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At least 721 records · Page 40Linked to original sources
PubMed1998

pH drift of "physiological buffers" and culture media used for cell incubation during in vitro studies.

In pharmacological or toxicological studies performed at room atmosphere comparison of various media used for cell incubation revealed discrepancies among results due to pH instability when these media contain bicarbonate. With the classically used protocols, a relatively fast and notable rise of the pH of such media has been observed, and values higher than 8.5 could be reached after 1 h of incubation. A less important rise in pH was also observed for media containing low amounts of sodium bicarbonate, e.g., Hank's formula-derived media. Because Hepes-buffered media or media with abnormal osmolarity cannot always be used for such studies, our choice of media is limited.

Bicarbonates↗
PubMed1998

In vitro taurine uptake into cell culture influenced by using media with or without CO2.

Buffers used to incubate cells for pharmacological or toxicological studies are usually of very simple composition, far from the composition of biological fluids or cell culture media. Comparative studies on taurine uptake levels by cultured cells show that a new CO2-Independent Medium (CIM) is suitable for incubating cells in place of the Krebs-Ringer buffer (KR) usually used. Basal uptake level of taurine was lower for cells incubated in CIM or in other culture media when compared to those incubated whether in KR or in other "physiological buffers." Isoproterenol depressed similarly the taurine uptake in cells incubated in CIM or KR. The same uptake modulation by beta-alanine, GES, GABA, or HEPES was observed for cells incubated in CIM or KR. C6 cells growth in CIM was dependent on the starting cell density when classically vented T-flasks were used, growth being notably reduced at low density. In tightly closed flasks cells grew in CIM similarly to control cultures maintained in M199 medium or DMEM.

Adrenergic beta-Agonists↗
PubMed2006

Reagentless, reusable, ultrasensitive electrochemical molecular beacon aptasensor.

A bifunctional derivative of the thrombin-binding aptamer with a redox-active Fc moiety and a thiol group at the termini of the aptamer strand was synthesized. The ferrocene-labeled aptamer thiol was self-assembled through S-Au bonding on a polycrystalline gold electrode surface and the surface was blocked with 2-mercaptoethanol to form a mixed monolayer. By use of a fluorescent molecular beacon, the effect of counterions on quadruplex formation was established. The aptamer-modified electrode was characterized electrochemically by cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS). The modified electrode showed a voltammetric signal due to a one-step redox reaction of the surface-confined ferrocenyl moiety of the aptamer immobilized on the electrode surface in 10 mM N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) buffer of pH 8.0. An increase in the DPV current signal was evident after blocking with 2-mercaptoethanol, effectively removing aptamer nonspecifically absorbed rather than bound to electrode surface or due to the formation of the aptamer-thrombin affinity interaction. The impedance measurement, in agreement with the differential pulse voltammetry (DPV), showed decreased Faradaic resistances in the same sequence. The "signal-on" upon thrombin association could be attributed to a change in conformation from random coil-like configuration on the probe-modified film to the quadruplex structure. The DPV of the modified electrode showed a linear response of the Fc oxidation signal to the increase in the thrombin concentration in the range between 5.0 and 35.0 nM with a linear correlation of r = 0.9988 and a detection limit of 0.5 nM. The molecular beacon aptasensor was amenable to full regeneration by simply unfolding the aptamer in 1.0 M HCl, and could be regenerated 25 times with no loss in electrochemical signal upon subsequent thrombin binding.

Aptamers, Nucleotide↗
PubMed1987

Multiple metabolic pools of phosphoinositides and phosphatidate in human erythrocytes incubated in a medium that permits rapid transmembrane exchange of phosphate.

