Glycolipids and myelin proteins in human oligodendrogliomas

We studied myelin proteins and glycolipids in 24 human oligodendrogliomas (16 pure, eight mixed), including two grade I, 13 grade II, five grade III, and four grade IV Tumours with a 1b ganglioside content (GD1b, GT1b and GQ1b) over 30% of total gangliosides occur more frequently in the WHO grade I II and (47%) and grade III (40%) than in the grade IV (25%) group; there was no difference in the amounts of total ganglioside or individual gangliosides between pure and mixed oligodendrogliomas. The presence of 6-LM1 correlated with higher grades of tumours (² p0.02); however, 3-LM1 and total neolacto-series gangliosides did not correlate with grade. Immunohistochemical studies of oligodendrocyte and myelin markers (GalCer, sulfatide, 2,3-cyclic nucleotide phosphodiesterase, myelin basic protein and proteolipid protein) using specific antibodies showed only a very small proportion of tumour cells staining. These data do not support the hypothesis that tumours classified as oligodendrogliomas are derived from mature oligodendrocytes.     

In vivo growth conditions suppress the expression of ganglioside GM2 and favour that of lacto series gangliosides in the human glioma D-54MG cell line

The human glioma D-54MG cell line grownin vitro primarily expresses ganglio series gangliosides, particularly GM2. Subcutaneous injection of these cells into nude mice produced xenografts with an increased content of the human glioma-associated lacto series gangliosides, primarily 3-isoLM1, an alteration that was dose dependent, with the highest dose (1×10⁸) resulting in a phenotype that was most like that of the inoculum. After one passagein vivo, the lacto series dominated and reached a proportional level that was kept throughout the 10 passages. The mRNA levels of the GM2-synthase clearly coincided with GM2 expression and was 20 times higher in cells grownin vitro than in those grownin vivo. These results support the view that ganglioside expression in human gliomas is strongly influenced by environmental factors. Abbreviations: The gangliosides have been designated according to Svennerholm (Eur J Biochem (1977)79: 11–21) GM3, II³NeuAc-LacCer; GM2, II³NeuAc-GgOse₃Cer; GM1, II³NeuAc-GgOse₄Cer; GD3, II³(NeuAc)₂-LacCer; GD2, II³(NeuAc)₂-GgOse₃Cer; GD1a, IV³NeuAc, II³NeuAc-GgOse₄Cer; GD1b, II³(NeuAc)₂-GgOse₄Cer; GT1b, IV³NeuAc, II³(NeuAc)₂-GgOse₄Cer; 3-LM1, IV³NeuAc-nLcOse₄Cer; 3-isoLM1, IV³NeuAc-LcOse₄Cer; 3,6-isoLD1, IV³NeuAc, III⁶NeuAc-LcOse₄Cer; 38-LM1, IV³(NeuAc)₂-nLcOse₄Cer. MAb(s), monoclonal antibody (ies); the designation LM1 is used when both 3-isoLM1 and 3-LM1 and LD1, when both 36-isoLD1 and 38-LD1 are included.     

Characterization of gangliosides from Ehrlich ascites tumour cells and their variants

Differences in the nature of the gangliosides present in two types of Ehrlich ascites tumour (EAT) cells, the adherent and non-adherent EAT cells, were studied. Gangliosides were isolated by DEAE Sephadex column chromatography and analysed by high-performance thin-layer chromatography (HPTLC). The non-adherent EAT (na-EAT) cells which grow in the peritoneal cavity of mice were selected for growth on basement membrane and tissue culture plastic to give the adherent EAT (a-EAT) cells. na-EAT cells contained 1.57 nmol lipid-bound sialic acid per mg protein and at least 12 different gangliosides, including major gangliosides such as GM3, GM2, GM1, GD3, GD1a and GT1b. On the other hand, the ganglioside pattern of a-EAT cells differed significantly from that of na-EAT cells, both quantitatively and qualitatively. The content of lipid-bound sialic acid in a-EAT cells was only 0.24 nmol per mg of protein. The gangliosides in a-EAT cells were characterized as GD1a and trisialogangliosides and, significantly, a-EAT cells did not contain monosialogangliosides. Neutral glycolipids were isolated from both cell lines and their patterns were compared. In contrast to the gangliosides pattern, their neutral glycolipid patterns were similar. Glucosylceramide and lactosylceramide were the major components in both types of cells. In addition to na- and a-EAT cells, a-EAT cells were passaged in mice by intraperitoneal injection, giving rise to a third variant (c/m EAT cells). We analysed the gangliosides in c/m EAT cells to determine whether there was a change in the ganglioside pattern found in na-EAT cells. After repeated passage of c/m EAT cells in mice, the pattern of gangliosides shifted to that of na-EAT cells. Alterations of ganglioside composition may be associated with the growth environment of the murine peritoneal cavity; alternatively, a selection process may have occurred.Abbreviations EAT cells Ehrlich ascites tumour cells - na-EAT cells non-adherent EAT cells - a-EAT cells adherent EAT cells - c/m EAT cells cultured a-EAT cells passaged in mice - HPTLC high-performance thin-layer chromatography - PBS 10 mM phosphate-buffered saline, pH 7.2, containing 0.15 M NaCl - EDTA ethylene-diaminetetraacetic acid - TFA trifluoroacetic acid - TG thioglycollate - Cer ceramide (N-fatty acyl sphingosine) - GM3 NeuAc2-3Gal1-4Glc-Cer - GM2 GalNAc1-4(NeuAc2-3)Gal1-4Glc-Cer - GM1a Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc-Cer - GD3 NeuAc2-8NeuAc2-3Gal1-4Glc-Cer - GD1a NeuAc2-3Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc-Cer - GT1b NeuAc2-3Gal1-3GalNAc1-4(NeuAc2-8NeuAc2-3)Gal1-4Glc-Cer - LacCer Gal1-4Glc-Cer - Gb3 Gal1-4Gal1-4Glc-Cer - Gb4 GalNAc1-4Gal1-4Gal1-4Glc-Cer This paper is dedicated to my esteemed colleague, Sen-itiroh Hakomori on the occasion of his 65th birthday.     

