Expression of GD2 and GD3 gangliosides in human embryonic neural stem cells

NSCs (neural stem cells) are undifferentiated neural cells endowed with a high potential for proliferation and a capacity for self-renewal with retention of multipotency to differentiate into neurons and glial cells. It has been recently reported that GD3, a b-series ganglioside, is a marker molecule for identifying and isolating mouse NSCs. However, the expression of gangliosides in human NSCs is largely unknown. In the present study, we analysed the expression of gangliosides, GD2 and GD3, in human NSCs that were isolated from human brains at gestational week 17 in the form of neurospheres, which are floating clonal aggregates formed by NSCs in vitro. Employing immunocytochemistry, we found that human NSCs were strongly reactive to anti-GD2 antibody and relatively weakly reactive to anti-GD3 antibody. Treatment of these cells with an organic solvent such as 100% methanol, which selectively removes glycolipids from plasma membrane, abolished the immunoreactivity with those antibodies, indicating that the reactivity was due to GD2 and GD3, but not to GD2-/GD3-like glycoproteins or proteoglycans. The immunoreactivity of human NSCs to antibody against SSEA-1 (stage-specific embryonic antigen-1), a well-known carbohydrate antigen of NSCs, was not decreased by the treatment with 100% methanol, indicating that SSEA-1 is mainly carried by glycoproteins and/or proteoglycans in human NSCs. Our study suggests that GD2 and GD3 can be marker gangliosides for identifying human NSCs.     

Blood group A glycolipid (Ax) with globo-series structure which is specific for blood group A1 erythrocytes: one of the chemical bases for A1 and A2 distinction

A new blood group A-active glycolipid fraction, termed Ax, showing a chromatographic mobility between Aa and Ab was found in blood group A1 erythrocytes but not in A2 erythrocytes. Ax was identified by its conversion to "globo H" by alpha-N-acetylgalactosaminidase and by 1H-NMR spectroscopy as GalNAc alpha l----3[Fuc alpha l----2]Gal beta l----3GalNAc beta l----3Gal alpha l----4Gal beta l----4Glc beta l----lCer. Globo-H (Fuc alpha l----2Gal beta l----3GalNac beta l----3Gal alpha l----4Gal beta l----4Glc beta l----lCer) was found in blood group A, and O but not in A1 erythrocytes. Thus, one of the A1-specific determinants must be an A determinant carried by globo-series structure.     

Monoclonal antibody directed to the stage-specific embryonic antigen (SSEA-1) reacts with branched glycosphingolipid similar in structure to Ii antigen

A monoclonal antibody reacting with early mouse embryos and murine embryonal carcinoma cells (F9) defines the stage-specific embryonic antigen (SSEA-1). We now report that the antigen (SSEA-1) is a complex glycolipid with the branched lacto-N-glycosyl series. Antibody to SSEA-1 reacts strongly with the branched H₄-glycosphingolipid but not with other various glycolipids so far tested. This reactivity was abolished by endo-β-galactosidase treatment. The homogeneous H₄-glycolipid not only reacted with the monoclonal antibody to SSEA-1 but also with antibody to I-(Ma), i-(Dench) and with anti-H specific lectin. Chemical analysis, including methylation, also indicates that the glycolipid antigen had a close resemblance to I-antigen.     

Expression of murine early embryonic antigens, SSEA-1 and antigenic determinant of EMA-1, in embryos and ovarian follicles of a teleost medaka (Oryzias latipes)

Stage-specific embryonic antigen-1 (SSEA-1) and the antigenic determinant of monoclonal antibody EMA-1 are expressed in a stage-specific manner in mouse early embryos. To study whether these antigens generally exist in fish, expression of the antigens was examined in embryos, ovarian follicles, and adult tissues of a teleost medaka (Oryzias latipes), using immunohistochemical techniques. In 1-cell-stage embryos, these carbohydrate antigens were found in numerous cytoplasmic granules in the blastodisc and the cortical cytoplasm. These granules gradually decreased in number as the embryos developed. In 4-cell-stage embryos, the antigens appeared on the cleavage planes and were located on the cleavage planes within the blastoderm in the following cleavage stages. In blastula-stage embryos, the expression was ubiquitously found on the cell surface of blastomeres. At the mid-gastrula stage, the antigens were restricted to the enveloping layer, yolk syncytial layer, and cortical cytoplasm, but were rarely found in deep cells that contribute to formation of the embryonic body. In later-stage embryos and adult fish, the antigens were located in various tissues. In ovarian follicles, the antigens were found in granules of oocytes and granulosa cells. These observations were basically consistent with those in mice; however, expression in 1-cell-stage embryos and ovarian follicles has not been observed in mice. This unexpected finding suggests that the antigens are produced in granulosa cells and transferred to 1-cell-stage embryos via oocytes, and that the antigens involved in the early developmental process are maternally prepared in teleosts.     