1. A Hepes-based medium has been devised which allows rapid Pi exchange across the plasma membrane of the human erythrocyte. This allows the metabolically labile phosphate pools of human erythrocytes to come to equilibrium with [32P]Pi in the medium after only 5 h in vitro. 2. After 5-7 h incubation with [32P]Pi in this medium, only three phospholipids, phosphatidic acid (PtdOH), phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P2) are radioactively labelled. The concentrations of PtdIns4P and PtdIns4,5P2 remain constant throughout the incubation, so this labelling process is a reflection of the steady-state turnover of their monoester phosphate groups. 3. During such incubations, the specific radioactivities of the monoesterified phosphates of PtdIns4, PtdIns4,5P2 and PtdOH come to a steady value after 5 h that is only 25-30% of the specific radioactivity of the gamma-phosphate of ATP at that time. We suggest that this is a consequence of metabolic heterogeneity. This heterogeneity is not a result of the heterogeneous age distribution of the erythrocytes in human blood. Thus it appears that there is metabolic compartmentation of these lipids within cells, such that within a time-scale of a few hours only 25-30% of these three lipids are actively metabolized. 4. The phosphoinositidase C of intact human erythrocytes, when activated by Ca2+-ionophore treatment, only hydrolyses 50% of the total PtdIns4,5P2 and 50% of 32P-labelled PtdIns4,5P2 present in the cells: this enzyme does not discriminate between the metabolically active and inactive compartments of lipids in the erythrocyte membrane. Hence at least four metabolic pools of PtdIns4P and PtdIns4,5P2 are distinguishable in the human erythrocyte plasma membrane. 5. The mechanisms by which multiple non-mixing metabolic pools of PtdOH, PtdIns4P and PtdIns4,5P2 are sustained over many hours in the plasma membranes of intact erythrocytes are unknown, although some possible explanations are considered.

2,3-Diphosphoglycerate↗
PubMed1998

Effects of calcium and bicarbonate on head-to-head agglutination in ejaculated boar spermatozoa.

The present study was conducted to reveal the effects of calcium and bicarbonate on the occurrence of head-to-head agglutination in ejaculated boar spermatozoa in vitro. Boar spermatozoa were washed and incubated in a modified Krebs-Ringer bicarbonate (mKRB) in a 37 degrees C CO2 incubator (5% CO2 in air) for 1-5 h. Before and after the incubation, aliquots of each sperm sample were fixed, smeared on glass slides, and stained with a phosphate-buffered solution of Giemsa to assess the percentages of head-to-head agglutinated spermatozoa. Before the incubation, only 5-12% of the spermatozoa were agglutinated. After the 1-h incubation, however, the percentage of head-to-head agglutinated spermatozoa rose to approximately 50%, followed by only minor increases thereafter. This rise was dependent on the concentrations of calcium chloride contained in the mKRB and was attenuated by the addition of 2 mM [ethylenebis(oxyethylenenitrilo)]tetra-acetic acid (EGTA) to the medium. Moreover, the replacement of sodium bicarbonate with 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (Hepes) in the medium and treatment with ruthenium red, which have both been shown previously to inhibit calcium uptake by boar spermatozoa, significantly reduced the rise. Based on these findings, it was concluded that extracellular calcium and bicarbonate are key factors regulating head-to-head agglutination in boar spermatozoa. The possible relationship between agglutinability and the fertilizing ability of boar spermatozoa is also discussed.

Animals↗
PubMed1998

Identification and characterization of a sialidase released by the salivary gland of the hematophagous insect Triatoma infestans.

Sialidases (EC 3.2.1.18) are commonly found in viruses, bacteria, fungi, protozoa, and vertebrates, but not in invertebrates. We have previously reported the presence of a new sialidase activity in the gut of exclusively hematophagous insects of the Triatoma genus, which transmit Chagas' disease (Amino, R., Acosta, A., Morita, O. M., Chioccola, V. L. P., and Schenkman, S. (1995) Glycobiology 5, 625-631). Here we show that this sialidase is present in the salivary gland of Triatoma infestans, and it is released with the saliva during the insect bite. The sialidase was purified to homogeneity (>5000 times) to a specific activity of more than 20 units/mg. It elutes from a gel filtration column with a volume corresponding to the size of 33 kDa, and it migrates as a single 26-kDa band in SDS-polyacrylamide gel electrophoresis, which is unusually smaller when compared with other known sialidases. T. infestans sialidase hydrolyzes preferentially alpha2-->3-linked sialic acids at pH 4-8, with maximal activity between pH 5.5 and 6.5, which is compatible with the optimal pH of secreted sialidases. The sialidase is competitively inhibited by 2-deoxy-2, 3-dehydro-N-acetyl-neuraminic acid (Ki = 0.075 mM) and differently from many sialidases, with exception of Salmonella typhimurium sialidase, it is inhibited competitively by HEPES (Ki = 15 mM). The fact that T. infestans sialidase is released with the saliva and can hydrolyze sialyl-LewisX blood groups, which are the ligands for selectins, suggests that it might have a role in the blood feeding.

Animals↗
PubMed1999

Regulation of connexin channels by pH. Direct action of the protonated form of taurine and other aminosulfonates.

