Linoleic acid hydroperoxide favours hypochlorite- and myeloperoxidase-induced lipid peroxidation

Liposomes composed of soybean phosphatidylcholine were peroxidized using the reagent sodium hypochlorite or the myeloperoxidase-hydrogen peroxide-Cl^- system. Linoleic acid hydroperoxide previously prepared from linoleic acid by means of lipoxidase was incorporated into liposomes. The yield of thiobarbituric acid reactive substances (TBARS) continuously increased with higher amounts of hydroperoxide groups after the initiation of lipid peroxidation by hypochlorous acid producing systems. The accumulation of TBARS was inhibited by scavengers of free radicals such as butylated hydroxytoluene and by the scavengers of hypochlorous acid, taurine and methionine. Lipid peroxidation was also prevented by sodium azide or chloride free medium in the myeloperoxidase-hydrogen peroxide-Cl^- system. Here we show for the first time that the reaction of hypochlorous acid with a biologically relevant hydroperoxide yields free radicals able to cause further oxidation of lipid molecules.     

Transport characteristics of system A in the rat exocrine pancreatic epithelium analyzed using the specific non-metabolized amino acid analogue alpha-methylaminoisobutyric acid

The selectivity and kinetics of system A amino acid transport in the rat exocrine pancreatic epithelium were characterized using the specific analogue alpha-methylaminoisobutyric acid. Unidirectional influx of alpha-methylaminoisobutyric acid was measured in isolated perfused pancreata by rapid dual tracer dilution. In cross-inhibition experiments DL-methylalanine, L-serine, L-cysteine, glycine, L-phenylalanine and L-glutamine were effective inhibitors of influx, whereas L-glutamate and L-lysine were less effective. In the presence of sodium alpha-methylaminoisobutyric acid influx was saturable with an apparent Kt = 1.7 +/- 0.2 mM and Vmax = 0.49 +/- 0.03 mumol/min per g (mean +/- S.E., n = 6). Influx of alpha-methylaminoisobutyric acid at 50 microM and 100 microM concentrations was significantly inhibited as the perfusate sodium concentration was gradually decreased from 156 mM to 26 mM by isoosmolar choline replacement. Estimated Kt values for sodium at these two methylaminoisobutyric acid concentrations approximated 200 mM. System A activity in the basolateral membrane of the exocrine pancreatic epithelium exhibits a high transport affinity, a wide tolerance for different amino acids and a dependency upon the extracellular sodium concentration.     

Low-affinity gamma-aminobutyric acid transport in rat brain

The low-affinity (Km = 100-200 microM) gamma-aminobutyric acid (GABA) transporter from membrane vesicles from rat brain has been characterized and found to be in many aspects similar to the well-known sodium- and chloride-coupled high-affinity gamma-aminobutyric acid transporter (Km = 2-4 microM). Influx by this system is sodium and chloride dependent and stimulated by an interior negative membrane potential. Steady-state levels obtained by both systems are lowered by the sodium channel openers veratridine and aconitine. However, while the channel blocker tetrodotoxin fully reverses this inhibition with the high-affinity system, this is not the case for its low-affinity counterpart. Furthermore, the toxin from the scorpion Androctonus australis Hector inhibited high-affinity transport only. Efflux of gamma-aminobutyric acid taken up by the high-affinity system displayed a Km of about 100 microM. Exchange catalyzed by the low-affinity system was observed in the absence of external sodium and chloride. Furthermore, both activities copurified in the fractionation procedure developed to purify the high-affinity transporter. All these observations are consistent with the idea that both activities are manifestations of only one gamma-aminobutyric acid transporter. The high-affinity binding site represents the extracellular and the low-affinity site the cytosolic aspect of the transporter. In addition, it was found that right-side-out synaptosomes also contain a low-affinity GABA transporter. This apparently represents a different transport protein.     