New ganglioside analogs that inhibit influenze virus sialidase

Synthetic thioglycoside-analogs of gangliosides such as Neu5Ac)2-S-6)Glc-(1-1)Ceramide (1) and the GM3 analog Neu5Ac(2-S-6)Gal-(1–4)Glc(1-1)Ceramide (2), competitively inhibited GM3 hydrolysis by the sialidase of different subtypes of human and animal influenza viruses with an apparent K_i value of 2.8×10^–6 and 1.5×10^–5 M, respectively. The inhibitory activity of the ganglioside GM4 analog [Neu5Ac-(2-S-6)Gal-(1-1)Ceramide (3)], in which the glucose of 1 was substituted by galactose, was lower than that of 1 (K_i =1.0×10^–4 M). The thioglycoside-analogs (1, 2, 3) of the gangliosides were nonhydrolyzable substrates for influenza virus sialidase. The inhibitory activity of 1 to bacterial sialidases fromClostridium perfringens andArthrobacter ureafaciens was considerably lower than that to influenza virus sialidase, indicating that the structure of the active site in bacterial and influenza virus sialidase may be different and the analogs may be useful to determine the orientation of the substrate to the active site of sialidases, especially of influenza viruses.Abbreviations Cer ceramide - GM3 Neu5Ac(2–3)Gal(1–4)Glc(1-1)Cer - GM4 Neu5Ac(2–3)Gal(1-1)Cer Gangliosides were abbreviated according to Svennerholm [1] and the recommendation of the IUPAC-IUB Commission on Biochemical Nomenclature [2].     

Human plasma trans-sialidase donor and acceptor specificity

Earlier we have isolated from human plasma desialylated low density lipoproteins (dLDL) and showed that, first, dLDL induce cholesterol esters accumulation—the main process accompanying atherosclerosis development. Second, the process of lipoprotein desialylation took place in plasma, and, finally, sialic acids removed from LDL are transferred to other serum glycoconjugates. In this study we have isolated from human plasma an enzyme transferring sialic acid residues (trans-sialidase) by affinity chromatography and studied its donor and acceptor specificity. Isolated enzyme in the presence of saccharide acceptor can remove sialic acids from different lipoproteins, glycoproteins (fetuin, transferrin), and gangliosides (GM3, GD3, GM1, GD1a, GD1b). Plasma enzyme translocates 2-6, 2-3 and to a lower extent 2-8 bonded sialic acids. Sialoglycoconjugates of human serum erythrocytes, serum lipoproteins, glycoproteins, and gangliosides can serve as donors of sialic acid for trans-sialidase. Desialylated lipoproteins, especially dLDL,are more preferable sialic acid acceptors. Transferred sialic acid is found to be 2-6, 2-3,and 2-8 connected.     

