Uptake and utilization of lipids by Paramecium tetraurelia

Uptake rates of a variety of 14C-labeled fatty acids and complex lipids by Paramecium tetraurelia during 48 h of log-phase growth varied. Fatty acid uptake was maximal during lag phase of growth when phagosome (food vacuole) formation was minimal. Food vacuole formation was shown to be suppressed by the presence of exogenous lipids and by starvation. The rates of uptake of lipids were significantly greater than those of small organic compounds such as amino acids, cyclitols, fatty acid precursors and metabolic intermediates. Significant amounts of radioactivity from 14C-labeled fatty acids were metabolized to 14CO2. The uptake rates of different saturated, straight-chain fatty acids of even carbon numbers were different and were not correlated with chain length, results suggesting that the primary mechanism for uptake of these compounds was neither by bulk transport nor simple diffusion and that carrier-mediated processes could possibly be involved.     

Regression of human melanoma xenografts in nude mice injected with methotrexate linked to monoclonal antibody 225.28 to human high molecular weight-melanoma associated antigen

Summary Intravenous injections into nude mice of 5 mg/kg methotrexate (MTX) linked to the antibody to human high molecular weight-melanoma associated antigen (HMW-MAA), monoclonal antibody (mAb) 225.28, an IgG_2a, on days 1, 4, 7, 10 and 14, starting 24 h after subcutaneous inoculation of 2 × 10⁶ cultured human M21 melanoma cells inhibited mean tumor volume by 90% on day 14 and by 65% on day 50 after the beginning of the treatment. Injections of equimolar amounts of free MTX and MTX linked to normal mouse IgG or to an isotypematched myeloma protein did not inhibit tumor growth significantly. MTX linked to mAb 225.28 did not inhibit the xenograft of a subline of human melanoma cell line M21 without detectable expression of HMW-MAA. In a clonogenic assay, the MTX-225.28 conjugate was three times more potent in inhibiting the growth of M21 melanoma cells than free MTX, but did not inhibit the growth of kidney carcinoma cells Caki-1, which do not express high-M _r MAA. In contrast, MTX linked to the mAb DAL K29, reacting with kidney carcinoma cells Caki-1, inhibited their growth but did not affect that of melanoma cells. M21 melanoma cells isolated from the residual tumor of a mouse treated with the MTX-225.28 conjugate did not differ in their reactivity with mAb 225.28 and in their sensitivity to MTX when compared with M21 cells from an untreated mouse.     

Potentiation of growth suppression and modulation of the antigenic phenotype in human melanoma cells by the combination of recombinant human fibroblast and immune interferons

Summary Administration of interferon as a single therapeutic regimen in cancer patients with various neoplasias has had only limited efficacy in ameliorating the negative clinical course of their disease. In the present study, we have evaluated the effect of recombinant human fibroblast (IFN) and immune (IFN) interferon, alone and in combination, on growth, differentiation and the expression of class I and II histocompatibility locus antigens (HLA) and melanoma-associated antigens on the human melanoma cell line H0-1. The effect of combinations of interferons on the antigenic profile of human melanoma cells displaying different organ colonization and spontaneous metastatic potential in athymic nude mice was also determined. H0-1 cells were more sensitive to the antiproliferative activity of IFN than to IFN and the combination of interferons resulted in a potentiation of growth suppression. The antiproliferative effect of both interferons was greater in later-passage than in earlier-passage H0-1 cells, possibly reflecting alterations in the evolving tumor cell population as a result of long-term in vitro propagation and/or the selective outgrowth of cells with an increased growth rate. The enhanced growth suppression observed in H0-1 cells treated with the combination of IFN plus IFN was not associated with a significant increase in the level of melanin, a marker of melanoma differentiation, above that observed with either interferon used alone. IFN and IFN differentially modulated the expression of class I and II HLA and melanoma-associated antigens in H0-1 cells and a series of melanoma cells with different organ colonization and metastatic potential, including MeWo, MeM 50-10, MeM 50-17, 3S5 and 70W. No consistent potentiation or antagonism in the expression of any specific antigen was observed in any of the melanoma cell lines exposed to the combination of interferons. The present study demonstrates that the combination of IFN plus IFN can potentiate growth suppression in H0-1 human melanoma cells and that this effect is not associated with an increase in differentiation or a potentiation in antigenic modulation. In addition, no direct correlation between the expression of any specific antigen or its modulation by IFN or IFN, alone or in combination, and organ colonization and metastatic potential in nude mice was observed in the different melanoma cell lines.     

