sepharose and Lymphoma

A 17.5-kDa trypsin inhibitor was purified from

Topics: Animals; Cell Line, Tumor; Cell Proliferation; DEAE-Cellulose; Fungi; Humans; Leukemia; Lymphoma; Mice; Phaseolus; Seeds; Sepharose; Trypsin; Trypsin Inhibitors

An antifungal peptide with a defensin-like sequence and exhibiting a molecular mass of 7.3kDa was purified from dried seeds of Phaseolus vulgaris 'Cloud Bean'. The isolation procedure entailed anion exchange chromatography on DEAE-cellulose, affinity chromatography an Affi-gel blue gel, cation exchange chromatography on SP-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. Although the antifungal peptide was unadsorbed on DEAE-cellulose, it was adsorbed on both Affi-gel blue gel and SP-Sepharose. The antifungal peptide exerted antifungal activity against Mycosphaerella arachidicola with an IC(50) value of 1.8 μM. It was also active against Fusarium oxysporum with an IC(50) value of 2.2 μM. It had no inhibitory effect on HIV-1 reverse transcriptase when tested up to 100 μM. Proliferation of L1210 mouse leukemia cells and MBL2 lymphoma cells was inhibited by the antifungal peptide with an IC(50) of 10 μM and 40 μM, respectively.

Topics: Adsorption; Animals; Antifungal Agents; Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Defensins; Fungi; Inhibitory Concentration 50; Leukemia; Lymphoma; Mice; Neoplasms; Phaseolus; Phytotherapy; Plant Extracts; Seeds; Sepharose; Triazines

Purified plasma membranes of mouse EL4 lymphoma cells were fractionated by means of affinity chromatography on concanavalin A-Sepharose into two subfractions; one (MF1) eluted freely from the affinity column, the second (MF2) adhered specifically to Con A-Sepharose. Both membrane subfractions proved to be of plasma membrane origin, as evidenced by the following criteria. (i) The ratio of cholesterol to phospholipid was nearly identical in plasma membrane and both subfractions. (ii) When isolated plasma membranes were labelled with tritiated NaBH4, both subfractions exhibited identical specific radioactivities. (iii) After enzymatic radioiodination of the cells, the total content of labelled proteins was very similar in isolated plasma membranes and in both subfractions. (iv) Some plasma membrane marker enzymes exhibited nearly identical specific activities in plasma membranes, MF1 or MF2 including gamma-glutamyl transpeptidase, 5'-nucleotidase and Mg2+-ATPase. Both subfractions exhibited characteristic differences. Thus the specific activities of (Na+ + K+)-ATPase, Ca2+-ATPase and lysophosphatidylcholine acyltransferase were several-fold enriched in MF2 compared to MF1. SDS-polyacrylamide gel electrophoresis revealed a different polypeptide composition of the two subfractions. Polypeptides of apparent molecular mass of 116, 95, 42, 39, 30 and 28 kDa were highly enriched in MF2, whereas MF1 contained another set of proteins, of apparent molecular mass of 70, 55 and 24 kDa. The phospholipid fatty acid composition of the subfractions proved to be different, as well, MF2 contained more saturated fatty acids than MF1. The data suggest the existence of plasma membrane domains in the plasma membranes of the mouse EL4 lymphoma cells, containing a set of polypeptides, among others membrane bound enzymes, embedded in a different phospholipid milieu.

Topics: Animals; Cell Line; Cell Membrane; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Enzymes; Lymphoma; Membrane Proteins; Mice; Molecular Weight; Peptides; Sepharose

The relationship between the tumorigenic potential and the cloning efficiency of T lymphoma BW5147 in semi-solid media has been studied. Two stable variants exhibiting a 30-fold decrease of their cloning efficiencies in agarose an methylcellulose media were independently isolated by negative selection with FuDr. These variants show no alteration of their growth properties in liquid medium and are still able to proliferate in liquid suspension over a bottom layer of agarose. This new phenotype is not correlated with any decreased tumorigenicity in syngeneic AKR/J mice.

Topics: Animals; Cell Division; Cell Line; Cell Separation; Culture Media; Lymphoma; Methylcellulose; Mice; Neoplasm Transplantation; Phenotype; Sepharose

A protocol has been developed that consistently gives high cloning efficiencies for human lymphoid cell lines (lymphoblastoid and lymphoma) in agarose without the use of feeder layers. This procedure utilizes a cloning medium that contains horse serum, alpha-ketoglutarate or oxalacetate, and high levels of glutamine.

Topics: Cell Line; Clone Cells; Culture Media; Cytological Techniques; Humans; Lymphocytes; Lymphoma; Sepharose

We present a general technique for fractionating cell-derived asparagine-linked oligosaccharides on the basis of oligosaccharide structure. This procedure has been applied to the study of [2-3H]mannose-labeled mouse lymphoma cells (BW5147). The fractionation scheme involves serial chromatography on concanavalin A-Sepharose, pea lectin-Sepharose, and leukoagglutinating phytohemagglutinin-agarose. Approximately 85% of the labeled glycopeptides was retained on one or more of the affinity columns. The various fractions eluted from the columns contain relatively pure populations of glycopeptides which were used for structural analysis. The recovery of the glycopeptides was quantitative. The procedure was used to estimate the overall spectrum of Asn-linked oligosaccharides synthesized by the lymphoma cell line. We conclude that serial lectin-agarose affinity chromatography is a rapid, sensitive, and specific technique for fractionating and analyzing Asn-linked oligosaccharides. A general fractionation scheme employing additional lectins is presented.

Topics: Animals; Asparagine; Carbohydrate Conformation; Carbohydrate Sequence; Cell Line; Chromatography, Affinity; Glycopeptides; Glycoside Hydrolases; Lectins; Lymphoma; Mice; Oligosaccharides; Sepharose; Structure-Activity Relationship

We have studied the growth in vitro of a lymphoma x fibroblast hybrid and several melanoma x fibroblast hybrids in which malignancy is suppressed. The parental cells, the hybrids, and malignant segregants derived from the hybrids were analysed for serum requirement, cloning efficiency in soft agarose, density-dependent inhibition of growth, and secretion of plasminogen-activating enzyme. One malignant segregant from the lymphoma x fibroblast cross was found by a number of criteria to have a more highly 'transformed' phenotype than the hybrid from which it was derived. However, in the case of the melanoma x fibroblast crosses, none of the parameters examined could be correlated in a direct way with malignancy.

Topics: Clone Cells; DNA; Fibroblasts; Hybrid Cells; Lymphoma; Melanoma; Plasminogen Activators; Sepharose

Topics: Animals; Ascitic Fluid; Cell Migration Inhibition; Cell-Free System; Cells, Cultured; Hypersensitivity, Delayed; Lymphoma; Macrophage Migration-Inhibitory Factors; Macrophages; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Mineral Oil; Mycobacterium tuberculosis; Neoplasms, Experimental; Sepharose; Spleen