Studies of the action of ceramide-like substances (d- andl-PDMP) on sphingolipid glycosyltransferases and purified lactosylceramide synthase

We have studied the effects ofD-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) and itsL-enantiomer on glycosphingolipids in cultured normal human kidney proximal tubular cells. We found thatD-PDMP exerted a concentration-dependent reduction in the metabolic labelling and cellular levels of glucosylceramide (GlcCer), lactosylceramide (LacCer), and the globo-series glycosphingolipids, GbOse₃Cer and GbOse₄Cer. It also directly inhibited the activity of UDP-glucose:ceramide 1 4-glucosyltransferase (GlcT-1) and UDP-galactose: GlcCer 1 4 galactosyltransferase (GalT-2). In contrast,L-PDMP had opposite effects on the metabolic labelling of GlcCer, LacCer, and GbOse₃Cer. The levels of GlcCer and LacCer were increased, while the labelling and level of GbOse₄Cer were strongly reduced. Purified GalT-2 from human kidney was inhibited byD-PDMP and stimulated byL-PDMP. It appears likely that the different glycosphingolipid glycosyltransferases possess similar binding sites for the ceramide moiety, which are blocked by binding toD-PDMP and, in the case of GbOse₄Cer synthase, byL-PDMP as well. The stimulatory effects ofL-PDMP on GlcCer and LacCer synthases may be the result of binding to a modulatory site on the glycosyltransferases; in intact cells, the enzyme-analog complex may afford protection against the normal catabolic inactivation of the enzymes.Abbreviations GalT-2 UDP-galactose:GlcCer -galactosyltransferase - GbOse₃Cer Gal1 4Gal1 GlcCer - GbOse₄Cer GalNAc1 3Gal1 4Gal1 GlcCer - GlcCer glucosylceramide - GlcT-1 UDP-glucose:ceramide -glucosyltransferase - GSLs glycosphingolipids - LacCer lactosylceramide - PDMP threo-1-phenyl-2-decanolyamino-3-morpholino-1-propanol     

Immobilization of Proteus vulgaris for the reduction of 2-oxo acids with hydrogen gas or formate to d-2-hydroxy acids

For the stereoselective reduction of 2-oxo acids by hydrogen gas or formate to d-2-hydroxy acids, anaerobically grown Proteus vulgaris cells were immobilized in alginate, -carrageenan, chitosan, polyurethane and polyacrylamide acylhydrazide. With the exception of the last matrix, immobilization led to a decrease in the apparent activity, probably caused by diffusional limitations. Chitosan or polyurethane-entrapped cells kept their initial catalytic activity for over more than 600 h in a continuous working period. In both matrices the cells could be partially reactivated by incubation of the immobilisates in growth medium. Polyurethane-immobilized cells (and also cell membranes) were repeatedly usable. After 30 batch operations, 30–40% of the initial reduction rate was still detectable. Chitosan-immobilized cells did not lose any activity during 17 months of storage at 4° C under exclusion of oxygen. Correspondence to: H. Simon     

Synaptosomal release of newly-synthetized or recently accumulated amino acids. Differential effects of kainic acid on naturally occurring excitatory amino acids and on [d-3H]aspartate

Kainic acid, a powerful neuroexcitant and neurotoxin, stimulates the release of naturally occurring excitatory amino acids, l-glutamate and l-aspartate, from hippocampal synaptosomes. The release stimulation affects in a similar way both the general pool of the two amino acids and the fraction of l-glutamate and l-aspartate, newly-synthetized from precursors or recently accumulated through the high-affinity uptake mechanism. Kainic acid exerts its stimulatory action on the basal release of the two amino acids as well as on the high K⁺-stimulated release of l-glutamate. Kainic acid has, however, different effects on the release of exogenously accumulated [d-³H]aspartate. In particular, the high K⁺-stimulated release of this false transmitter is strongly inhibited by 1 mM kainic acid. The present data confirm the presynaptic action of kainic acid on the general as well as on the recently-formed pools of naturally occurring excitatory amino acids. At the same time, our results suggest that [d-³H]aspartate is not a reliable substitute for l-glutamate and l-aspartate, in release studies and that the radioactivity released after preloading with [d-³H]aspartate does not necessarily reflect the release of naturally occurring excitatory amino acids.     