Sodium-dependent high-affinity uptake of taurine by isolated rat brain capillaries

Transport of taurine has been demonstrated in capillary preparations from adult rat brains using [3H]taurine. Taurine transport is mediated by a saturable high-affinity system which is entirely dependent on sodium ions. The apparent maximal influx (Vmax) and half-saturation concentration (Km) corresponded to 1.06.10(-4) mumol/min per mg protein and 27.5 microM, respectively. Competition experiments in the presence of sodium ion showed that [3H]taurine uptake was strongly inhibited by 0.1 mM unlabeled structural analogues of taurine such as beta-alanine and hypotaurine as well as unlabeled taurine. gamma-Aminobutyric acid (GABA) (0.1 mM) inhibited the uptake of labeled taurine by 30%, whereas isethionic acid, L-methionine, L-2,4-diaminobutyric acid, glycine, L-cysteinesulfonic acid and cystamine did not exhibit any inhibitory effect. The results suggest that the Na+ gradient is the principal source of energy for taurine transport into isolated brain capillaries. This transport system may play an active role in the regulation of taurine concentration in the brain extracellular space.     

Effects of meconic and comenic acids on slow sodium channels of secondary neurons

Effects of comenic and meconic acids on cultured dorsal root ganglion cells were investigated by the whole-cell patch clamp technique. The acids, having a well-known antiinflammatory and antibacterial action, decreased effective charge transfer in the activation gating system of TTX-resistant (slow) sodium channels in a dose-dependent manner. The effects were described by Hill's equation. The dissociation constant and Hill coefficient values were K(D) = 100 nM and X = 0.5 (for comenic acid) and K(D) = 10 nM and X = 0.34 (for meconic acid). The nonspecific antagonist of opioid receptors naltrexone totally blocked the effects. We suggest that the acids studied activate a subpopulation of opioid receptors negatively coupled to TTXr sodium channels.     

Conversion of deoxycorticosterone to 3-keto-4-etienic acid, catalyzed by a partially purified preparation from bovine adrenocortical mitochondria.

Bovine adrenocortical mitochondria were sonicated and subjected to extraction with sodium cholate. The extract contained not only cytochrome P-450 activities, but also an activity which catalyzed the conversion of deoxycorticosterone to an unknown steroid (designated X). The latter activity was concentrated by (NH4)2SO4 fractionation in the presence of sodium cholate, and separated from P-450 by taking advantage of their different solubilities in phosphate buffer without sodium cholate. The specific activity of the partially purified enzyme fraction was 70 times higher than that of sonicated mitochondria. The conversion of deoxycorticosterone to steroid X required NAD or NADP. The conversion rate was dependent on the concentration of deoxycorticosterone. The major product, steroid X, was isolated from the reaction mixture by means of silicic acid and Iatrobeads column chromatography. The steroid was characterized as 3-keto-4-etienic acid (3-oxoandrost-4-ene-17beta-carboxylic acid). This result suggests that an enzyme system for the conversion of deoxycorticosterone to 3-keto-4-etienic acid exists in adrenocortical mitochondria.     

Neurohumoral mechanisms of sodium-dependent hypertension

The contribution of neurohumoral factors to arterial pressure has been studied in several models of sodium-dependent hypertension including the deoxycorticosterone-saline, Dahl salt-sensitive rats, and reduced renal mass-saline. Observations from these animals have largely pointed to the sympathetic nervous system and arginine vasopressin (AVP) as the critical factors responsible for mediating the increased arterial pressure. Our work has indicated that the one-kidney, figure-8 renal wrap model of experimental hypertension is also sodium dependent. In these rats, prior sodium depletion prevented the development of hypertension whereas high sodium intake exacerbated the increase in arterial pressure. An activation of the sympathetic nervous system and increased AVP activity appeared to be responsible for the hypertension in rats maintained on normal and high sodium intake. Stimulation of the AVP and sympathetic nervous systems in sodium-dependent hypertension may be associated with a suppression of cardiovascular gamma-aminobutyric acid (GABA)-ergic function in the central nervous system. The inhibitory neurotransmitter, GABA, and an inhibitor of GABA uptake, nipecotic acid, lowered arterial pressure in a sodium-stimulated model of hypertension.     

