The Sphingenine/Sphinganine Ratio in Sphingolipids of Transplantable Rat Tumors Depends on a Transplantation Organ

The content of sphingenine (sphingosine) and sphinganine was determined in the total pool of sphingomyelin and ceramide in the rat tumors transplanted subcutaneously and intrahepatically. The sphingenine/sphinganine ratio in the subcutaneously transplanted sarcoma M1 and cholangiocellular carcinoma RS1 was lower than that in the sphingolipids of the intrahepatically transplanted tumors. However, the sphingenine/sphinganine ratio in the subcutaneously transplanted rat hepatoma 27 was higher than in the intrahepatically transplanted hepatoma. These observations indicate that the sphingenine/sphinganine ratio in sphingolipids of tumors depends on the tumor type and its cellular microenvironment.     

The changes in the sphingenine/sphinganine ratio in sphingolipids of transplantable rat tumors depends on a transplantation organ

The content of sphingenine (sphingosine) and sphinganine was determined in the total pool of sphingomyelin and ceramide in the rat tumors transplanted subcutaneously and intrahepatically. The sphingenine/sphinganine ratio in the subcutaneously transplanted sarcoma M1 and cholangiocellular carcinoma RS1 was lower than that in the sphingolipids of the intrahepatically transplanted tumors. However, the sphingenine/sphinganine ratio in the subcutaneously transplanted rat hepatoma 27 was higher than in the intrahepatically transplanted hepatoma. These observations indicate that the sphingenine/sphinganine ratio in sphingolipids of tumors depends on the tumor type and its cellular microenvironment.     

A ferredoxin from the thermohalophilic bacterium Rhodothermus marinus

The substrate specificity of human sphingosine kinase was investigated using a bacterially expressed poly(His)-tagged protein. Only the D-erythro isomer of the sphingoid bases, sphinganine and sphingenine, was effectively phosphorylated. Long chain 1-alkanols, alkane-1,2-diols, 2-amino-1-alkanol or 1-amino-2-alkanol and short chain 2-amino-1,3-alkanediols were very poor substrates, indicating that the kinase is recognizing the chain length and the position of the amino and secondary hydroxy group. A free hydroxy group at carbon 3 is not a prerequisite, however, since 1-O-hexadecyl-2-desoxy-2-amino-sn-glycerol was an efficient substrate with an apparent K(m) value of 3.8 microM (versus 15.7 microM for sphingenine). This finding opens new perspectives to design sphingosine kinase inhibitors. It also calls for some caution since it cannot be excluded that this ether lipid analogue is formed from precursors that are frequently used in research on platelet activating factor or from phospholipid analogues which are less prone to degradation.     

N-acyl migration in ceramides

Upon exposure of truncated ceramides, such as N-acetyl-sphingenine, and long-chain ceramides to moderate acidic conditions, three derivatives are formed. Two of them turned out to be O-acylated sphingenine, 1-O- and 3-O-acyl-sphingenine, and the other was identified as sphingenine. Truncated dihydroceramides (e.g., N-acetyl- and N-hexanoyl-sphinganine) also show this type of rearrangement, which is therefore not related to the presence of the allylic hydroxy group or to the length of the N-acyl chain. Of particular concern is the fact that the O-acylated compounds, which can be considered sphingoid base analogs, can be formed in chloroform or chloroform-methanol mixtures upon storage. For long-term storage, methanol or dichloromethane is the preferred solvent. A procedure to document the presence/formation of such O-acylated sphingoid bases in ceramide solutions in the picomole range, based on reversed-phase chromatography after derivatization of their amino group with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, is presented.     

Content and structure of ceramide and sphingomyelin and sphingomyelinase activity in mouse hepatoma-22

The relative content of phosphatidylcholine is lower and that of sphingomyelin is higher in transplantable fast growing mouse hepatoma-22, thus decreasing their ratio approximately 2.5-fold versus normal liver. The ceramide content and the neutral sphingomyelinase activity is markedly higher (3- and 6.5-fold, respectively), whereas the acid sphingomyelinase activity is 4-fold lower in hepatoma-22 versus normal liver. The content of saturated fatty acids in ceramide and sphingomyelin of hepatoma-22 is higher than in normal liver. All sphingolipids of hepatoma-22 contain a considerable amount (25-37%) of sphinganine (dihydrosphingosine) along with sphingenine (sphingosine), whereas sphingolipids of normal liver contain predominantly sphingenine (over 95%). These results indicate that the activity of enzymes involved in sphingolipid biosynthesis and catabolism is disturbed in the transplantable mouse hepatoma-22 compared to normal liver.     

Do sphingoid bases interact with the peroxisome proliferator activated receptor alpha (PPAR-alpha)?

