM). The data indicate that the translocation rate of the chloride-loaded transport protein is greater than that of bicarbonate-loaded transport protein and that bicarbonate has a higher affinity for the transport protein than chloride. Our results validate the use of SPQ to measure quantitatively chloride-bicarbonate exchange in red cell ghosts. The methods we describe should be applicable to other systems exhibiting chloride-bicarbonate exchange.

Anions↗
PubMed1993

Induction of tolerance to freeze-thaw (FT) damage in mammalian cells by pre-FT hypothermia treatment.

Attached asynchronous exponential phase V79 Chinese hamster cells were pretreated by hypothermic cycling in Hepes growth medium by Method 3 (48 h at 25 degrees C + trypsin) or Method 4 (48 h at 25 degrees C + 3 h at 37 degrees C + trypsin) prior to a freeze-thaw (FT) cycle in Hepes growth medium. Pretreatment by Method 3 or 4 increased the FT survival by a factor of 3.4. This implies that the 3 h at 37 degrees C, after the 25 degrees C exposure, is not necessary to confer resistance to the subsequent FT cycle in the case of V79 cells. However, with RIF-1 mouse cells, the 3 h at 37 degrees C confers increased resistance. The increase in FT survival of V79 cells after the above pretreatments cannot be accounted for by changes in cell cycle age distribution. No heat shock proteins are produced by this pretreatment. Since pretreatment by Method 3 or 4 also makes the cells resistant to hyperthermia, three other pretreatments, making the cells thermotolerant, were tried. None of these pretreatments resulted in a change in FT survival of the cells. Interaction analysis of FT data, when pretreatment by Method 4 is combined with the presence of DMSO during the FT cycle, indicates that the pretreatment and DMSO act synergistically whether exponential or stationary phase cells are used. Furthermore, the pretreatments and L-glutamine also act synergistically. These pretreatments also increase the FT survival of the RIF-1 mouse cell line; again, pretreatment and DMSO act synergistically. Hence, the method is not limited to cells of hibernating mammals.

Animals↗
PubMed1993

Separation of the rabbit ciliary body epithelial layers in viable form: identification of differences in bicarbonate transport.

Sections of whole ciliary (CB) dissected from Dutch belted rabbits were incubated for 2 hr at 36 degrees C in a 30 microM Ca2+ Ringer's. This incubation resulted in the spontaneous dissociation of the two cell layers comprising this epithelium, each remaining firmly cohesive with its own basement membrane. The inner limiting membrane with its adherent non-pigmented epithelium (NPE) was then mechanically removed from the surface exposing the apical surface of the pigmented epithelium (PE). Ultrastructural examination revealed no noxious effects in most cells although gross morphological changes in the NPE cells were noted. The newly separated layers were loaded with the cell-permeable acetoxymethyl ester form of the fluorescent probe BCECF. Most cells of both layers acquired stable BCECF fluorescence indicating viability. To achieve a preliminary evaluation of differences in PE and NPE bicarbonate transport, dye-loaded tissues were perfused in a flow-through chamber which was mounted on a microscope equipped for quantitative epifluorescence. The intracellular pH (pHi) of groups of cells (5-10) was derived from the ratio of emission intensities generated by excitations at 490 and 440 nm. In Hepes-buffered Ringer's the pHis for the PE and NPE were 7.20 +/- 0.10 and 7.33 +/- 0.14 (+/- S.D., n = 6), respectively. Replacement of 28 mM Hepes by 28 mM HCO3-/5% CO2 led to a 0.13 pHi increase in the PE and a decrease of 0.27 U in the NPE. The pHi responses of the two cell layers to removal and/or reintroduction of Na+ and/or Cl- were also highly dissimilar.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Physical Adsorption of Bilayer Membranes on Silica

Isotherms for adsorption of bilayer-forming synthetic amphiphiles or phospholipids from vesicles onto hydrophilic silica particles (Aerosil OX-50) are obtained over a range of experimental conditions. Phosphatidylcholine (PC), dipalmitoylphosphatidylcholine (DPPC), dihexadecylphosphate (DHP), and dioctadecyldimethylammonium bromide (DODAB) dispersed in 10 mM Tris, pH 7.4, as small unilamellar vesicles present affinities for silica following the sequence DODAB > DPPC > PC > DHP. Among these, only DHP adsorption is below that expected for one bilayer deposition. Interaction at 65degreesC for 1 h between DPPC (or, at 25degreesC, for PC) vesicles and silica efficiently leads to bilayer deposition at maximal adsorption, if Tris is the buffer used. Preliminary centrifugation of the PC or DPPC vesicle dispersion is necessary to prevent overestimated adsorption. PC affinity for silica and its deposition as a bilayer depend on the nature of buffer used being much higher for Tris than for Hepes at pH 7.4. Formation of ion pairs between protonated amino groups in Tris and silanol groups on silica may lead to Tris adsorption and an increase in density of -OH groups on the solid surface. Therefore, formation of cooperative hydrogen bridges between -P=O in the phosphatidyl of the phospholipid bilayer and the -OH groups of Tris adsorbed on silica increases PC affinity for silica in the presence of Tris. For Hepes as buffer, PC affinity for silica is much lower and no plateau indicative of bilayer deposition is observed in the adsorption isotherm. Stabilization of supported phospholipid bilayers on solid surfaces requires several cooperative hydrogen bridges between the phospholipid and the solid surface. DODAB adsorption was unaffected by vesicle age and physical state of the bilayer vesicle. Adsorption isotherms for DODAB are of the high-affinity type with a maximum indicative of competition between intervesicle interactions and DODAB deposition on silica. Stabilization of DODAB bilayer deposition requires surface charge densities on silica higher than -1 μC/cm2. Copyright 1997Academic Press

Journal Article↗
PubMed1993

A novel allosteric mechanism in haemoglobin. Structure of bovine deoxyhaemoglobin, absence of specific chloride-binding sites and origin of the chloride-linked Bohr effect in bovine and human haemoglobin.

