Tamotsu Taketomi

Usefulness of MALDI/TOF mass spectrometry of immunoprecipitated serum variant transthyretin in the diagnosis of familial amyloid polyneuropathy.

A matrix-assisted laser desorption ionization/time-of-flight (MALDI/TOF) mass spectrometry (MS) system was used to detect variant transthyretin (TTR) in immunoprecipitated serum TTR molecules obtained from 6 patients with familial amyloid polyneuropathy (FAP) who were already proven not to have ATTR Val30Met. This simple and quick method showed six different patterns of mass spectra of TTR-related immunoprecipitates from these patients, and in each patient the clearly identified characteristic doublet-shaped ion peaks consisted of normal and variant TTR apart from each other peak with a mass difference between them. DNA sequencing confirmed that the patterns of variant TTR corresponded respectively to ATTR Val30Leu, ATTR Phe33Val, ATTR Asp38Ala, ATTR Ser50Arg, ATTR Ala97Gly and ATTR Ala97Ser. ATTR Asp38Ala and ATTR Ala97Ser are previously unknown variants of TTR leading to the development of FAP. ATTR Phe33Val was found in a Chinese FAP patient and ATTR Ala97Ser in a Taiwanese. Serum analysis using immunoprecipitation and MALDI/TOF MS system can provide useful information when investigating FAP patients with diverse types of variant TTR.

Glycolipids of gastric cancer: The presence of blood group A-active glycolipids in cancer tissues from blood group O patients

Glycolipids were isolated from human gastric cancer tissues and normal mucosae. Sulfogalactosylceramide, ganglioside and neutral glycolipid fractions were separated by DEAE-Sephadex and silica gel column chromatography. Sulfogalactosylceramide contents were higher in the cancer tissues than in the normal mucosae. Ganglioside contents showed considerable variations but in the cancer tissues in mole percentage of ganglioside GM3 was higher than in the normal mucosae. The cancer tissues contained more neutral glycolipids than normal tissues. Glycolipids of lacto-series, including fucolipids, were markedly increased in the cancer tissues. Blood group A-active glycolipids were found in the cancer tissues from two patients with blood group O but not found in the uninvolved tissue associated with the cancer tissue.

Application of delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry for analysis of sphingolipids in tissues from sphingolipidosis patients

Sphingolipidosis is due to defects in enzymes involved in hydrolysis of sphingolipids. We analyzed sphingolipids in tissues from patients with sphingolipidosis, including Farber disease (FD, acid ceramidase deficiency), Gaucher disease (GD), Niemann-Pick disease type C (NPDC), and GM1-gangliosidosis (GM1G), using delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry (DE MALDI-TOF-MS). Crude lipids were extracted from about 100 mg wet weight of autopsied tissues, including liver, spleen, cerebrum or cerebellum. After mild alkaline treatment, a sphingolipid fraction was prepared from the crude lipids and analyzed by DE MALDI-TOF-MS. The results were as follows: (a) In FD liver both the ceramide/sphingomyelin and ceramide/monohexosylceramide ratios were significantly high; (b) in both liver and spleen from a GD patient, the glucosylceramide/sphingomyelin ratio was raised; (c) in liver from a NPDC patient, the monohexosylceramide/sphingomyelin ratio was markedly low, suggesting an increase of sphingomyelin; and (d) in all tissues examined in the GM1G patient, GM1-gangliosides or asialo-GM1-gangliosides, that are undetectable in a normal control, were increased. In conclusion, sphingolipids in human tissues could be directly determined by DE MALDI-TOF-MS, with only a small amount of specimens. This method will be useful for the diagnosis and biochemical evaluation of sphingolipidosis patients.

