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Abstract: To identify post-transcriptionally modulated genes at the translational level by phorbol 12-myristate 13-acetate (PMA), we investigated mRNA profiles in the polysomal and the cytoplasmic fractions of U937 cells before and after PMA stimulation using microarrays with 15017 oligonucleotide probes. Global comparison of the profiles showed that the cytoplasmic distribution of mRNAs was considerably modulated upon PMA stimulation. The results also indicate that PMA post-transcriptionally regulated at least 0.7% of detectable genes in U937 cells. Thus, besides transcriptional modulation by PMA, changes in the translational state of transcripts seem to play a critical role in PMA-induced differentiation of U937 cells. [Copyright &y& Elsevier]

TNFα has dramatic effects on iron metabolism contributing to the generation of hypoferraemia in the anaemia of chronic disease. Interestingly, TNFα is also synthesised and released within the intestinal mucosa, suggesting that this pro-inflammatory cytokine may play a role in regulating dietary iron absorption. To investigate this possibility, we stimulated intestinal Caco-2 cells with TNFα (10 ng/ml). In TNFα-treated cells, apical iron uptake was significantly decreased and this was accompanied by a reduction in divalent metal transporter protein and mRNA expression. Our data suggest that TNFα could regulate dietary iron absorption and that the apical transport machinery is the target for these actions. [Copyright &y& Elsevier]

We recently published the genomic characterization of the STAT5A and STAT5B paralogous genes that are located head to head in the 17q21 chromosome and share large regions of sequence identity. We here demonstrate by transient in vitro transfection that STAT5A and STAT5B promoters are able to direct comparable levels of transcription. The expression of basal promoters is enhanced after Sp1 up-regulation in HeLa and SL2 cells while DNA methylation associated to the recruitment of MeCP2 methyl CpG binding protein down-regulates STAT5A and B promoters by interfering with Sp1-induced transcription. In addition, cross-species sequence comparison identified a bi-directional negative cis-acting regulatory element located in the STAT5 intergenic region. [Copyright &y& Elsevier]

The poly(A)-binding protein (PABP) is a highly conserved eukaryotic protein whose synthesis is regulated at the post-transcriptional level. The binding of PABP to the poly(A)-rich element found in the 5′-untranslated region (5′UTR) of PABP mRNA specifically inhibits its own translation. In this report, we show that similar adenosine-rich elements in the 5′UTR of the chloramphenicol acetyl-transferase (CAT) gene can significantly reduce the reporter mRNA abundance and translation in human 293 cells. The reduction in mRNA level, but not CAT expression, is dependent on the size of the 5′UTR poly(A) element. Furthermore, one 5′UTR-tethered PABP molecule is enough to inhibit CAT expression without affecting its mRNA level. We propose that the control of PABP synthesis may involve mRNA decay and the repression of translation. [Copyright &y& Elsevier]

The mitogen-activated protein kinase p38 pathway was originally identified as a signalling cascade activated by pro-inflammatory stimuli and cellular stresses, and playing a critical role in the translational regulation of pro-inflammatory cytokine synthesis. In almost a decade since this discovery, a great deal has been learned about the role of the p38 pathway in the post-transcriptional regulation of pro-inflammatory gene expression. However, important questions remain to be answered concerning the specificity and mechanism or mechanisms of action of p38. This review describes recent progress and remaining puzzles in the field of post-transcriptional regulation by p38. [Copyright &y& Elsevier]

Triticum aestivum endoxylanase inhibitors (TAXIs) are wheat proteins that inhibit family 11 endoxylanases commonly used in different (bio)technological processes. Here, we report on the identification of the TAXI-I gene which encodes a mature protein of 381 amino acids with a calculated molecular mass of 38.8 kDa. When expressed in Escherichia coli, the recombinant protein had the specificity and inhibitory activity of natural TAXI-I, providing conclusive evidence that the isolated gene encodes an endoxylanase inhibitor. Bioinformatical analysis indicated that no conserved domains nor motifs common to other known proteins are present. Sequence analysis revealed similarity with a glycoprotein of carrot and with gene families in Arabidopsis thaliana and rice, all with unknown functions. Our data indicate that TAXI-I belongs to a newly identified class of plant proteins for which a molecular function as glycoside hydrolase inhibitor can now be suggested. [Copyright &y& Elsevier]

