Characterization of the stimulatory effects of PGF2alpha on the release of arachidonic acid

Fibroblasts derived from a rat carrageenin granuloma were cultured in the presence of radioactive arachidonic acid, palmitic acid and linoleic acid. More than 90% of each labeled fatty acid was incorporated into a phospholipid fraction by the cells in 18 hrs. Arachidonic acid was evenly incorporated into phosphatidylcholine and phosphatidylethanolamine, while both palmitic acid and linoleic acid were almost entirely incorporated into phosphatidylcholine. The position of phosphatidylcholine where the fatty acids were incorporated was different for each fatty acid. The ratio of the amount of fatty acid incorporated into the 2-position to the amount incorporated into the 1-position of phosphatidylcholine for each fatty acid was greater than 90% for arachidonic acid, 2:1 for palmitic acid and 5:1 for linoleic acid. In the case of phosphatidylethanolamine, most arachidonic acid (greater than 90%) was incorporated into the 2-position. PGF2alpha caused the stimulation of arachidonic acid release but not of palmitic acid and linoleic acid from pre-labeled fibroblasts. The serum in the medium was completely replaceable by bovine serum albumin. The effect of PGF2Alpha increased with an increasing concentration of bovine serum albumin, suggesting that serum only acts as a "trap" for released arachidonic acid. The effect of PGF2Alpha was greater than bradykinin, and no synergistic effect was seen, although an additive effect was observed. The effect of PGF2Alpha depended on the concentration of calcium ions under magnesium-supplemented conditions.     

Metabolism of 4-O-methyl-N-acetylneuraminic acid a synthetic sialic acid

1. 4-O-Methyl-N-acetylneuraminic acid shows a strong positive periodate-thiobarbiturate reaction. The mechanism of dye formation in this test for sialic acids is discussed in view of the studies already published. 2. An efficient preparation of a tritium-labelled 4-O-methyl-N-acetylneuraminic acid, with high specific radioactivity, by an oxymercuration-demercuration procedure is presented. 3. Sialytransferase activities in microsomal fractions of equine liver using desialylated fetuin are studied. The enzyme activity, assayed in a radioactive procedure, shows an apparent Km value for CMP-N-acetylneuraminic acid of 0.7 mM, whereas this value is 3.4 mM for CMP-4-O-methyl-N-acetylneuraminic acid. Differences are also observed in the maximal velocity for the two substrates. 4. The equine liver system can be used to prepare substantial amounts of fetuin containing radioactive N-acetylneuraminic acid or 4-O-methyl-N-acetylneuraminic acid. The isolated reaction products show similar sialic acid release by treatments with acid or fowl-plague virus neuraminidase. In contrast, 4-O-methyl-N-acetylneuraminic acid-fetuin displays a marked resistance to desialylation by Vibrio cholerae neuraminidase. 5. Free 4-O-methyl-N-acetylneuraminic acid is completely resistant to the action of acylneuraminate pyruvate-lyase. It does not inhibit the enzymic cleavage reaction of N-acetylneuraminic acid. 6. The influence of a substitution at C-4 neuraminic acid on the enzymatic reaction mechanisms is discussed.     

Fatty acid profiles of nitrite-oxidizing bacteria reflect their phylogenetic heterogeneity

