Cloning and characterization of an mRNA encoding a novel G protein alpha-subunit abundant in mantle and gill of pearl oyster Pinctada fucata

Nacre formation is an ideal model to study biomineralization processes. Although much has been done about biomineralization mechanism of nacre, little is known as to how cellular signaling regulates this process. We are interested in whether G protein signaling plays a role in mineralization. Degenerate primers against conserved amino acid regions of G proteins were employed to amplify cDNA from the pearl oyster Pinctada fucata. As a result, the cDNA encoding a novel G(s)alpha (pfG(s)alpha) from the pearl oyster was isolated. The G(s)alpha cDNA encodes a polypeptide of 377 amino acid residues, which shares high similarity to the octopus (Octopus vulgaris) G(s)alpha. The well-conserved A, C, G (switch I), switch II functional domains and the carboxyl terminus that is a critical site for interaction with receptors are completely identical to those from other mollusks. However, pfG(s)alpha has a unique amino acid sequence, which encodes switch III and interaction sites of adenylyl cyclase respectively. In situ hybridization and Northern blotting analysis revealed that the oyster G(s)alpha mRNA is widely expressed in a variety of tissues, with highest levels in the outer fold of mantle and epithelia of gill, the regions essential for biomineralization. We also show that overexpression of the pfG(s)alpha in mammalian MC3T3-E1 cells resulted in increased cAMP levels. Mutant pfG(s)alpha that has impaired CTX substrate diminished its ability to induce cAMP production. Furthermore, the alkaline phosphatase (ALP) activity, an indicator for mineralization, is induced by the G(s)alpha in MC3T3-E1 cells. These results indicated that G(s)alpha may be involved in regulation of physiological function, particularly in biological biomineralization.     

The influence of transposable elements on genome size

Genome size displays an important variability between species without any direct link to complexity. This paradox, so-called "C value paradox", now becomes understood as resulting from a differential abundance of numerous repeated sequences, among which transposable elements. Genomes indeed contain a important proportion of such sequences (95 % of DNA in man, about 45 % of which are transposable elements, up to 99 % of DNA in some plants). While most investigations until now are focalized on genes or coding sequences, which thus represent a small part of the genome, more attention now is dedicated on so-called non-coding sequences. Transposable elements, which are capable of moving around in genomes, inducing mutations, chromosomal rearrangements, gene expression regulations, thus appear as major actors in diversity and evolution. We present here a brief review of the most prominent acquisition in this expanding domain.     

Partial purification of α-amylase from culture supernatant of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> in aqueous two-phase systems

A study was made of the partition and purification of -amylase from a culture supernatant of Bacillus subtilis in the polyethylene glycol (PEG)—citrate aqueous two-phase system (ATPS). Factors that influenced the partition of the protein in this system, including the molecular weight of the PEG, the tie line length of ATPS, the pH value and the sodium chloride concentration, were investigated. Purification of -amylase was attained with a purification factor (PF) of 1.8 and 90% yield at pH 6.0 in a PEG1000-citrate ATPS with short tie line length. By utilizing the salt-out effect of neutral salt, the purification of -amylase was further improved to 2.0 of PF and 80% yield in a PEG3350-citrate ATPS with 4% sodium chloride.     

Telomere protection without a telomerase; the role of ATM and Mre11 in Drosophila telomere maintenance

The conserved ATM checkpoint kinase and the Mre11 DNA repair complex play essential and overlapping roles in maintaining genomic integrity. We conducted genetic and cytological studies on Drosophila atm and mre11 knockout mutants and discovered a telomere defect that was more severe than in any of the non-Drosophila systems studied. In mutant mitotic cells, an average of 30% of the chromosome ends engaged in telomere fusions. These fusions led to the formation and sometimes breakage of dicentric chromosomes, thus starting a devastating breakage-fusion-bridge cycle. Some of the fusions depended on DNA ligase IV, which suggested that they occurred by a nonhomologous end-joining (NHEJ) mechanism. Epistasis analyses results suggest that ATM and Mre11 might also act in the same telomere maintenance pathway in metazoans. Since Drosophila telomeres are not added by a telomerase, our findings support an additional role for both ATM and Mre11 in telomere maintenance that is independent of telomerase regulation.     

Characterization of a novel toxin-antitoxin module, VapBC, encoded by Leptospira interrogans chromosome

Comparative genomic analysis of the coding sequences (CDSs) of Leptospira interrogans revealed a pair of closely linked genes homologous to the vapBC loci of many other bacteria with respect to both deduced amino acid sequencesand operon organizations. Expression of single vapC gene in Escherichia coli resulted in inhibition of bacterial growth,whereas co-expression of vapBC restored the growth effectively. This phenotype is typical for three other character-ized toxin-antitoxin systems of bacteria, i.e., mazEF[1], reIBE[2] and chplK[3]. The VapC proteins of bacteria and a thermophilic archeae, Solfolobus tokodaii, form a structurally distinguished group of toxin different from the other known toxins of bacteria. Phylogenetic analysis of both toxins and antitoxins of all categories indicated that althought oxins were evolved from divergent sources and may or may not follow their speciation paths (as indicated by their 16s RNA seouences）, co-evolution with their antitoxins was obvious.     

