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Mesoderm-specific transcript (Mest) is a distinct gene associated with adipocyte differentiation and proliferation. The mechanisms regulating expression of the Mestgene are not established. Therefore, we investigated Mestgene expression during adipogenic differentiation in murine 3T3-L1 preadipocytes and adipose-derived stromal cells (ADCs) from C57BL/6J mouse adipose tissue. Expression of MestmRNA increased significantly in 3T3-L1 cells during differentiation. Additionally, MestmRNA expression levels were additively enhanced by the inhibition of DNA methylation. Expression levels of the Mestgene were also markedly elevated in differentiating ADCs in vitro. Additionally, we showed that MestmRNA can be upregulated by increasing intracellular cAMP, and that Mestexpression is suppressed by inhibition of protein kinase A (PKA). Mestexpression was regulated through cAMP-dependent PKA pathways during differentiation of preadipocytes into adipocytes in vitro, supporting the critical role of Mestin proliferation and differentiation of adipocytes.

Mesoderm-specific transcript (Mest) is a distinct gene associated with adipocyte differentiation and proliferation. The mechanisms regulating expression of the Mestgene are not established. Therefore, we investigated Mestgene expression during adipogenic differentiation in murine 3T3-L1 preadipocytes and adipose-derived stromal cells (ADCs) from C57BL/6J mouse adipose tissue. Expression of MestmRNA increased significantly in 3T3-L1 cells during differentiation. Additionally, MestmRNA expression levels were additively enhanced by the inhibition of DNA methylation. Expression levels of the Mestgene were also markedly elevated in differentiating ADCs in vitro. Additionally, we showed that MestmRNA can be upregulated by increasing intracellular cAMP, and that Mestexpression is suppressed by inhibition of protein kinase A (PKA). Mestexpression was regulated through cAMP-dependent PKA pathways during differentiation of preadipocytes into adipocytes in vitro, supporting the critical role of Mestin proliferation and differentiation of adipocytes.

Cathepsin A is a mammalian lysosomal enzyme that catalyzes the hydrolysis of the carboxy-terminal amino acids of polypeptides and also regulates -galactosidase and neuraminidase-1 activities through the formation of a multienzymic complex in lysosomes. Human cathepsin A (hCathA), yeast carboxypeptidase (CPY), and wheat carboxypeptidase II (CPW) belong to the /-hydrolase fold family. They have structurally similar active-site clefts, but there are small differences in the amino acid residues comprising their active sites that might determine the substrate specificity and sensitivity to microbial inhibitors including chymostatin. To examine the selectivity and binding mechanism of chymostatin as to hCathA, CPY, and CPW at the atomic level, we analyzed the interaction energy between chymostatin and each protein quantitatively by semiempirical molecular orbital calculation AM1 with the continuum solvent model. We predicted the electrostatic repulsion between the P3 cyclic arginine residue of the inhibitor and the Arg344 in the S3 active substite of hCathA. Genetic conversion of Arg344 of the wild-type hCathA to Ile also caused an increase in its sensitivity to chymostatin, which was correlated with the decrease in the interaction energy calculated with the molecular orbital method. The present results suggest that such molecular calculation should be useful for evaluating the interactions between ligands, including inhibitors and homologous enzymes, in their docking models.

Sialidosis and galactosialidosis are lysosomal storage diseases caused by the genetic defects of lysosomal sialidase (neuraminidase-1; NEU1) and lysosomal protective protein/cathepsin A (PPCA), respectively, associated with a NEU1 deficiency, excessive accumulation of sialylglycoconjugates, and development of progressive neurosomatic manifestations; in addition, the latter disorder is accompanied by simultaneous deficiencies of β-galactosidase and cathepsin A. We demonstrated that a few soluble N-glycosylated proteins carrying sialyloligosaccharides sensitive to glycopeptidase F (GPF) can be specifically detected in cultured fibroblasts from sialidosis and galactosialidosis cases by blotting with a Maackia amurensis (MAM) lectin. We also examined the therapeutic effects of normal gene transfer and enzyme replacement by evaluating the decreases in sialylglycoconjugates accumulated in fibroblasts with these NEU1 deficiencies. The specific N-glycosylated proteins detected on MAM lectin blotting as well as the granular lysosomal fluorescence due to an avidin–FITC/biotinylated MAM lectin conjugate in sialidosis and galactosialidosis fibroblasts disappeared in parallel with the restoration of the intracellular NEU1 activity after transfection of the recombinant NEU1 fused to HA tag sequence and the wild-type PPCA cDNA as well as administration of the recombinant PPCA precursor protein. The detection method for the abnormal sialylglycoproteins in cultured cells involving MAM lectin was demonstrated to be useful not only for biochemical and diagnostic analyses of NEU1 deficiencies but also for therapeutic evaluation of these conditions.