Protonated aminosulfonate compounds directly inhibit connexin channel activity. This was demonstrated by pH-dependent connexin channel activity in Good's pH buffers (MES (4-morpholineethanesulfonic acid)), HEPES, and TAPS (3-({[2-hydroxy-1, 1-bis(hydroxymethyl)ethyl]amino]-1-propanesulfonic acid)) that have an aminosulfonate moiety in common and by the absence of pH-dependent channel activity in pH buffers without an aminosulfonate moiety (maleate, Tris, and bicarbonate). The pH-activity relation was shifted according to the pKa of each aminosulfonate pH buffer. At constant pH, increased aminosulfonate concentration inhibited channel activity. Taurine, a ubiquitous cytoplasmic aminosulfonic acid, had the same effect at physiological concentrations. These data raise the possibility that effects on connexin channel activity previously attributed to protonation of connexin may be mediated instead by protonation of cytoplasmic regulators, such as taurine. Modulation by aminosulfonates is specific for heteromeric connexin channels containing connexin-26; it does not occur significantly for homomeric connexin-32 channels. The identification of taurine as a cytoplasmic compound that directly interacts with and modulates connexin channel activity is likely to facilitate understanding of cellular modulation of connexin channels and lead to the development of reagents for use in structure-function studies of connexin protein.

Animals↗
PubMed2004

Impaired regulatory volume decrease in freshly isolated cholangiocytes from cystic fibrosis mice: implications for cystic fibrosis transmembrane conductance regulator effect on potassium conductance.

Various K(+) and Cl(-) channels are important in cell volume regulation and biliary secretion, but the specific role of cystic fibrosis transmembrane conductance regulator in cholangiocyte cell volume regulation is not known. The goal of this research was to study regulatory volume decrease (RVD) in bile duct cell clusters (BDCCs) from normal and cystic fibrosis (CF) mouse livers. Mouse BDCCs without an enclosed lumen were prepared as described (Cho, W. K. (2002) Am. J. Physiol. 283, G1320-G1327). The isotonic solution consisted of HEPES buffer with 40% of the NaCl replaced with isomolar amounts of sucrose, whereas hypotonic solution was the same as isotonic solution without sucrose. The cell volume changes were indirectly assessed by measuring cross-sectional area (CSA) changes of the BDCCs using quantitative videomicroscopy. Exposure to hypotonic solutions increased relative CSAs of normal BDCCs to 1.20 +/- 0.01 (mean +/- S.E., n = 50) in 10 min, followed by RVD to 1.07 +/- 0.01 by 40 min. Hypotonic challenge in CF mouse BDCCs also increased relative CSA to 1.20 +/- 0.01 (n = 53) in 10 min but without significant recovery. Coadministration of the K(+)-selective ionophore valinomycin restored RVD in CF mouse BDCCs, suggesting that the impaired RVD was likely from a defect in K(+) conductance. Moreover, this valinomycin-induced RVD in CF mice was inhibited by 5-nitro-2'-(3-phenylpropylamino)-benzoate, indicating that it is not from nonspecific effects. Neither cAMP nor calcium agonists could reverse the impaired RVD seen in CF cholangiocytes. Our conclusion is that CF mouse cholangiocytes have defective RVD from an impaired K(+) efflux pathway, which could not be reversed by cAMP nor calcium agonists.

1-Methyl-3-isobutylxanthine↗
PubMed2005

Functional interaction of carbonic anhydrase and chloride/bicarbonate exchange in human platelets.

Recently, our laboratory has reported the presence of one acidifying Cl-/HC exchange mechanism in human platelets. This paper demonstrates that this exchanger decreases its activity after inhibition of carbonic anhydrase. BCECF-loaded platelets, previously equilibrated in a bicarbonate/CO2 buffered solution, were resuspended in a Hepes-buffered, chloride-free (glucuronate) medium to produce a pHi increase. After addition of 50 mM NaCl, pHi fell rapidly reaching steady state in the succeeding 400 s. The recovery in chloride-containing solution was in contrast to the effect of a similar change in osmolarity by addition of 50 mM sodium glucuronate that produced a significantly slower variation of pHi. Alkali loads produced by 25 mM TMA were also counteracted by HC equivalent efflux via Cl-/HC exchange. The present study shows that the efflux of HC was slower when the platelets were previously incubated in 100 microM methazolamide. As a conclusion, the recovery of pHi from alkalosis by Na-independent Cl-/HC exchange is facilitated in platelets by the enzymatic activity of the carbonic anhydrase.

Bicarbonates↗
PubMed2000

Superoxide, amine buffers and tetranitromethane: a novel free radical chain reaction.