Pxl1p, a paxillin-like protein in Saccharomyces cerevisiae, may coordinate Cdc42p and Rho1p functions during polarized growth

Rho-family GTPases Cdc42p and Rho1p play critical roles in the budding process of the yeast Saccharomyces cerevisiae. However, it is not clear how the functions of these GTPases are coordinated temporally and spatially during this process. Based on its ability to suppress cdc42-Ts mutants when overexpressed, a novel gene PXL1 was identified. Pxl1p resembles mammalian paxillin, which is involved in integrating various signaling events at focal adhesion. Both proteins share amino acid sequence homology and structural organization. When expressed in yeast, chicken paxillin localizes to the sites of polarized growth as Pxl1p does. In addition, the LIM domains in both proteins are the primary determinant for targeting the proteins to the cortical sites in their native cells. These data strongly suggest that Pxl1p is the "ancient paxillin" in yeast. Deletion of PXL1 does not produce any obvious phenotype. However, Pxl1p directly binds to Rho1p-GDP in vitro, and inhibits the growth of rho1-2 and rho1-3 mutants in a dosage-dependent manner. The opposite effects of overexpressed Pxl1p on cdc42 and rho1 mutants suggest that the functions of Cdc42p and Rho1p may be coordinately regulated during budding and that Pxl1p may be involved in this coordination.     

CCK-A receptor activates RhoA through G alpha 12/13 in NIH3T3 cells

Cholecystokinin (CCK) is a major regulator of pancreatic acinar cells and was shown previously to be capable of inducing cytoskeletal changes in these cells. In the present study, using NIH3T3 cells stably transfected with CCK-A receptors as a model cell, we demonstrate that CCK can induce actin stress fibers through a G₁₃- and RhoA-dependent mechanism. CCK induced stress fibers within minutes similar to those induced by lysophosphatidic acid (LPA), the active component of serum. The effects of CCK were mimicked by active RhoV14 and blocked by dominant-negative RhoN19, Clostridium botulinum C3 transferase, and the Rho-kinase inhibitor Y-27632. CCK rapidly induced active Rho in cells as shown with a pull-down assay using the Rho binding domain of rhotekin and by a serum response element (SRE)-luciferase reporter assay. To evaluate the G protein mediating the action of CCK, cells were transfected with active -subunits; G₁₃ and G₁₂ but not G_q induced stress fibers and in some cases cell rounding. A p115 Rho guanine nucleotide exchange factor (GEF) regulator of G protein signaling (RGS) domain known to interact with G_12/13 inhibited active _12/13-and CCK-induced stress fibers, whereas RGS2 and RGS4, which are known to inhibit G_q, had no effect. Cotransfection with plasmids coding for the G protein -subunit carboxy-terminal peptide from ₁₃ and, to a lesser extent ₁₂, also inhibited the effect of CCK, whereas the peptide from _q did not. These results show that in NIH3T3 cells bearing CCK-A receptors, CCK activates Rho primarily through G₁₃, leading to rearrangement of the actin cytoskeleton. actin; cholecystokinin; Rho; Rho-kinase; stress fibers     

Regulation of septin organization and function in yeast

Septins are a conserved eukaryotic family of GTP-binding filament-forming proteins with functions in cytokinesis and other processes. In the budding yeast Saccharomyces cerevisiae, septins initially localize to the presumptive bud site and then to the cortex of the mother-bud neck as an hourglass structure. During cytokinesis, the septin hourglass splits and single septin rings partition with each of the resulting cells. Septins are thought to function in diverse processes in S. cerevisiae, mainly by acting as a scaffold to direct the neck localization of septin-associated proteins.     

TRAIL-R2 (DR5) mediates apoptosis of synovial fibroblasts in rheumatoid arthritis

TRAIL has been proposed as an anti-inflammatory cytokine in animal models of rheumatoid arthritis (RA). Using two agonistic mAbs specific for TRAIL-R1 (DR4) and TRAIL-R2 (DR5), we examined the expression and function of these death receptors in RA synovial fibroblast cells. The synovial tissues and primary synovial fibroblast cells isolated from patients with RA, but not those isolated from patients with osteoarthritis, selectively expressed high levels of cell surface DR5 and were highly susceptible to anti-DR5 Ab (TRA-8)-mediated apoptosis. In contrast, RA synoviocytes did not show increased expression of TRAIL-R1 (DR4), nor was there any difference in expression of Fas between RA and osteoarthritis synovial cells. In vitro TRA-8 induced apoptosis of RA synovial cells and inhibited production of matrix metalloproteinases induced by pro-inflammatory cytokines. In vivo TRA-8 effectively inhibited hypercellularity of a SV40-transformed RA synovial cell line and completely prevented bone erosion and cartilage destruction induced by these cells. These results indicate that increased DR5 expression and susceptibility to DR5-mediated apoptosis are characteristic of the proliferating synovial cells in RA. As highly proliferative transformed-appearing RA synovial cells play a crucial role in bone erosion and cartilage destruction in RA, the specific targeting of DR5 on RA synovial cells with an agonistic anti-DR5 Ab may be a potential therapy for RA.