Search

Edible fungi, mushrooms, are a popular food in Japan and over 15 cultured mushroom species are available at the food markets. Recently, constituents or ingredients of edible mushrooms have drawn attention because possibilities have been seen for their medical usage. Mycoglycolipids (basidiolipids) of higher mushrooms have been characterized as glycosylinositolphosphoceramides, having a common core structure of Manalpha1-2Ins1-[PO(4)]-Cer and extensions of Man, Gal, and/or Fuc sugar moieties. Seven mycoglycolipids were purified from the edible mushroom Hypsizygus marmoreus by successive column chromatography on ion exchange Sephadex (DEAE-Sephadex) and silicic acid (Iatrobeads). Their structures were characterized to be Ins1-[PO(4)]-Cer (AGL0), Manalpha1-2Ins1-[PO(4)]-Cer (AGL1), Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL2), Fucalpha1- 2Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL3), Galalpha1-3(Fucalpha1-2)Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL4), Galalpha1-2Galalpha1-3(Fucalpha1-2)Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL5), and Galalpha1-2Galalpha1-2Galalpha1-3(Fucalpha1-2)Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL6) by sugar compositional analysis, methylation analysis, periodate oxidation, partial acid hydrolysis, enzymatic hydrolysis, immunochemical analysis, gas-liquid chromatography (GC), gas chromatography-mass spectrometry (GC-MS), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and (1)H-nuclear magnetic resonance spectroscopy (NMR). Ceramide constituents of their mycoglycolipids were composed of phytosphingosine as the sole sphingoid, and mainly 2-hydroxy C22:0 and C24:0 acids as the fatty acids. By immunochemical detection, the terminal structure of AGL4, Galalpha1-3(Fucalpha1-2)Galbeta-, was shown to have blood group type B activity. Galalpha1-2 and its repeating sequence in AGL5 and AGL6 are novel structures on the nonreducing sugar end in mycoglycolipids. These two mycoglycolipids in H. marmoreus distinguish it from other basidiomycetes.

For chemical characterization of glycosphingolipids, it is necessary to determine the chemical compositions of three constituents, i.e., sugars, fatty acids, and sphingoids. A new rapid analytical method is described using a one-pot reaction in a household microwave oven, producing sugars, fatty acids, and especially sphingoids free of by-products, from a single aliquot of a biological sample. Glycosphingolipids were hydrolyzed by microwave exposure with 0.1 M NaOH/CH(3)OH for 2 min followed by 1 M HCl/CH(3)OH for 45 s. The alkaline methanolysis step produced intermediate lysoglycosphingolipids virtually free of by-products such as the O-methyl ethers usually seen. The fatty acid methyl esters were extracted with n-hexane, and other reaction products were dried, taken up in aqueous alkaline methanol, and shaken with chloroform. Sphingoids partitioned into the organic phase under these conditions, whereas the sugar portion that partitioned into the aqueous phase was re-N-acetylated and remethanolyzed for 30 s by microwave exposure. Analysis of the profiles of glycosphingolipid constituents obtained using the microwave oven method showed that they were quantitatively and qualitatively comparable to those obtained by time-consuming conventional methods, which require reaction for several hours. Analysis of the three constituents, including analysis by gas chromatography, may be obtained within 1 day using the method described here.

A novel glycosphingolipid containing inositol phosphate as an acidic group has been demonstrated in whole tissues of the porcine roundworm, Ascaris suum. The thin layer chromatographic pattern of the total acidic glycolipid revealed the presence of several components, of which a major component (named AGL) with positive reactions toward both orcinol-sulfuric acid (sugar) and molybdate (phosphate) spray reagents was isolated and purified by the use of successive column chromatography on DEAE-Sephadex and silicic acid (latrobeads). From structural studies including compositional sugar analysis, hydrogen fluoride degradation, methylation analysis, periodate oxidation, proton magnetic resonance spectroscopy and fast atom bombardment mass spectrometry, the structure of AGL was deduced to be Gal alpha 1-2Ins(1-->)-P-Cer. Aliphatic constituents were lignoceric acid and its 2-hydroxy homologue as the principal fatty acids, and octadecasphinganine and branched heptadecasphinganine as the major sphingoids.