The amine buffer Tris slowly reduces tetranitromethane (TNM) to the nitroform anion in a non-accelerating reaction. The amine buffers HEPES and MOPS also (slowly) react with TNM but the dialkylaminoalkyl radicals formed from these two buffers undergo further reactions resulting in a rapid, accelerating, free radical chain process whereby the amine is oxidized and TNM reduced. The chemical functionality in any reaction component, not necessarily the buffer, required for this radical chain mechanism is >N-CH<. In the presence of such groups, the quantification of superoxide by TNM is impossible.

Amines↗
PubMed2000

Pore block versus intrinsic gating in the mechanism of inward rectification in strongly rectifying IRK1 channels.

The IRK1 channel is inhibited by intracellular cations such as Mg(2+) and polyamines in a voltage-dependent manner, which renders its I-V curve strongly inwardly rectifying. However, even in excised patches exhaustively perfused with a commonly used artificial intracellular solution nominally free of Mg(2+) and polyamines, the macroscopic I-V curve of the channels displays modest rectification. This observation forms the basis of a hypothesis, alternative to the pore-blocking hypothesis, that inward rectification reflects the enhancement of intrinsic channel gating by intracellular cations. We find, however, that residual rectification is caused primarily by the commonly used pH buffer HEPES and/or some accompanying impurity. Therefore, inward rectification in the strong rectifier IRK1, as in the weak rectifier ROMK1, can be accounted for by voltage-dependent block of its ion conduction pore by intracellular cations.

Animals↗
PubMed2002

IRK1 inward rectifier K(+) channels exhibit no intrinsic rectification.

In intact cells the depolarization-induced outward IRK1 currents undergo profound relaxation so that the steady-state macroscopic I-V curve exhibits strong inward rectification. A modest degree of rectification persists after the membrane patches were perfused with artificial solutions devoid of Mg(2+) and polyamines, which has been interpreted as a reflection of intrinsic channel gating and led to the view that inward rectification results from enhancement of the intrinsic gating by intracellular cations rather than simple pore block. Furthermore, IRK1 exhibits significant extracellular K(+)-sensitive relaxation of its inward current, a feature that has been likened to the C-type inactivation observed in the voltage-activated Shaker K(+) channels. We found that both these current relaxations can be accounted for by impurities in some common constituents of recording solutions, such as residual hydroxyethylpiperazine in HEPES and ethylenediamine in EDTA. Therefore, inherently, IRK1 channels are essentially ohmic at the macroscopic level, and the voltage jump-induced current relaxations do not reflect IRK1 gating but the unusually high affinity of its pore for cations. Furthermore, our study helps define the optimal experimental conditions for studying IRK1.

Animals↗
PubMed1990

Voltage-dependent block of anthrax toxin channels in planar phospholipid bilayer membranes by symmetric tetraalkylammonium ions. Effects on macroscopic conductance.

In a recent paper (Blaustein, R. O., T. M. Koehler, R. J. Collier, and A. Finkelstein, 1989. Proc. Natl. Acad. Sci. USA. 86:2209-2213) we described the general channel-forming properties of the PA65 fragment of anthrax toxin in planar phospholipid bilayer membranes. In the present paper we extend our previous studies of the permeability properties of this channel, using a series of symmetric tetraalkylammonium (TAA) ions. Our main finding is that at micromolar concentrations on either the cis (toxin-containing) or trans side of a membrane containing many (greater than 1,000) channels, these ions, ranging in size from tetramethylammonium to tetrahexylammonium, induce a voltage-dependent reduction of membrane conductance. (We attribute a similar voltage-dependent reduction of membrane conductance by millimolar concentrations of HEPES to a cationic form of this buffer present at micromolar concentrations.) In going from large negative to large positive voltages (on the TAA side) one sees that the conductance first decreases from its value in the absence of TAA, reaches a minimum, and then rises back at larger positive voltages toward the level in the absence of TAA. Our interpretation of this behavior is that these symmetric TAA ions block the cation-selective PA65 channel in a voltage-dependent manner. We postulate that there is a single site within the channel to which TAA ions can bind and thereby block the passage of the major current-carrying ion (potassium). A blocking ion is driven into the site by modest positive voltages, but is driven off the site and through the channel by larger positive voltages, thus explaining the relief of block. (In the accompanying paper [Blaustein, R. O., E. J. A. Lea, and A. Finkelstein. 1990. J. Gen. Physiol. 96:921-942] we confirm this interpretation of the data by analysis at the single-channel level.) This means that these blocking ions can pass through the channel; the permeability to tetrahexylammonium, the largest ion studied, implies that the narrowest part of the channel has a diameter of at least 11 A.