Ganglioside-cholera toxin interactions: a binding and lipid monolayer study

Summary On t.l.c. plates ¹²⁵I-cholera toxin binds to a disialoganglioside tentatively identified as GD_lb with about 10 times less capacity than to ganglioside GM₁. Binding of labeled toxin to both gangliosides was abolished in presence of excess amounts of unlabeled B subunit. Ganglioside extracts from human or pig intestinal mucosa showed toxin binding to gangliosides GM₁ and GD_1b. In ganglioside-containing lipid monolayers the penetration of the toxin was independent of the ganglioside binding capacity.Abbreviations GM₂ Gal-NAc14Gal(3-2NeuAc)14G1c1Cer - GM₁ Gal3Ga1-NAc14Gal(32NeuAc)14G1c11Cer - GD_1a NeuAc23Ga113Gal-NAc14Gal(32NeuAc)14G1c11Cer - GD1b Gall3Gal-NAcl4Gal(32NeuAc82NeuAc)14Glc11Cer - GT_1b NeuAc23Ga113Ga1-NAcal4Gal(3-2NeuAc82NeuAc)14G1c11Cer - dpPC 1,2-hexadecanoyl-sn-glycero-3-phosphocholine - dpPE 1,2-hexadecanoyl-sn-glycero-3-phosphoethanolamine     

Locus-control-region-coupled betaS Antilles- and alpha2-hemoglobin genes select for high alpha2-hemoglobin expression in adult transgenic mice

Two transgenic lines of mice were produced which contained the ^S _Antilles- and ₂-hemoglobin genes trandemly coupled to the micro locus control region (LCR). The LCR^S _Antilles2-hemoglobin transgenic mice expressed high levels of ₂-hemoglobin while ^S _Antilles-hemoglobin expression was virtually undetectable. Abundant ₂-hemoglobin protein was observed in the blood of transgenic mice, while ^S _Antilles-hemoglobin chains could not be detected. Transgenic red blood cells had substantially decreased sensitivity to osmotic lysis. Attempts to produce homozygotes containing the transgene were unsuccessful. The phenotype of these mice closely resembles that of -thalassemic mice. The LCR^S _Antilles2 transgenic mice demonstrate that if the LCR is coupled to the ^S _Antilles- and ₂-hemoglobin genes in tandem, only the distal ₂-hemoglobin gene is selected for expression to significant levels in adult mice. These results support a reciprocally competitive model for LCR-hemoglobin developmental switching.     

Glycosphingolipid expression in human skeletal and heart muscle assessed by immunostaining thin-layer chromatography

In this study the comparative TLC immunostaining investigation of neutral GSLs and gangliosides from human skeletal and heart muscle is described. A panel of specific polyclonal and monoclonal antibodies as well as the GM1-specific choleragenoid were used for the overlay assays, combined with preceding neuraminidase treatment of gangliosides on TLC plates. This approach proved homologies but also quantitative and qualitative differences in the expression of ganglio-, globo- and neolacto-series neutral GSLs and gangliosides in these two types of striated muscle tissue within the same species. The main neutral GSL in skeletal muscle was LacCer, followed by GbOse3Cer, GbOse4Cer, nLcOse4Cer and monohexosylceramide, whereas in heart muscle GbOse3Cer and GbOse4Cer were the predominant neutral GSLs beside small quantities of LacCer, nLcOse4Cer and monohexosylceramide. No ganglio-series neutral GSLs and no Forssman GSL were found in either muscle tissue. GM3(Neu5Ac) was the major ganglioside, comprising almost 70% in skeletal and about 50% in cardiac muscle total gangliosides. GM2 was found in skeletal muscle only, while GD3 and GM1b-type gangliosides (GM1b and GD1) were undetectable in both tissues. GM1a-core gangliosides (GM1, GD1a, GD1b and GT1b) showed somewhat quantitative differences in each muscle; lactosamine-containing IV3Neu5Ac-nLcOse4Cer was detected in both specimens. Neutral GSLs were identified in TLC runs corresponding to e.g. 0.1 g muscle wet weight (GbOse3Cer, GbOse4Cer), and gangliosides GM3 and GM2 were elucidated in runs which corresponded to 0.2 g muscle tissue. Only 0.02 g and 0.004 g wet weight aliquots were necessary for unequivocal identification of neolacto-type and GM1-core gangliosides, respectively. Muscle is known for the lowest GSL concentration from all vertebrate tissues studied so far. Using the overlay technique, reliable GSL composition could be revealed, even from small muscle probes on a sub-orcinol and sub-resorcinol detection level. Abbreviations: ATCC, American Type Culture Collection; GSL(s), glycosphingolipid(s); HPLC, high performance liquid chromatography; HPTLC, high performance thin layer chromatography; Neu5Ac, N-acetylneuraminic acid; Neu5Gc, N-glycolylneuraminic acid [78]; PBS, phosphate buffered saline. The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations [79] and the ganglioside nomenclature system of Svennerholm [80]. Lactosylceramide or LacCer, Gal1-4Glc1-1Cer; gangliotriaosylceramide or GgOse3Cer, GalNAc1-4Gal1-4Glc1-1Cer; gangliotetraosylceramide or GgOse4Cer, Gal1-3GalNAc1-4Gal1-4Glc1-1Cer; globotriaosylceramide or GbOse3Cer, Gal1-4Gal1-4Glc1-1Cer; globoside or globotetraosylceramide or GbOse4Cer, GalNAc1-3Gal1-4Gal1-4Glc1-1Cer; Fo or Forssman GSL, GalNAc1-3GalNAc1-3Gal1-4Gal1-4Glc1-1Cer; paragloboside or lacto-N-neotetraosylceramide or nLcOse4Cer, Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer; lacto-N-norhexaosylceramide or nLcOse6Cer, Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer; GM3, II3Neu5Ac-LacCer; GM2, II3Neu5Ac-GgOse3Cer; GM1 or GM1a, II3Neu5Ac-GgOse4Cer; GM1b, IV3Neu5Ac-GgOse4Cer; GD3, II3(Neu5Ac)2-LacCer; GD1a, IV3Neu5Ac,II3Neu5Ac-GgOse4Cer; GD1b, (II3Neu5Ac)2-GgOse4Cer; GD1, IV3Neu5Ac,III6Neu5Ac-GgOse4Cer; GT1b, IV3Neu5Ac,II3(Neu5Ac)2-GgOse4Cer; GQ1b, IV3(Neu5Ac)2, II3(Neu5Ac)2-GgOse4Cer.     