The novel amphoteric glycosphingolipids containing a choline phosphate were purified from whole tissues of the earthworm, Pheretima hilgendorfi. Their chemical structures were completely characterized as cholinephosphoryl-->6(Man alpha 1-4)Gal beta 1-6Gal beta 1-1Cer (cholinephosphorylmannosylneogalabiaosylceramide, named PGL3a) and cholinephosphoryl-->6Gal beta 1-6Gal beta 1-6Gal beta 1-1Cer (cholinephosphorylneogalatriaosylceramide, named PGL3b) by compositional sugar, fatty acid and sphingoid analyses, hydrogen fluoride degradation, partial acid hydrolysis, methylation analysis, exoglycosidase degradation, proton magnetic resonance spectroscopy and fast atom bombardment mass spectrometry. The ceramide moieties of these lipids consisted of 22:0, 23:0 and 24:0 acids as major fatty acids, and branched octadeca- and nonadeca-4-sphingenines and octadeca-4-sphingenine as main sphingoids. Since the oligosaccharides and the ceramide moieties of PGL3a and PGL3b were identical with those of neutral glycosphingolipids found in this organism, the biosynthesis of the amphoteric glycolipids may occur by the addition of a choline phosphate residue to the corresponding neutral glycolipids, Man alpha 1-4Gal beta 1-6Gal beta 1-1Cer or Gal beta 1-6Gal beta 1-6Gal beta 1-1Cer.

The enzyme defect in Farber disease, a deficiency of acid ceramidase, has been demonstrated in cultured skin fibroblasts, which provides a means of confirming the diagnosis during life. The assay can also be performed using cultured amniotic fluid cells and is a potential tool for detection of carriers of the disease.

The high-performance liquid chromatographic (HPLC) method for the determination of unsaturated sulfated disaccharides is a comprehensive and reliable method which expedites ensymatic studies of isomeric chondroitin sulfates. Responses for these unsaturated disaccharides derived from urinary chondroitin sulfates were linear from 100 ng to 10 micrograms injected and good quantitation was obtained for 25 microliters or less of samples placed on the column. This method which is more sensitive and accurate than methods now being used has considerable potential for the chemical diagnosis of patients with mucopolysaccharidoses and for the clarification of glycosaminoglycan structure. The isomeric chondroitin sulfates in urines from patients with mucopolysaccharidoses were studied by enzyme digestion with chondroitinases followed by HPLC determination of the sulfated unsaturated disaccharides produced. Evaluation by HPLC of the unsaturated 4-sulfated disaccharide produced by digestion of the urinary GAG with chondroitinases ABC and AC revealed rapidly and quantitatively the large amounts of dermatan sulfate present in Hurler, Hunter, and Maroteaux-Lamy urines. Chondroitin 4-sulfate predominated in Sanfilippo urinary isomeric chondroitin sulfates whereas chondroitin 6-sulfate and chondroitin 4-sulfate were shown to be present in nearly equal amounts in Morquio urine. An oversulfated chondroitin sulfate was detected in small amounts in some of these urines. This was demonstrated by the detection of an unsaturated disulfated disaccharide after digestion with chondroitinase ABC but not with chondroitinase AC.

Conditions have been determined for the benzoylation of ceramides containing nonhydroxy and hydroxy fatty acids, and a high performance liquid chromatography system for the separation and measurement of these derivatives has been devised that is capable of good resolution and high sensitivity. These methods have been used to determine quantitatively the levels of ceramides in human tissues, and in serum and urine, and to demonstrate elevated amounts of ceramide in Farber's disease urine and tissues.

Two novel branched-chain fatty acids, which appear to be unsaturated analogs of phytanic acid, have been observed in sera and urine of patients with Refsum's disease. They occur in both phospholipids and neutral lipids, and have been isolated and characterized.

It has been shown that tissues of patients with Farber's disease characteristically lack acid (pH 4.0) ceramidase. In normal cerebellum, however, ceramide cleavage and the reverse reaction, free fatty acid-dependent ceramide synthesis, both occur not only at pH 4.0 but also at pH 9.0, although normal kidney exhibits these activities only at pH 4.0. Both tissues are capable of snythesizing ceramide via an acyl-COA-dependent pathway at neutral pH. The synthetic analog of ceramide, N-oleoyl-ethanolamine, is a potent inhibitor of ceramidase.

The mean specific activity of alph-L-iduronidase in leukocytes from six obligate heterozygotes for Hurler's syndrome was found to be slightly less than one-half of the mean in normal controls; no overlap of normal and known heterozygote values was encountered. The assay has been applied with success to six potential heterozygotes, siblings of a child with Hurler's syndrome. Thus heterozygote detection of Hurler's syndrome is clearly possible; this finding, as well as the ready availability of leukocytes for screening tests, recommends their use for examination of potential carrier status in this disorder.