Antigens, Bacterial↗
PubMed2002

Sperm binding to epithelial oviduct explants in bulls with different nonreturn rates investigated with a new in vitro model.

A new in vitro method was developed for analyzing the capacity of sperm to bind to oviductal epithelium to determine whether this binding capacity could be used to predict nonreturn rates (NRR). Sperm binding was evaluated by counting 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1)-labeled spermatozoa attached to oviductal epithelium and by measuring the surface area of the oviduct explants by means of an image analysis program. Hepes + Tyrode albumin lactate pyruvate (TALP) was a more useful medium than in vitro fertilization (IVF)-TALP, TCM-199 medium + 10% fetal calf serum, and TCM-199 medium alone for the investigation of sperm binding to oviductal explants. Oviduct explants with a surface area of < 20 000 micro m(2) provided more consistent results than did explants with a surface area of >100 000 micro m(2). A positive association was found between the log(e) transformed number of spermatozoa bound to 0.1 mm(2) oviductal epithelium and the NRR of the respective sires after 24 h of coincubation, provided that the membrane integrity of the sperm sample was >60%. Determination of the capacity of sperm to bind to oviductal explants could become a reliable in vitro method for predicting the NRR of a given sire.

Animals↗
PubMed1977

Secretory capacity and ultrastructure of rat pancreatic islets after preservation of pancreas in different conditions.

Experiments were performed with rat pancreas to investigate optimum conditions for obtaining and preserving the pancreas for subsequent isolation of islets that were viable by both functional and morphological criteria. After only 30 min of warm ischaemia, the yield and viability of islets that could be isolated were poor. However, if the pancreas was removed and placed immediately in a cold bicarbonate-buffered medium, which was supplemented with HEPES to maintain the pH and Trasylol to inhibit proteolytic activity, then viable islets could be isolated consistently after over 8 hr of storage. These results demonstrate that even short periods of warm ischaemia will render the pancreas unsuitable for islet isolation. Once the pancreas is placed in a suitable cold medium, however, considerable delay may be permissible without adversely affecting the viability of the islets which can be obtained. The same conditions might prove to be applicable when human pancreas is obtained for the purpose of transplantation into diabetic subjects.

Animals↗
PubMed1978

Some characteristics of Ureaplasma urealyticum. Urease activity in a simple buffer: effect of metal ions and sulphydryl inhibitors.

Urealytic activity of the cytoplasmic fraction of Ureaplasma urealyticum prepared by digitonin lysis was assayed in a simple buffer system (HEPES plus EDTA) by measuring the release of 14CO2 from [14C]urea. The Km of this preparation agreed with our previous observations of the same activity measured in a more complex reaction mixture. The substrate concentration at which maximum velocity occurred was approximately 20 mM. The activity was sensitive to heavy metals and inhibitors which react with sulphydryl groups such as N-ethylmaleimide and p-chloromercuribenzoate. It was not inhibited by Ca2+ or Mg2+ or by the reaction products, ammonia and carbon dioxide.

Culture Media↗
PubMed2003

Crystallization and preliminary crystallographic analysis of DJ-1, a protein associated with male fertility and parkinsonism.

DJ-1 was identified as a novel oncogene product that transformed mouse NIH3T3 cells in cooperation with activated Ras. DJ-1 was also correlated with male infertility and parkinsonism. DJ-1 was crystallized using sodium citrate and HEPES at pH 7.5. The crystal belongs to space group P3(1) or P3(2), with unit-cell parameters a = 75.04, c = 74.88 A and contains two molecules in an asymmetric unit. An intensity data set was collected to 2.00 A resolution.

Buffers↗
PubMed2005

Structure of myelin P2 protein from equine spinal cord.

Equine P2 protein has been isolated from horse spinal cord and its structure determined to 2.1 A. Since equine myelin is a viable alternative to bovine tissue for large-scale preparations, characterization of the proteins from equine spinal cord myelin has been initiated. There is an unusually high amount of P2 protein in equine CNS myelin compared with other species. The structure was determined by molecular replacement and subsequently refined to an R value of 0.187 (Rfree=0.233). The structure contains a molecule of the detergent LDAO and HEPES buffer in the binding cavity and is otherwise analogous to other cellular retinol-binding proteins.

Amino Acid Sequence↗