Shizuo Handa

Coomassie brilliant blue staining of lipids on thin-layer plates.

Coomassie brilliant blue staining of lipids on silica gel thin-layer chromatography plates is described. This stain proved to be useful for the wide-range detection of simple and complex lipids on thin-layer plates. It can stain several classes of lipids, including cholesterol, cholesterol esters, glycerides, phospholipids, ceramides, and neutral and acidic glycosphingolipids. It stains the spots evenly without a corrosive reagent, and is simple to use and suitable for storage. The visual detection limits of this stain for lipids were 0.05 to 0.5 microgram.

Ceramide induces apoptosis via CPP32 activation

Although both ceramide and interleukin‐1β converting enzyme (ICE) family proteases are key molecules during apoptosis, their relationship remains to be elucidated. We report here that cell‐permeable ceramide induced cleavage and activation of CPP32, a Ced‐3/ICE‐like protease, but not ICE. Ceramide‐induced apoptosis of Jurkat cells was blocked by the CPP32‐specific tetrapeptide inhibitor DEVD‐CHO, but not by the ICE inhibitor YVAD‐CHO. Furthermore, variant Jurkat cells with defective CPP32 activation were resistant to both antiFas‐ and ceramide‐induced apoptosis. These results indicate that CPP32 activation is required for ceramide‐induced apoptosis, and suggest sphingomyelin‐ceramide pathway functions upstream of CPP32.

Close association of Guillain–Barré syndrome with antibodies to minor monosialogangliosides GM1b and GM1α

Cumulative evidence supports the theory that anti-ganglioside antibodies function in the development of Guillain-Barré syndrome (GBS). Some patients have developed GBS after the administration of monosialoganglioside extracted from bovine brain. To clarify the pathogenesis of GBS associated with and without administration of the monosialoganglioside fraction, we investigated serum antibodies to the minor monosialogangliosides GM1b and GM1 alpha in patients with GBS and in control patients. GM1b and GM1 alpha were recognized specifically by the IgG antibody from the GBS patients. Twelve of 20 GBS patients who had high IgG anti-GM1b antibody titers had a preceding gastrointestinal infection. To evaluate the hypothesis that GM1b could be an immunogen, we determined whether a GM1b epitope was present in Campylobacter jejuni isolated from a patient with GBS associated with anti-GM1b antibody. Immunostaining with the monoclonal anti-GM1b antibody indicated that the lipopolysaccharide of the C. jejuni strain has the GM1b epitope. We speculate that an injection of bovine GM1 fraction that contains GM1b, as well as infection by an agent that bears the GM1b epitope, induces production of the anti-GM1b antibody which functions in the development of GBS in some patients.

Antibody to Ga1NAc-GD1a and Ga1NAc-GM1b in Guillain—Barré syndrome subsequent to Campylobacter jejuni enteritis

N-Acetylgalactosaminyl GD1a (GalNAc-GD1a) is a proposed target molecule for serum antibody in some patients with Guillain-Barré syndrome (GBS) (Kusunoki et al., 1994). We examined autoantibody to GalNAc-GD1a in sera from 58 GBS patients. Eight GBS patients had high IgG anti-GalNAc-GD1a antibody titers, 3 of whom also had high IgM anti-GalNAc-GD1a antibody titers. These 8 patients had experienced gastrointestinal infection before the onset of their neurological symptoms. Campylobacter jejuni was isolated from 4 of them. An absorption test indicated the presence of the GalNAc-GD1a epitope in lipopolysaccharides of C. jejuni. Sera that had anti-GalNAc-GD1a antibody reacted with several acidic glycolipids in bovine peripheral nerve, one of which was identified as N-acetylgalactosaminyl GM1b (GalNAc-GM1b). Serum binding to GalNAc-GM1b was decreased by absorption with GalNAc-GD1a. The presence of GalNAc-GM1b as well as GalNAc-GD1a has been reported in human peripheral nerves. We assume that C. jejuni, which bears the [GalNAc beta 1-4 (NeuAc alpha 2-3) Gal beta 1-3 GalNAc beta 1-] epitope, is the immunogen and that the glycoconjugates with the epitope are target molecules for the autoantibody in peripheral nerves of some GBS patients.

Autoantibodies to peripheral nerve glycosphingolipids SPG, SLPG, and SGPG in Guillain–Barré syndrome and chronic inflammatory demyelinating polyneuropathy

Unlike CNS myelin, human peripheral nerve myelin has the acidic glycosphingolipids sialosyl paragloboside (SPG), sialosyl lactosaminyl paragloboside (SLPG), and sulfated glucuronyl paragloboside (SGPG). To elucidate the pathogenesis of Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating neuropathy (CIDP), we investigated the autoantibodies to peripheral nerve molecules in patients with these diseases and compared the frequency of the autoantibodies with that of autoantibody to GM1 which is present in both the CNS and PNS. The report of Sheikh et al. (Ann. Neurol. 1995; 38: 350) that Campylobacter jejuni bears the SGPG epitope led us to study whether sera from patients with GBS subsequent to C. jejuni enteritis have anti-SGPG antibody; but, high anti-SGPG antibody titers were not found in the GBS patients from whom C. jejuni was isolated. Although the frequency of the anti-SPG, anti-SLPG and anti-SGPG antibodies were lower than that of the anti-GM1 antibody in GBS, 5 patients with demyelinating GBS had high IgG anti-SPG antibody titers. IgG anti-SPG antibody may function in the development of demyelinating GBS. We found that 6 CIDP patients had elevated IgM anti-SGPG antibody titers. Immunoelectrophoresis failed to detect IgM M-protein in 3 of the patients. IgM anti-SGPG antibody could be a diagnostic marker for a subgroup of CIDP with or without paraprotein.

