TBL1 and TBLR1 phosphorylation on regulated gene promoters overcomes dual CtBP and NCoR/SMRT transcriptional repression checkpoints

Cellular differentiation entails loss of pluripotency and gain of lineage- and cell-type-specific characteristics. Using a murine system that progresses from stem cells to lineage-committed progenitors to terminally differentiated neurons, we analyzed DNA methylation and Polycomb-mediated histone H3 methylation (H3K27me3). We show that several hundred promoters, including pluripotency and germline-specific genes, become DNA methylated in lineage-committed progenitor cells, suggesting that DNA methylation may already repress pluripotency in progenitor cells. Conversely, we detect loss and acquisition of H3K27me3 at additional targets in both progenitor and terminal states. Surprisingly, many neuron-specific genes that become activated upon terminal differentiation are Polycomb targets only in progenitor cells. Moreover, promoters marked by H3K27me3 in stem cells frequently become DNA methylated during differentiation, suggesting context-dependent crosstalk between Polycomb and DNA methylation. These data suggest a model how de novo DNA methylation and dynamic switches in Polycomb targets restrict pluripotency and define the developmental potential of progenitor cells.     

Comparison of respiratory burst activity of inflammatory neutrophils in ayu (Plecoglossus altivelis) and carp (Cyprinus carpio)

Neutrophils of ayu (Plecoglossus altivelis) were previously shown to have unusually high respiratory burst activity (RBA). To understand this unique character of ayu neutrophils, the RBAs of resting and inflammatory neutrophils of ayu and carp (Cyprinus carpio) were compared. Inflammation was induced in the peritoneal cavity by injecting killed-bacteria. The RBA of peritoneal-exudate (inflammatory) neutrophils was measured after stimulation with phorbol myristate acetate (PMA). Resting neutrophils were obtained from kidney and blood of non-injected fish. In carp, the RBA of inflammatory neutrophils was much higher than that of resting neutrophils. On the other hand, in ayu no significant difference was observed. The RBA of neutrophils was already high in the kidney stock. The process of inflammation did not further enhance RBA. In addition to PMA, other stimulants (zymosan, opsonized-zymosan, and zymosan-treated serum) were used to measure RBA. Even with these stimulants, the RBA of inflammatory neutrophils was always higher than that of kidney neutrophils in carp. On the other hand in ayu, the RBA of kidney neutrophils was already high in the kidney stock, and no significant difference was observed between peritoneal and kidney neutrophils in ayu. These results indicate ayu neutrophils have spontaneously activated characteristics with the respect to the ROS generation in the kidney hematopoietic-stock.     

On a salmon (Oncorhynchus [corrected] keta) liver RNase, belonging to RNase T2 family: primary structure and some properties

A base-nonspecific and acid ribonuclease (RNase Ok2) was purified from the liver of a salmon (Oncorhnchus keta) to a homogeneous state by SDS-PAGE. The primary structure of RNase Ok2 was determined by protein chemistry and molecular cloning. The RNase Ok2 was a glycoprotein and consisted of 216 amino acid residues. Its molecular mass of protein moiety was 25,198, and its amino acid sequence showed that it belongs to the RNase T2 family of enzymes. The optimal pH of RNase Ok2 was around 5.5. The base preferences at the B1 and B2 sites were estimated from the rates of hydrolysis of 16 dinucleoside phosphates to be G>A>U, C, and G>A>U>C respectively. In this enzyme, one of the three histidine residues which have been thought to be important for catalysis of RNase Rh, a typical RNase of this family of enzymes, His104 was replaced by tyrosine residue. Based on the results, the role of H104, which has been proposed to be a phosphate binding site with a substrate, was reconsidered, and we proposed a revised role of this His residue in the hydrolysis mechanism of RNase T2 family enzymes.     

Molecular cloning and preliminary expression analysis of banded dogfish (Triakis scyllia) CC chemokine cDNAs by use of suppression subtractive hybridization

Suppression subtractive hybridization was carried out by using cDNAs of peripheral white blood cells (PWBCs) of banded dogfish (Triakis scyllia) after phorbol 12-myristate 13-acetate (PMA) stimulation. The Trsc-SCYA107, MIP31 and MIP32 clones contained an open reading frame encoding 97, 99 and 97 amino acids, respectively. Comparison of the deduced amino acids showed that the banded dogfish MIP31 and MIP32 sequences shared 42.3% and 40.0% identity with human SCYA20, respectively, while the Trsc-SCYA107 sequence shared 50.6, 44.2 and 42.0% identity with the catshark (Scyliorhinus canicula) Scca-SCYA107, rainbow trout (Oncorhynchus mykiss) CK4A and CK4B, respectively. The genomic sequences of banded dogfish Trsc-SCYA107, MIP31 and MIP32 contain four exons and three introns, and MIP31 and MIP32 shared the same intron/exon organization with that of human. The MIP31 and MIP32 genes of lipopolysaccharide (LPS)-unstimulated banded dogfish were expressed in gill, kidney and liver, while Trsc-SCYA107 mRNA was detected in various tissues except for brain. However, the constitutive expression of MIP32 gene was much lower than the Trsc-SCYA107 and MIP31 genes. RT-PCR analysis of the Trsc-SCYA107 expression in tissues of LPS-stimulated fish showed enhanced expression at 24 h poststimulation in the gill, heart, leydig, spleen and testes, while the expression of MIP31 and MIP32 was not influenced by LPS-stimulation in vivo. Furthermore, a relative increase in the expression of the Trsc-SCYA107 and MIP32 genes in PWBCs was observed at 1–12 h poststimulation with PMA and LPS, with maximal expression observed at 3 h, while MIP31 expression was observed at 3–12 h poststimulation only with PMA.     