The fatty acid profiles of all described species of the nitrite-oxidizing genera Nitrobacter, Nitrococcus, Nitrospina and Nitrospira were analyzed. The four genera had distinct profiles, which can be used for the differentiation and allocation of new isolates to these genera. The genus Nitrobacter is characterized by vaccenic acid as the main compound with up to 92% of the fatty acids and the absence of hydroxy fatty acids. The genus Nitrococcus showed cis-9-hexadecenoic acid, hexadecanoic acid and vaccenic acid as main parts. Small amounts of 3-hydroxy-dodecanoic acid were detected. The genus Nitrospina possessed tetradecanoic acid and cis-9-hcxadecenoic acid as main compounds, also 3-hydroxy-hexadecanoic acid was detected for this genus. The genus Nitrospira showed a pattern with more variations among the two described species. These organisms are characterized by the cis-7 and cis-11-isomers of hexadecenoic acid. For Nitrospira moscoviensis a specific new fatty acid was found, which represented the major constituent in the fatty acid profiles of autotrophically grown cultures. It was identified as 11-methyl-hexadecanoic acid. Since this compound is not known for other bacterial taxa, it represents a potential lipid marker for the detection of Nitrospira moscoviensis relatives in enrichment cultures and environmental samples. A cluster analysis of the fatty acid profiles is in accordance with 16S rRNA sequence-based phylogeny of the nitrite-oxidizing bacteria.     

Correlation between high-performance liquid chromatography and automated fluorimetric methods for the determination of dopamine, 3, 4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindoleacetic acid in nervous tissue and cerebrospinal fluid

The correlation between automated fluorimetric methods and high-performance liquid chromatography is described for the determination of homovanillic acid and 5-hydroxyindoleacetic acid in cerebrospinal fluid, and for dopamine, 3, 4-dihydroxyphenylacetic acid and homovanillic acid in striata of rat brain. The automated fluorimetric methods for 3, 4-dihydroxyphenylacetic acid and homovanillic acid showed a good correlation with the high-performance liquid chromatographic methodology. The fluorimetric determination for dopamine was somewhat less reliable than the high-performance liquid chromatographic assay. The fluorimetric assay for 5-hydroxyindoleacetic acid correlated poorly with the chromatographic method.     

An inducible lactone hydrolase yielding 2,5-diphenyl-3-hydroxy-4-oxo-2-hexendioic acid from pulvinic acid

The metabolism of vulpinic acid by an unclassified soil micro-organism was studied. A new compound, 2,5-diphenyl-3-hydroxy-4-oxo-2-hexendioic acid (DHOHA) was isolated from the reaction mixture of a cell-free preparation and pulvinic acid. The existence of a hydrolase which catalyses the conversion of vulpinic acid to pulvinic acid was detected in cell-free preparation, and an inducible lactone hydrolase capable of converting pulvinic acid to DHOHA was purified 130-fold and characterized. This enzyme had a MW of ca 34 000, a K_m for pulvinic acid at pH optimum (pH 7.0) less than 10 ^{? 6} M, pI = 5.0, and was inhibited by p-chloromercuriphenylsulfonate and diethylpyrocarbonate. The enzyme was highly specific for pulvinic acid. The initial degradative steps proposed for this organism are vulpinic acid → pulvinic acid → DHOHA.     

Acid hydrolysis of fibers from dairy manure

Concentrated acid hydrolysis of lignocellulosic materials is a conventional treatment process for the production of mono-sugars. However, this method has been proved ineffective and undesirable for the treatment of dairy manure due to the high nitrogen content of dairy manure and the environmental issues caused by the use of highly concentrated acid solution. In an effort to overcome these barriers, a modified acid hydrolysis process with short reaction time was introduced that involved a nitrogen-removing pretreatment followed by decrystallization with concentrated acid and then hydrolysis using dilute acid. The effects of nitrogen, acid concentration, reaction time, and temperature were investigated. A pretreated manure with a low nitrogen content of 1.3% was used as the substrate. The results indicated that the optimal conditions for fiber decrystallization were 75% acid concentration, 3:5 sample to acid ratio (weight basis), and 30 min of reaction time; while the optimal conditions for acid hydrolysis were 12.5% acid and 10% dry sample at 135 degrees C for 10 min. These conditions produced 26 g/L glucose at a yield of 84% and 11 g/L hemicellulose-sugars at a yield of 80%.     

Maturation of membrane function: transport of amino acid by rat erythroid cells.

