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PubMed1997

Fatty acid composition, eicosanoid production and permeability in skin tissues of rainbow trout (Oncorhynchus mykiss) fed a control or an essential fatty acid deficient diet.

Rainbow trout (Oncorhynchus mykiss) were fed either a control diet containing fish oil or an essential fatty acid (EFA) deficient diet containing only hydrogenated coconut oil and palmitic acid as lipid source (93.4% saturated fatty acids) for 14 weeks and the fatty acid compositions of individual phospholipid classes from skin and opercular membrane (OM) determined. The permeability of skin and OM to water and the production of eicosanoids in skin and gills challenged with the Ca2+ ionophore A23187 were also measured. Phospholipid (PL) fatty acid compositions were substantially modified in EFA-deficient fish, with increased saturated fatty acids and decreased polyunsaturated fatty acids (PUFA), especially arachidonic acid (AA) and eicosapentaenoic acid (EPA), while docosahexaenoic acid (DHA) was largely retained. The onset of EFA deficiency was shown by the appearance of n-9 PUFA, particularly 20:3n-9. The main effects of EFA deficiency on phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were to increase saturated fatty acids and monoenes, especially 16:1 and 18:1, and to decrease EPA and DHA. The content of DHA in phosphatidylserine (PS) was high in control animals (40% in skin and 35% in opercular membrane) and was mostly retained in EFA deficient animals. Arachidonic acid (AA) was the most abundant PUFA esterified to phosphatidylinositol (PI) and was significantly reduced in EFA deficient animals (from 31% to 13% in skin), where a large amount of 20:3n-9 (9% in skin) was also present. Influxes and effluxes of water through skin and opercular membrane were measured in vitro. No differences were detected between rainbow trout fed the control or the EFA deficient diet. 12-Hydroxyeicosatetraenoic acid (12-HETE), 12-hydroxyeicosapentaenoic acid (12-HEPE) and 14-hydroxydocosahexaenoic acid (14-HDHE) could not be detected in skin from control or EFA deficient fish. There was no difference between control and EFA deficient trout in the levels of leukotriene C4 (LTC4) and leukotriene C5 (LTC5) in skin cells challenged with the calcium ionophore A23187, and of prostaglandin F2alpha (PGF2alpha), 12-HETE and 12-HEPE in gill cells challenged similarly. Prostaglandin F3alpha (PGF3alpha) production by ionophore stimulated gill cells was significantly reduced in fish fed the EFA-deficient diet. 14-HDHE produced by gill cells was 3.3 fold higher in EFA deficient fish compared to controls.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1999

Biosensor for direct determination of organophosphate nerve agents. 1. Potentiometric enzyme electrode.

A potentiometric enzyme electrode for the direct measurement of organophosphate (OP) nerve agents was developed. The basic element of this enzyme electrode was a pH electrode modified with an immobilized organophosphorus hydrolase (OPH) layer formed by cross-linking OPH with bovine serum albumin (BSA) and glutaradehyde. OPH catalyses the hydrolysis of organophosphorus pesticides to release protons, the concentration of which is proportional to the amount of hydrolysed substrate. The sensor signal and response time was optimized with respect to the buffer pH, ionic concentration of buffer, temperature, and units of OPH immobilized using paraoxon as substrate. The best sensitivity and response time were obtained using a sensor constructed with 500 IU of OPH and operating in pH 8.5, 1 mM HEPES buffer. Using these conditions, the biosensor was used to measure as low as 2 microM of paraoxon, ethyl parathion, methyl parathion and diazinon. The biosensor was completely stable for at least one month when stored in pH 8.5, 1 mM HEPES + 100 mM NaCl buffer at 4 degrees C.

Animals↗
PubMed1999

Trout thrombocytes contain 12- but not 5-lipoxygenase activity.