Isolation of Iodide-Oxidizing Bacteria from Iodide-Rich Natural Gas Brines and Seawaters

Iodide-oxidizing bacteria (IOB), which oxidize iodide (I⁻) to molecular iodine (I₂), were isolated from iodide-rich (63 μM to 1.2 mM) natural gas brine waters collected from several locations. Agar media containing iodide and starch were prepared, and brine waters were spread directly on the media. The IOB, which appeared as purple colonies, were obtained from 28 of the 44 brine waters. The population sizes of IOB in the brines were 10² to 10⁵ colony-forming units (CFU) mL⁻¹. However, IOB were not detected in natural seawaters and terrestrial soils (fewer than 10 CFU mL⁻¹ and 10² CFU g wet weight of soils⁻¹, respectively). Interestingly, after the enrichment with 1 mM iodide, IOB were found in 6 of the 8 seawaters with population sizes of 10³ to 10⁵ CFU mL⁻¹. 16S rDNA sequencing and phylogenetic analyses showed that the IOB strains are divided into two groups within the α-subclass of the Proteobacteria. One of the groups was phylogenetically most closely related to Roseovarius tolerans with sequence similarities between 94% and 98%. The other group was most closely related to Rhodothalassium salexigens, although the sequence similarities were relatively low (89% to 91%). The iodide-oxidizing reaction by IOB was mediated by an extracellular enzyme protein that requires oxygen. Radiotracer experiments showed that IOB produce not only I₂ but also volatile organic iodine, which were identified as diiodomethane (CH₂I₂) and chloroiodomethane (CH₂ClI). These results indicate that at least two types of IOB are distributed in the environment, and that they are preferentially isolated in environments in which iodide levels are very high. It is possible that IOB oxidize iodide in the natural environment, and they could significantly contribute to the biogeochemical cycling of iodine.     

Enzymatic properties of glutamine 32 mutants of RNase Rh from Rhizopus niveus,a trial to alter the most preferential inter-nucleotidic linkages of RNase Rh

In order to investigate the effects of mutation of Gln32, a component of a base recognition site (B2 site) of a base-nonspecific RNase from Rhizopus niveus, we prepared several enzymes mutant at this position, Q32F, Q32L, Q32V, Q32T, Q32D, Q32N, and Q32E, and their enymatic activities toward RNA and 16 dinucleoside phosphates were measured. Enzymatic activities of the mutant enzymes towards RNA were between 10-125% of the native enzyme. From the rates of hydrolysis of 16 dinucleoside phosphates by mutant enzymes, we estimated the base specificity of both B1 and B2 sites. The results indicated that mutation of Gln32 to Asp, Asn, and Glu caused the B2 site to prefer cytosine more and to a less extent, to prefer uracil (Q32N), and that Q32F made the enzyme more guanine-base preferential. The results suggested that we are able to construct an enzyme that preferentially cleaves internucleotidic linkages, at the 5'-side of cytosine residues (Q32D, Q32N, and Q32E) and guanine residues (Q32F and Q32T), thus, cleaves purine-C(Q32D, Q32N, Q32E) and GpG and ApG (Q32F, and Q32T) most easily. The results seemed to suggest converting a base-non-specific RNase to a base-specific one.     

Substrate specificities of deuterolysin from Aspergillus oryzae and electron paramagnetic resonance measurement of cobalt-substituted deuterolysin

The substrate specificities of deuterolysin, a 19-kDa zinc-protease (EC 3.4.24.39) from Aspergillus oryzae, were investigated at pH 9.0 with various fluorogenic acyl-peptide-4-methylcoumaryl-7-amides (peptide-MCAs). N-Butoxycarbonyl-Arg-Val-Arg-Arg-MCA was the best substrate for deuterolysin. We therefore measured its kinetic parameters. Deuterolysin had high activity toward the peptide bonds next to pairs of basic residues in calf thymus histone H4. The specificity of cobalt-substituted deuterolysin (Co-deuterolysin) for peptide-MCAs was similar to that of native deuterolysin. The CD spectrum of Co-deuterolysin was similar to that of the native deuterolysin. The metal coordination sphere of Co-deuterolysin was analyzed by Q-band (33.9570 GHz) electron paramagnetic resonance (EPR) spectroscopy. Using computer simulation of EPR, we found the g principal values to be g(xx) = 5.20, g(yy) = 4.75, and g(zz) = 2.24; the metal center was a divalent cobalt ion in a high spin state.     

Tbx5 specifies the left/right ventricles and ventricular septum position during cardiogenesis

Extensive misexpression studies were carried out to explore the roles played by Tbx5, the expression of which is excluded from the right ventricle (RV) during cardiogenesis. When Tbx5 was misexpressed ubiquitously, ventricular septum was not formed, resulting in a single ventricle. In such heart, left ventricle (LV)-specific ANF gene was induced. In search of the putative RV factor(s), we have found that chick Tbx20 is expressed in the RV, showing a complementary fashion to Tbx5. In the Tbx5-misexpressed heart, this gene was repressed. When misexpression was spatially partial, leaving small Tbx5-negative area in the right ventricle, ventricular septum was shifted rightwards, resulting in a small RV with an enlarged LV. Focal expression induced an ectopic boundary of Tbx5-positive and -negative regions in the right ventricle, at which an additional septum was formed. Similar results were obtained from the transient transgenic mice. In such hearts, expression patterns of dHAND and eHAND were changed with definitive cardiac abnormalities. Furthermore, we report that human ANF promoter is synergistically activated by Tbx5, Nkx2.5 and GATA4. This activation was abrogated by Tbx20, implicating the pivotal roles of interactions among these heart-specific factors. Taken together, our data indicate that Tbx5 specifies the identity of LV through tight interactions among several heart-specific factors, and highlight the essential roles of Tbx5 in cardiac development.