A novel gap junction forming protein, mouse connexin-30.2 (Cx30.2) contains 278 amino acid residues, and is 79% identical to human Cx31.9 (GJA11). Northern analysis showed that Cx30.2 is ubiquitously expressed, most prominently in testis. Polyclonal antibodies against Cx30.2 detected a 30 kDa protein in cells overexpressing Cx30.2, and in mouse testis. Immunofluorescence showed that Cx30.2 was expressed in vascular smooth muscle, but also in cell types where Cx31.9 was not detected. These data demonstrate that Cx30.2 is a bona fide gene, and suggest that it is the orthologue of Cx31.9, but that it may have additional roles compared with Cx31.9. [Copyright &y& Elsevier]

Our previous evidence suggests that heterogeneous nuclear ribonucleoprotein (hnRNP) A1 plays a part in the regulation of the Cyp2a5 gene by interacting with the 3′ untranslated region (UTR) of the CYP2A5 mRNA. However, the exact role of this interaction is not clear. The aim of the present work was to gain further insight into the regulation process of Cyp2a5. For this purpose the 3′ UTR of CYP2A5 was fused to the coding region of luciferase mRNA. Luciferase recombinants containing either the full length 3′ UTR, or the 3′ UTR lacking a previously described 71 nucleotide (nt) region (the hnRNP A1 primary binding site), were transiently expressed in cells expressing or lacking hnRNP A1. The expression of the luciferase recombinants was examined both at mRNA and enzyme activity levels. The results disclosed that the presence of hnRNP A1 was required for the high expression of the recombinant carrying the full length 3′ UTR of CYP2A5. Deletion of the hnRNP A1 primary binding site dramatically modified the expression pattern: the mRNA levels and luciferase activities of the deletion mutant were independent from hnRNP A1. These results conclusively demonstrate that the 71 nt region in the 3′ UTR of CYP2A5 mRNA can confer hnRNP A1-dependent regulation to a gene. In addition, comparison of RNA levels and luciferase activities suggested that regions flanking the hnRNP A1 binding site could regulate translation of the CYP2A5 mRNA. These results are consistent with a model in which the binding of hnRNP A1 to the 71 nt putative hairpin-loop region in the CYP2A5 mRNA 3′ UTR upregulates mRNA levels possibly by protecting the mRNA from degradation. [Copyright &y& Elsevier]

We describe a cis element that dramatically increases the expression levels of exogenous genes in baculovirus-infected insect cells. This 21 bp sequence element is derived from a 5′ untranslated leader sequence of a lobster tropomyosin cDNA (L21). By using a transfer vector carrying L21, the expression levels of tropomyosin and luciferase were 20- and seven-fold higher with L21 than without L21, respectively. L21 has both the Kozak sequence and the A-rich sequence found in the polyhedrin leader sequence. We assume that both sequence elements are essential for the enhancement of protein expression in the baculovirus-based expression system. [Copyright &y& Elsevier]

Copper is an essential dietary trace metal, however the mechanisms involved in intestinal copper uptake are unclear. Two putative copper transporters are expressed in Caco-2 cells, the divalent metal transporter (DMT1) and copper transporter (Ctr1). Our data demonstrate that copper could compete with iron for uptake via DMT1 and that DMT1 protein and mRNA expression were decreased following exposure (24 h) to high copper. Expression of Ctr1, which acts as a copper transporter in transfected cell lines, was unaffected by copper treatment. Interestingly, exposure to copper increased iron efflux from Caco-2 cells and up regulated IREG1 (iron-regulated mRNA) expression. [Copyright &y& Elsevier]

We have identified and characterised a cDNA encoding a novel gene, designated myocyte stress 1 (ms1), that is up-regulated within 1 h in the left ventricle following the application of pressure overload by aortic banding in the rat. The deduced ms1 protein of 317 amino acids contains several putative functional motifs, including a region that is evolutionarily conserved. Distribution analysis indicates that rat ms1 mRNA expression is predominantly expressed in striated muscle and progressively increases in the left ventricle from embryo to adulthood. These findings suggest that ms1 may be important in striated muscle biology and the development of pressure-induced left ventricular hypertrophy. [Copyright &y& Elsevier]

A divalent metal transporter, DMT1, located on the apical membrane of intestinal enterocytes is the major pathway for the absorption of dietary non-haem iron. Using human intestinal Caco-2 TC7 cells, we have shown that iron uptake and DMT1 protein in the plasma membrane were significantly decreased by exposure to high iron for 24 h, in a concentration-dependent manner, whereas whole cell DMT1 protein abundance was unaltered. This suggests that part of the response to high iron involved redistribution of DMT1 between the cytosol and cell membrane. These events preceded changes in DMT1 mRNA, which was only decreased following 72 h exposure to high iron. [ABSTRACT FROM AUTHOR]