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The embryonic pattern of global DNA methylation is first established in the inner cell mass (ICM) of the mouse blastocyst The methyl donor S-adenosylmethionine (SAM) is produced in most cells through the f olate cycle, but only a few cell types generate SAM from betaine (N, N,N-trimethylglycine) via betaine-homocysteine meth-yltransferase (BHMT), which is expressed in the mouse ICM. Here, mean ICM cell numbers decreased from 18-19 in controls to 11-13 when the f olate cycle was inhibited by the antifolate methotrexate and to 12-14 when BHMT expression was knocked down by antisense morpholinos. Inhibiting both pathways, however, much more severely affected ICM development (7-8 cells). Total SAM levels in mouse blastocysts decreased significantly only when both pathways were inhibited (from 3.1 to 1.6 pmol/100 blastocysts). DNA methylation, detected as 5-methylcytosine (5-MeC) immunofluorescence in isolated ICMs, was minimally affected by inhibition of either pathway alone but decreased by at least 45-55% when both BHMT and the folate cycle were inhibited simultaneously. Effects on cell numbers and 5-MeC levels in the ICM were completely rescued by methionine (immediate SAM precursor) or SAM. Both the f olate cycle and betaine/BHMT appear to contribute to a methyl pool required for normal ICM development and establishing initial embryonic DNA methylation. [ABSTRACT FROM AUTHOR]

Alix (ALG-2-interacting protein X), a cytoplasmic adaptor protein involved in endosomal sorting and actin cytoskeleton assembly, is required for the maintenance of fibroblast morphology. As Alix has sequence similarity to adhesin in Entamoeba histolytica, and we observed that Alix is secreted, we determined whether extracellular Alix affects fibroblast morphology. Here, we demonstrate that secreted Alix is deposited on the substratum of non-immortalized WI38 fibroblasts. Antibody binding to extracellular Alix retards WI38 cell adhesion and spreading on fibronectin and vitronectin. Alix knockdown in WI38 cells reduces spreading and fibronectin assembly, and the effect is partially complemented by coating recombinant Alix on the cell substratum. Immortalized NIH/3T3 fibroblasts deposit less Alix on the substratum and have defects in α5β1-integrin functions. Coating recombinant Alix on the culture substratum for NIH/3T3 cells promotes α5β1-integrin-mediated cell adhesions and fibronectin assembly, and these effects require the aa 605–709 region of Alix. These findings demonstrate that a sub-population of Alix localizes extracellularly and regulates integrin-mediated cell adhesions and fibronectin matrix assembly. [ABSTRACT FROM AUTHOR]

Reports that anti-peptide sera against cell-CAM 105 determine high molecular-mass variants of the long isoform in rat hepatocytes. Isolation and sequencing of the L-isoform; Production of polyclonal antiserum by immunization with synthetic hexapeptide; Evidence of the specificity of anti-peptide serum for the S-isoform.