The membrane changes which occur during cellular maturation of erythroid cells have been investigated. The transport of alpha-aminoisobutyric acid, alanine, and N-methylated-alpha-aminoisobutyric acid have been studied in the erythroblastic leukemic cell, the reticulocyte, and the erythrocyte of the Long-Evans rat. The dependence of amino acid transport on extracellular sodium concentration was investigated. Erythrocytes were found to transport these amino acids only by Na-independent systems. The steady state distribution ratio was less than 1. Reticulocytes were found to transport alpha-aminoisobutyric acid and alanine by Na-dependent systems, but only small amounts of N-methylated-alpha-aminoisobutyric acid. Small amounts of these amino acids were transported by Na-independent systems. The steady state distribution ratio was greater than one for Na-dependent transport. The erythroblastic leukemia cell, a model immature erythroid cell, showed marked Na-dependence (greater than 90%) for alpha-aminoisobutyric acid and alanine transport, and greater than 80% for the Na-dependent transport of N-methyl-alpha-aminoisobutyric acid. The steady state distribution ratio for the Na-dependent transport was greater than 4. In the erythroblastic leukemic cell, at least three Na-dependent systems are present: one includes alanine and alpha-aminoisobutyric acid, but excludes N-methyl-alpha-aminoisobutyric acid; one is for alpha-aminoisobutyric acid, alanine and also N-methyl-alpha-aminoisobutyric acid; and one is for N-methyl-alpha-aminoisobutyric acid alone. In the reticulocyte, the number of Na-dependent systems are reduced to two: one for alpha-aminoisobutyric acid and alanine; one for N-methyl-alpha-aminoisobutyric acid. In the erythrocytes, no Na-dependent transport was found. Therefore, maturation of the blast cell to the mature erythrocyte is characterized by a systematic loss in the specificity and number of transport system for amino acids.     

Novel biomarkers of the metabolism of caffeic acid derivatives in vivo

The purpose of this study was to investigate biomarkers of the bioavailability and metabolism of hydroxycinnamate derivatives through the determination of the pharmacokinetics of their urinary elimination and identification of the metabolites excreted. Coffee was used as a rich source of caffeic acid derivatives and human supplementation was undertaken. The results show a highly significant increase in the excretion of ferulic, isoferulic, dihydroferulic acid (3-(4-hydroxy-3-methoxyphenyl)-propionic acid), and vanillic acid postsupplementation relative to the levels presupplementation. Thus, ferulic, isoferulic, and dihydroferulic acids are specific biomarkers for the bioavailability and metabolism of dietary caffeic acid esters. Isoferulic acid is a unique biomarker as it is not a dietary component, however, dihydroferulic acid may well derive from other flavonoids with a structurally related B-ring. 3-Hydroxyhippuric acid has also been identified as an indicator for bioavailability and metabolism of phenolic compounds, and shows a highly significant excretion increase postsupplementation. The results reveal isoferulic acid (and possibly dihydroferulic acid) as novel markers of caffeoyl quinic acid metabolism.     

Inhibition of chymase activity by long chain fatty acids

The activity of chymase was markedly inhibited by fatty acids with carbon chain lengths of 14-22 at doses greater than 0.02 microM, irrespective of the number of double bonds. Cis acids with a carbon chain length of 18, such as stearic acid, oleic acid, linoleic acid, and linolenic acid were potent inhibitors, whereas the trans isomer of oleic acid, elaidic acid, showed less inhibitory activity. The extent of inhibition by oleyl alcohol was almost the same as that by oleic acid, suggesting that the acid moiety itself was not necessary for the inhibition; but a fatty acid with a terminal functional amide, oleamide, showed little inhibitory activity. The inhibition was noncompetitive and was reversible, and the Ki value of oleic acid was 2.7 microM. Stearic acid and oleic acid inhibited all chymotrypsin-type serine endopeptidases tested. The ID50 values of these fatty acids for atypical mast cell protease were higher than those for the other chymotrypsin-type serine endopeptidases tested. Other proteases, such as papain, trypsin, collagenase, and carboxypeptidase A, except cathespin D, were not affected by stearic or oleic acid.     

