The human MYOD1 transgene is suppressed by 5-bromodeoxyuridine in mouse myoblasts

5-Bromodeoxyuridine (BrdU) immediately and clearly suppresses expression of the mouse Myod1 and human MYOD1 genes in myoblastic cells. Despite various studies, its molecular mechanism remains unknown. We failed to identify a BrdU-responsive element of the genes in experiments in which reporter constructs containing known regulatory sequences were transferred to mouse C2C12 myoblasts. Therefore, we transferred human chromosome 11 containing the MYOD1 gene to the cells by microcell-mediated chromosome transfer. In the resulting microcell hybrids, BrdU suppressed expression of the transgene, as determined by quantitative real-time RT-PCR analysis. We then transfected human PAC clones containing the MYOD1 gene to the cells. In the resulting transfectants, BrdU suppressed the transgene similarly. Deletion analysis suggested that a BrdU-responsive element or chromatin structure exists between 24 and 47 kb upstream of the gene. These results are the first demonstrating BrdU-responsiveness of a transgene for the known BrdU-responsive genes and facilitating determination of its precise responsible structure.     

A structure-based strategy for discovery of small ligands binding to functionally unknown proteins: combination of in silico screening and surface plasmon resonance measurements

In the postgenomic era, many researchers and organizations have been engaged in structural and functional analyses of proteins. As a part of these efforts, searching for small organic compounds that bind specifically to target proteins is quite important. In this study, we have developed a rational strategy for ligand discovery based on the three-dimensional structures of target proteins, which were elucidated by X-ray crystallography and nuclear magnetic resonance spectroscopy. The strategy has three features: (i) rapid selection of candidate compounds by in silico screening, (ii) automated preparation of sample solutions with robotics, and (iii) reliable evaluation of the candidates with surface plasmon resonance. Applying the strategy to a protein, At2g24940 from Arabidopsis thaliana, we discovered four small ligands out of a commercially available library of about 150 000 compounds. Although these compounds had only weak affinities to the target protein, with dissociation constants ranging from 68 to 120 microM, they apparently possess common structural features. They would be leads for the development of specific inhibitors/drugs for At2g24940, and provide important clues toward elucidation of the protein function.     

The ascidian Mesp gene specifies heart precursor cells

Understanding the molecular basis of heart development is an important research area, because malformation of the cardiovascular system is among the most frequent inborn defects. Although recent research has identified molecules responsible for heart morphogenesis in vertebrates, the initial specification of heart progenitors has not been well characterized. Ascidians provide an appropriate experimental system for exploring this specification mechanism, because the lineage for the juvenile heart is well characterized, with B7.5 cells at the 110-cell stage giving rise to embryonic trunk ventral cells (TVCs) or the juvenile heart progenitors. Here, we show that Cs-Mesp, the sole ortholog of vertebrate Mesp genes in the ascidian Ciona savignyi, is specifically and transiently expressed in the embryonic heart progenitor cells (B7.5 cells). Cs-Mesp is essential for the specification of heart precursor cells, in which Nkx, HAND and HAND-like (NoTrlc) genes are expressed. As a result, knockdown of Cs-Mesp with specific morpholino antisense oligonucleotides causes failure of the development of the juvenile heart. Together with previous evidence obtained in mice, the present results suggest that a mechanism for heart specification beginning with Mesp through Nkx and HAND is conserved among chordates.     

Angiotensin II and III upregulate body fluid volume of the clam worm Perinereis sp. via angiotensin II receptors in different manners

Angiotensin III (Ang III) as well as angiotensin II (Ang II) suppressed body weight loss of the clam worm Perinereis sp. under a hyper-osmotic condition, and enhanced body weight gain under a hypo-osmotic condition. Under a drying condition where the water inflow from outside the body was eliminated, Ang II suppressed body weight loss, but Ang III did not. Under these conditions, angiotensins I, IV, and (1–7) had no effect, and saralasin blocked the effects of Ang II and Ang III. It is concluded that Ang II and Ang III upregulate body fluid volume of the clam worm via Ang II receptors in different ways.     

The cell death machinery controlled by Bax and Bcl-XL is evolutionarily conserved in <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

Bax and Bcl-XL are key regulators of apoptosis in mammals. Here we report the functional characterization of two Bcl-2 homologues, ciBax and ciBcl-XL, in a basal invertebrate-chordate ascidian Ciona intestinalis. CiBax is a Ciona homologue of the BH1-3 pro-apoptotic protein Bax, whereas ciBcl-XL is a Bcl-XL-like anti-apoptotic protein. Molecular modeling analysis showed that ciBax and ciBcl-XL share both sequence and structural similarities to human Bax and Bcl-XL, respectively. Like their human counterparts, ciBax could form a homodimer or oligomers as well as heterodimerize with ciBcl-XL, and overexpression of ciBax caused apoptosis that could be attenuated by ciBcl-XL. Mutagenesis studies showed that the BH3 domain of ciBax is critical for its cell death-inducing function and also for its interaction with ciBcl-XL. In Ciona embryos, ectopic expression of ciBax but not its BH3 deletion mutant resulted in cell dissociation and apoptosis after late gastrula stage of embryonic development. Moreover, not only wild type ciBcl-XL but also a mutant ciBcl-XL(F101V), which is unable to interact with ciBax, could block cell dissociation and developmental deficit in Ciona embryos induced by overexpression of ciBax. Taken together, these findings suggest that functional homologues of both the BH1-3 death effector Bax and the pro-survival Bcl-XL exist in sea squirt Ciona intestinalis, and they control the cell death machinery independent of their heterodimerization.