Fish thrombocytes are thought to be the evolutionary forerunners of mammalian platelets. Thrombocyte preparations made by conventional methods, such as density gradient centrifugation, contain other cell types such as neutrophilic granulocytes and lymphocytes that could interfere with subsequent experiments. In this study, rainbow trout thrombocytes were separated by density gradient centrifugation and further purified by magnetic cell sorting (MACS) using the thrombocyte specific monoclonal antibody, 30D8. Thrombocyte purity was assessed by reactivity to 30D8 using flow cytometry and immunocytochemistry. Following purification by density gradient centrifugation, thrombocytes were 66.9+/-9.2% (mean value+/-S.E.M., n=3) pure. Further purification by MACS significantly increased thrombocyte purity to 97.3+/-0.6%, whereas only 1.4% of the MACS -ve fraction were identified as these cells. Incubation of thrombocytes isolated by density gradient alone with calcium ionophore, A23187, generated a range of eicosanoids derived from arachidonic or eicosapentaenoic acids, namely, leukotriene (LT)B4, LTB5, lipoxin (LX)A4, LXA5, 12-hydroxyeicosatetraenoic acid (12-HETE) and 12-hydroxyeicosapentaenoic acid (12-HEPE). A similar eicosanoid generation profile was observed for cells in the MACS -ve fraction; however, MACS +ve cells (thrombocytes) generated no 4 or 5 series LT or LX but did generate significant amounts of the 12-lipoxygenase (LO) products, 12-HETE and 12-HEPE. These results indicate that trout thrombocytes contain no demonstrable 5-LO activity and like their mammalian counterparts possess 12-LO activity.

Animals↗
PubMed1989

Effect of different oxygen concentrations in the gas phase on biochemical activities of goat mammary tissue in organ culture.

Non-secretory mammary explants from virgin goats showed higher RNA and protein synthesis in a low O2 gas phase (air) than in high O2 (95% O2). Lipid and casein synthesis was not affected significantly by the concentration of O2 in the atmosphere during culture. On the other hand, the more developed mammary tissue from primigravid goats showed higher lipid, casein and protein synthesis in 95% O2. The relative response of mammary tissue to hormones was not substantially different when cultured in the presence of a low or high O2 gas phase. As Hepes-buffered medium was found not to need a supply of CO2 to maintain the correct pH and as Hepes did not interfere with biochemical activities of cells, it is recommended to use it for cultures in a low O2 gas phase.

Animals↗
PubMed2004

Effects of sperm concentrations and culture media on fertilization and development of in vitro matured pig oocytes.

This study was carried out to investigate the effects of sperm concentrations and culture media on fertilization and development of in vitro matured pig oocytes. The concentrations of frozen-thawed sperm were 0.2 x 10(7), 2 x 10(7), 20 x 10(7) and 200 x 10(7)/ml, respectively. Culture media were NCSU-23, HEPES-buffered (25 mM) NCSU-23, PZM-3 and PZM-4, respectively. Increasing the sperm concentration from 0.2 x 10(7) to 2 x 10(7)/ml, significantly increased the penetration rate. Also, increasing the sperm concentration from 20 x 10(7) to 200 x 10(7)/ml increased the penetration rate from 62.1% to 69.9%, respectively, with no differences between these two concentrations. A similar pattern was observed for polyspermic penetration and male pronucleus formation. The mean number of sperm per oocyte significantly increased in the 20 x 10(7)/ml and again in the 200 x 10(7)/ml sperm concentrations. The percentage of blastocysts from cleaved oocytes at the 2 x 10(7)/ml sperm concentration was significantly higher than that at the 0.2 x 10(7), 20 x 10(7) and 200 x 10(7)/ml sperm concentrations. The percentage of blastocysts from cleaved oocytes and the cell numbers per blastocyst were significantly higher in the HEPES-buffered NCSU-23 culture medium than in the NCSU-23, PZM-3 and PZM-4 culture media under a gas atmosphere of 5% CO2 in air.

Animals↗
PubMed2000

Sequence-specific DNA binding by the glucocorticoid receptor DNA-binding domain is linked to a salt-dependent histidine protonation.

We used isothermal titration calorimetry in the temperature range 21-25 degrees C to investigate the effect of pH on the calorimetric enthalpy (delta H(cal)) for sequence specific DNA-binding of the glucocorticoid receptor DNA-binding domain (GR DBD). Titrations were carried out in solutions containing 100 mM NaCl, 1 mM dithiothreitol, 5% glycerol by volume, and 20 mM Tris, Hepes, Mops, or sodium phosphate buffers at pH 7.5. A strong dependence of delta H(cal) on the buffer ionization enthalpy is observed, demonstrating that the DNA binding of the GR DBD is linked to proton uptake at these conditions. The apparent increase in the pK(a) for an amino acid side chain upon DNA binding is supported by the results of complementary titrations, where delta H(cal) shows a characteristic dependence on the solution pH. delta H(cal) is also a function of the NaCl concentration, with opposite dependencies in Tris and Hepes buffers, respectively, such that a similar delta H(cal) value is approached at 300 mM NaCl. This behavior shows that the DNA-binding induced protonation is inhibited by increased concentrations of NaCl. A comparison with structural data suggests that the protonation involves a histidine (His451) in the GR DBD, because in the complex this residue is located close to a DNA phosphate at an orientation that is consistent with a charged-charged hydrogen bond in the protonated state. NMR spectra show that His451 is not protonated in the unbound protein at pH 7.5. The pH dependence in delta H(cal) can be quantitatively described by a shift of the pK(a) of His451 from approximately 6 in the unbound state to close to 8 when bound to DNA at low salt concentration conditions. A simple model involving a binding competition between a proton and a Na(+) counterion to the GR DBD-DNA complex reproduces the qualitative features of the salt dependence.

