Transcriptional Induction of Two Genes for CCaPs, Novel Cytosolic Proteins, in Arabidopsis thaliana in the Dark

Ca2+-signaling in downstream effectors is supported by many kinds of Ca2+-binding proteins, which function as a signal mediator and a Ca2+-buffering protein. We found in Arabidopsis thaliana a new type of Ca2+-binding protein, CCaP1, which consists of 152 amino acid residues, and binds (45)Ca2+ even in the presence of a high concentration of Mg2+. We found two other proteins with similar motifs, CCaP2 and CCaP3. These three proteins had no organelle localization signal and their green fluorescent protein (GFP) fusions were detected in the cytosol. Real-time PCR and histochemical analysis of promoter-beta-glucuronidase fusions revealed that CCaP1 was predominantly expressed in petioles while CCaP2 was expressed in roots. CCaP3 was hardly expressed. Expression of CCaP1 and CCaP2 was enhanced in darkness and became maximal after 24 h. Immunoblotting revealed petiole-specific accumulation of CCaP1. Expression of CCaP1 and CCaP2 was suppressed by a high concentration of Ca2+ and other metal ions. Deletion of sucrose from the medium markedly increased the mRNA levels of CCaP1 and CCaP2 within 2 h. Gibberellic acid enhanced the expression of CCaP1 and CCaP2 by 5- and 2.5-fold, respectively, after 6 h. CCaP1 and CCaP2 were suppressed in the petiole and the root, respectively, by light and the product of photosynthesis (sucrose) or both. These results suggest that CCaP1 functions as a mediator in response to continuous dark or gibberellic acid.     

Hypoxia-induced apoptosis and tube breakdown are regulated by p38 MAPK but not by caspase cascade in an in vitro capillary model composed of human endothelial cells

In order to improve medical treatment of ischemic injury such as myocardial infarction, it is important to elucidate hypoxia-induced changes to endothelial cells. An in vitro blood vessel model, in which HUVECs are stimulated to form a network of capillary-like tubes, was used to analyze hypoxia-induced morphological and biochemical changes. When exposed to hypoxia, the network of capillary tubes broke down into small clusters. This tube breakdown was accompanied by chromatin condensation and cell nuclear fragmentation, morphological markers of apoptosis, and activation of two apoptotic signals, caspase-3 and p38. We investigated what roles caspase cascade and p38 play in hypoxia-induced apoptosis and tube breakdown by using zVAD-fmk and SB203580, specific inhibitors of these two apoptotic signals, respectively. Chromatin condensation and cell nuclear fragmentation and tube breakdown were effectively inhibited by SB203580, but not by zVAD-fmk. SB203580 caused dephosphorylation of p38, which indicates that p38 was autophosphorylated. Inhibition by zVAD-fmk caused slight MW increase in p17 and emergence of p19, which indicates that the inhibitor caused partial processing of caspase-3. Inhibition of p38 suppressed activation of caspase-3 but not vice versa. In addition, these two inhibitors were shown to differentially inhibit cleavage of so-called caspase substrates. SB203580 inhibited cleavage of PARP and lamin A/C, while zVAD-fmk inhibited cleavage of lamin A/C but not that of PARP. Taken together, these results show that p38 is located upstream of caspase cascade and that, although caspase-3 is activated, a p38-regulated caspase-independent pathway is crucial for the execution of hypoxia-induced apoptosis and tube breakdown.     

Protein kinase C delta regulates the phosphorylation of heat shock protein 27 in human hepatocellular carcinoma

We have recently reported that attenuated phosphorylation of heat shock protein (HSP) 27 correlates with tumor progression in patients with hepatocellular carcinoma (HCC). In the present study, we investigated what kind of kinase regulates phosphorylation of HSP27 in human HCC-derived HuH7 cells. 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1-oleoyl-2-acetylglycerol, direct activators of protein kinase C (PKC), markedly strengthened the phosphorylation of HSP27. Bisindorylmaleimide I, an inhibitor of PKC, suppressed the TPA-induced levels of HSP27 phosphorylation in addition to its basal levels. Knock down of PKCdelta suppressed HSP27 phosphorylation, as well as p38 mitogen-activated protein kinase (MAPK) phosphorylation. SB203580, an inhibitor of p38 MAPK, suppressed the TPA-induced HSP27 phosphorylation. Our results strongly suggest that activation of PKCdelta regulates the phosphorylation of HSP27 via p38 MAPK in human HCC.     

