Photochemical labeling of human erythrocyte membranes with radioiodinatable azidosalicylic acid derivative of globoside.

In an attempt to define glycolipid functions we have prepared photoactivatable, iodinatable derivative of globoside and used it for photoaffinity labeling of human erythrocyte membranes. Lysogloboside (Gb4Sph) was prepared from globoside through deacylation in methanolic KOH followed by re-N-acetylation of galactosaminyl residue. The NH2 group of sphingosine residue in Gb4 Sph reacted with N-hydroxysuccinimidyl-4-azidosalicylic acid resulting in the formation of Gb4Sph-ASA which was purified by preparative tlc and column chromatography. It migrated on tlc as a single spot in two solvent systems, was susceptible to leech ceramide glycanase and could be radioiodinated to a specific radioactivity of about 200 Ci/mmol. Gb4Sph-[125I]ASA was incorporated into human erythrocytes in a time and concentration-dependent manner. Before photolysis 96% of the Gb4Sph-ASA could be removed with albumin but not with trypsin. After photolysis about 50% of the label was firmly bound to erythrocytes being resistant to albumin and trypsin treatment. The label was distributed between membrane proteins and lipids in about 1:2.3 ratio. Photolabeled proteins were analyzed by SDS-PAGE followed by autoradiography and immunostaining. Most of the radioactivity was detected in band 3 and its proteolytic fragments irrespective of the duration of photolysis. Photolabeling of erythrocyte lipids was demonstrated by Sephadex LH-20 column chromatography.

Anti-fucosyl-GM1 ganglioside IgG and IgM autoantibodies in human serum: no link to pathology.

Anti-fucosyl-GM1 ganglioside antibodies were detected in sera of five persons: four patients with autoimmune neuropathies and more recently, IgG antibodies in one with Graves' disease (Adler et al., Autoimmunity 18, 149-152, 1994) [1]. In the latter case, we were unable to find any relation between the occurrence of antibodies and thyroid disease. Now we report a detailed study on the anti-glycolipid antibodies in this patient. We found that her serum contained not only IgG but also a high level of anti-FucGM1 IgM antibodies, with a titer stable over a period of 5 years of treatment and follow-up. The carbohydrate structure of the epitope recognized by IgG and some of IgM antibodies seems to consist of Fuc-Gal-GalNAc-Gal- or a part of this sequence. Moreover, this patient's serum contained other IgM antibodies active against FucGM1 and also asialo GM1 glycolipids. Our results indicate that anti-FucGM1 ganglioside antibodies of G and M classes occur in serum of this patient with no apparent adverse health effects.

Small cell lung cancer is not associated with the presence of anti-fucosyl-GM1 ganglioside autoantibodies reactive in immunoenzymatic test.

The characteristic feature of small cell lung cancer carcinoma (SCLC) is the aberrant expression and abundant presentation of fucosyl-GM1 ganglioside (FucGM1). In the present study we searched for the presence of anti-FucGM1 ganglioside, as well as anti-GM1, GM2 and GD3 ganglioside autoantibodies in the sera of patients with SCLC and as a control, in sera of patients with renal cell cancer (RC) and healthy blood donors. The autoantibodies against FucGM1 were present at low titer in only three of 36 SCLC patients, and with similar titer in two of 36 RC patients and four of 36 healthy controls. Likewise, the autoantibodies against GM2 and GM3 gangliosides were found only sporadically and with the same titer and frequency in cancer patients as in healthy persons. Anti-GD3 autoantibodies could not be detected in any of the screened sera.

Photochemical labeling of human erythrocyte membrane proteins with radioiodinated 4-azidosalicylic acid derivatives of G(M3), G(D3), G(M1), and FucG(M1) gangliosides.