Studies on platelet lipoxygenase specificity towards icosapolyenoic and docosapolyenoic acids

Two docosapolyenoic acids (22:5(n-3) and 22:5(n-6)) were isolated from the liver of normal and 18:3(n-3)-deficient trout, respectively. They were prepared by combined thin-layer chromatography (TLC) and reversed-phase high performance liquid chromatography (HPLC). Their purity, checked by capillary gas liquid chromatography, was greater than 95%. Each fatty acid was oxygenated into monohydroxy derivatives by human platelets. The hydroxy compounds were purified by TLC and HPLC and then derivatized for gas chromatography-mass spectrometry analysis. Whereas 22:5(n-6) was only converted into 14-OH-22:5, three hydroxy derivatives (11, 13 and 14) were obtained from 22:5(n-3). However, 13-hydroxy was not formed in the presence of aspirin, indicating that platelet lipoxygenase catalyses the formation of both 11- and 14-hydroxy derivatives from 22:5(n-3), as described previously, from 22:6(n-3). Further studies showed that 22:4(n-6) and 20:5(n-3) were only converted into 14- and 12-hydroxy derivatives. We conclude then that, besides the well-known n-9 oxygenation, lipoxygenase of human platelets is able to catalyse an n-12 oxygenation on docosapolyenoic acids of the n-3 family.     

Studies on the ribonucleic acids of fresh and processed tea leaves

1. A marked decrease in the total RNA content during the withering process of tea leaves was found. During the fermentation process, there was a small but significant decrease in the total RNA content. 2. During isolation of RNA from tea leaf tissues, the action of leaf ribonuclease was minimized by the addition of sodium dodecyl sulphate during extraction; 1% (w/v) sodium dodecyl sulphate in 0.2m-tris-hydrochloric acid buffer, pH8.0, containing 0.005% EDTA was found to be most efficient for the extraction and gave about 93% yield. 3. The total RNA preparations isolated from fresh, withered and fermented tea leaves were compared with regard to nucleotide composition and spectral characteristics. The total RNA preparations from all three sources contained more purines than pyrimidines (purine/pyrimidine ratio 1.47-1.52).     

Studies on plant growth-regulating substances XXVI. Isatic and anthranilic acids

The plant growth-regulating activities of isatic acid and twenty-six of its derivatives, together with the twenty-seven corresponding anthranilic acids, have been assessed in the wheat cylinder, the pea segment and the pea curvature tests. Activity was sustained by substitution in the 4- and 5-positions of isatic acid but decreased by substitution in the 3- and 6-positions. In the anthranilic acid series, the parent acid was inactive but the introduction of a large grouping (bromine or iodine) into the 5-position conferred activity. The 3,6- and 5,6-dichloro and the 3,6-dibromo acids were also active; compounds substituted in the 4-position to the carboxyl group or disubstituted in the 3,5-positions, were, as expected, inactive. In metabolism experiments on wheat and pea tissues with isatic and 5-chloroisatic acids the corresponding anthranilic acid was formed, together with an unidentified non-acidic metabolite in each case. There was no evidence that the growth regulating activity of isatic acids was related to this breakdown and it is concluded that the acids possess activity per se. The results are briefly discussed in terms of recent theories relating chemical structure to plant growth-regulating activity.     

Studies on dissimilatory sulfate-reducing bacteria that decompose fatty acids

Three strains (2ac9, 3ac10 and 4ac11) of oval to rodshaped, Gram negative, nonsporing sulfate-reducing bacteria were isolated from brackish water and marine mud samples with acetate as sole electron donor. All three strains grew in simple defined media supplemented with biotin and 4-aminobenzoic acid as growth factors. Acetate was the only electron donor utilized by strain 2ac9, while the other two strains used in addition ethanol and/or lactate. Sulfate served as electron acceptor and was reduced to H₂S. Complete oxidation of acetate to CO₂ was shown by stoichiometric measurements with strain 2ac9 in batch cultures using sulfate, sulfite or thiosulfate as electron acceptors. With sulfate an average growth yield of 4.8 g cell dry weight was obtained per mol of acetate oxidized; with sulfite or thiosulfate the growth yield on acetate was about twice as high. None of the strains contained desulfoviridin. In strain 2ac9 cytochromes of the b- and c-type were detected. Strain 2ac9 is described as type strain of the new species and genus, Desulfobacter postgatei.     