Two pregnancies at risk for Farber's disease were monitored with amiocentesis at 15 and 16 weeks' gestation. In the first pregnancy tested, cultured amniotic-cell ceramidase activity was 7.8% of the control mean and an affected fetus was predicted. The pregnancy was terminated at 22 weeks' gestation and the diagnosis was confirmed by the demonstration of considerably elevated renal and hepatic ceramide concentrations and severe reduction of ceramidase activity in fetal brain and cultured fibroblasts. In the second pregnancy tested, cultured amniotic-cell ceramidase activity was within the control range, and the prediction of an unaffected fetus was confirmed in the newborn.

The livers of four patients with metachromatic leukodystrophy contained galactosyl sulfatide and lactosyl sulfatide, whereas these substances were undetectable in normal human liver. On the basis of methanolysis and permethylation studies, both sulfatides were shown to be substituted with sulfate at the C-3 position of the galactose moiety. Examination of the fatty acid compositions of these sulfatides showed that C(22:0) and higher 2-hydroxy and nonhydroxy fatty acids predominated in both. Both sulfatides contained the same long-chain bases, predominantly sphingosine, dihydrosphingosine, and phytosphingosine. Using as criteria the proportion of lactosyl sulfatide to galactosyl sulfatide, and the fatty acid and long-chain base compositions, the liver sulfatides from subjects with metachromatic leukodystrophy closely resemble those in the kidney and differ from those in brain and peripheral nerve.

A monounsaturated and a triunsaturated form of phytanic acid (3,7,11,15-tetramethylhexacosanoate) were isolated from plasma lipids of a patient with Refsum disease. Both were converted to their methyl esters, oxidized to polyhydroxy acids by treatment with OsO4 and converted to their vicinal trimethylsilyl ethers. These derivatives were analyzed by gas chromatography-mass spectrometry using both electron impact ionization (at 21 and 70 eV) and chemical ionization conditions to obtain clear evidence to establish the structure of the monounsaturated form of phytanic acid as 3,7,11,15-tetramethylhexadec-15-monoenoic acid and that of the triunsaturated form of phytanic acid as 3,7,11,15-tetramethylhexadec-6,10,14-trienoic acid. The possible metabolic and dietary sources for these novel fatty acids are discussed.

Since plant lectins were used to help define differences between normal and transformed cell surfaces (reviewed in References 1-4), they have been employed in many other situations where their sugar-recognition specificities could be used to advantage. One of these applications has been the purification and characterization of enzymes and other proteins; this work is reviewed here in order to define some of the variables that affect binding of glycoproteins to lectins, as well as to demonstrate how this technique has been profitably exploited for isolation of purified glycoproteins, and for their better understanding.

Amines, diamines and polyamines inhibit the erythropoietin-stimulated incorporation of 59Fe into newly-synthesized heme by fetal mouse liver cells in short-term culture. As assessed by cellular retention of lactic dehydrogenase, most of these compounds affect the viability of the cells very little at concentrations that substantially inhibit heme synthesis. In the families of amines tested, compounds of chain length greater than five carbons are more effective inhibitors the longer the chain, and monoamines are more inhibitory than diamines. The naturally-occurring polyamines spermine and spermidine are among the most potent amine inhibitors tested.

Ceramidase activity could not be demonstrated in the kidney and cerebellum from a deceased patient with Farber's disease, whereas the activities of six control acid hydrolase enzymes appeared normal. This enzyme defect presumably accounts for the accumulation that has been described in two patients and may represent the biochemical basis of this disorder.

Nucleotide pyrophosphatase and phosphodiesterase I of rat liver have been found to be localized primarily in cell particulates highly enriched with respect to the most commonly accepted plasma membrane marker, 5'-nucleotidase, and therefore should themselves be assigned a plasma membrane localization. The observation that plasma membranes sediment in isotonic sucrose with both nuclear and microsomal fractions was exploited to obtain plasma membrane preparations from each fraction. Both preparations are similar in chemical and enzymic composition. Moreover, the preparative method developed in this study appears to give the best combination of yield, purity, and reproducibility available. The question of the possible identity of nucleotide pyrophosphatase and phosphodiesterase I is considered, and evidence is presented suggesting that these activities may be manifestations of the same enzyme.