Dynamics of Docosahexaenoic Acid Metabolism in the Central Nervous System: Lack of Effect of Chronic Lithium Treatment

Using a method and model developed in our laboratory to quantitatively study brain phospholipid metabolism, in vivo rates of incorporation and turnover of docosahexaenoic acid in brain phospholipids were measured in awake rats. The results suggest that docosahexaenoate incorporation and turnover in brain phospholipids are more rapid than previously assumed and that this rapid turnover dilutes tracer specific activity in brain docoshexaenoyl-CoA pool due to release and recycling of unlabeled fatty acid from phospholipid metabolism. Fractional turnover rates for docosahexaenoate within phosphatidylinositol, choline glycerophospholipids, ethanolamine glycerophospholipids and phosphatidylserine were 17.7, 3.1, 1.2, and 0.2 %.h^–1, respectively. Chronic lithium treatment, at a brain level considered to be therapeutic in humans (0.6 mol.g^–1), had no effect on turnover of docosahexaenoic acid in individual brain phospholipids. Consistent with previous studies from our laboratory that chronic lithium decreased the turnover of arachidonic acid within brain phospholipids by up to 80% and attenuated brain phospholipase A₂ activity, the lack of effect of lithium on docosahexaenoate recycling and turnover suggests that a target for lithium's action is an arachidonic acid-selective phospholipase A₂.     

A PHAGOTROPHICALLY DERIVABLE GROWTH FACTOR IN THE PLASTIDIC DINOFLAGELLATE GYRODINIUM RESPLENDENS (DINOPHYCEAE)

The marine dinoflagellate Gyrodinium resplendens Hulburt is a mixotroph. It possesses chloroplasts and is photosynthetic, and it also feeds phagotrophically on another dinoflagellate, Prorocentrum minimum (Pavillard) Schiller. The species could be cultivated only in food-replete cultures. When kept in cultures without food, cellular chl a content and photosynthetic activity of G. resplendens decreased and growth ceased after a few days. In food-replete cultures, G. resplendens could grow strictly heterotrophically in darkness, but growth rate was then three times lower than in food-replete cultures kept in light. It is suggested that the main importance of phagotrophy is to acquire a growth factor essential to photosynthetic growth. The addition of soil extract or amino acids to the growth medium induced enhanced photosynthetic growth of the species even without the presence of particulate food, but only for approximately 2 weeks. Long-term starvation of G. resplendens led to loss of the ability to feed, and therefore starved cells eventually reached a point of no return where neither photosynthesis nor phagotrophy could sustain further growth. Light microscopical observations on G. resplendens revealed new morphological and behavioral details of the species.     

Free amino acids mimic the anabolic but not the proliferative effect of albumin in OK proximal tubular cells

When plasma proteins leak from circulation into the renal tubular lumen in the proteinuric renal diseases, nephrotoxicity of filtered albumin (and/or molecules bound to it) may be important in the subsequent development of tubulo-interstitial damage which contributes to the progression of the disease. When cultured opossum kidney (OK) proximal tubular cells were exposed to bovine serum albumin for 3 days in vitro, increased cell division ([3H]-thymidine incorporation) and cellular hypertrophy (increased protein/DNA ratio) were observed. Both effects were halved if defatted albumin was used. A trivial explanation for the growth responses is that free fatty acids carried on the albumin, and amino acids generated by intracellular degradation of the albumin, are exerting a non-specific growth effect as metabolic fuels which are oxidized to generate ATP. However, the water-soluble free fatty acid octanoate (1 mmol l(-1)) had no significant effect on protein/DNA ratio and a very variable stimulatory effect on [3H]-thymidine incorporation, whereas an essential amino acid mixture or 1 mmol/l(-1) l-Ala or l-Phe only increased the protein/DNA ratio. Furthermore no carnitine was added to the culture medium. This absence would have impaired mitochondrial transport (and hence oxidation) of long-chain free fatty acids derived from the albumin. l-Phe is also a poor substrate for mitochondrial oxidation in kidney. It is therefore concluded that the growth effects of albumin in OK proximal tubular cells are specific effects of the albumin protein and of the free fatty acids and amino acids derived from it, and not a non-specific effect on metabolic fuel supply.     