Ganglioside-like epitopes of lipopolysaccharides from Campylobacter jejuni (PEN 19) in three isolates from patients with Guillain-Barré syndrome

Sera from patients with Guillain-Barré syndrome (GBS) frequently have anti-GM1 antibody. We earlier showed that an lipopolysaccharides (LPS) from Campylobacter jejuni (PEN 19) isolated from a GBS patient has a GM1 ganglioside-like structure. Aspinall et al. (Biochemistry, 61 (1994) 335-337) reported that OH 4382 has an LPS that bears a CD3 ganglioside-like structure and that OH 4384 has an LPS that bears a GT1a-like structure; both strains were isolated from patients with GBS. They also suggested a GM1-like structure is present in the LPSs from OH 4384, but failed to show the presence in the LPSs from OH 4382. To clarify the pathogenesis of GBS after infection by C. jejuni (PEN 19), we investigated the carbohydrate structures of the three strains by thin-layer chromatography immunostaining with cholera toxin and monoclonal anti-ganglioside antibodies. We found that both OH 4382 and OH 4384 have an LPS with the GM1 epitope as well as one with the GT1a or GD3 epitope.

Pathogenesis of the neurotoxicity caused by anti-GD2 antibody therapy

After treatment of melanomas with anti-GD2 monoclonal antibody (MAb) (14G2a), some patients develop sensorimotor demyelinating polyneuropathy with and without the syndrome of inappropriate antidiuretic hormone (SIADH). To clarify what causes the neurotoxicity of anti-GD2 MAb, we investigated the immunohistochemical localization of GD2 in the human nervous system. Anti-GD2 MAb (14G2a) reacted with the myelin sheaths in the peripheral nerves as well as with the pituicyte cytoplasm in the posterior lobe of the pituitary gland. We assume that the binding of anti-GD2 MAb to peripheral nerve myelin and the pituicytes in the posterior pituitary causes sensorimotor demyelinating neuropathy and SIADH.

Preparation of peptides which mimic glycosphingolipids by using phage peptide library and their modulation on β‐galactosidase activity

We describe the use of a phage‐displayed random pentadecamer peptide library for searching glycosphingolipid mimicking peptides. Two phage clones (AD‐1 and AD‐2) were selected by biopanning using monoclonal antibody AD117m, directed to lactotetraosylceramide (Lc4Cer). The amino acid sequences of the selected clones showed high homology (VPPXFXXXY) in 9‐mer. Three phage clones were selected by using monoclonal antibody H11, directed to neolactotetraosylceramide (nLc4Cer), the linkage isomer of Lc4Cer, and the displayed amino acid sequences were compared. One of these peptides showed the same amino acid sequence as that of AD‐2 except for one amino acid substitution. Pentadecamer, 9‐mer and point mutated 9‐mer peptides were synthesized on the basis of the displayed amino acid sequences. Binding activity of the peptides to the monoclonal antibodies or Ricinus communis lectin showed that 9‐mer peptides are enough to mimic the epitope carbohydrate structure. Furthermore, six of the synthesized peptides inhibited Jack bean β‐galactosidase activity towards nLc4Cer at a high concentration of the enzyme, whereas at lower enzyme concentrations some peptides showed potent activation of the enzyme activity. This is the first report of carbohydrate mimicking peptides which modulate glycosidase activity.

Antibodies to GT1a ganglioside in patients with Guillain–Barré syndrome

Serum antibodies from 8 (13%) of 62 patients with the acute Guillain-Barré syndrome (GBS) and 1 of 3 patients with the Miller Fisher syndrome (MFS) recognized a minor ganglioside in bovine and human brain trisialoganglioside fractions. The ganglioside antigen migrated between GD1a and GD1b on thin-layer chromatograms. The structure of this ganglioside was established to be GT1a by thin-layer chromatography blotting and mass spectrometry. GT1a a ganglioside was also detected in human and bovine peripheral nerves by thin-layer chromatogram immunostaining. Serum from the GBS patients had IgM, IgG, or IgA antibodies against GT1a detectable by enzyme-linked immunosorbent assay (ELISA). Serum from the MFS patient also had elevated levels of IG against GT1a. None of the sera from 43 patients with other neurological diseases or from 24 healthy controls reacted with GT1a. Sera from 6 of 8 GBS patients with anti-Gt1a antibodies also reacted with GQ1b. There was no difference in the incidence of anti-GT1a immunoglobulins in acute GBS patients with or without oculomotor abnormalities. Levels of anti-GT1a antibodies correlated temporally wit clinical symptoms in GBS patients. Although the incidence of dysphagia was slightly higher in GBS patients with anti-GT1a antibodies than in those without, the number of patients studied may have been too small to detect an association between anti-GT1a antibodies and an a specific clinical variant of GBS. Our data demonstrate that a proportion of GBS patients have antibodies against GT1a ganglioside and suggest that these antibodies may play a role in the pathogenesis of neuropathy in GBS.

Direct analysis of glycolipids on thin-layer plates by matrix-assisted secondary ion mass spectrometry: Application for glycolipid storage disorders

The lipids accumulated in organs of patients with Gauchers, Tay-Sachs, and Fabrys disease were identified by means of the combination of thin-layer chromatography and matrix-assisted secondary ion mass spectrometry. The total lipid extract of each lipidosis tissue was chromatographed on a TLC plate and then analyzed directly by mass spectrometry without elution of the sample from the TLC plate. The amount of material needed to obtain an adequate spectrum is in the order of a few micrograms of lipids per band for both positive and negative ion detection. By scanning the plates, mass spectral and chromatographic information can be obtained simultaneously, which was shown to be useful for the qualitative identification of the components on the plates.