New genetic variants identified in donkey's milk whey proteins

Novel genetic variants for donkey milk lysozyme and -lactoglobulins I and II have been identified by the combined use of peptide mass mapping and sequencing by tandem mass spectrometry in association with database searching. The novel donkey lysozyme variant designated as lysozyme B (Mr 14,631 Da) differed in three amino acid exchanges, N⁴⁹ D, Y⁵² S, and S⁶¹ N, from the previously published sequence. Three novel genetic variants for donkey -lactoglobulins were identified. One of them is a type -lactoglobulin I with three amino acid exchanges at E³⁶ S, S⁹⁷ T, and V¹⁵⁰ I (-lactoglobulin I B, Mr 18,510 Da). The two others are type -lactoglobulins II with two amino acid exchanges at C¹¹⁰ P and M¹¹⁸ T (-lactoglobulin II B, Mr 18,227 Da) and with three amino acid exchanges at D⁹⁶ E, C¹¹⁰ P, and M¹¹⁸ T (-lactoglobulin II C, Mr 18,241 Da). All these primary structures are closely related to those of homologous proteins in horse milk (percent identity >96%).     

Untersuchungen zum C′3-Polymorphismus (β 1c-Globulin)

Zusammenfassung Serumproben von 1322 Blutspendern aus Hessen, 40 Familien mit 89 Kindern, 20 Mutter-Kind-Kombinationen und 268 Sera einer Bantupopulation aus Portugiesisch Angola wurden mit einer modifizierten Technik der Hochspannungs-Dünnschichtelektrophorese auf Agarosegel hinsichtlich des C3-Polymorphismus untersucht. Die Genfrequenzen für Weiße (C3^S=0.779, C3^F=0.215) und Neger (C3^S=0.95, C3^F=0,048) stimmen gut mit den Werten anderer Autoren überein. Insgesamt ließen sich bei Weißen 9 Phänotypen sicher abgrenzen, bei Negern 3. Familienstudien bestätigten den für die Allele C3^S und C3^F angenommenen Vererbungsmodus (autosomal codominant) ausnahmslos. Die Frage der Lagerungsstabilität des C3 wurde abschließend untersucht.

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Investigations on C3-polymorphism ( _1c-Globulin)Gene frequencies and family studies in blood donors from Hessen and a Bantu population

Summary Serum samples of 1322 unrelated individuals from Hessen (Germany), 40 families with 89 children, 20 mother-child-combinations and 268 sera of a Bantu population from Angola were examined for C3 polymorphism using a modified technique of high voltage agarose gel electrophoresis. The gene frequencies for Caucasians (C3^S=0.779, C3^F=0.215) and negroes (C3^S=0.95, C^F=0.048) are in good accordance with those obtained by other authors. In total 9 different phenotypes were observed in Caucasians, 3 phenotypes in negroes. Family studies verify the supposed way of inheritance (autosomal codominant for C3^S and C3^F) without exception. Finally the problem of C3-inactivation by storage was investigated.