Photoreactive gangliosides of high specific radioactivity may prove useful for studies on glycosphingolipid functions. We prepared 4-azidosalicylic acid (ASA) acylated derivatives of GM3, GD3, GM1, and FucGM1 gangliosides (gangliosides-ASA). Gangliosides-ASA were characterized by their TLC mobility, UV spectra, carbohydrate composition, and digestion with leech endoceramidase. After radioiodination to about 200 Ci/mmole gangliosides-ASA were used for photochemical labeling of human erythrocytes. Radioiodinated gangliosides-ASA were incorporated into erythrocytes in a time and concentration dependent manner, the kinetics and extent of incorporation being similar for all the gangliosides-ASA used. Radioiodinated gangliosides-ASA incorporated into erythrocytes were resistant to trypsin digestion while treatment with 1% BSA removed about 90% of the label. Incubation with cholera toxin protected radioiodinated GM1-ASA and, to a lesser extent, FucGM1-ASA but not GM3-ASA and GD3-ASA, against removal with BSA. After photolysis about 40-50% of radioactivity was firmly bound to erythrocyte lipids and proteins. The ratio of lipid- to protein-bound radioactivity ranged from 2.2:1 to 3.2:1. Photolabeled proteins were analyzed by SDS/PAGE followed by autoradiography. Band 3 was the most extensively photolabeled protein with all the radioiodinated gangliosides-ASA used. DIDS, an inhibitor of band 3 protein activity, caused reduction in photolabeling of this protein by about 20%.

Photochemical labeling of HL-60 cell membrane proteins with radioiodinated, 4-azidosalicylic acid acylated derivatives of gangliosides.

To detect HL-60 human promyelocytic leukemia cell proteins involved in the uptake of gangliosides from the culture medium we used photoreactive, 4-azidosalicylic acid (ASA) acylated and radioiodinated (200 Ci/mmole) derivatives of GM3, GD3, GM1, and FucGM1 gangliosides. Gangliosides-ASA, added to the medium at 15-20 nM concentration, followed a similar time course of uptake. After 1 min incubation cell bound gangliosides-ASA could not be removed with trypsin, but only 5-10% remained after incubation with BSA. The proportion of cell bound gangliosides-ASA resistant to BSA treatment increased with time of incubation up to 76% after 20 h. As shown on TLC, GM3- and GD3-ASA were catabolized to LacSph-ASA and ceramide-ASA, while GM1-ASA was hydrolyzed to GM2-ASA. FucGM1-ASA was converted to GM1-ASA very slowly. Upon irradiation with UV lamp, cell bound gangliosides-ASA crosslinked to and photolabeled many proteins but the distribution of radioactivity after SDS/PAGE was very uneven and did not correlate with Coomassie staining. In all experiments the 42 kDa protein bands were most intensely photolabeled. Photolabeling of 42 kDa proteins decreased with time of incubation as compared to lower molecular mass pro teins. With all gangliosides-ASA used similar but not identical protein photolabeling patterns were obtained. Photolabeling patterns with GM3- and GD3-ASA differed from those with GM1- and FucGM1-ASA.

[Thoracic radiation myelopathy]. / Popromienna mielopatia piersiowa.

In a 62-year-old patient 8 month after radiotherapy for right pulmonary hilus carcinoma signs of thoracis cord involvement with ascending course from Th10 to Th7. Cerebrospinal fluid and myelogram were normal. The patient died after about 12 months. Radiation myelopathy was confirmed by neurohistological examination. The results of experimental studies are quoted which could predict the possibility of early diagnosis of radiation myelopathy by rising level of basic myelin protein in cerebrospinal fluid.

Biological properties and clinical application of propolis. IV. The action of ethanol extract of propolis (EEP) on cells cultured in vitro.

An increase of total cell number was shown in the cell culture in vitro under the influence of ethanol extract of propolis (EEP). Addition of EEP to the nutrient medium of the cells caused a strong activation of mitoses. Besides, distinctly intensified metabolism of these cells expressed by a strong activation of NADH2-reductase was also observed.