Preparation of a cell-free extract from rat brain which can initiate protein synthesis in vitro

A cell-free protein synthesis system, derived from brains of 3 mo-old male Fischer-344 rats, has been characterized. The optimum conditions for amino acid incorporation in the system were 5 mM magnesium ion and 200 mM potassium ion. Incorporation depended on the addition of ATP, GTP, and an enegy-generating system, and was sensitive to addition of the drugs aurintricarboxylic acid and sodium fluoride, inhibitors of initiation of protein synthesis. Both 40S and 80S initiation complexes were labeled in vitro, using [³⁵S]methionine. Such labeling was sensitive to the protein synthesis inhibitors, aurintricarboxylic acid and sodium fluoride. The system, which can initiate protein synthesis, should be of use for examining mechanisms which underlie alterations in rat brain protein synthesis induced by various treatments.     

Synthesis,characterization and swelling behaviors of sodium alginate-g-poly(acrylic acid)/sodium humate superabsorbent

A novel sodium alginate-g-poly(acrylic acid)/sodium humate superabsorbent was prepared by graft copolymerization with sodium alginate, acrylic acid and sodium humate in aqueous solution, using N,N’-methylenebisacrylamide as a crosslinker and ammonium persulfate as an initiator. The effects of crosslinker, sodium alginate and sodium humate content on water absorbency of the superabsorbent were studied. The swelling behavior in solutions of various pH and the swelling kinetics in saline solutions (5 mmol/L NaCl and CaCl₂) were also investigated. The results from IR analysis showed that both sodium alginate and sodium humate react with the acrylic acid monomer during the polymerization process. The introduction of sodium humate into the sodium alginate-g-poly(acrylic acid) system could enhance the water absorbency and the superabsorbent containing 10 wt% sodium humate acquired the highest water absorbency (1380 g/g in distilled water and 83 g/g in 0.9 wt% NaCl solution).     

The effect of bile salts on human vascular endothelial cells

The uptake and release of radiochromium from adult human vascular endothelial cells in culture was employed to determine the relative toxicity of different bile salts. Endothelial cells after pre-incubation with 51Cr for 18 h were incubated with bile salts for 24 h and percentage chromium release was taken as a measure of toxicity to cells. Lithocholic acid (LC) (potassium salt) was cytotoxic at concentrations greater than 50 microM. However, LC glucuronide, sulfate and the beta-epimer were progressively less toxic with toxicity seen at concentrations of 60, 110 and 180 microM, respectively. The greatest cytotoxic effect was observed with glycolithocholic acid (GLC) (potassium salt) which was toxic at every concentration tested (20-200 microM). Sulfation abolished the toxic effect of GLC. At the concentrations employed for the assay (between 20 and 240 microM) GLC sulfate (disodium salt), taurolithocholic acid sulfate (disodium salt), cholic acid (sodium salt), glycocholic acid (sodium salt), deoxycholic acid (sodium salt) and ursodeoxycholic acid (sodium salt) were not cytotoxic. The 51Cr release cytotoxicity assay was validated with lactate dehydrogenase leakage from endothelial cells with a good correlation (r = 0.87). These data confirm in a human cellular system that LC and its conjugates were the most toxic of the bile salts tested and explains its pathophysiological importance in hepatobiliary disease. It also suggests that biotransformation by either sulfation or beta-epimerisation of bile salts especially of LC, as occurs in patients with intrahepatic or extrahepatic biliary obstruction or severe cholestasis, is hepatoprotective.     

Effect of acid-base status on plasma phosphorus response to lactate

The mechanism(s) underlying the hyperphosphatemia of lactic acidosis is uncertain. We assessed the interacting influence of the acid anion and acid-base status on plasma phosphorus concentration by administering lactic acid alone, lactic acid plus sodium bicarbonate, sodium bicarbonate alone, and sodium lactate alone to four different groups of dogs. The findings of (1) no increase in plasma phosphorus concentration with lactic acid plus sodium bicarbonate versus a marked increment with lactic acid alone, and (2) no difference in the plasma phosphorus response to sodium lactate versus sodium bicarbonate indicate that acidemia is necessary for the expression of lactate-induced hyperphosphatemia. The apparent greater propensity for marked hyperphosphatemia in lactic acidosis than in other types of metabolic acidosis remains unexplained, but conceivably might relate to differences in intracellular pH and in the rate of glycolysis.     