     

Genetic variation in the subunits of globulin-1 storage protein of French bean

Summary Charge and molecular weight heterogeneity of globulin-1 (G1) polypeptides of the bean, Phaseolus vulgaris L., were revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Different bean cultivars were classified into three groups: Tendergreen, Sanilac, and Contender on the basis of their protein subunit composition. Nine distinct major bands: 51,49, 48.5,48^T, 48^S, 47, 45.5, 45^S, and 45^C, and two minor bands: 46^T and 46^S were found to account for the three profiles seen on one-dimensional SDS-PAGE. Two-dimensional analysis revealed these eleven protein bands to be composed of a minimum of fourteen distinct protein subunits. The Tendergreen and Sanilac types differ in their G1 polypeptide composition. The protein patterns of the Contender types are intermediate, containing many protein subunits found in the patterns of the Tendergreen and Sanilac types suggesting a genetic and evolutionary relationship.     

Total synthesis of a cholinergic neuron-specific ganglioside GT1aα: A high affinity ligand for myelin-associated glycoprotein (MAG)

An efficient total synthesis of a cholinergic neuron-specific ganglioside GT1a (IV³NeuAcIII⁶NeuAcII³NeuAc-GgOse4Cer) is described. The suitably protected sialyl-(26)-gangliotriose (III⁶NeuAc-GgOse3) derivative was glycosylated with the phenyl 2-thioglycoside of sialic acid in the presence of N-iodosuccinimide (NIS) and trimethylsilyl trifluoromethanesulfonate (TMSOTf) in acetonitrile medium, giving the disialogangliotriose (III⁶NeuAcII³NeuAc-GgOse3) derivative which contains both sialyl-(26)-GalNAc and sialyl-(23)-Gal structures (Route I). This pentasaccharide was efficiently synthesized also by the coupling of (methyl 5-acetamido-4,7,8,9-tetra--acetyl-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosylonate)-(26)-2-deoxy-3,4--isopropylidene-2-phthalimido-D-galactopyranosyl trichloroacetimidate with 2-(trimethylsilyl)ethyl (methyl 5-acetamido-4,7,8,9-tetra--acetyl-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosylonate)-(23)-(2,6-di--benzyl--D-galactopyranosyl)-(14)-2,3,6-tri--benzyl--D-glucopyranoside, followed by conversion of the phthalimido group to the acetamido group (Route II). -Deisopropylidenation and further glycosylation with methyl (methyl 5-acetamido-4,7,8,9-tetra--acetyl-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosylonate)-(23)-2,4,6-tri--benzoyl-1-thio--D-galactopyranoside, promoted by dimethyl(methylthio)sulfonium triflate (DMTST), gave the desired trisialogangliotetraose (IV³NeuAcIII⁶NeuAcII³NeuAc-GgOse4) derivative, which was converted stepwise into the title ganglioside GT1a by the introduction of the ceramide part and then complete deprotection. The ganglioside obtained was shown to be identical with the native GT1a on TLC-immunostaining.     

On the action of hydroxylamine,hydrazine and their derivatives on the water-oxidizing complex

Photosynthetic water oxidation proceeds by a four-step sequence of one-electron oxidations which is formally described by the transitions S₀ S₁, S₁ S₂, S₂ S₃, S₃ (S₄) S₀. State S₁ is most stable in the dark. Oxygen is released during S₃ (S₄) S₀. Hydroxylamine and hydrazine interact with S₁. They cause a two-digit shift in the oxidation sequence as observed from the dark equilibrium, i.e. from S₁ S₂ : S₂ S₃ : S₃ (S₄) S₀ : S₀ S₁ :... in the absence of the agents, to S₁ ^* S₀ : S₀ S₁ : S₁ S₂ : S₂ S₃ :... in the presence of hydroxylamine or hydrazine.We measured the concentration dependence of this two-digit shift via the pattern of proton release which is associated with water oxidation. At saturating concentrations hydroxylamine and hydrazine shift the proton-release pattern from OH⁺(S₁ S₂) : 1H⁺(S₂ S₃) : 2H(S₃ S₀) : 1H⁺(S₀ S₁) :... to 2H⁺(S₁ ^* S₀) : 1H⁺(S₀ S₁) : OH⁺(S₁ S₂) : 1H⁺(S₂ S₃) : 2H⁺(S₃ S₀) :... The 2H⁺ were released upon the first excitation with a half-rise time of 3.1 ms, both with hydroxylamine and withydrazine. The concentration dependence of the shift was rather steep with an apparent Hill coefficient at half saturation of 2.43 with hydroxylamien (Förster and Junge (1985) FEBS Lett. 186, 53–57) and 1.48 with hydrazine. The concentration dependence could be explained by cooperative binding of n3 molecules of hydroxylamine and of n2 molecules of hydrazine, respectively. Tentatively, we explain the interaction of hydroxylamine and hydrazine with the water-oxidizing complex (WOC) as follows: Two bridging ligands, possible Cl^- or OH^-, which normally connect two Mn nuclei, can be substituted by either 4 molecules of hydroxylamine or 2 molecules of hydrazine when the WOC resides in state S₁.Abbreviations DNP-INT dinitrophenylether of iodonitrothymol - FWHM full width at half maximum - NR neutral red (3-amino-7-dimethylamino-2-methylphenazine-HCI) - PS II photosystem II - WOC or (in formulas:) W water-oxidizing complex Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.     