The structure of bovine deoxyhaemoglobin has been determined at 2.2 A resolution and refined to an R-factor of 0.193 for all 32,583 reflections, and a free R-factor of 0.249 for 1527 reflections excluded from the refinement. The structure shows no significant differences between the alpha-carbon positions of bovine and human haemoglobin, except at the N-terminal segment and the first helix (A) which are closer to the dyad symmetry axis and pushed more tightly against the rest of the beta-subunits in the bovine form. In a search for the predicted chloride-binding sites, three-dimensional data were collected from crystals suspended in 50% polyethylene glycol buffered either with 50 mM Na phosphate (pH 7.3) +/- 0.1 M NaCl or with 0.1 M Hepes (pH 7.3) +/- 0.1 M NaBr. Difference electron density maps with and without NaCl or NaBr showed no evidence of specific halide ion-binding sites. Oxygen equilibria were measured in 10 mM Hepes buffer without added NaCl, with 0.1 mM NaCl, 0.1 M NaCl + 1 mM 2,3-diphosphoglycerate, and 0.1 M NaCl + 1 mM inositol hexaphosphate. Without added chloride, P50 of stripped bovine haemoglobin was similar to that of human haemoglobin with 0.1 M NaCl. With 0.1 M NaCl it was similar to that of human haemoglobin saturated with 2,3-diphosphoglycerate. In 0.1 M NaCl neither organic phosphate significantly affected the oxygen affinity. Titration of P50 with NaCl showed delta log P50/delta log[Cl-] of bovine and human haemoglobin to be identical. Analysis of the oxygen equilibrium curves showed the low intrinsic oxygen affinity of bovine haemoglobin to be due to a larger oxygen dissociation constant from the T-structure. The influence of chloride on P50 and on the alkaline Bohr effect is the same in bovine and human haemoglobins. It is proposed that this is due to the excess positive charges in the central cavity and its widening in the transition from the R to the T-structure. The widening would allow more chloride ions to enter and neutralize the positive charges, but these ions would remain mobile and therefore do not show up as peaks of high electron density. Repulsion between excess positive charges in the central cavity raises the free energy of the T-structure relative to the R-structure, thereby raising the oxygen affinity. Conversely, entry of chloride ions on widening of the cavity reduces the free energy of the T-structure and therefore lowers the oxygen affinity.(ABSTRACT TRUNCATED AT 400 WORDS)

Allosteric Regulation↗
PubMed2000

Augmentation of calcium current by hypoxia in carotid body glomus cells.

Several lines of evidence indicate that transduction of the hypoxic stimulus at the carotid body involves an increase in cytosolic Ca2+ ([Ca2+]i) via activation of voltage-gated Ca2+ channels in the glomus cells. However, reported responses to hypoxia include either no effect on or inhibition of Ca2+ current in glomus cells. The apparent discrepancy between the effects of hypoxia on [Ca2+]i and Ca2+ channel activity prompted us to re-examine the effects of low oxygen on Ca2+ currents in glomus cells. Experiments were performed on freshly dissociated glomus cells from rabbit carotid bodies. Ca2+ channel activity was monitored using the whole-cell configuration of the patch clamp technique with Ba2+ as the charge carrier. Hypoxia (pO2 = 40 mmHg) augmented the Ca2+ current by 24% (at 0 mV). This augmentation was seen in a CO2/HCO3- but not in a HEPES buffered extracellular solution. However, when the extracellular pH (pHo) of a HEPES buffered solution is lowered from 7.4 to 7.0, then the Ca2+ current in glomus cells is augmented by hypoxia by 20%. Nisoldipine, an L-type Ca2+ channel blocker (2 microM), prevented augmentation of the Ca2+ current by hypoxia. On the other hand, an N- and P-type Ca2+ channel blocker (2 microM omega-conotoxin MVIIC) did not prevent the augmentation of the Ca2+ current by hypoxia. Protein kinase C (PKC) inhibitors, staurosporine (100 nM) and bisindolylmaleimide (2 microM), prevented augmentation by hypoxia. Okadaic acid (100 nM), an inhibitor of serine/threonine phosphatases also prevented augmentation of Ca2+ current by hypoxia; whereas, norokadaone, an inactive analog of okadaic acid, had no effect. These results suggest that hypoxia augments Ca2+ current through L-type Ca2+ channels via a PKC and/or phosphatase-sensitive pathways.

Animals↗
PubMed1995

An improved method for in vitro susceptibility testing of Trichomonas vaginalis.

A protocol is presented for in vitro susceptibility testing of Trichomonas vaginalis. A 100 ml culture of the microorganisms is prepared for inoculation into antibiotic dilutions and controls by centrifugation, washing with 10 mM HEPES (pH 6.2) plus 1.5 x 10(-1) M NaCl, a second centrifugation and a resuspension in the HEPES-saline buffer. Inclusion of the gelling agent carrageenan in the culture medium permits an ease of harvesting the trichomonads and a reproducible initial cell density of 1-4 x 10(4) cells per ml. Following inoculation, tubes with antibiotic dilutions and controls are incubated anaerobically at 35 degrees C for 48 h, which corresponds to late exponential phase. Inclusion of a negative control helps determine Minimum Lethal Concentration (MLC) values.

Animals↗