Characterization of visna virus envelope neuraminic acid.

Visna virus particles inhibit influenza virus hemagglutination in an assay for neuraminic acid-containing viruses. Pretreatment of visna virus with neuraminidase abolished hemagglutination inhibition activity but did not significantly affect attachment, infectivity, or virus-induced cell fusion in sheep choroid plexus cell monolayers.     

A micromethod for the estimation of oligosaccharides containing glycosidically linked sialic acid or hexoses, or both, in glycoproteins

The peeling reaction, the process by which oligosaccharides are degraded in alkali, was used as the basis for an assay to provide structural information about glycosidically linked oligosaccharides in glycoproteins. Glycoproteins were treated with 0.05 M NaOH at 50 degrees to induce release, and subsequent degradation ("peeling"), of glycosidically linked, but not of N-glycosydically linked, oligosaccharides. Among the degradation products generated from O-linked chains were three 3-deoxy sugar acids whose formation was correlated with certain structural features of the oligosaccharides. N-Acetylneuraminic acid was released from terminal positions in the oligosaccharides, and iso- and meta-saccharinic acids were derived from the degradation of 4-O- and 3-O-substituted hexoses, respectively. All of these sugar acids were detected colorimetrically by periodate oxidation and reaction of the product with 2-thiobarbituric acid. The ability of the method to generate 3-deoxy sugar acids was tested in 8 alkali-treated glycoproteins. 3-Deoxy sugar acids were detected only in those glycoproteins whose glycosidically linked carbohydrates contained N-acetylneuraminic acid, or 3-O- or 4-O-substituted hexoses, or both. As little as 0.12 microgram of 3-deoxy sugar acid produced from 5 micrograms of human chorionic gonadotropin was sufficient for detection. This method is novel in its ability to distinguish sialylation of glycosidically linked carbohydrates. Furthermore, it combines the specificity of beta-elimination with the sensitivity of the 2-thiobarbituric acid assay in targeting degradation products of the peeling reaction as candidates for an assay method.     

Characterization of the human melanoma nerve growth factor receptor

Monoclonal antibodies to the human nerve growth factor receptor have been used to biochemically characterize the receptor in the human melanoma cell line A875. Labeling of A875 cell proteins by culture with [35S]cysteine or labeling of cell surface proteins with 125I followed by immunoprecipitation with anti-nerve growth factor receptor antibody reveals a receptor protein with an apparent Mr of 70,000-75,000 and an isoelectric point of 4.9-5.2. Incorporation of [3H]glucosamine into this species indicates it is a glycoprotein. The receptor becomes phosphorylated on serine residues in intact cells and in isolated membranes incubated with [gamma-32P]ATP. The receptor appears to exist, at least partially, in the form of a disulfide-linked oligomer (probably a dimer) of Mr = 75,000 subunits. Kinetic [35S]cysteine labeling studies reveal an Mr = 59,000 core protein which is glycosylated via N-linked and probably also O-linked sugar moieties to produce the mature (Mr = 70,000-75,000) receptor.     

Characterization of phosphatidylinositol phospholipase C activity in human melanoma

Phosphoinositide phospholipase C activity was investigated in human melanoma grown as solid tumor xenografts in nude mice. The enzyme was dependent on calcium for activity and was stimulated by the detergent deoxycholate. The pH optimum was 5.5 in the absence of detergent, and in the presence of deoxycholate two pH maxima were present, 5.5 and 7.2. Phospholipase C activity was inhibited by the sulfhydryl reagent dithionitrobenzoate with an IC50 in the micromolar range. Phospholipase C activity was distributed widely in mouse tissues. The enzyme showed a progressive increase in activity from heart, liver, lung, colon, spleen, to brain tissue. Mouse and human melanomas grown as solid tumors had higher phospholipase C activity than mouse brain. The relatively high activity of this enzyme in melanoma may suggest a biological role for phospholipase C in solid tumor growth.     

Berberine increases the expression of cytokines and proteins linked to apoptosis in human melanoma cells

Molecular Biology Reports - Melanoma is the most lethal form of skin cancer, and its incidence has increased considerably in the last decades. Melanoma presents difficult treatment with strong...     

Role of neuraminic acid in the heterogeneity of alkaline phosphatase in sheep brain

1. Comparative studies of the polyuridylic acid-directed phenylalanine-incorporating activity of cell-free systems derived from rat and chicken livers demonstrated markedly lower activity in the chicken liver system. 2. The chicken liver cell sap contained the factor(s) responsible for this lower activity. Ribosomes from chicken and rat performed equally well in the presence of rat liver cell sap. Chicken liver cell sap, when mixed with rat liver cell sap, caused an inhibition of incorporation of phenylalanine into acid-insoluble material. 3. Though ribosomal preparations and cell sap from both rat and chicken liver degraded polyuridylic acid to some extent, the chicken liver cell sap contained the largest amount of activity. 4. Rat liver cell sap inhibited the nuclease activities of ribosomal preparations, but no such nuclease inhibition could be demonstrated with chicken liver cell sap.     

Characterization of IIB-MEL-J: a new and highly heterogenous human melanoma cell line

A highly heterogeneous cell line, IIB-MEL-J, was established from a human metastatic melanoma. This cell line contains small cells, dendritic cells, and megacells with multiple nuclei. IIB-MEL-J expresses S 100, cytokeratin intermediate filaments and the gangliosides GD2 and GD3. It requires growth factors (insulin, EGF, and transferrin) and antioxidants for optimal growth. When plated under optimal conditions, IIB-MEL-J grows with a doubling time of 70-80 hours. The cells may be fractionated by Percoll gradient centrifugation into several subpopulations (A, B, and C) with different characteristics. Subpopulation A is the slowest growing, and most of the DNA-synthesizing cells are concentrated in fractions B and C. Every subpopulation expresses S 100 and cytokeratin intermediate filaments, whereas only subpopulation B and C express GD2 and GD3. Pigmented cells are concentrated mainly in subpopulation C. Cytogenetic analysis of IIB-MEL-J revealed extensive chromosomal alterations, including a highly heterogeneous chromosome number and chromosomal rearrangements, gains, losses, isochromosomes, and double minutes. This highly heterogeneous cell line may be helpful to study cellular differentiation and interaction between different subpopulations in human melanoma.     

Characterization of human D-amino acid oxidase

D-Amino acid oxidase (DAAO) has been proposed to be involved in the oxidation of D-serine, an allosteric activator of the NMDA-type glutamate receptor in the brain, and to be associated with the onset of schizophrenia. The recombinant human DAAO was expressed in Escherichia coli and was isolated as an active homodimeric flavoenzyme. It shows the properties of the dehydrogenase-oxidase class of flavoproteins, possesses a low kinetic efficiency, and follows a ternary complex (sequential) kinetic mechanism. In contrast to the other known DAAOs, the human enzyme is a stable homodimer even in the apoprotein form and weakly binds the cofactor in the free form.