alpha-Carboxyl-linked glutamates in the folylpolyglutamates of Escherichia coli

Folate cofactors in most cells contain polyglutamate side chains, which since the late 1940s have been assumed to be linked via their gamma-COOH groups. We report here an investigation of the structure of the polyglutamate chain attached to the folates of Escherichia coli. Folates were extracted from E. coli grown with [7-14C] p-aminobenzoate and cleaved to p-aminobenzoyl polyglutamates of varying chain lengths (pAB(Glu)n) by the method of Foo et al. (Foo, S. K., Cichowicz, D. J., and Shane, B. (1980) Anal. Biochem. 107, 109-115). The pAB(Glu)n derived from E. coli did not co-chromatograph with chemically synthesized pAB(gamma-Glu)n-Glu on several high performance liquid chromatography (HPLC) systems, except for the triglutamate which did elute with pAB(gamma-Glu)2-Glu. E. coli-derived pAB(Glu)3-8 were purified by HPLC on C18 columns eluted with acetonitrile/trifluoroacetic acid, and the structures were determined through mass spectrometry, chiral amino acid analysis, and peptidase digestion experiments. Molecular weight determinations on the methyl ester derivatives of E. coli-derived pAB(Glu)n by liquid secondary ion mass spectrometry and sequence analysis using collision-activated dissociation on a tandem mass spectrometer confirmed the structures as pAB(Glu)3-8. Chiral HPLC of hydrolyzed and dansylated E. coli-derived materials, on a beta-cyclodextrin column, identified the glutamate as the L-enantiomer. pAB(Glu)n were digested with carboxypeptidase Y, which specifically cleaved glutamates linked at their alpha-carboxyls; E. coli-derived pAB(Glu)4-8 (but not synthetic pAB(gamma-Glu1-6-Glu) were sequentially digested to pAB(gamma-Glu)2-Glu. Thus, in E. coli folylpolyglutamates, glutamate residues 4-8 were each linked to the polyglutamate chain at the alpha-carboxyl of the preceding glutamate.     

Idiotypic diversity and variable region gene usage by mouse anti-HLA-DQ3 mAb

The anti-HLA-DQ3 monoclonal antibodies (mAb) KS13, SO1, SO2, SO3, SO4, and SO5 recognize spatially close but distinct antigenic determinants, since they crossinhibit each other in their binding to HLA-DQ3 antigens, but do not share idiotopes recognized in their antigen combining site by syngeneic and anti-id antisera and mAb. Furthermore, mAb SO1, SO3, SO4, and SO5 react also with HLA-DQ allospecificities other than HLA-DQ3. Sequence analysis of the heavy (V _H ) and light (V _L ) chain variable region of the six mAb revealed preferential usage of V _H 36–60 and V _K 12/13 gene families. However, the individual V _H and V _L germline gene usage by the six mAb is diverse and the utilization of D, J _H , and J _L gene segments is heterogeneous. The diverse usage of V _H and V _L gene segments and heterogeneous amino acid sequences of V _H and V _L CDR, together with the heterogeneous idiotypic profile, may reflect the complexity of the determinants recognized by the six mAb on HLA-DQ3 antigens. The results we have presented provide for the first time information about the structural basis of the diversity of antibodies recognizing human histocompatibility antigens.The nucleotide sequence data reported in this Papershave been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers L20499, L20957, L20961, L24557, L24558 and L20962, respectively, for V _H region genes, and L20956, L20958, L24555, L24556, L20959, and L20960, respectively, for V _L region genes     

Cross-reactivity between human and murine lymphocyte antigens

The anti-H-2 alloantiserum D-32 [(BlO.A(2R) × C3H.SW) anti-C3H] is cytolytic to human lymphocytes. Fab₂ blocking assays, indirect immunoprecipitation and sequential immunoprecipitation experiments showed that the anti-H-2 alloantiserum D-32 recognizes antigenic determinants which are expressed on the heavy chain of subpopulations of HLA-A, B antigens. These determinants are different from those defining the serological polymorphism of the HLA-A, B, C system, are the same as or spatially close to those recognized by the anti-HLA-A, B monoclonal antibody Q6/64 and are expressed on rabbit, rat or guinea pig lymphocytes.     

Structural relationship between W4/W6 antigens and other surface markers on cultured human lymphoid cells as determined by the ‘lysostrip’ method

The structural relationship of histocompatibility antigens W4 and W6 with other surface markers on cultured human lymphoid cells of the B type has been investigated, utilizing the lysostrip technique. W4 and W6 antigens were found to be structurally associated with antigens coded for by theB locus of theHLA region, but independent of receptors for breakdown products of the third complement component and xenogeneic red blood cells, and of antigenic moieties reacting with antibodies present in H-2 alloantisera.