Animals↗
PubMed1992

Action of derivatives of mu-conotoxin GIIIA on sodium channels. Single amino acid substitutions in the toxin separately affect association and dissociation rates.

We have studied binding and block of sodium channels by 12 derivatives of the 22-residue peptide mu-conotoxin GIIIA (mu-CTX) in which single amino acids were substituted as follows: Arg or Lys by Gln, Gln-18 by Lys, Asp by Asn, and HO-Pro by Pro. Derivatives were synthesized as described by Becker et al. [(1989) Eur. J. Biochem. 185, 79]. Binding was measured by displacement of labeled saxitoxin from eel electroplax membranes (100 mM choline chloride, 10 mM HEPES-NaOH, pH 7.4). Blocking kinetics were evaluated from steady-state, single-channel recordings from rat skeletal muscle sodium channels incorporated into planar, neutral phospholipid/decane bilayers (200 mM NaCl, 10 mM HEPES-NaOH, pH 7.0). Blocking events generally appeared as periods of seconds to minutes in which current through the single channel was completely eliminated. A notable exception was seen for the substitution Arg-13-Gln for which the "blocked" events showed measurable conductances of about 20-40% of the open state. The substitution of Arg-13 reduced binding to electroplax membranes to undetectable levels and increased the apparent dissociation constant determined for skeletal muscle channels by greater than 80-fold compared with the native peptide. Other substitutions caused smaller decreases in affinity. The decreased potency of the toxin derivatives resulted both from increases in the rates of dissociation from the channel, and from decreases in association rates. Our data support the suggestion by Sato et al. [(1991) J. Biol. Chem. 265, 16989] that Arg-13 associates intimately with the binding site on the channel. In addition, our results suggest that certain residues affect almost exclusively the approach and docking of the toxin with its binding site, others appear to be important only to the strength of the association once binding has taken place, and yet others affect both.

Amino Acid Sequence↗
PubMed1992

Cleavage of the Lys196-Ser197 bond of prolyl oligopeptidase: enhanced catalytic activity for one of the two active enzyme forms.

Prolyl oligopeptidase, a representative of a new family of serine proteases, is remarkably sensitive to ionic strength and has two catalytically active forms, which interconvert with changing pH [Polgár, L. (1991) Eur. J. Biochem. 197, 441-447]. To reveal whether conformational changes are associated with these effects, prolyl oligopeptidase was digested with trypsin. SDS gel electrophoresis studies demonstrated that tryptic digestion of the 75-kDa native protein generated two fragments, one having a molecular mass of 51 kDa and the other of 26 kDa. The digestion was markedly dependent on the ionic strength. Specifically, the digestion proceeded more rapidly in 0.05 M Hepes buffer than in 0.05 M Hepes buffer containing 0.5 M NaCl. Moreover, the nicked enzyme formed at low ionic strength was not stable but degraded and inactivated during an extended incubation. The digestion experiments suggested that alteration in the ionic strength elicits conformational changes in native prolyl oligopeptidase, and this may account for the enhanced catalytic activity observed at higher ionic strength. The two fragments of the nicked prolyl oligopeptidase did not separate during size-exclusion chromatography under nondenaturing conditions on a Superose 12 column and eluted in place of the native enzyme, indicating that they were strongly associated. The reactive serine residues of the nicked enzyme was labeled with tritiated diisopropyl phosphofluoridate, and the fragments were separated by size-exclusion chromatography in urea.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2001

The vnd/NK-2 homeodomain: thermodynamics of reversible unfolding and DNA binding for wild-type and with residue replacements H52R and H52R/T56W in helix III.