Application of delayed extraction–matrix-assisted laser desorption ionization time-of-flight mass spectrometry for analysis of sphingolipids in pericardial fluid, peritoneal fluid and serum from Gaucher disease patients

Gaucher disease is a glycolipid storage disorder characterized by the accumulation of glucosylceramide. Using delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry (DE-MALDI-TOF-MS), we analyzed sphingolipids in pericardial fluid, peritoneal fluid, and serum from two patients with Gaucher disease. Crude lipids were extracted from 1 ml each of pericardial fluid, peritoneal fluid, and serum with chloroform and methanol. After mild alkaline treatment of the crude lipids, a sphingolipid fraction was prepared and analyzed by DE-MALDI-TOF-MS. The results were as follows: (a) in all the specimens, peaks of ceramide monohexoside and sphingomyelin were detected in both the controls and Gaucher disease patients; (b) in pericardial fluid, peritoneal fluid, and serum, the ceramide monohexoside/sphingomyelin ratio was increased in the Gaucher disease patients compared with in the controls. It was indicated that the accumulation of ceramide monohexoside in such samples from Gaucher disease patients can be easily detected with this DE-MALDI-TOF-MS method.

SULFATIDE PROLONGS BLOOD-COAGULATION TIME AND BLEEDING TIME BY FORMING A COMPLEX WITH FIBRINOGEN

Sulfatides (galactosylceramide I3-sulfate), which are found in serum lipoproteins of various mammals, effectively increased prothrombin time (anticoagulant effect) and also effectively prolonged bleeding time (anti-platelet effect). When equal volumes of a homogeneous micellar solution of sulfatide and fibrinogen in phosphate-buffered saline were mixed, an insoluble complex precipitated. Analysis of the precipitated complex showed that the molar ratio of sulfatide to fibrinogen was about 400:1. These results indicate that the sulfatide micelle binds tightly to fibrinogen and thereby interferes with both fibrin gel formation (anticoagulant activity) and platelet function.

Heterophile Hanganutziu-Deicher antigen in ganglioside fractions of human melanoma tissues.

Gangliosides were purified from melanoma tissue extracts obtained from 5 patients. GM3 and GD3 were identified as the major components in ganglioside fractions of the melanoma tissues. Following thin-layer chromatography, enzyme immunostaining with Hanganutziu-Deicher (H-D) antigen-specific chicken antisera demonstrated the presence of NeuGc-neolactotetraosylceramide (H-D5) and NeuGc-lacto-N-norhexa-osylceramide (H-D7) in all 5 melanoma extracts.

Effect of Exogenous Gangliosides on Amino Acid Uptake and Na+,K+-ATPase Activity in Superior Cervical and Nodose Ganglia of Rats

Abstract: The effects of some gangliosides on active uptake of nonmetabolizable a‐aminoisobutyric acid (AIB) and Na+,K+‐ATPase and Ca2+,Mg2+‐ATPase activities in superior cervical ganglia (SCO) and nodose ganglia (NG) excised from adult rats were examined during aerobic incubation at 37°C for 2 h. In NG, amino acid uptake was greatly acceler‐a.ed with addition of galactosyl‐N‐acetylgalactosaminyl‐[N‐acetylneuraminyl]‐galactosylglucosyl ceramide (GM1) (85%) and also with N‐acetylgalactosaminyl‐[N‐acetylneuraminyl]‐galactosylglucosyl ceramide (GM2) or [N‐acetylneuraminyl]‐galactosyl‐N‐acetylgalactosaminyl‐[N‐acetylneuraminyl]‐galactosylglucosyl ceramide (GD1a) (43% each) compared with a nonaddition control at a 5 nM concentration. Under identical conditions, Na+,K+‐ATPase activity was strongly stimulated with GM1 (180%) and GD1a (93%), whereas Ca,Mg2+‐ATPase activity showed no change. In SCG, on the other hand, AIB uptake was apparently inhibited (‐27%) by addition of GM1, with a slight decrease in Na+,K+‐ATPase but no change in Ca2+,Mg2+‐ATPase activity in the tissue. Both asialo‐GM1, in which W‐acetylneuraminic acid is deficient, and Forssman glycolipid, which is not present in nervous tissue, failed to produce any significant increase in both SCG and NG not only in amino acid uptake, but also in Na+,K+‐ATPase activity. A kinetic study of active AIB uptake showed that GM1 ganglioside produced an increase in Km with no change in Vmax in SCG, whereas it caused a decrease in Km with a slight increase in Vmax in NG. Treatment of NG and SCG with neuraminidase from Vibrio cholerae, an enzyme that split off sialic acid from polysialoganglioside, leaving GM1 intact, caused little inhibition of the amino acid uptake. GM1‐evoked amino acid uptake increase in NG and decrease in SCG seem both to be independent of the effect of nerve growth factor (NGF). Glia maturation factor (GMF)‐induced AIB uptake was further increased by GM1 application in NG. These results suggest that exogenous brain gangliosides may modulate membrane transport of amino acid by processes somewhat different from those of NGF or GMF, with or without affecting the activity of a transport enzyme, Na+,K+‐ATPase.

