Tamio Yamakawa

Glycolipids at the cell surface and their biological functions

A large variety of sphingoglycolipids occur at the surface of animal cells. The carbohydrate moieties vary, and each glycolipids may exhibit a special function, as an annular lipid, receptor or surface marker and matrix lipid.

Serum Concentrations of Bile Acid Glucuronides in Hepatobiliary Diseases

Bile acid glucuronides in the serum in various hepatobiliary diseases (36 cases) were quantitated by mass fragmentography and their clinical significance was discussed. Serum was added to defined amounts of deuterium-labeled bile acids and their glucuronide and sulfate derivatives, and the bile acids were separated into unconjugated, glucuronidated and sulfated groups after enzymatic cleavage of amide bonds. The liberated bile acids were quantitated by mass fragmentography. Bile acid glucuronides comprised about 7-8% of the total bile acids in the serum of various patients. Chenodeoxycholic acid was the major glucuronidated bile acid while cholic acid was mostly unconjugated. Lithocholic acid was almost all either sulfated or glucuronidated. In patients with obstructive jaundice, glucuronidated bile acids also comprised about 5%, although their absolute amounts were increased. In patients with liver cirrhosis, bile acid glucuronides were decreased, especially in decompensated cases, possibly as a result of hepatocellular dysfunction.

Genetic Regulation of GM2 Expression in Liver of Mouse

GM2 containing NeuGc was found to be a major ganglioside in the liver of most inbred strains of mouse we have examined so far. They were DBA/2, C3H/He, C57BL/6, BALB/c, and CBA mice. However, WHT/Ht mouse does not contain a detectable amount of GM2 (NeuGc) but has GM3 containing NeuGc as the major ganglioside in the liver. The positive expression of GM2 (NeuGc) was demonstrated in the liver of F1 hybrids between WHT/Ht and DBA/2 mice, as one of the possible combinations. Moreover, the expression of GM2 (NeuGc) in the liver was proved to be an autosomal dominant trait by ganglioside analysis of 34 individual mice of the F2 and backcross generations. WHT/Ht mouse was demonstrated to be a recessive homozygote lacking GM2 (NeuGc) expression. During the analysis, interestingly enough, the enhanced elongation of the sugar chain in the gangliosides from GM2 (NeuGc) to GM1 (NeuGc) and GD1 was observed in the liver of F1 mice and this elongation was segregated only in half members of F2 and backcross generations.

A reflection on the early history of glycosphingolipids

In the textbooks of biochemistry printed before 1950, we can find brief descriptions for phrenosine, kerasine, nervone, and hydroxynervone, but nothing more for glycosphingolipid related substances. These names are not used now but these four names indicate galactosylceramides containing C24 normal fatty acid, C24 o~-hydroxy fatty acid, C24 monounsaturated fatty acid, and C24 monounsaturated a-hydroxy fatty acid, respectively. Phrenosine and kerasine were isolated from human brain and named by J.L.W. Thudichum in 1884 [1]. Thudichum was a German clinician with training in chemistry. He also isolated and named several lipids such as cerebroside, sphingosine, ceramide, sphigomyelin, and cephalin from human brain. Thus, Thudichum can be considered the father of sphingolipid research. Glycosphingolipids and sphingolipids were originally derived from the brain, thus, these molecules were postulated to be important for neural functions. Thudichum seems to have been quite a determined person. He was not accepted by German academic society and moved to London where he started his own laboratory in his house and did experiments by himself. He published his lifes work in a book entitled A Treatise on the Chemical Constitution of the Brain in 1884 [1]. In May, t965, the 16th Colloquium of The German Society for Physiological Chemistry was held in Mosbach, a small city by the Neckar river, and I was invited to this meeting by E. Klenk. After the meeting, a ceremony took place in Biidingen, near Frankfurt, to dedicate a memorial plate indicating the house where Thudichum was born on

Mouse liver gangliosides.

The major gangliosides from mouse liver were purified and characterized by t.l.c., g.l.c., sialidase treatment, and a methylation study. GM3(NeuAc), GM3(NeuGc), GM2(NeuGc), GM1(NeuGc), and GDla(NeuGc, NeuGc) were identified. The structural identification of three of the gangliosides, GM2(NeuGc), GM1(NeuGc), and GDla(NeuGc, NeuGc), was supported by the results of 1H-n.m.r. analysis, and the structures of GM3(NeuGc), GM2(NeuGc), and GM1(NeuGc) were further confirmed by negative-ion fast-atom bombardment mass spectrometry. Ganglioside mapping showed that there was polymorphic variation of gangliosides in the liver of inbred strains of mice and that the major gangliosides were GM3(NeuGc) in WHT/Ht, GM2(NeuGc) in BALB/c and C3H/He, and GM2(NeuGc), GM1(NeuGc), and GDla(NeuGc, NeuGc) in ICR mice. Gangliosides containing N-acetylneuraminic acid, except for GM3(NeuAc), were not detected as major gangliosides in the strains of mice we analyzed.

Isolation of cerebroside containing glucose (Glucosyl ceramide) and its possible significance in ganglioside synthesis

A small amount of cerebroside containing glucose (glucosyl ceramide) was isolated from bovine brain by Florisil column chromatography and thin-layer chromatography. The fatty acids of the glucosyl ceramide were palmitic and stearic acids; small amounts of oleic and linoleic acids were present.Rat brain tissue slices, incubated with U-14C-glucose, incorporated more radioacivity into glucosyl ceramide than into galactosyl ceramide. From these results the possible metabolic significance of the brain glucosyl ceramide in ganglioside metabolism is discussed.