The conformational stabilities of the vnd (ventral nervous system defective)/NK-2 homeodomain [HD(wt); residues 1-80 that encompass the 60-residue homeodomain] and those harboring mutations in helix III of the DNA recognition site [HD(H52R) and HD(H52R/T56W)] have been investigated by differential scanning calorimetry (DSC) and ellipticity changes at 222 nm. Thermal unfolding reactions at pH 7.4 are reversible and repeatable in the presence of 50-500 mM NaCl with DeltaC(p) = 0.52 +/- 0.04 kcal K(-1) mol(-1). A substantial stabilization of HD(wt) is produced by 50 mM phosphate or by the addition of 100-500 mM NaCl to 50 mM Hepes, pH 7.4, buffer (from T(m) = 35.5 degrees C to T(m) 43-51 degrees C; DeltaH(vH) congruent with 47 +/- 5 kcal mol(-1)). The order of stability is HD(H52R/T56W) > HD(H52R) > HD(wt), irrespective of the anions present. Progress curves for ellipticity changes at 222 nm as a function of increasing temperature are fitted well by a two-state unfolding model, and the cooperativity of secondary structure changes is greater for mutant homeodomains than for HD(wt) and also is increased by adding 100 mM NaCl to Hepes buffer. A 33% quench of the intrinsic tryptophanyl residue fluorescence of HD(wt) by phosphate binding (K(D)' = 2.6 +/- 0.3 mM phosphate) is reversed approximately 60% by DNA binding. Thermodynamic parameters for vnd/NK-2 homeodomain proteins binding sequence-specific 18 bp DNA have been determined by isothermal titration calorimetry (10-30 degrees C). Values of DeltaC(p) are +0.25, -0.17, and -0.10 +/- 0.04 kcal K(-1) mol(-1) for HD(wt), HD(H52R), and HD(H52R/T56W) binding duplex DNA, respectively. Interactions of homeodomains with DNA are enthalpically controlled at 298 K and pH 7.4 with corresponding DeltaH values of -6.6 +/- 0.5, -10.8 +/- 0.1, and -9.0 +/- 0.6 kcal mol(-1) and DeltaG' values of -11.0 +/- 0.1, -11.0 +/- 0.1, and -11.3 +/- 0.3 kcal mol(-1) with a binding stoichiometry of 1.0 +/- 0.1. Thermodynamic parameters for DNA binding are not predicted from homeodomain structural changes that occur upon complexing to DNA and must reflect also solvent and possibly DNA rearrangements.

Amino Acid Substitution↗
PubMed2004

Influence of monovalent cation identity on parvalbumin divalent ion-binding properties.

Rat alpha- and beta-parvalbumins have distinct monovalent cation-binding properties [Henzl et al. (2000) Biochemistry 39, 5859-5867]. Beta binds two Na(+) or one K(+), and alpha binds one Na(+) and no K(+). Ca(2+) abolishes these binding events, suggesting that the monovalent ions occupy the EF-hand motifs. This study compares alpha and beta divalent ion affinities in Na(+) and K(+) solutions. Solvent cation identity seriously affects alpha. In Hepes-buffered NaCl, at 5 degrees C, the macroscopic Ca(2+)-binding constants are 2.6 x 10(8) and 6.4 x 10(7) M(-1) and the Mg(2+) constants, 1.8 x 10(4) and 4.3 x 10(3) M(-1). In Hepes-buffered KCl, the Ca(2+) values increase to 2.9 x 10(9) and 6.6 x 10(8) M(-1) and the Mg(2+) values to 2.2 x 10(5) and 3.7 x 10(4) M(-1). Monte Carlo simulation of alpha binding data-employing site-specific constants and explicitly considering Na(+) binding-yields a K(Na) of 630 M(-1) and indicates that divalent ion-binding is positively cooperative. NMR data suggest that the lone Na(+) ion occupies the CD loop. Solvent cation identity has a smaller impact on beta. In Na(+), the Ca(2+) constants for the EF and CD sites are 2.3 x 10(7) and 1.5 x 10(6) M(-1), respectively; the Mg(2+) constants are 9.2 x 10(3) and 1.7 x 10(2) M(-1). In K(+), these values shift to 3.1 x 10(7) and 3.8 x 10(6) M(-1) and the latter to 1.4 x 10(4) and 2.9 x 10(2) M(-1). These data suggest that parvalbumin divalent ion affinity, particularly that of rat alpha, can be significantly attenuated by increased intracellular Na(+) levels.

Animals↗
PubMed1999

Product catalyzes the deamidation of D145N dehalogenase to produce the wild-type enzyme.