Le(x) glycan mediates homotypic adhesion of embryonal cells independently from E-cadherin: a preliminary note

Le(x) glycan and E-cadherin (Ecad) are co-expressed at embryonal stem (ES) cells and embryonal carcinoma (EC) cells. While the structure and function of Ecad mediating homotypic adhesion of these cells have been well established, evidence that Le(x) glycan also mediates such adhesion is weak, despite the fact that Le(x) oligosaccharide inhibits the compaction process. To provide stronger evidence, we knocked out Ecad gene in EC and ES cells to establish F9 Ecad (-/-) and D3M Ecad (-/-) cells, which highly express Le(x) glycan but do not express Ecad at all. Both F9 Ecad (-/-) and D3M Ecad (-/-) cells displayed strong autoaggregation in the presence of Ca(2+), while PYS-2 cells, which express trace amount of Ecad and undetectable level of Le(x) glycan, did not display autoaggregation. In addition, F9 Ecad (-/-) and D3M Ecad (-/-) cells displayed strong adhesion to plates coated with Le(x) glycosphingolipid (III(3)FucnLc4Cer), in dose-dependent manner, in the presence of Ca(2+). Thus, ES or EC cells display autoaggregation and strong adhesion to Le(x)-coated plates in the absence of Ecad, further supporting the notion of Le(x) self-recognition (i.e., Le(x)-to-Le(x) interaction) in cell adhesion.     

Human cancer-associated gangliosides defined by a monoclonal antibody (IB9) directed to sialosyl alpha 2 leads to 6 galactosyl residue: a preliminary note

A new monoclonal antibody (IB9) was prepared by hybridoma technique directed specifically to sialosyl alpha 2 leads to 6 galactosyl residue. With this reagent, accumulation of two major gangliosides in human colonic and liver adenocarcinoma has been detected, and these gangliosides were isolated and characterized as structures A and B (below). Another ganglioside with a ceramide nonasaccharide structure, which reacted to anti-X-hapten antibody after desialylation, was also isolated and partially characterized. (formula; see text) These gangliosides were absent or present in very small quantity in normal tissue and may represent human cancer-associated markers.     

Comparative study of the composition of waxes extracted from isolated leaf cuticles and from whole leaves of Citrus: Evidence for selective extraction

The effect of different extraction methods on the composition of samples of soluble cuticular lipids (SCL) of Citrus aurantium L. was investigated. The variation of extraction yields, when whole leaves were immersed in solvent, was studied as a function of solvent type and duration of immersion. Cuticular waxes were also quantitatively extracted from isolated cuticular membranes of C. aurantium and their composition was compared to that of samples obtained by the immersion method. Significant differences were observed. Higher carbon number homologues of the aliphatic constituent classes were discriminated against when whole C. aurantium leaves were extracted by immersion. The alkyl ester fraction was almost entirely lacking in extracts from whole leaves. The dependence on carbon chain length of the saturation concentrations in chloroform of major aliphatic SCL constituents was determined. The results are discussed in terms of the major physico-chemical processes involved in the extraction of SCL.     

Location and dynamics of a membrane-bound fluorescent hapten. A spectroscopic study

In the preceding paper (Petrossian, A. and Owicki, J.C. (1984), Biochim. Biophys. Acta 776, 217–227), we describe the binding of a monoclonal anti-fluorescein antibody to a membrane bound fluorescein-lipid hapten. Those results suggest that some of the hapten fluorescein moiety is extended away from the membrane surface and is available for antibody binding, while some of the hapten is sequestered and not immediately available for antibody binding. In this paper, we carry out a spectroscopic study of the membrane-bound hapten and show that there is more than one physically distinct fluorophore environment, with the sequestered hapten associated with the phospholipid headgroup region. The amount of membrane-associated fluorophore depends upon the membrane lipid composition: most of the fluorophore is associated when the lipid is unsaturated or branched-chain phosphatidylcholines (PC), whereas the hapten is largely extended for PC/cholesterol mixtures. The effect of cholesterol on the availability of membrane-bound hapten to antibody binding is not unique to this system. The conversion between sequestered and extended hapten is slow (minutes).     