Effect of divalent cations on permeabilizer-induced lysozyme lysis of Pseudomonas aeruginosa

The presence of divalent (Mg²⁺) ions greatly reduced the lysis of Pseudomonas aeruginosa strain G48 in a system at pH 7·8 or 9·0 consisting of ethylenediamine tetraacetic acid (EDTA), lysozyme and tris. Similar reductions in lysis occurred when EDTA was replaced by nitrilotriacetic acid, sodium citrate or sodium polyphosphate. The effect depended on the cation concentration. Mg²⁺ may replace cations removed from the outer membrane, or may effectively remove the permeabilizer from the system. The results suggest that the permeabilizing activity associated with these agents against this organism has a common basis in affecting the outer membrane.     

Transport of 5-formyltetrahydrofolate into primary cultured rat astrocytes

Transport of 5-formyltetrahydrofolate (5-FTHF) into primary cultured rat astrocytes was studied. Uptake of 5-FTHF into astrocytes was linear in the first 60 min and is saturable with K(m)=3.3 microM and V(max)=27.5 pmol/mg protein/45 min in pH 7.4 medium. Uptake of 5-FTHF displayed the characteristics of countertransport. Uptake of 5-FTHF was inhibited by the structural analogs 5-methyltetrahydrofolate, methtrexate, and folic acid (K(i)=3.8, 2.7, and 18.4 microM, respectively). Uptake was significantly decreased by sodium azide but was increased by high concentration of sodium cyanide and low concentration of sodium arsenate. Uptake was also inhibited by p-chloromercuriphenylsulfonate and by the anions probenecid and 4,4(')-diisothiocyanostilbene-2,2(')-disulfonic acid. Acute exposure of the cells to ethanol (100mM) inhibited the uptake for 90 min of the experimental duration. It is concluded that astrocytes have a system for the uptake of 5-FTHF and folate analogs which is carrier mediated, this system is sensitive to energy inhibitors and alcohol exposure.     

Inhibition of the human sodium/bile acid cotransporters by side-specific methanethiosulfonate sulfhydryl reagents: substrate-controlled accessibility of site of inactivation

Mammalian sodium/bile acid cotransporters (SBATs) constitute a subgroup of the sodium cotransporter superfamily and function in the enterohepatic circulation of bile acids. They are glycoproteins with an exoplasmic N-terminus, seven or nine transmembrane segments, and a cytoplasmic C-terminus. They exhibit no significant homology with other members of the sodium cotransporter family and there is limited structure/function information available for the SBATs. Membrane-impermeant methanethiosulfonates (MTS) inhibited bile acid transport by alkylation of cysteine 270 (apical SBAT)/266 (basolateral SBAT) that is fully conserved among the sodium/bile acid cotransporters. The accessibility of this residue to MTS reagent is regulated by the natural substrates, sodium and bile acid. In experiments with the apical SBAT, sodium alone increases the reactivity with the thiol reagents as compared to sodium-free medium. In contrast, bile acids protect the SBATs from inactivation, although only in the presence of sodium. The inhibition and protection data suggest that cysteine 270/266 lies in a sodium-sensitive region of the SBATs that is implicated in bile acid transport.     

Characterization of amino acid transport in membrane vesicles from the thermophilic fermentative bacterium Clostridium fervidus.

Amino acid transport was studied in membrane vesicles of the thermophilic anaerobic bacterium Clostridium fervidus. Neutral, acidic, and basic as well as aromatic amino acids were transported at 40 degrees C upon the imposition of an artificial membrane potential (delta psi) and a chemical gradient of sodium ions (delta microNa+). The presence of sodium ions was essential for the uptake of amino acids, and imposition of a chemical gradient of sodium ions alone was sufficient to drive amino acid uptake, indicating that amino acids are symported with sodium ions instead of with protons. Lithium ions, but no other cations tested, could replace sodium ions in serine transport. The transient character of artificial membrane potentials, especially at higher temperatures, severely limits their applicability for more detailed studies of a specific transport system. To obtain a constant proton motive force, the thermostable and thermoactive primary proton pump cytochrome c oxidase from Bacillus stearothermophilus was incorporated into membrane vesicles of C. fervidus. Serine transport could be driven by a membrane potential generated by the proton pump. Interconversion of the pH gradient into a sodium gradient by the ionophore monensin stimulated serine uptake. The serine carrier had a high affinity for serine (Kt = 10 microM) and a low affinity for sodium ions (apparent Kt = 2.5 mM). The mechanistic Na+-serine stoichiometry was determined to be 1:1 from the steady-state levels of the proton motive force, sodium gradient, and serine uptake. A 1:1 stoichiometry was also found for Na+-glutamate transport, and uptake of glutamate appeared to be an electroneutral process.     