3′-Azidothymidine significantly alters glycosphingolipid synthesis in melanoma cells and decreases the shedding of gangliosides

In this report, we establish that 3-azido-3-deoxythymidine (AZT) treatment of melanoma cells greatly alters the pattern of glycosphingolipid biosynthesis. In SK-MEL-30 cells, synthesis of the gangliosides GM3 and GD3 was significantly inhibited (60% and 50% of control, respectively) and the production of their precursor, lactosylceramide, was stimulated by 2.5-fold. Control experiments established that phospholipid synthesis was not affected by AZT treatment, consistent with AZT treatment only affecting lipid biosynthetic reactions that involve glycosylation. Likely as a consequence of decreased rates of ganglioside synthesis, AZT treatment of SK-MEL-30 cells also significantly suppressed the amount of gangliosides shed from the membranes of these cells. Since shedding of gangliosides has been proposed to allow melanoma cells to avoid destruction by the immune system and alterations of glycosphingolipid levels are likely important for the malignant cell phenotype, these results may have important implications regarding the potential use of AZT or related glycosylation inhibitors as cancer chemotherapeutics.Abbreviations AZT 3-azido-3-deoxythymidine - Cer ceramide - Crbr cerebroside - Gal galactose - GalNAc N-acetylgalactosamine - Glc glucose - GlcNAc N-acetylglucosamine - GD3 disialyl lactosylceramide - GM3 sialyl lactosylceramide - HPTLC high-performance thin layer chromatography - LacCer lactosylceramide - MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide - NeuAc N-acetylneuraminic acid - PA phosphatidic acid - PBS phosphate-buffered saline - PC phosphatidylcholine - PDMP 1-phenyl-2-decanoylamino-3-morpholino-1-propanol - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - SM sphingomyelin     

Heritable variation in the phaseolin protein of nondomesticated common bean,Phaseolus vulgaris L.

Summary Crude protein extracts from single seeds of nondomesticated Mexican bean accessions were analysed by SDS polyacrylamide gel electrophoresis for variability in phaseolin protein. Six new phaseolin types; M1, M2, M3, M4, M5, M6, which contained polypeptides within the same range of molecular weights (51,000 to 45,000 daltons) as occur in the S, T and C phaseolin types of cultivated beans were identified. No T and C types were found among the non-domesticated Mexican accessions, and the S type occurred in less than 7% of the seeds screened. Genetic analyses of F₂ progenies from crosses between Sanilac (S), and five of the M types showed that each M phaseolin phenotype was allelic to the S type and expressed codominantly.     

Ganglioside alterations in YAC-1 cells cultivated in serum-supplemented and serum-free growth medium

Gangliosides of the G_M1b-pathway (G_M1b and GalNAc-G_M1b) have been found to be highly expressed by the mouse T lymphoma YAC-1 grown in serum-supplemented medium, whereas G_M2 and G_M1 (G_M1a-pathway) occurred only in low amounts [Müthing, J., Peter-Katalini, J., Hanisch, F.-G., Neumann, U. (1991)Glycoconjugate J 8:414–23]. Considerable differences in the ganglioside composition of YAC-1 cells grown in serum-supplemented and in well defined serum-free medium were observed. After transfer of the cells from serum-supplemented medium (RPMI 1640 with 10% fetal calf serum) to serum-free medium (RPMI 1640 with well defined supplements), G_M1b and GalNAc-G_M1b decreased and only low amounts of these gangliosides could be detected in serum-free growing cells. The expression of G_M1a was also diminished but not as strongly as that of G_M1b and GalNAc-G_M1b. These growth medium mediated ganglioside alterations were reversible, and the original ganglioside expression was achieved by readaptation of serum-free growing cells to the initial serum-supplemented medium. On the other hand, a new ganglioside, supposed to represent GalNAc-G_D1a and not expressed by serum-supplemented growing cells, was induced during serum-free cultivation, and increased strongly after readaptation. These observations reveal that the ganglioside composition ofin vitro cultivated cells can be modified by the extracellular environment due to different supplementation of the basal growth medium. Abbreviations: BSA, bovine serum albumin GSL(s), glycosphingolipid(s); HPTLC, high-performance thin-layer chromatography; LDL, low density lipoprotein; NeuAc,N-acetylneuraminic acid; NeuGc,N-glycoloylneuraminic acid. The designation of the following glycosphingolipids follows IUPAC-IUB recommendations. GgOse₃Cer or gangliotriaosylceramide, GalNAc1-4Gal1-4GlcCer; GgOse₄Cer or gangliotetraosylceramide, Gal1-3GalNAc1-4Gla1-4GlcCer; GgOse₅Cer or gangliopentaosylceramide, GalNAc1-4Gal1-3GalNAc1-4Gal1-4GlcCer; GgOse₆Cer or gangliohexaosylceramide, Gal1-3GalNAc1-4Gal1-3GalNAc1-4Gal1-4GlcCer or GgOse₆Cer; II³NeuAc-GgOse₃Cer or G_M2; II³NeuAc-GgOse₄Cer or G_M1 or G_M1a; IV³NeuAc-GgOse₄Cer or G_M1b; IV³NeuAc-GgOse₅Cer or GalNAc-G_M1b; IV³NeuAc-GgOse₆Cer or Gal-GalNAc-G_M1b; IV³NeuAc, II³NeuAc-GgOse₄Cer or G_D1a; II³(NeuAc)₂-GgOse₄Cer or G_D1b; IV³NeuAc, III⁶NeuAc-GgOse₄Cer or G_D1a; IV³NeuAc, II³NeuAc-GgOse₅Cer or GalNAc-G_D1a. Enzymes: Vibrio cholerae andArthrobacter ureafaciens neuraminidase (EC 3.2.1.18).     