Presence of coenzyme M derivatives in the prosthetic group (coenzyme MF430) of methylcoenzyme M reductase from Methanobacterium thermoautotrophicum

2-Mercaptoethanesulfonic acid (coenzyme M), or a derivative of it, and a yellow chromophore, known as the nickel-containing tetrapyrrole factor F₄₃₀, occur in the prosthetic group of methylcoenzyme M reductase in an equimolar amount, and bound to each other; this enzyme catalyzes the final step of methane production. The prosthetic group, which is called coenzyme MF₄₃₀, was isolated from the purified enzyme and was extracted from cells. The presence of coenzyme M was confirmed by a bioassay using Methanobrevibacter ruminantium and by the use of chemical and physicochemical analyses.     

The effect of manganese and ferric ions on the in vitro formation of dihydrodihydroxy metabolites of benzo(a)pyrene

The effect of ferric and manganese ions on the in vitro metabolism of benzo(a)pyrene (BP) to dihydrodihydroxy (diol) metabolites by rat liver microsomal preparations was studied. Of the 3 diols separated by high-pressure liquid chromatography (HPLC) and called diols 1, 2 and 3 in order of elution, diol 1 was identified by its U.V. spectrum as the 9,10-diol; diols 2 and 3 have not yet been identified positively but are probably the 4,5- and 7,8-diols respectively. Higher concentrations of both metals altered the diol profile; 10 and 50 mumol Fe3+ per incubation caused the disappearance of diols 1 and 2 and an increase in diol 3; 10 mumol Mn2+ caused a significant decrease in diol 2 while 50 mumol reduced diol 2 to a negligible amount and inhibited the formation of diol 1; both concentrations caused a relative increase in diol 3. If the tentative identification of diol 3 as the 7,8-diol is correct, manganese and ferric ions could be significant in the metabolism of BP to the active metabolite, the 7,8-diol-9,10-epoxide.     

Genetic networks regulated by ASYMMETRIC LEAVES1 (AS1) and AS2 in leaf development in Arabidopsis thaliana: KNOX genes control five morphological events

The asymmetric leaves 1 ( as1 ) and as2 mutants of Arabidopsis thaliana exhibit pleiotropic phenotypes. Expression of a number of genes, including three class-1 KNOTTED -like homeobox ( KNOX ) genes ( BP , KNAT2 and KNAT6 ) and ETTIN / ARF3 , is enhanced in these mutants. In the present study, we attempted to identify the phenotypic features of as1 and as2 mutants that were generated by ectopic expression of KNOX genes, using multiple loss-of-function mutations of KNOX genes as well as as1 and as2 . Our results revealed that the ectopic expression of class-1 KNOX genes resulted in reductions in the sizes of leaves, reductions in the size of sepals and petals, the formation of a less prominent midvein, the repression of adventitious root formation and late flowering. Our results also revealed that the reduction in leaf size and late flowering were caused by the repression, by KNOX genes, of a gibberellin (GA) pathway in as1 and as2 plants. The formation of a less prominent midvein and the repression of adventitious root formation were not, however, related to the GA pathway. The asymmetric formation of leaf lobes, the lower complexity of higher-ordered veins, and the elevated frequency of adventitious shoot formation on leaves of as1 and as2 plants were not rescued by multiple mutations in KNOX genes. These features must, therefore, be controlled by other genes in which expression is enhanced in the as1 and as2 mutants.     

Synthesis of trehalose dimycolate (cord factor) by a cell-free system of Mycobacteriumsmegmatis

A 105,000 x g residue fraction of $\text{Mycobacterium}\text{smegmatis}$ contains an enzyme (acyl transferase) that transfers endogenous mycolic acid to trehalose monomycolate to yield trehalose dimycolate. This enzyme activity is stable to repeated freezing and thawing and is unaffected by the antimycobacterial drug, ethambutol. These results show that trehalose monomycolate is a direct precursor of trehalose dimycolate and suggest the presence of activated mycolic acids (acyl donor) in the cell-free system.     

Some properties of vicinal diol-epoxides derived from benzo(a)anthracene and benzo(a)pyrene

The alkylating properties of pairs of syn- and anti-isomers of 2 diol-epoxides derived from benzo(a)pyrene (BP) and of 1 derived from benz(a)anthracene (BA) have been investigated. Of the anti-diol-epoxides, anti-BP 7,8-diol-9,10-oxide was the most reactive compound towards DNA, towards sodium p-nitrothiophenolate in a non-aqueous solvent system, and towards 4-(p-nitrobenzyl)pyridine in aqueous solution; anti-BP 9,10,-diol-7,8-oxide was of intermediate reactivity and anti-BA 8,9-diol-10,11-oxide was least reactive. The syn-diol-epoxides gave unsatisfactory results with DNA and 4-(p-nitrobenzyl)pyridine because of their rapid solvolysis in aqueous solution, but with sodium p-nitrothiophenolate showed the order of reactivity syn-BP 7,8-diol-9,10-oxide greater than syn-BA 8,9-diol-10,11-oxide greater than syn-BP 9,10-diol-7,8-oxide. The products of the reaction between diol-epoxides and nucleic acids were examined by Sephadex LH-20 chromatography followed by high-pressure liquid chromatography (HPLC) and the diol-epoxides were shown to react principally with the guanosine and adenosine moieties of RNA.     