Temperature and nutrient availability control growth rate and fatty acid composition of facultatively psychrophilic <Emphasis Type="Italic">Cobetia marina</Emphasis> strain L-2

A facultative psychrophilic bacterium, strain L-2, that grows at 0 and 5°C as minimum growth temperatures in complex and defined media, respectively, was isolated. On the basis of taxonomic studies, strain L-2 was identified as Cobetia marina. The adaptability of strain L-2 to cold temperature was higher than that of the type strain and of other reported strains of the same species. When the bacterium was grown at 5–15°C in a defined medium, it produced a high amount of trans-unsaturated fatty acids. By contrast, in a complex medium in the same temperature range it produced a low amount of trans-unsaturated fatty acids. In the complex medium at 5°C, the bacterium exhibited a three-fold higher growth rate than that obtained in the defined medium. Following a temperature shift from 11 to 5°C, strain L-2 grew better in complex than in defined medium. Furthermore, when the growth temperature was shifted from 0 to 5°C both the growth rate and the yield of strain L-2 growing in complex medium was markedly enhanced. These phenomena suggest that an upshift of the growth temperature had a positive effect on metabolism. The effects of adding complex medium components to the defined medium on bacterial growth rate and fatty acid composition at 5°C were also studied. The addition of yeast extract followed by peptone was effective in promoting rapid growth, while glutamate addition was less effective, resulting in a cis-unsaturated fatty acid ratio similar to that of cells grown in the complex medium. These results suggest that the rapid growth of strain L-2 at low temperatures requires a high content of various amino acids rather than the presence of a high ratio of cis-unsaturated fatty acids in the cell membrane.     

Saturation transfer difference (STD) 1H-NMR experiments and in silico docking experiments to probe the binding of N-acetylneuraminic acid and derivatives to Vibrio cholerae sialidase

Saturation transfer difference (STD) (1)H NMR experiments were used to probe the epitope binding characteristics of the sialidase [EC 3.2.1.18] from the bacterium Vibrio cholerae, the causative agent of cholera. Binding preferences were investigated for N-acetylneuraminic acid (Neu5Ac, 1), the product of the sialidase catalytic reaction, for the known sialidase inhibitor 5-acetamido-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-non-2-enoic acid (Neu5Ac2en, 2), and for the uronic acid-based Neu5Ac2en mimetic iso-propyl 2-acetamido-2,4-dideoxy-alpha-L-threo-hex-4-enopyranosiduronic acid (3), in which the native glycerol side-chain of Neu5Ac2en is replaced with an O-iso-propyl ether. The STD experiments provided evidence, supporting previous studies, that Neu5Ac (1) binds to the sialidase as the alpha-anomer. Docking experiments using DOCK (version 4.0.1) revealed further information regarding the binding characteristics of the enzyme active site in complex with Neu5Ac2en (2) and the Neu5Ac2en mimetic (3), indicating an expected dominant interaction of the acetamide moiety with the protein.     

Phosphorus-31 Transverse Relaxation Rate Measurements by NMR Spectroscopy: Insight into Conformational Exchange Along the Nucleic Acid Backbone

Phosphorus ((31)P) NMR spectroscopy can provide important information about the dynamics of nucleic acids. In this communication, we propose an inversely detected (31)P transverse relaxation rate ( R (2)) measurement experiment. This experiment enables fast measurement of accurate (31)P transverse relaxation rates and provides the possibility to detect slow motions mapped by the phosphorus nuclei along the nucleic acid backbone. Dispersion curves show some (31)P nuclei experiencing chemical exchange in the millisecond time scale. Under the assumption of a two-state exchange process, the reduced lifetimes of the exchanging sites (tau(ex)) obtained are in accordance with base pair lifetime estimates deduced from imino proton exchange measurements.     