Nuclear Fraction of Bacillus subtilis as a Template for Ribonucleic Acid Synthesis

A "nuclear fraction" prepared from Bacillus subtilis was a more efficient template than purified deoxyribonucleic acid for the synthesis of ribonucleic acid by exogenously added ribonucleic acid polymerase isolated from B. subtilis. The initial rate of synthesis with the nuclear fraction was higher and synthesis continued for several hours, yielding an amount of ribonucleic acid greater than the amount of deoxyribonucleic acid used as the template. The product was heterogenous in size, with a large portion exceeding 23S. When purified deoxyribonucleic acid was the template, a more limited synthesis was observed with a predominantly 7S product. However, the ribonucleic acids produced in vitro from these templates were very similar to each other and to in vivo synthesized ribonucleic acid as determined by the competition of ribonucleic acid from whole cells in the annealing of in vitro synthesized ribonucleic acids to deoxyribonucleic acid. Treatment of the nuclear fraction with heat (60 C for 15 min) or trypsin reduced the capacity of the nuclear fraction to synthesize ribonucleic acid to the level observed with purified deoxyribonucleic acid.     

Isolation and sequencing of a cDNA clone encoding acid phosphatase in rat liver lysosomes

A full length cDNA for acid phosphatase in rat liver lysosomes was isolated and sequenced. The predicted amino acid sequence comprises 423 residues (48,332 Da). A putative signal peptide of 30 residues is followed by the NH2-terminal sequence of lysosomal acid phosphatase (45,096 Da). The deduced NH2-terminal 18-residue sequence is identical with that determined directly for acid phosphatases purified from the rat liver lysosomal membranes. The primary structure deduced for acid phosphatase contains 9 potential N-glycosylation sites and a hydrophobic region which could function as a transmembrane domain. It exhibits 89% and 67% sequence similarities in amino acids and nucleic acids, respectively, to human lysosomal acid phosphatase. The amino acid sequence of the putative transmembrane segment shows a complete similarity to that of the human enzyme. Northern blot hybridization analysis identified a single species of acid phosphatase mRNA (2.2 kbp in length) in rat liver.     

Metabolism of Benzoic Acid by Bacteria: 3,5- Cyclohexadiene-1,2-Diol-1-Carboxylic Acid Is an Intermediate in the Formation of Catechol

3,5-Cyclohexadiene-1,2-diol-1-carboxylic acid (1,2-dihydro-1,2-dihydroxy-benzoic acid) is converted enzymatically to catechol in cell extracts from Acinetobacter, Alcaligenes, Azotobacter, and three Pseudomonas species. This enzymatic activity is present only in cultures which have been grown in the presence of benzoic acid, and which convert benzoic acid to catechol rather than to protocatechuic acid. The reaction is assayed by the concomitant formation of reduced nicotinamide adenine dinucleotide from nicotinamide adenine dinucleotide. The conversion of [(14)C]benzoic acid to [(14)C]dihydrodihydroxybenzoic acid is demonstrated in cell extracts. A scheme for the conversion of benzoic acid to catechol in bacteria is presented, involving the formation of dihydrodihydroxybenzoic acid from benzoic acid by a dioxygenase which is unstable in cell extracts, followed by the dehydrogenation and decarboxylation of dihydrodihydroxybenzoic acid to catechol by a previously undescribed enzyme. Experiments with anthranilic acid and phthalic acid suggest that dihydrodihydroxybenzoic acid is a metabolite unique to benzoic acid metabolism. Two new methods for assaying benzoic acid dioxygenase are suggested.     