Evolution of shoot apical meristem structures in vascular plants with respect to plasmodesmatal network

Vascular plants have evolved shoot apical meristems (SAMs), whose structures differ among plant groups. To clarify the evolutionary course of the different structural types of SAMs, we compared plasmodesmatal networks in the SAMs for 17 families and 24 species of angiosperms, gymnosperms, and pteridophytes, using transmission electron microscopy (TEM). The plasmodesmata (PD) in almost all cell walls in median longitudinal sections of SAMs were counted, and the PD density per unit area was calculated for each cell wall. Angiosperm and gymnosperm SAMs have low densities, with no difference between stratified (tunica-corpus) and unstratified structures. SAMs of ferns, including Psilotum and Equisetum, have average densities that are more than three times higher than those of seed plants. Interestingly, microphyllous lycopods have both the fern and seed-plant types of PD networks; Selaginellaceae SAMs with single apical cells have high PD densities, while SAMs of Lycopodiaceae and Isoetaceae with plural initial cells have low PD densities, equivalent to those of seed plants. In summary, PD networks are strongly correlated to SAM organizations-SAMs with single and plural initial cells have the fern and seed-plant types of PD, respectively. The two SAM organizations may have evolved separately in lycophytes and euphyllophytes and may be associated with gain or loss of the ability to form secondary PD.     

A Calcium-Receptor Agonist Induces Gustatory Neural Responses in Bullfrogs

The effect of calcium-sensing receptor (CaR) agonists on frog gustatory responses was studied using glossopharyngeal nerve recording and whole-cell patch-clamp recording of isolated taste disc cells. Calcimimetic NPS R-467 dissolved in normal saline solution elicited a large transient response in the nerve. The less active enantiomer of the compound, NPS S-467 induced only a small neural response. The EC₅₀ for NPS R-467 was about 20 μM. Cross-adaptation experiments were performed to examine the effect of 30 μM NPS R-467 and 100 μM quinine on the gustatory neural response. The magnitude of the R-467-induced response after adaptation to quinine was approximately equal to that after adaptation to normal saline solution, indicating that the receptor site for NPS R-467 is different from the site for quinine. NPS R-467 (100 μM) also induced an inward current accompanied with conductance increase and large depolarization in two (13%) of 15 rod cells, and a sustained decrease in outward current and small depolarization in six (40%) other rod cells. NPS S-467 (100 μM) induced a sustained decrease in outward current and depolarization in five (50%) of 10 rod cells. Another calcimimetic cinacalcet (100 μM) induced an inward current accompanied with conductance increase in three (27%) of 11 rod cells. The results suggest that NPS R-467 induces neural responses through cell responses unrelated to a resting K⁺ conductance decrease.     

Parasporin-2Ab,a Newly Isolated Cytotoxic Crystal Protein from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

A novel crystal protein that exhibited potent cytotoxicity against human leukemic T-cells was cloned from the Bacillus thuringiensis TK-E6 strain. The protein, designated as parasporin-2Ab (PS2Ab), was a polypeptide of 304 amino acid residues with a predicted molecular weight of 33,017. The deduced amino acid sequence of PS2Ab showed significant homology (84% identitiy) to parasporin-2Aa (PS2Aa) from the B. thuringiensis A1547 strain. Upon processing of PS2Ab with proteinase K, the active form of 29 kDa was produced. The activated PS2Ab showed potent cytotoxicity against MOLT-4 and Jurkat cells and the EC₅₀ values were estimated as 0.545 and 0.745 ng/mL, respectively. The cytotoxicity of PS2Ab was significantly higher than that of PS2Aa reported elsewhere. Although both cytotoxins were structurally related, it was thought that the minor differences found were responsible for the different cytotoxicities of PS2Ab and PS2Aa.     