In a search for possible endogenous ligands of nuclear receptors that are activated by peroxisome proliferators (PPARs), a solid phase binding assay was developed employing recombinant mouse PPAR-alpha, containing a myc-epitope, a histidine repeat and a kinase A domain. After in vitro labelling with 32P-gamma-ATP, the binding of purified 32P-PPAR-alpha to a panel of different natural and synthetic lipids, immobilized on silica layers, was evaluated. Autoradiographs of the silica layers revealed binding to two main classes of lipophilic compounds. A first class comprised (poly)unsaturated fatty acids. Compounds belonging to a second class were characterized by the presence of an overall positive charge such as long chain amines, sphingoid bases (sphingenine), and lysoglycosphingolipids (psychosine). PPAR-alpha did not bind to N-acylated sphingoid bases (ceramides) or to sphingenine phosphorylated at the primary hydroxy group (sphingenine-1-phosphate). The binding of PPAR-alpha to sphingoid bases might be of interest given the role of PPAR-alpha and sphingolipids in various cellular processes.     

Occurrence of galactosylceramide in pig epidermal cells

Monoglycosylceramides were isolated from pig epidermal cells which had been prepared free from dermal elements. The most polar glycolipid among the five isolated monoglycosylceramides was galactosylceramide. The galactosylceramide was composed of alpha-hydroxypalmitic acid and 16- and 18-carbon chain sphingenine, being quite different from epidermal glucosylceramides. This is the first report demonstrating the occurrence of galactosylceramide in mammalian epidermal cells.     

Synthesis of ceramides using N-hydroxysuccinimide esters

Fatty acyl esters of N-hydroxysuccinimide have been used to directly N-acylate sphingenine or sphinganine, forming the corresponding ceramides. The reaction proceeds in excellent yield (84-96%) from small amounts of starting material (10-20 mg). The product ceramides are pure after one recrystallization.     

Human blood group glycosphingolipids of porcine erythrocytes.

Two glycosphingolipids with human blood group A and H antigenicity were isolated from porcine erythrocyte membranes which were obtained from the pooled blood. The yield of the A- and H-antigenic glycolipids was approximately 0.2 and 0.1% of total neutral glycolipids, respectively. No B antigen was detected. Through several methods the porcine erythrocyte antigens were all found to belong to lactoseries (type 1 chain), IV2Fuc alpha, IV3GalNAc alpha Lc4Cer for type A and IV2-Fuc alpha Lc4Cer for type H, in contrast to the antigenic glycolipids in human erythrocytes, which mostly belong to neolactoseries (type 2 chain). The constituent fatty acids of the A antigen were 75% normal acids and 25% 2-hydroxy acids, and the long chain base was 95% sphingenine. This is the first demonstration of the A- and H-antigenic glycolipids on erythrocytes of pig in whose gastric mucin the human blood group A and H substances have been demonstrated.     

Synthesis of threo-4,5-dihydroxy diastereomers of sphinganine;.

The threo-4,5-dihydroxy diastereomers of sphinganine were prepared by the following sequence of reactions: (a) benzoylation of sphingenine to the tribenzoyl derivative; (b) osmylation followed by resolution of the mixture of threo-4,5-dihydroxy tribenzoyl (DHTBS) diastereomers; and (c) alkaline hydrolysis to yield the threo-4,5-dihydroxysphinganines (DHS). Carbon atoms 4 and 5 of the high and low melting threo-4,5-DHTBS diastereomers and the compounds derived from them were tentatively assigned 4R, 5S and 5R configurations, respectively;     

Synthesis and degradation of long-chain base phosphates affect fumonisin B<Subscript>1</Subscript>-induced cell death in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Fumonisin B₁ (FB₁), an inducer of cell death, disrupts sphingolipid metabolism; large accumulations of de novo synthesized free long-chain bases (LCBs) are observed. However, it remains unclear whether tolerance to FB₁ toxicity in plants is connected with preventing the accumulation of free LCBs through their phosphorylation. Here a workflow for the extraction, detection and quantification of LCB phosphates (LCBPs) in Arabidopsis thaliana was developed. We studied the effect of expression of genes for three enzymes involved in the synthesis and degradation of LCBPs, LCB kinase (LCBK1), LCBP phosphatase (SPP1) and lyase (DPL1) on FB₁-induced cell death. As expected, large accumulations of saturated free LCBs, dihydrosphingosine and phytosphingosine, were observed in the FB₁-treated leaves. On the other hand, a high level of sphingenine phosphate was found in the FB₁-treated leaves even though free sphingenine was found in low amounts in these leaves. In comparison of WT and spp1 plants, the LCBP/LCB ratio is likely to be correlated with the degree of FB₁-induced cell death determined by trypan blue staining. The FB₁-treated leaves in dpl1 plants showed severe cell death and the elevation of free LCBs and LCBPs. LCBK1-OX and -KD plants showed resistance and sensitivity to FB₁, respectively, whereas free LCB and LCBP levels in FB₁-treated LCBK1-OX and -KD plants were moderately different to those in FB₁-treated WT plants. Overall, the findings described here suggest that LCBP/LCB homeostasis is an important topic that participates in the tolerance of plant cells to FB₁.     