Ascorbic acid within chromaffin granules. In situ kinetics of norepinephrine biosynthesis

Ascorbic acid requirements for norepinephrine biosynthesis were investigated in intact bovine chromaffin granules using the physiologic substrate dopamine and a novel coulometric electrochemical detection high pressure liquid chromatography system for ascorbic acid. 10 mM external dopamine, 1 mM Mg-ATP, and 1 mM ascorbic acid produced maximal norepinephrine biosynthesis without granule lysis. When external ascorbic acid was omitted, intragranular ascorbic acid was consumed in a 1:1 ratio with respect to norepinephrine biosynthesis. The initial concentration of intragranular ascorbic acid was 10.5 mM, which was depleted in stepwise fashion to 15 lower concentrations over the range of 9.2-0.2 mM. Chromaffin granules containing these varying concentrations of intragranular ascorbic acid were then incubated with 1 mM exogenous ascorbic acid, and norepinephrine biosynthesis from dopamine was determined. The apparent Km of norepinephrine biosynthesis for intragranular ascorbic acid was 0.57 mM by Eadie-Hofstee analysis and 0.68 mM by Lineweaver-Burk analysis. These data indicate that intragranular ascorbic acid is available and required for norepinephrine biosynthesis, that ascorbic acid is a true co-substrate for dopamine beta-monooxygenase, and that intragranular ascorbic acid is maintained by extragranular ascorbic acid. Continued norepinephrine biosynthesis in granules is dependent on both intragranular and extragranular concentrations of the vitamin. Furthermore, in situ kinetics of dopamine beta-monooxygenase for ascorbic acid may be most accurately determined using intact granules and the true physiologic substrate.     

Arachidonic acid-induced release of calcium in permeabilized human neutrophils

The addition of arachidonic acid to a suspension of digitonin-permeabilized human neutrophils was found to induce, in a dose-dependent manner (ED50 about 15 microM), the release of calcium from internal stores. Arachidic acid was without effect, while linoleic acid and linolenic acid were (on a concentration basis) at least 5-times less active than arachidonic acid. The activity of arachidonic acid appears to be due to the fatty acid itself and not to one of its metabolites. The pool of calcium mobilized by arachidonic acid includes that sensitive to inositol 1,4,5-triphosphate. These results demonstrate a significant intracellular role for arachidonic acid at the level of the internal mobilization of calcium in human neutrophils.     

Characterization of the stimulatory effects of PGF2α on the release of arachidonic acid

Fibroblasts derived from a rat carrageenin granuloma were cultured in the presence of radioactive arachidonic acid, palmitic acid and linoleic acid. More than 90% of each labeled fatty acid was incorporated into a phospholipid fraction by the cells in 18 hrs. Arachidonic acid was evenly incorporated into phosphatidylcholine and phosphatidylethanolamine, while both palmitic acid and linoleic acid were almost entirely incorporated into phosphatidylcholine. The position of phosphatidylcholine where the fatty acids were incorporated was different for each fatty acid. The ratio of the amount of fatty acid incorporated into the 2-position to the amount incorporated into the 1-position of phosphatidylcholine for each fatty acid was >90% for arachidonic acid, 2:1 for palmitic acid and 5:1 for linoleic acid. In the case of phosphatidylethanolamine, most arachidonic acid (>90%) was incorporated into the 2-position. PGF_2^α caused the stimulation of arachidonic acid release but not of palmitic acid and linoleic acid from pre-labeled fibroblasts.The serum in the medium was completely replaceable by bovine serum albumin. The effect of PGF_2^α increased with an increasing concentration of bovine serum albumin, suggesting that serum only acts as a ‘trap’ for released arachidonic acid. The effect of PGF_2^α was greater than bradykinin, and no synergistic effect was seen, although an additive effect was observed.The effect of PGF_2^α depended on the concentration of calcium ions under magnesium-supplemented conditions.     