Acyl chain conformational ordering in liquid-crystalline bilayers: comparative FT-IR and 2H-NMR studies of phospholipids differing in headgroup structure and chain length

FT-IR spectroscopy has been used to evaluate the acyl chain conformational ordering of DMPC, DMPE, DMPA (pH 6 and 12), DMPG (pH 1 and 7), and DPPC, DPPE, DPPA (pH 6). The frequencies of the symmetric and antisymmetric methylene stretching vibrations were determined as a function of temperature. In the liquid-crystalline phase the frequencies show a qualitative dependence on the amount of chain disorder. Quantitative data for trans-gauche isomerization were obtained from the integral intensities of the conformation sensitive methylene wagging absorptions at ca. 1368 cm^–1 (gtg and gtg sequences), 1356 cm ^–1 (double gauche) and 1342 cm^–1 (end gauche). The integral band intensities were converted to the number of gauche conformers per acyl chain using the calibration factors published by Senak et al. (1991). At 69°C the highest number of gauche conformers excluding contributions from single gauche conformers and jogs (gtttg) are found for PCs (DMPC: 2.6; DPPC: 2.4), followed by DMPG^– (2.0), phosphatidylethanolamines (DMPE: 1.4; DPPE: 2.0), protonated DMPG (1.5), and phosphatidic acids (DPPA^–: 1.7; DMPA^–: 1.4, DMPA^2–: 1.7). From ²H-NMR measurements of perdeuterated samples of DMPC, DMPA, DPPC, and DPPA the quadrupolar splittings _Qi and the order parameter S _CDi of the CD₂-segments close to the chain ends could be determined whereas splittings in the plateau region of the chains could not be resolved. The quadrupolar splittings are affected by trans-gauche isomerization, long axis rotation, and restricted wobbling motions of the acyl chains. In the simplest assumption, the order parameter S_CD can be expressed as a product of a segmental order parameter S and a lhain order parameter S . For comparison of the different lipids we used average order parameters S_CD, obtained by averaging over all values, and S determined from the total number of gauche conformers per chain by FT-IR-spectroscopy, to calculate an empirical average chain order parameter S. The combination of ²H-NMR and FT-IR results allows the estimation of the relative extent of chain wobbling for the different lipid molecules. S is lowest for PCs (S 0.475) while PEs (S 0.51) and PAs (S0.52) show less chain wobbling.Abbreviations FT-IR Fourier transform infrared - ²H-NMR deuterium nuclear magnetic resonance - DMPC(–d₅₄) (perdeuterated) dimyristoyl-phosphatidylcholine - DMPE(–d₅₄) (perdeuterated) dimyristoyl-phosphatidylethanolamine - DMPA(–d₅₄) (perdeuterated) dimyristoyl-phosphatidic acid - DMPG dimyristoyl-phosphatidylglycerol - DPPC(–d₆₂) (perdeuterated) dipalmitoyl-phosphatidylcholine - DPPE(–d₆₂) (perdeuterated) dipalmitoyl-phosphatidylethanolamine - DPPA(–d₆₂) (perdeuterated) dipalmitoyl-phosphatidic acid - gtg gauche ^±-trans-gauche^± - gtg gauche^±-trans-gauche^± - dg double gauche - eg end gauche Correspondence to: A. Blume     

The Structural Basis for the Susceptibility of Gangliosides to Enzymatic Degradation