HIV/AIDS in Asia： The Shape of Epidemics and Their Molecular Epidemiology

The Asia-Pacific region is a home to 60% of the population in the world and to approximately one quarter of people with HIV/AIDS. Close to a million of people has been infected and a half million people died of AIDS annually in Asia,becoming the second largest epicenter of global AIDS epidemic. Molecular epidemiology has been useful tool to track a course of HIV spread. In-depth knowledge from the studies on molecular epidemiology elucidates the dynamics of HIV spread and the interrelationship of epidemics in the different regions in Asia.     

Structural basis of the initial binding of tRNA(Ile) lysidine synthetase TilS with ATP and L-lysine

In the bacterial genetic-code system, the codon AUA is decoded as isoleucine by tRNA(Ile)(2) with the lysidine residue at the wobble position. Lysidine is derived from cytidine, with ATP and L-lysine, by tRNA(Ile) lysidine synthetase (TilS), which is an N-type ATP pyrophosphatase. In this study, we determined the crystal structure of Aquifex aeolicus TilS, complexed with ATP, Mg2+, and L-lysine, at 2.5 A resolution. The presence of the TilS-specific subdomain causes the active site to have two separate gateways, a large hole and a narrow tunnel on the opposite side. ATP is bound inside the hole, and L-lysine is bound at the entrance of the tunnel. The conserved Asp36 in the PP-motif coordinates Mg2+. In these initial binding modes, the ATP, Mg2+, and L-lysine are held far apart from each other, but they seem to be brought together for the reaction upon cytidine binding, with putative structural changes of the complex.     

Molecular characterization of T4-type bacteriophages in a rice field

Bacteriophages, the viruses that infect bacteria, are the most abundant biological entities in the biosphere and play a key role in global biogeochemical cycling. All T4-type bacteriophage isolates tested so far have a conserved genetic module that encodes the virion components including gene 23 (g23), the major capsid protein. Molecular analysis of the g23 sequence revealed a remarkable level of diversity of T4-type bacteriophages isolated from rice straw and surface soil in a Japanese rice field. It was found that g23 sequences obtained from the rice field were quite distinctive from those obtained in marine environments. Phylogenetic analysis showed that most of these g23 sequences belonged to two novel subgroups of T4-type bacteriophages, although some of them were related to well-studied subgroups of T4-type bacteriophages, such as marine cyanophage isolates of exoT-evens.     

Small‐molecule auxin inhibitors that target YUCCA are powerful tools for studying auxin function

Auxin is essential for plant growth and development, this makes it difficult to study the biological function of auxin using auxin‐deficient mutants. Chemical genetics have the potential to overcome this difficulty by temporally reducing the auxin function using inhibitors. Recently, the indole‐3‐pyruvate (IPyA) pathway was suggested to be a major biosynthesis pathway in Arabidopsis thaliana L. for indole‐3‐acetic acid (IAA), the most common member of the auxin family. In this pathway, YUCCA, a flavin‐containing monooxygenase (YUC), catalyzes the last step of conversion from IPyA to IAA. In this study, we screened effective inhibitors, 4‐biphenylboronic acid (BBo) and 4‐phenoxyphenylboronic acid (PPBo), which target YUC. These compounds inhibited the activity of recombinant YUC in vitro, reduced endogenous IAA content, and inhibited primary root elongation and lateral root formation in wild‐type Arabidopsis seedlings. Co‐treatment with IAA reduced the inhibitory effects. Kinetic studies of BBo and PPBo showed that they are competitive inhibitors of the substrate IPyA. Inhibition constants (K_i) of BBo and PPBo were 67 and 56 nm , respectively. In addition, PPBo did not interfere with the auxin response of auxin‐marker genes when it was co‐treated with IAA, suggesting that PPBo is not an inhibitor of auxin sensing or signaling. We propose that these compounds are a class of auxin biosynthesis inhibitors that target YUC. These small molecules are powerful tools for the chemical genetic analysis of auxin function.