Metabolism and nucleic acid binding of 7-fluoro-2-acetamidofluorene in rats: oxidative defluorination and apparent dissociation from hepatocarcinogenesis of 8-(N-arylamide) guanine adducts on DNA

The significance in hepatocarcinogenesis of various arylamine/amide adducts with nucleic acid was investigated by the use of comparison studies on several different parameters. Female Fischer and Sprague-Dawley rats are comparably sensitive to hepatocarcinogenesis by 2-acetamidofluorene (AAF), while male rats are more sensitive. 7-Fluoro-AAF is more carcinogenic in Sprague-Dawley rats than is AAF, but is strikingly so toward the liver of the female rat. Based on these observations, binding of both compounds to liver nucleic acids was determined for male and female Fischer rats at 1 and 3 days after a single injection of carcinogen, and in female Sprague-Dawley rats from 1 to 28 days after a single injection. As shown by others, no 8-(N-2-fluorenylacetamido)guanine adduct could be found in RNA or DNA of female Sprague-Dawley rats treated with AAF (nor was the corresponding 7-fluoro derivative detectable). These adducts were present, however, in comparable amounts in both male and female Fischer rats. The binding of 7-fluoro-AAF derivatives was higher than that of AAF derivatives in female Sprague-Dawley rats. Feeding of either AAF or 7-fluoro-AAF to Sprague-Dawley rats for 4 weeks before a single injection of [3H]7-fluoro-AAF resulted in reduction of the 8-(N-2-(7-fluoro)fluorenylacetamido)guanine adduct in males to undetectable levels in DNA and to 10% of control level in RNA. Non-acetylated adducts were increased in males, but decreased in females by AAF prefeeding; 7-fluoro-AAF prefeeding resulted in little change in adduct formation in females and in a major increase in non-acetylated adducts in males. AAF adducts disappeared from DNA more rapidly than did 7-fluoro-AAF adducts. Assay of the urinary metabolites from the animals in the prefeeding experiment showed that all compounds fed (including the non-hepatocarcinogens 4-acetamidobiphenyl and 2-acetamidophenanthrene) increased the proportion of N-hydroxy-7-fluoro-AAF among the metabolites. Defluorination of 7-fluoro-AAF to 7-hydroxy-AAF was also demonstrated and the ratio of 7-hydroxy-AAF to 5-hydroxy-7-fluoro-AAF was comparable to that observed for 7-hydroxy-AAF/5-hydroxy-AAF and AAF itself, suggesting that fluoro substitution does not increase activity by preventing detoxication.     

Synthesis and metal complexes of 2,6-bis(1-triazolo)pyridine: X-ray structure of 2,6-bis(1-triazolo)pyridine dihydrochloride

2,6-Bis(1-triazolo)pyridine (1) was synthesized and characterized via IR and NMR. The regiochemistry of the compound was confirmed via single crystal X-ray analysis of the hydrochloride salt. A series of insoluble complexes of the general formulae M(1)₂(X)₂ or M(1)(X)₂ [M=Co(II), Ni(II), Cu(II); X=ClO₄, BF₄, Cl] were prepared and their structures interpreted in light of infrared spectra and composition analysis. The results suggest that first row transition metals are not chelated by 1, but rather form extended coordination polymeric networks. A second family of complexes was prepared using 2,6-bis(1-imidazolo)pyridine to support this interpretation.     

New syntheses of N-(guanosin-8-yl)-4-aminobiphenyl and its 5′-monophosphate

N-Acetoxy-4-trifluoroacetylaminobiphenyl (N-acetoxy-TFAABP) reacted readily with Guo and GMP at neutrality in a one-step fashion to yield N-(guanosin-8-yl)4-aminobiphenyl (Guo-ABP) (I) and N(guanosin-8-yl)-4-aminobiphenyl-5′-monophosphate (GMP-ABP) (II), respectively.GMP-ABP could also be formed in much lower yield from the reaction of N-acetoxy-4-formylaminobiphenyl (N-acetoxy-FABP) with GMP (pH 7.0) under more rigorous conditions.Enzymatic hydrolysis of GMP-ABP with alkaline phosphatase in Tris buffer (pH 8.0) at 37°C yielded Guo-ABP.Guo-ABP showed a brilliant blue fluorescence on exposure to 366 nm UV light and its UV absorption spectrum was identical to that of Guo-ABP prepared by Kriek via a different route. Elemental analysis and nuclear magnetic resonance (NMR) data further confirmed the identity of this compound.     