Augmenting effect of acetic acid for acidification on bactericidal activity of hypochlorite solution

AIMS: Bactericidal activity of chlorine solution is enhanced by weak acidification. We compared the effects of various acids on the bactericidal activity of hypochlorite solution to establish a method for safe and effective use of an acidic hypochlorite solution. METHODS AND RESULTS: The bactericidal activities of acidic hypochlorite solutions that had been adjusted to pH 5.0 with hydrochloric acid, acetic acid, citric acid, lactic acid, formic acid, phosphoric acid or sulphuric acid against Bacillus subtilis spores were compared. The acidic solutions prepared with hydrochloric acid and acetic acid showed the highest bactericidal activity, and all of the spores (5 x 106 cfu ml(-1)) were killed within 10 min. On the other hand, the solutions prepared with citric acid and lactic acid showed no bactericidal activity against any bacterial strains tested in this study despite the low pH. The amount of chlorine gas produced by the preparation using acetic acid was sixfold less than that produced from the preparation using hydrochloric acid. CONCLUSIONS: Acetic acid is the most suitable and safe acid for the preparation of an acidic hypochlorite solution. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study provide useful information for establishing a method for safe and effective use of an acidic hypochlorite solution.     

果胶d-半乳糖醛酸生物转化制备黏酸研究进展

黏酸属于己糖二酸,可以由果胶的主要成分D-半乳糖醛酸氧化制备.黏酸结构、性质与葡萄糖二酸类似,可应用于重要平台化合物、聚合物、高分子材料的制备.与目前受到广泛关注的葡萄糖二酸合成相比,黏酸合成的研究工作尚处于起步阶段.果胶是一种廉价、丰富的可再生生物质资源,以果胶为原料生物转化制备黏酸具有重要的经济价值和环保意义.文中...     

Bacterial degradation of 3-chloroacrylic acid and the characterization of cis- and trans-specific dehalogenases

A coryneform bacterium that is able to utilize cis- and trans-3-chloroacrylic acid as sole carbon source for growth was isolated from freshwater sediment. The organism was found to produce two inducible dehalogenases, one specific for the cis- and the other for trans-3-chloroacrylic acid. Both dehalogenases were purified to homogeneity from cells induced for dehalogenase synthesis with 3-chlorocrotonic acid. The enzymes produced muconic acid semialdehyde (3-oxopropionic acid) from their respective 3-chloroacrylic acid substrate. No other substrates were found. The cis-3-chloroacrylic acid dehalogenase consisted of two polypeptide chains of a molecular weight 16.2 kDa. Trans-3-chloroacrylic acid dehalogenase was a protein with subunits of 7.4 and 8.7 kDa. The subunit and amino acid compositions and the N-terminal amino acid sequences of the enzymes indicate that they are not closely related.     

Low- and high-affinity transport systems for citric acid in the yeast Candida utilis.

Citric acid-grown cells of the yeast Candida utilis induced two transport systems for citric acid, presumably a proton symport and a facilitated diffusion system for the charged and the undissociated forms of the acid, respectively. Both systems could be observed simultaneously when the transport was measured at 25 degrees C with labelled citric acid at pH 3.5 with the following kinetic parameters: for the low-affinity system, Vmax, 1.14 nmol of undissociated citric acid s-1 mg (dry weight) of cells-1, and Km, 0.59 mM undissociated acid; for the high-affinity system, Vmax, 0.38 nmol of citrate s-1 mg (dry weight) of cells-1, and Km, 0.056 mM citrate. At high pH values (above 5.0), the low-affinity system was absent or not measurable. The two transport systems exhibited different substrate specificities. Isocitric acid was a competitive inhibitor of citric acid for the high-affinity system, suggesting that these tricarboxylic acids used the same transport system, while aconitic, tricarballylic, trimesic, and hemimellitic acids were not competitive inhibitors. With respect to the low-affinity system, isocitric acid, L-lactic acid, and L-malic acid were competitive inhibitors, suggesting that all of these mono-, di-, and tricarboxylic acids used the same low-affinity transport system. The two transport systems were repressed by glucose, and as a consequence diauxic growth was observed. Both systems were inducible, and not only citric acid but also lactic acid and malic acid may induce those transport systems. The induction of both systems was not dependent on the relative concentration of the anionic form(s) and of undissociated citric acid in the culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)