The conformational properties of GM2, GalNac-4(Neu5Ac-3) Gal-4Glc-1Cer have been compared to those of 6-GM2, in which the linkage between the GalNAc and Gal was altered from GalNac-4Gal- to GalNac-6Gal-, and to those of GD1a, Neu5Ac-3Gal-3GalNAc-4(Neu5Ac-3)Gal-4Glc-1Cer, and GalNAc-GD1a.Our results revealed that unlike the compact and rigid oligosaccharide head group found in GM2, where the Neu5Ac and the GalNAc residues interact, the sugar chain of 6-GM2 is in an open spatial arrangement, with the Neu5Ac no longer interacting with GalNAc, freely accessible to external interactions.The structure of GD1a can be regarded as that of GM2 with an extension of the terminal Neu5Ac-3Gal-disaccharide. The inner portion of GD1a is that of GM2 comprising the very rigid GalNAc-[Neu5Ac-]Gal trisaccharide. The terminal Neu5Ac-Gal linkage is flexible and fluctuates between two limiting conformations. In GalNAc-GD1a the outer sialic acid gains conformational rigidity due to the presence of the outer GalNAc in position 4 of galactose. This ganglioside has two core GalNAc-[Neu5Ac-]Gal trisaccharide linked in tandem.     

α-Thalassemia and the production of different α chain variants in heterozygotes

The production of five chain variants (Hb G-Georgia, Hb St. Luke's, Hb Lloyd, Hb Montgomery, and Hb G-Philadelphia) in heterozygotes was evaluated through hematological observations, hemoglobin quantification, and biosynthetic studies. All heterozygotes for Hb St. Luke's and Hb Lloyd and most heterozygotes with Hb G-Georgia and Hb Montgomery had normal hematology and average / values of about 1.1. They were assigned a normal genotype (^G/), although the proportions of Hb St. Luke's and Hb G-Georgia were low (10 to 13%) and those of Hb Lloyd and Hb Montgomery twice as high (20%). Data from short-term incubations confirmed this genotype for some of these heterozygotes. Isolated Hb St. Luke's and Hb G-Georgia gave low ^G/ values (0.2 and 0.3) indicating that these Hb variants were defective at the level of Hb assembly. Isolated Hb Montgomery and Hb G-Philadelphia, however, gave higher ^G/ values of 0.6 and 0.8, respectively. A second type of variability existed among Hb G-Georgia (20 vs. 13%), Hb Montgomery (28 vs. 20%), and Hb G-Philadelphia (47 vs. 34%) heterozygotes, in whom the levels of Hb G differed. The occurrence of higher levels of these three chain heterozygosities was associated with hematological or biosynthetic evidence of a mild or moderate chain deficiency due to an -thalassemia-2 heterozygosity (^G/⁰ or ^0G/) or a homozygosity (^0G/⁰), respectively.This study was supported in part by USPHS Research Grants HLB-05168 and HLB-15158.     

Foam behavior of biological media

Summary The surface tension and foaminess of (a) unlimited, (b) substrate limited, and (c) oxygen transfer limited growth media of Hansenula polymorpha were measured using methanol, ethanol or glucose as a substrate.The time dependence of can be described by the Avrami-Überreiter relationship: log (2.3 log V)=n log t+log b, where V = (_O – _eq/(_t – _eq, and _O, _t and _eq are at t_M=0, t_M=t and t_M (equilibrium value).The constants n and b are functions of the fermentation time t_F as long as the growth is unlimited but they are constant in the state of limited growth. With glucose substrate, the foaminess can be presented as a definite function of the time, t_DG, which is necessary to attain _eq. With alcohol as a substrate no definite (t_DG) function was found.Symbols b constant in Eq. (1) - n constant in Eq. (1) - S substrate concentration - T temperature - t_M time h (measured from the beginning of the determination of the surface tension ) - t_F cultivation time h (measured from the time of inoculation) - t_DG time (min) necessary to attain the equilibrium surface tension ) - X dry biomass concentration (gl^–1) - V (_O – _eq)/(_t – _eq) - V_S equilibrium volume of the foam (cm³) - V_G volumetric gas flow rate during the estimation of (cm³ s^–1) - vvm volumetric gas flow rate with regard to the volume of the medium (min^–1) - w_SG superficial gas velocity (cm s^–1) - _m maximum specific growth rate (h^–1) - V_S/V_G foaminess (s) - surface tension, mMm^–1 (milli Newton m^–1) - _O at t_M=0 - _eq equilibrium surface tension ( at t_M) - _t at t_M=t - HP probes from Hansenula polymorpha cultivation - NLG non limited growth - OTLG oxygen transfer limited growth - SLG substrate limited growth