Aspartate 145 plays an essential role in the active site of 4-chlorobenzoyl-CoA dehalogenase, forming a transient covalent link at the 4-position of the benzoate during the conversion of the substrate to 4-hydroxybenzoyl-CoA. Replacement of Asp 145 by residues such as alanine or serine results in total inactivation, and stable complexes can be formed with either substrate or product. The Raman spectroscopic characterization of some of the latter is described in the preceding publication (Dong et al.). The present work investigates complexes formed by D145N dehalogenase and substrate or product. Time-resolved absorption and Raman difference spectroscopic data show that these systems evolve rapidly with time. For the substrate complex, initially the absorption and Raman spectra show the signatures of the substrate bound in the active site of the asparagine 145 form of the enzyme but these signatures are accompanied by those for the ionized product. After several minutes these signatures disappear to be replaced with those closely resembling the un-ionized product in the active site of wild-type dehalogenase. Similarly, for the product complex, the absorption and Raman spectra initially show evidence for ionized product in the active site of D145N, but these are rapidly replaced by signatures closely resembling the un-ionized product bound to wild-type enzyme. It is proposed that product bound to the active site of asparagine 145 dehalogenase catalyzes the deamidation of the asparagine side chain to produce the wild-type aspartate 145. For the complexes involving substrate, the asparagine 145 enzyme population contains a small amount of the WT enzyme, formed by spontaneous deamidation, that produces product. In turn, these product molecules catalyze the deamidation of Asn 145 in the major enzyme population. Thus, conversions of substrate to product and of D145N to D145D dehalogenase go on simultaneously. The spontaneous deamidation of asparagine 145 has been characterized by allowing the enzyme to stand at RT in Hepes buffer at pH 7.5. Under these conditions deamidation occurs with a rate constant of 0.0024 h-1. The rate of product-catalyzed deamidation in Hepes buffer at 22 degrees C was measured by stopped-flow kinetics to be 0.024 s-1, 36000 times faster than the spontaneous process. A feature near 1570 cm-1 could be observed in the early Raman spectra of both substrate and product-enzyme complexes. This band is not associated with either substrate or product and is tentatively assigned to an ester-like species formed by the attack of the product's 4-O- group on the carbonyl of asparagine's side chain and the subsequent release of ammonia. A reaction scheme is proposed, incorporating these observations.

Amides↗
PubMedDate not supplied

Reaction of chromium(VI) with ascorbate produces chromium(V), chromium(IV), and carbon-based radicals.

Reaction of potassium dichromate with sodium ascorbate was studied by EPR spectroscopy at room temperature, in 0.10 M N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), phosphate, cacodylate, and tris(hydroxymethyl)aminomethane hydrochloride (Tris.HCl) buffers at pH 7.0, in the presence of 0.10 M spin trap [5,5-dimethyl-1-pyrroline 1-oxide or 2-methyl-N-(4-pyridinylmethylene)-2-propanamine N,N'-dioxide]. Chromium(V), ascorbate radical, CO2-, and other carbon-based spin trap-radical adducts were observed. Chromium(V), CO2-, and the carbon-based radicals were observed at low ratios of ascorbate to chromium, and ascorbate radical was observed at high ratios of ascorbate to chromium. The presence of Cr(IV) was detected indirectly by reaction with Mn(II) and a subsequent decrease in the Mn(II) EPR signal. More Cr(IV) was found for the higher reaction ratios of ascorbate to Cr(VI). The only buffer effect observed was a relative decrease of the Cr(V) signal in Tris.HCl vs HEPES, phosphate, and cacodylate buffers, no change in the radical adducts was observed. There was no evidence for reactive oxygen species an intermediates in this reaction. Addition of the singlet oxygen trap 2,2,6,6-tetramethyl-4-piperidone hydrochloride showed no 2,2,6,6-tetramethyl-1-piperidinyloxy radical formation. The Cr(V) species did not react with dioxygen, and dioxygen did not affect the formation of carbon-based radicals. A mechanism consistent with these observations is discussed.

Ascorbic Acid↗
PubMed2000

The intestinal uptake of phenol from micellar systems does not conform to the aqueous transfer model.