Mild encephalitogenic activity of basic protein—acidic lipid complex from myelin and detection of immune complexes in experimental allergic encephalomyelitis

Further biochemical and pathological investigation of basic protein--acidic lipid complex from canine cerebral myelin show the presence of sulfatide, phosphatidylserine, ganglioside and basic protein in the molar ratio of approximately 6 : 3 : 1 : 1 and that it is associated with mild encephalitogenic activity in guinea pigs in comparison to intact myelin and basic protein. Circulating immune complexes were detected in the sera of guinea pigs with clinical signs of experimental allergic encephalomyelitis and immunofluorescent staining showed the deposition of immune complexes of immunoglobulin and complement in vessels of white matter and meninges and in the choroid plexus.

Synthesis of azacrown ethers modified with side-chains containing germanium

Abstract The reaction between ethanolamine ( 4 ) and tri- or tetraethylene tosylate ( 5 ) and ( 13 ) gave 9- and 12-membered monoazacrowns with a CH 2 CH 2 OH residue on nitrogen, A-OH and B-OH , together with 18- and 24-membered diazacrown ethers with the same substituent on nitrogen, E-OH and F-OH. If 2-(2-aminoethoxy)ethanol ( 12 ) was used in the reaction described above, instead of 4 , the final products, G-OH and H-OH , and K-OH and L-OH , have a longer side chain, CH 2 CH 2 OCH 2 CH 2 OH, on nitrogen (Route 1). A series of reactions involving bromoethanol ( 6 ) and diethanolamine ( 9 ) formed, with 5 or 13 , 18- and 24-membered monoazacrowns with the shorter substituent as described above, a 15-membered monoazacrown ether C-OH and an 18-membered D-OH . If 2-(2-chloroethoxy)ethanol ( 14 ) is used instead of 6 , I-OH and J-OH are obtained (Route 2′). All these hydroxy azacrown ethers were made to react with 3-trimethylgermylpropionic acid ( 18 ) to give the corresponding germanium-containing A – L . A preliminary investigation was carried out on the cation transport capability of these germanium-containing azacrown ethers to observe whether germanium might enhance their cation transporting capability.

Effects of various lysosphingolipids on cell growth, morphology and lipid composition in three neuroblastoma cell lines

The cell numbers of three mouse neuroblastoma cell lines were decreased upon incubation with lysosphingolipids in the following order of effectiveness: lysosulfatide (lysoCS) greater than psychosine (Ps) greater than sphingosylphosphocholine (SPC). The different cell lines showed characteristic sensitivities to various concentrations of lysolipids less than 150 microM. Interestingly, only SPC induced neurite outgrowth and changed the lipid composition, modifying the amounts of cholesterol, sphingomyelin (SM) and ganglioside GM3 in all cell lines. The effect of SPC on these cell lines was comparable to the effect of N-acetyl SPC (NAcSPC) rather than that of SM.