Radioimmunoassay and chemical properties of glucose-6-phosphate dehydrogenase and of a specific NADP(H)-binding protein (FX) from human erythrocytes

Two sensitive radioimmunoassays, based on a double-antibody technique, were developed which allow detection of nanogram amounts of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and of a so far unknown NADP(H)-binding protein present in human erythrocytes (designated FX).The two proteins isolated in homogeneous form from human erythrocytes were iodinated with ¹²⁵I by means of lactoperoxidase. Antisera to both purified proteins were raised in rabbits and sequentially adsorbed on human erythrocytes and on human serum before use. No cross-reaction between the two proteins was apparent.Hemolysates from normal as well as from glucose-6-phosphate dehydrogenase-deficient subjects were investigated for their content in both immunoreactive proteins using the two radioimmunoassay methods. This preliminary study showed significantly lowered levels of immunoreactive glucose-6-phosphate dehydrogenase in erythrocytes from subjects carrying the Mediterranean variant of this enzyme (characterized by severe deficiency of catalytic activity), compared with normal subjects. This figure was reversed as concerns the content of immunoreactive FX which was found to be twice as high in glucose-6-phosphate dehydrogenase Mediterranean erythrocytes as in normal ones.The two purified proteins were submitted to a comparative analysis of their chemical properties including NH₂-terminal residues, CNBr peptides and tryptic fingerprints. These studies revealed significant differences in the primary structures of the two proteins and therefore tend to exclude FX'x being a discrete product arising from degradation of native glucose-6-phosphate dehydrogenase. Moreover, amino axid analysis and tryptic fingerprints indicated that FX, as well as glucose-6-phosphatase dehydrogenase, is composed of very similar and possibly identical polypeptide chains.     

Antifungal mechanism of SMAP-29 (1-18) isolated from sheep myeloid mRNA against Trichosporon beigelii

The antifungal activity and mechanism of SMAP-29 (1-18) (SMAP-29), a cathelicidin-derived antimicrobial peptide deduced from N-terminal sequence of sheep myeloid mRNA, were investigated. SMAP-29 displayed a strong antifungal activity against various fungi. To understand the antifungal mechanism(s) of SMAP-29, we examined the interaction of SMAP-29 with the pathogenic fungus Trichosporon beigelii. Confocal microscopy showed that SMAP-29 was localized in the plasma membrane. The antifungal effects of SMAP-29 were further confirmed by using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a plasma membrane probe. Flow cytometric analysis revealed that SMAP-29 acted in an energy-dependent manner. This interaction is also dependent on the ionic environment. Furthermore, SMAP-29 caused significant morphological changes when testing the membrane disrupting activity using liposomes (phosphatidylcholine/cholesterol; 10:1, w/w), as shown by scanning electron microscopy. The results suggest that SMAP-29 may exert its antifungal activity by disrupting the structure of cell membranes, via direct interaction with the lipid bilayers and irregularly disrupted fungal membranes in an energy- and salt-dependent manner.     

The synthesis, structure, and characterization of three novel 1D nitrogen-heterocyclic copper(I)-diphosphine polymers

At ambient temperature, three 1D nitrogen-heterocyclic Cu(I)-diphosphine polymers, {[Cu₂(dppm)₂(BF₄)₂(pyz)](CH₂Cl₂)₂}_n (1), {[Cu₂(dppm)₂(4,4′-bpy)(CF₃SO₃)](CF₃SO₃)(CH₃OH)}_n (2), {[Cu₂(dppe)₂ (phen)₂](ClO₄)₂(CH₂Cl₂)}_n (3) (dppm = bis(diphenylphosphino)methane, dppe = bis(diphenylphosphino)ethane, pyz = pyrazine, 4,4′-bpy = 4,4′-bipyridine, phen = 1,10-phenanthroline) have been synthesized and characterized by X-ray crystallography, luminescence, IR, ¹H, and ³¹P NMR. Structure analysis shows that 1 is a 1D linear polymer, 2 is a 1D stair-shaped polymer, and 3 is a 1D W-shaped polymer. A photoluminescent study of them shows that they exhibit fluorescent emission bands at ca. 555 nm, 535 nm and 557 nm, respectively.