Sphinganine in Sphingomyelins of Tumors and Mouse Regenerating Liver

Contents of sphingenine (sphingosine) and sphinganine were studied in sphingomyelins of transplantable mouse tumors (hepatoma-22, melanoma B16, Lewis lung carcinoma, intestine carcinoma) and rat nephroma RA. The content of sphinganine was increased in sphingomyelins of hepatoma-22 and nephroma RA compared to sphingomyelins of liver and kidneys. Significant contents of sphinganine were also found in sphingomyelins of other studied tumors. The content of sphinganine in regenerating mouse liver (30 h after hepatectomy) was normal. The data suggest that disorders should exist in biosynthesis of sphingoid bases in tumors but not in normal rapidly proliferating tissue.     

Characterization of the lacto series of gangliosides, especially of disialo-lacto-N-norhexaosyl ceramide isolated from adult bovine nasal cartilage

A disialosylganglioside was isolated from adult bovine nasal cartilage, and its structure was determined by analysis of sugar composition, permethylation analysis, exoglycosidase treatment, and mild acid hydrolysis. The structure of this ganglioside was identified as disialo-lacto-N-norhexaosyl ceramide, NeuNAc(alpha 2-8)NeuNAc-(alpha 2-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(1-4)Glc(1-1)Cer. Furthermore, we also isolated from this cartilage gangliosides whose structures were presumed to be monosialo-lacto-N-norhexaosyl ceramide, and mono- and disialo-lacto-N-neotetraosyl ceramide. The major fatty acids of the four gangliosides isolated were palmitic, stearic, behenic and lignoceric acids. The predominant long chain bases were sphingenine, heptadecasphingenine and hexadecasphingenine.     

Apparatus for Preparation of Bacterial Extracellular Enzymes

A method is described for the growth of bacteria within dialysis tubing to yield extracellular enzymes, or, possibly, other nondialyzable extracellular products, in concentrated form.     

A new simple method of preparing actin from chicken gizzard

A simple method for preparing actin from chicken gizzard was described. This method takes advantage of a property of gizzard tropomyosin, that is, that it does not form Mg paracrystals readily.     

Visual detection of fructose-1,6-P2 aldolase after electrophoresis by its oxidative paracatalytic reaction

A simple method for detecting aldolase activity is described which is based on the reduction of the oxidation-reduction indicator porphyrindin to its leuco form by paracatalytic oxidation of an aldolase substrate intermediate.     

Colorimetric determination of agarose-immobilized proteins by formation of copper-protein complexes

A simple, reliable, and sensitive colorimetric procedure for the determination of proteins bound to agarose is described. The procedure utilizes the capacity of diethyldithiocarbamate to react with cupric ions resulting in a complex of dark yellow color. The extent of reduction in the color, due to chelation of Cu2+ by the immobilized proteins, indicates the amount of protein. The optimum conditions for the determination of immobilized proteins are investigated. The formation of Cu2+-protein complex proceeds stepwise until enough excess of Cu2+ is present to form the final complex. The reliability of the procedure requires that all the protein species, samples and standards, are in the final Cu2+-protein complex form. A comparative study on the determination of different proteins bound to agarose using this method as well as other methods, such as protein balance, modified Lowry reaction, and direct ultraviolet spectrophotometry, is described. The method is substantially more reliable, accurate, and simple than previously described methods.     

In vivo incorporation of palmitic acid and galactose in glycolipids of Trypanosoma cruzi epimastigotes

Incubation of epimastigote forms of Trypanosoma cruzi with (3H)-palmitic acid and (3H)-galactose, respectively, results in the incorporation of both precursors into the lipopeptidophosphoglycan (LPPG) and in at least two glycosphingolipids. Palmitic acid was incorporated into sphinganine and sphingenine, identified after hydrolysis, respectively, of the major and the minor glycosphingolipid. The purified glycosphingolipids labeled with either precursor migrated with a Rf similar to that of a sample labeled by periodate oxidation and borotritide reduction in which sialic acids have been previously characterized. This, together with the fact that the palmitic acid labeled glycosphingolipids were partially hydrolysed with neuraminidase favors a ganglioside-like structure for these compounds.     

A sensitive and specific method for quantitative estimation of carbamylation in proteins

A simple, rapid, reproducible, and specific micromethod for the estimation of carbamylation of proteins is described. The method is based on permanganate oxidation of radioactive carbamyl derivatives of protein to form urea and on the specific decomposition of urea by urease.     

Determination of half-reaction equilibrium in a ping-pong enzyme mechanism

Substituted enzyme (or ping-pong) mechanisms usually involve enzymes that exist in two forms that alternate during the catalytic reaction. A method is described here for determining the position of the equilibrium of a half reaction in a ping-pong enzyme mechanism that is based on the kinetics of the burst reaction which occurs upon addition of reactants that recycle the enzyme from one form to another. The theoretical basis for the analysis is developed, and the method is applied to the half reaction of the aldimine form of aspartate transaminase with difluoro-oxaloacetate. Special issue dedicated to Herman Bachelard