Stereochemical studies of hydrogen incorporation from nucleotides with fatty acid synthetase from Brevibacterium ammoniagenes.

The biosynthesis of fatty acids from malonyl-CoA and acetyl-CoA was investigated with an enzyme preparation which was purified 100-fold from Brevibacterium ammoniagenes. Fatty acids synthesized in the presence of D2O and stereospecifically deuterated NADPH and NADH were isolated and analyzed by mass chromatography to examine the localization of deuterium in the molecule. The following results were obtained: 1) HB hydrogen of NADPH was used for beta-ketoacyl reductase. 2) HB hydrogen of NADH was used for enoyl reductase. 3) Hydrogen atoms from water were found on the even-numbered methylene carbon atoms (2-hydrogen atoms per carbon atom) and some were also found on the odd-numbered methylene carbon. 4) Hydrogen atoms from NADPH were found on odd-numbered methylene carbon atoms (1-hydrogen per carbon). 5) Hydrogen atoms from NADH were also found on the odd-numbered methylene carbon atoms, but the number of incorporated hydrogen atoms was less than expected. The exchange of HB hydrogen of NADH with water catalyzed by enoyl reductase was suspected. 6) The exchange of methylene hydrogen atoms of malonyl-CoA with proton of water was suggested by 13C NMR analysis.

A locus controlling the activity of UDP-galactose:GM2(NeuGc) galactosyltransferase in mouse liver is linked to the H-2 complex

Genetic polymorphism in the expression of the GM1(NeuGc) ganglioside has been shown in the liver of inbred strains of mice. Through analysis of the gangliosides of H-2 congenic and recombinant strains, this polymorphism was demonstrated to be controlled by a locus mapped left outside of the H-2 complex on chromosome 17, and the locus was assumed to control the level of the activity of GM1(NeuGc) synthetase, UDP-galactose:GM2(NeuGc) galactosyltransferase (E.C.2.4.1.62) [Hashimotoet al., J Biochem (1983) 94:2049-54].In the present study we analyzed the genetic linkage between the activity of the galactosyltransferase and the H-2 haplotype. For this purpose, we selected two inbred strains of mice, WHT/Ht and BALB/c, because they have different levels of the transferase activity and show different H-2 haplotypes; the specific activity of the transferase obtained with BALB/c was one-eighth of that with WHT/Ht, and BALB/c expressed the la.7 antigen as one of the products encoded in their H-2d complex, whereas WHT/Ht did not. To analyze the linkage between these two phenotypes, WHT/Ht were mated with BALB/c to obtain the F1 mice, and the female F1 mice were then backcrossed to WHT/Ht. It was found that one half of the backcross generation expressed the la.7 antigen derived from BALB/c and had a significantly lower specific activity of the transferase than that of WHT/Ht, while the other half did not express the la.7 antigen but had the same specific activity of the transferase as that obtained with WHT/Ht.These results suggest that the locus controlling the level of the transferase activity in mouse liver is linked to the H-2 complex on chromosome 17.

Serum bile acid profiles in cerebrotendinous xanthomatosis

Non-sulfated bile acid concentrations in sera of 10 cases of cerebrotendinous xanthomatosis (CTX) were determined by mass fragmentography. Total bile acid (TBA) in serum was 0.492 +/- 0.436 microgram/ml (mean +/- SD) which was significantly lower than that (1.481 +/- 0.571) in healthy control sera. Cholic acid was 0.342 +/- 0.291 microgram/ml and was the dominant bile acid, which constituted 69.5% of TBA in serum. Chenodeoxycholic acid was 0.111 +/- 0.133 microgram/ml being a minor component in CTX sera, although it was the major bile acid in healthy control sera. Other bile acids such as deoxycholic acid, lithocholic acid and ursodeoxycholic acid were scarcely detected. Subnormal TBA level and deranged bile acid composition in CTX sera may reflect the defect of bile acid biosynthesis in CTX patients. Determination of serum bile acid may be useful in the diagnosis of CTX.

THE INCORPORATION OF LABELLED ACETATE INTO CEREBROSIDE AND OTHER LIPIDS OF THE DEVELOPING MOUSE BRAIN

—Cerebroside in the brain is highly localized in myelin and has a relatively slow turnover rate. The aim of this study was to evaluate the true cerebroside biosynthetic activity under conditions in which the degradation and reutilization of brain lipids were as small as possible. The 3‐week‐old mice were decapitated at 0·5, 1, 2·5, 5 and 15 min after the intraperitoneal injection of labelled acetate and the incorporation of radioactivity into each lipid class was examined. Even at 0·5 min, a considerable amount of radioactivity was found in simple lipids, especially in the free fatty acid fraction, and in the course of time the radioactivity of complex lipids increased. On the other hand, the incorporation of radioactivity into cerebrosides was extremely small throughout the experimental period. Results indicated that the low radioactivity of cerebroside might be due to its high content of long‐chain fatty acids which were weakly labelled. The radioactivity of the sphingosine moiety was also low. In short, one of the rate‐limiting steps of cerebroside synthesis in brain might exist in long‐chain fatty acid and sphingosine synthesis. In addition, the incorporation curves of each component of cerebroside were compared with each other and the difference of the incorporation pattern of non‐hydroxy fatty acids of cerebroside was noted.