PURPOSE: To evaluate the aqueous transfer model as the mechanism for the micelle-mediated uptake of phenol in the rat in situ intestinal perfusion model. METHODS: Phenol in isotonic HEPES buffer was perfused through the jejunal segment at two flow rates and at various concentrations. Phenol was then dispersed in two, distinct mixed micelle systems composed of sodium taurocholate and phosphatidylcholine at 10 mM:2.5 mM (10:2.5 system) and at 10 mM: 10 mM (10:10 system) and its uptake studied in each case. Equilibrium dialysis was done to determine the aqueous fraction of phenol in each system. RESULTS: The P(eff) of phenol in isotonic HEPES buffer at a low flow rate (n = 6) was 1.7 +/- 0.4 x 10(-4) cm/s and at a high flow rate (n = 13) was 1.8 +/- 0.5 x 10(-4) cm/s. The P(eff) for the 10:2.5 system at the high flow rate (n = 3) was 1.5 +/- 0.4 x 10(-4) cm/s and at the low flow rate (n = 3) was 1.4 +/- 0.3 x 10(-4) cm/s. Uptake was membrane rate-limited in both the non-micellar and 10:2.5 systems. P(eff) at a high flow rate (n = 3) in the 10:10 system was 1.3 +/- 0.1 x 10(-4) cm/s. Equilibrium dialysis (n = 4) revealed free fractions of 0.60 +/- 0.05 and 0.50 +/- 0.03 for the 10:2.5 and 10:10 systems. CONCLUSIONS: The uptake of micellized phenol did not follow the aqueous transfer model of uptake.

Animals↗
PubMed2002

Interactions of the 18.5 kDa isoform of myelin basic protein with Ca2+-calmodulin: in vitro studies using gel shift assays.

The interactions of the 18.5 kDa isoform of myelin basic protein (MBP) with calmodulin (CaM) in vitro have been investigated using glutaraldehyde or dithiobis[succinimidylpropionate] (DSP) cross-linking, and SDS-polyacrylamide gel electrophoresis. The following forms of MBP were used: the natural bovine C1 charge isomer (bMBP/C1) and a recombinant murine product (rmMBP), and their fragments generated by digestion with cathepsin D (EC 3.4.23.5). In physiological buffers (10 mM HEPES-NaOH, pH 7.4, 5 mM CaCl2, 0.0035% glutaraldehyde; or 50 mM HEPES-NaOH, pH 7.4, 100 mM NaCl, 1 mM CaCl2, 0.0035% DSP), MBP and CaM interacted primarily in a 1:1 molar ratio, consistent with previous studies that used 6 M urea, i.e. denaturing conditions. Moreover, the appearance of higher-order bands (not previously observed) suggested that the mechanism of interaction of the two proteins involved a series of relatively complex equilibria, resulting in 2:1 ratios of MBP to CaM. This observation would explain the cooperativity of association inferred from fluorescence studies [13]. Our results demonstrated further that the interaction involved the C-terminal domain of MBP, again in a primarily 1:1 molar ratio with CaM, consistent with our identification of a CaM-binding motif at the C-terminus.

Calcium↗
PubMed1996

Effect of sodium bicarbonate on intracellular pH under different buffering conditions.

Previous in vitro studies have reported a paradoxical exacerbation of intracellular acidosis following bicarbonate therapy due to the generated CO2 entering the cytoplasm. However, these studies were conducted in nonphysiological Hepes-buffered media. We compared the effect of a sodium bicarbonate load on the intracellular pH (pHi) of hepatocytes placed in nonbicarbonate (NBBS) or bicarbonate (BBS) buffering systems. The pHi of isolated rat hepatocytes was measured using the fluorescent pH sensitive dye BCECF and a single-cell imaging technique. Cells were placed in medium buffered with HCO3-/CO2 or Hepes. All media were adjusted to pH 7 with L-lactic acid or HCl. An acute 45 mM sodium bicarbonate load was added to each medium and the changes in pHi were measured every three seconds for 90 seconds. The sodium bicarbonate load caused rapid cytoplasmic acidification of cells in NBBS (N = 50, P < 0.001). In contrast, hepatocytes in BBS underwent a marked increase in pHi (N = 50, P < 0.001) without any initial decrease in pHi. These differences were highly significant for the buffer (P < 0.01), but not for the acid used. We conclude that sodium bicarbonate exacerbates intracellular acidosis only in a NBBS. Hence, in vitro studies reporting a paradoxical intracellular acidosis following bicarbonate therapy cannot be extrapolated to the in vivo buffering conditions, and should not be used to argue against bicarbonate therapy.

Acidosis↗
PubMed1998

Dual effects of histamine and substance P on intracellular calcium levels in human U373 MG astrocytoma cells: role of protein kinase C.