Studies on dissimilatory sulfate-reducing bacteria that decompose fatty acids

Gliding motility, ultrastructure and nutrition of two newly isolated filamentous sulfate-reducing bacteria, strains 5ac10 and 4be13, were investigated. The filaments were always attached to surfaces. Growth was supported by addition of insoluble aluminium phosphate or agar as substrata for gliding movement. Electron microscopy of ultrathin sections revealed cell walls characteristic of Gramnegative bacteria; the undulated structure of the outer membrane may pertain to the translocation mechanism. Intracytoplasmic membranes were present. Acetate, higher fatty acids, succinate or fumarate served as electron donors and carbon sources. Strain 5ac10 grew also with lactate, but not with benzoate that was used only by strain 4be13. Strain 5ac10 was able to grow slowly on H₂ plus CO₂ or formate in the presence of sulfate without additional organic carbon source. The capacity of complete oxidation was shown by stoichiometric measurements with acetate plus sulfate. Both strains contained b- and c-type cytochromes. Desulfoviridin was detected only in strain 5ac10. The two filamentous gliding sulfate reducers are described as new species of a new genus, Desulfonema limicola and Desulfonema magnum.     

Studies on the secondary structure of nuclear ribonucleic acids

Rat liver nuclei were separated into two fractions, the nucleolus and the nucleoplasm, by a combined salt-enzymic extraction. RNA was purified from both sources and analysed on sucrose density gradients. In both fractions a prominent heterogeneous RNA class with mean sedimentation coefficient of 18S was found. This material was analysed by measuring the rate of reaction with formaldehyde, the ultraviolet absorbance-temperature profile, the spectrophotometric observation of conformational changes as a function of pH, the spectrophotometric titration of uracil and guanine residues and the effect of both temperature and ionic strength on the spectrophotometric titration of cytosine residues. Nucleoplasmic 18S RNA fraction exhibited, on heating and also by adjustment of the pH to 2.5, a hyperchromicity of about 16%, close to that observed, in control experiments, for ribosomal RNA (22%). Titration of cytosine residues in solutions containing 1mm-NaCl and 0.1m-NaCl yielded pK values equal to 4.41 and 3.84 respectively. These results suggest that this RNA fraction is composed of structurally complex polymers. The hypochromicity of the nucleolar 18S RNA fraction determined by heating or adjusting the pH to 2.5, was not greater than 6% of the initial value. The rate of reaction with formaldehyde was 88% of that observed for the hydrolysed 18S fraction which suggested only 12% hydrogen bonding. pK values for uracil and guanine residues were 10.1 and 10.05 respectively. Titration of cytosine residues yielded a pK of 4.10, which was found to be independent of temperature and ionic strength variations.     

Studies on furan fatty acids of salmon roe phospholipids

Mature salmon roe lipids were found to consist of triacylglycerols (63%), phospholipids (30%), sterols (4.2%), steryl esters (0.7%), and other minor components. In the steryl esters and phospholipids, furan fatty acids were detected instead of the triacylglycerols of the testes lipids in male fish. The representative 12,15-epoxy-13,14-dimethyleicosa-12,14-dienoic acid (F6) amounted to 3.8% and 0.6% of the total fatty acids in each fraction, respectively. However, the absolute amount of the acid in the phospholipid was much more than that contained in the steryl esters. The characteristic distribution of the furan acids found in the phospholipids was common to the steryl esters in the liver. Large amounts of furan acids were contained in phosphatidylcholine (PC) rather than in phosphatidylethanolamine. For positional analysis of furan fatty acids in PC, furan-containing species in the molecule were concentrated fourteenfold by using selective hydrogenation and repeated silica gel column chromatography. A series of furan fatty acids in PC was found to be exclusively linked to the sn-1 position. The amount of the acids in the roe exclusively linked to the sn-1 position. The amount of the acids in the roe phospholipids was comparable with that in the testes triacylglycerols. The physiological roles of furan fatty acids are discussed.