Alpha-(2----8)-polysialic acid immunoreactivity in voltage-sensitive sodium channel of eel electric organ

The voltage-sensitive sodium channel from eel electroplax is formed of a polypeptide of 208,321 Da, to which is attached ca. 85 kDa of carbohydrate. Sialic acid is a prominent constituent, contributing ca. 113 negative charges to the protein surface. We here demonstrate that antibodies raised against the bacterial antigen alpha-(2----8)-polysialic acid, specific for polymers of ten or more consecutive sialic acid residues, react specifically and with high affinity to the electroplax sodium channel. In extracts of electroplax membranes, the sodium channel is the only protein that demonstrates this immunoreactivity, suggesting the presence of a polysialosyl-sialyltransferase specifically committed to this unique post-translational modification of the sodium channel. Polysialic acid is rare in vertebrates, having previously been found only associated with neural-cell adhesion molecules, present in the developing neuromuscular system. The other prominent source is the capsular polysaccharide of highly pathogenic meningitis bacteria. Antibodies to the bacterial antigen thus provide highly specific affinity markers for the sodium channel. The high avidity of these antibodies and the ratio of sialic acid residues to consensus glycosylation sites suggest that the terminal chains are well over ten sialosyl residues in length, potentially extending 10-30 nm into the extracellular environment.     

Isocratic separation of PTH-amino acids at picomole level by reverse-phase HPLC in the presence of sodium dodecylsulfate

The phenylthiohydantoin (PTH) derivatives of protein amino acids have been separated by reverse-phase high performance liquid chromatography (HPLC) on a fully end-capped C18 column using an isocratic solvent system. The developing solvent was 0.01 M sodium acetate buffer (pH 4.5) containing 39.5% acetonitrile and 0.02% sodium dodecylsulfate (SDS). With an automated liquid chromatography equipped with a dual-channel detector, operating at 254 and 313 nm, the present isocratic separation system was quite useful for routine microanalysis of PTH-amino acids released with a "gas-phase" sequencer. The time for one run was approximately 23 min and the limit of analysis approximately 2.5 pmol of a PTH-amino acid.     

Optimization of acetic acid production from synthesis gas by chemolithotrophic bacterium--Clostridium aceticum using statistical approach

Efforts in optimizing reducing agents, cysteine-HCl.H2O and sodium sulfide in order to attain satisfactory responses during acetic acid fermentation have been carried out in this study. Cysteine-HCl.H2O each with five concentrations (0.00-0.50 g/L) was optimized one at a time and followed by sodium sulfide component (0.00-0.50 g/L). Response surface methodology (RSM) was used to determine the optimum concentrations of cysteine-HCl.H2O and sodium sulfide. The statistical analysis showed that the amount of cells produced and efficiency in CO conversion were not affected by sodium sulfide concentration. However, sodium sulfide is required as it does influence the acetic acid production. The optimum reducing agents for acetic acid fermentation was at 0.30 g/L cysteine-HCl.H2O and sodium sulfide respectively and when operated for 60 h cultivation time resulted in 1.28 g/L acetic acid production and 100% CO conversion.     

Lipase from Candida paralipolytica

The extracellular lipase from Candida paralipolytica required essential activators* (usually bile- or calcium salts) for the in vitro hydrolysis of triglycerides. The reaction systems emulsified with gum arabic, gelatin, lecithin, methyl cellulose, pectin, polyvinyl alcohol, sodium cholate, or without emulsifier were compared concerning requirement for essential activator, inhibition with sodium chloride and maximum reaction rate, and the following findings have been obtained. (1) The emulsions used can be classified into five groups by the essential activator requirement. (2) The inhibition with sodium chloride depended on reaction system. (3) Each reaction system gave a similar reaction rate at pH 8.2. (4) Long-chain fatty acid dissolved in substrate was necessary to the activation with calcium salts.