1. In human U373 MG astrocytoma cells agonist-induced increases in intracellular Ca2+ ([Ca2+]i) are rapidly returned towards prestimulated levels. Examination of the effect of histamine and substance P on [Ca2+]i in thapsigargin-treated cells has allowed a mechanism contributing to this effect to be characterized. 2. Histamine and substance P stimulated [3H]-inositol monophosphate ([3H]-IP1) accumulation in U373 MG cells. Concentration-response curves of [3H]-IP1 accumulation in suspensions of U373 MG cells in HEPES buffer containing 30 mM Li+ yielded best-fit EC50 values of 19.1+/-1.5 microM for histamine and 5.7+/-1.3 nM for substance P. 3. In confluent monolayers of fura-2 loaded U373 MG cells perfusion with 100 microM histamine resulted in a transient 597+/-50 nM increase in [Ca2+]i. The best-fit EC50 for histamine was 4.6+/-2.2 microM. The initial, transient, histamine response was often followed by further small transient increases in [Ca2+]i. 4. Treatment of U373 MG cells with 5 microM thapsigargin, followed by the readdition of 1.8 mM Ca2+ to the perfusion buffer, resulted in a steady-state level of [Ca2+]i 97+/-5 nM above pretreated levels (measured 400 s after readdition of Ca2+). Perfusion of histamine (100 microM, 100 s) caused a rapid decline in the thapsigargin-induced steady state level of [Ca2+]i. This effect of histamine was normally reversible upon washout. The best-fit EC50, for the histamine response was 0.8+/-0.2 microM. Substance P (10 nM, 100s) also caused a reduction in thapsigargin-induced steady-state levels of [Ca2+]i. 5. Neither 100 microM histamine nor 10 nM substance P inhibited the rate of quench of fura-2 fluorescence by Mn2+ in U373 MG cells pretreated with 5 microM thapsigargin, indicating that the depressant effect on steady-state raised [Ca2+]i was probably not due to a block of Ca2+ entry. 6. The depressant effect of histamine on [Ca2+]i was blocked by 1 microM mepyramine, and was partially reduced by pre-incubation with 1 microM staurosporine (61+/-7% reduction) and with Ro 31-8220 (24+/-10% and 50+/-6% reduction by 1 and 10 microM Ro 31-8220, respectively). Pre-incubation with H-89 did not alter the depressant effect of histamine. 7. Neither 1 microM staurosporine nor 10 microM KN-62 inhibited the binding of [3H]-mepyramine to guinea-pig cerebellar membranes, whereas it was reduced by 17+/-1% and 55+/-2% by 1 and 10 microM Ro 31-8220, respectively. However, [3H]-IP1 accumulation stimulated by histamine in U373 MG cells was not inhibited by 1 or 10 microM Ro 31-8220 and in 2 out of 3 experiments there was a significant potentiation of the response to histamine with both concentrations of Ro 31-8220. Staurosporine, 1 microM, similarly potentiated the response to 100 microM histamine in 3 out of 4 experiments. KN-62 (10 microM) did not stimulate histamine-induced [3H]-IP1 accumulation. 8. In HEPES buffer to which no Ca2+ had been added, histamine stimulated a transient 451+/-107 nM increase in [Ca2+]i. Pretreatment with 1 microM and 10 microM Ro 31-8220 did not significantly alter the initial peak response to histamine, but slowed the rate at which histamine-induced increases in [Ca2+]i were returned to prestimulated levels. Pretreatment with KN-62 had no significant effect on the response to histamine, but consistently inhibited the secondary slower phase of the decline in [Ca2+]i. H-89 did not alter the histamine response. 9. The effect of histamine in stimulating Ca2+ extrusion was not confined to U373 MG cells, since 100 microM histamine also caused a rapid decrease in steady-state levels of [Ca2+]i in thapsigargin-treated human HeLa cells. 10. The results indicate that agonists which increase [Ca2+]i via activation of phosphoinositide metabolism can also stimulate a homeostatic mechanism which acts to reduce [Ca2+]i. The balance of the evidence indicates that in U373 MG cells the latter effect most likely involves a PKC-mediated stimulation of a Ca2+-extrusion pump.

Animals↗
PubMed2002

Adeno-associated virus-mediated transfer of human acid maltase gene results in a transient reduction of glycogen accumulation in muscle of Japanese quail with acid maltase deficiency.