Stability of immobilized D-hydantoinase from Vigna angularis and repeated cycles of highly enantioenriched production of N-carbamoyl-D-phenylglycines

Summary. D-hydantoinase from Vigna angularis was immobilized by covalent linkage to aminopropyl glass beads. Thermal stability, resistance to storage at different pH values and temperatures of this biocatalyst were studied. This enzyme preparation was used as a catalyst to prepare enantioenriched N-carbamoyl-D-phenylglycine, N-carbamyl-D-p-fluorophenylglycine and N-carbamoyl-D-p-trifluoromethylphenylglycine, using a stirred batch reactor. Reactions were conducted during eight repeated reaction cycles, without loss of enzymatic activity or variation of the enantiomeric excess of the respective product (>98%).     

The facile HPLC enantioresolution of amino acids,peptides on naphthylethylcarbamate-β-cyclodextrin bonded phases using the acetonitrile-based mobile phase after their pre-column derivatization with phenyl isothiocyanate: factors that affect the resolution

Summary. A variety of -amino acids are enantioresolved for the first time on naphthylethylcarbamate--cyclodextrin bonded phases (i.e., SN- and RN--CD) using the acetonitrile-based mobile phase after their pre-column derivatization with phenyl isothiocyanate in alkaline medium. The resolution is better obtained on RN--CD phase and fails to reproduce if the amino acid is N-benzoylated or N-carbobenzyloxylated under the same chromatographic conditions. The enhanced resolution is believed to be due to the re-location of the hydrogen receptor site from sulfur to nitrogen on the isothiocyanyl fragment of derivatizing reagent, which in turn changes the enantioselectivity. Also, the sulfur atom is larger in size and subject to steric hindrance more significantly in comparison with oxygen. The carboxyl group of amino acid is essential toward a satisfactory resolution. The position of the amino group on the backbone affects the resolution as well. Finally, the resolution is either not observed or unsatisfactory in the reversed- or normal phase mode for most of the amino acids examined in this study.     

Analysis of 6-hydroxy-2-aminocaproic acid (HACA) as a specific marker of protein oxidation: The use of <Emphasis Type="Italic">N(O,S)</Emphasis>-ethoxycarbonyl trifluoroethyl ester derivatives and gas chromatography/mass spectrometry

Summary. An alteration of low density lipoprotein (LDL) apolipoprotein (apo) B-100 structure by direct oxidative modification is an important mechanism involved in atherogenesis. There is difficulty in quantifying this type of modification because a lack of specific assays. The use of N(O,S)-ethoxycarbonyl trifluoroethyl amino acid esters for a rapid and sensitive determination of 6-hydroxy-2-aminocaproic acid (HACA), a highly specific marker of metal catalyzed protein oxidation, by using standard gas chromatography/electron impact mass spectrometry, is discussed. The derivatives are formed by the unlabored reaction of amino acids with ethylchloroformate plus trifluoroethanol plus pyridine. Femtomole levels of HACA can be reproducible measured in different LDL preparations subjected to oxidative damage in the presence of iron or copper. HACA determination compares well with the measurement of carbonyl groups that are generally accepted as a nonspecific index of protein oxidation. Thus, the method could prove to be a sensitive assay for studying specific apoB-100 modification.     

A review of adsorption of amino acids on minerals: Was it important for origin of life?

Minerals more readily adsorb amino acids with charged R groups than uncharged R groups, so that the incorporation of amino acids with charged R groups into peptides would be more frequent than for amino acids with uncharged R groups. However, 74% of the amino acids in the proteins of modern organisms contain uncharged R groups. Thus, what could have been the mechanisms that produced peptides/proteins with more amino acids with uncharged R groups than precursors with charged R groups? Should we expect the composition of amino acids adsorbed on minerals to be similar to those of present proteins? Was the adsorption of amino acids on minerals important for the origin of life? The lipid world offers an alternative view of origin of life. Liposomes contributed to elongation of peptides as well as select hydrophobic amino acids and peptides. These experiments could be showing the mechanism, which hydrophobic amino acids have been selected. However, liposomes have no influence on the stereoselectivity in the oligomerization of amino acids. In the present paper, several other mechanisms are also discussed that could produce peptides with a greater proportion of amino acids with uncharged R groups.