Glycogen storage disease type II (GSD II), Pompe's disease, is caused by the deficiency of acid alpha-D-glucosidase (GAA) in lysosome and is the most common form of GSD in Taiwan. Most cases are the infantile form. The disease is relentless and most patients die of cardiac failure and respiratory tract infection in the first year of life. At present, no treatment has been proved effective for this fatal disease. The applicability of enzyme replacement therapy is under investigation. However, high price and transient efficiency are the major problems to be solved. Accordingly, gene therapy by viral method has been conducted. In this study we constructed a plasmid that contained 5'-shortened BglII-NotI fragment human GAA cDNA, downstream of CMV promoter and bovine growth hormone polyadenylation signal, as well as AAV ITR region. When fibroblasts obtained from GSD II patients were cultured and infected with rAAV-GAA, the GAA activity of the fibroblasts increased four- to five-fold. Using acid maltase deficient (AMD) Japanese quail as the animal model, rcAAV-GAA 0.1 ml per site (1 x 10(9)-10) particles), totally 10 different sites to make 1 ml (1 x 10(1)0-11) particles), was injected into unilateral deep pectoral muscle of AMD quails. Medium (hepes) was only injected in the same way into the contralateral deep pectoral muscle to serve as control. Four days after injection, PAS staining showed disappearance of the glycogenosomes with regeneration of myocytes surrounding the intramuscular injected area as compared with the contralateral muscle of the same birds. Using anti-GAA monoclonal antibody, GAA was demonstrated on the regenerated myocytes by immunohistochemical staining and absent on the contralateral muscle of the same birds. Nevertheless, T lymphocytes infiltration was noted in both the rcAAV-GAA and hepes (medium) injected muscles and more prominent in the rcAAV-GAA-injected site. Functional evaluation demonstrated that wing flapping movement improved with wide flapping in the rAAV-GAA injected side, but not in the counterpart. Unfortunately, these histochemical and functional improvements faded away in 14 days, probably due to destruction of rcAAV by cell-mediated immunity of infiltrated T cells. Taken together, the present study suggests that rAAV can enter either human or quail cells and express and effectively reduce the glycogen accumulation in the skeletal muscle of AMD quails. These preliminary results are similar to these of low-dose rGAA replacement therapy. The mechanisms underlying the induction of cell-mediated immunity are unknown. How to elevate the number of packaged AAV, enhance the infectivity of AAV and reduce cell-mediated immunity must be solved in the future.

Animals↗
PubMed1983

Mitochondrial-reticular cytostructure in liver cells.

This study examines the structural relationship of mitochondria and the endoplasmic reticulum in liver. Livers of rat and Japanese quail were homogenized and fractionated in media of 0.25 M-sucrose, either 5mM or 50 mM in sodium Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid], pH 7.4 (2.2 mM or 22 mM in Na respectively), designated here as low- and high-salt media. Three particulate fractions were prepared by sequential centrifugation. A nuclear pellet sedimenting at 300 g was obtained as described by Shore & Tata [(1977) J. Cell Biol. 72, 714-725], and from the resulting supernatant thereof a low-speed pellet (1100-1500 g) and a high-speed pellet (8000-10 000 g) were prepared. In the low-salt medium the yields of mitochondrial matrix enzymes (citrate synthase, glutamate dehydrogenase, ornithine carbamoyltransferase) and their specific activities in the low-speed pellet were over twice those in the high-speed pellet. In the high-salt medium the yield of matrix enzymes was 4-5 times, and the specific activities were up to 3 times, higher in the low-speed pellet than in the high-speed pellet. Oxygen uptake and respiratory control ratio were also much higher in the low-speed pellets in both media. Some 50-65% of the microsomal marker enzyme glucose 6-phosphatase was in the supernatant from the high-speed pellet, and the rest sedimented with the mitochondria. Repeated washing with the high-salt medium removes only a limited amount of reticulum. Washing with salt-free sucrose removes most of the reticulum, but a fraction remains strongly bound to mitochondria. Homogenates from quail and rat liver were fractioned isopycnically on Percoll gradients in either 0.25 M-sucrose or 0.25 M-sucrose/50 mM-sodium Hepes. Up to five particulate bands were separated and assayed. Mitochondria were present in two to three bands and were associated with endoplasmic reticulum. As seen in the phase-contrast microscope the mitochondria prepared in the low-salt medium consist of separate organelles. In the high-salt medium the mitochondria appear as chains of from three to ten organelles not touching each other. On addition of univalent ions at concentrations above 20 mM, the mitochondria aggregate into chains, and at higher ionic strength larger multidimensional aggregates are formed. The dispersion and aggregation of mitochondria are reversible. Negatively stained electron micrographs reveal a branched mitochondrial structure, with mitochondria held together by strands of reticulum.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