Maintenance of growth rate at low temperature in rice and wheat cultivars with a high degree of respiratory homeostasis is associated with a high efficiency of respiratory ATP production

Some plants have the ability to maintain similar respiratory rates (measured at the growth temperature) when grown at different temperatures. This phenomenon is referred to as respiratory homeostasis. Using wheat and rice cultivars with different degrees of respiratory homeostasis (H), we previously demonstrated that high-H cultivars maintained shoot and root growth at low temperature [Kurimoto et al. (2004) Plant Cell Environ., 27: 853]. Here, we assess the relationship between respiratory homeostasis and the efficiency of respiratory ATP production, by measuring the levels of alternative oxidase (AOX) and uncoupling protein (UCP), which have the potential to decrease respiratory ATP production per unit of oxygen consumed. We also measured SHAM- and CN-resistant respiration of intact roots, and the capacity of the cytochrome pathway (CP) and AOX in isolated mitochondria. Irrespective of H, SHAM-resistant respiration of intact roots and CP capacity of isolated root mitochondria were larger when plants were grown at low temperature, and the maximal activity and relative amounts of cytochrome c oxidase showed a similar trend. In contrast, CN-resistant respiration of intact roots and relative amounts of AOX protein in mitochondria isolated from those roots, were lower in high-H plants grown at low temperature. In the roots of low-H cultivars, relative amounts of AOX protein were higher at low growth temperature. Relative amounts of UCP protein showed similar trends to AOX. We conclude that maintenance of growth rate in high-H plants grown at low temperature is associated with both respiratory homeostasis and a high efficiency of respiratory ATP production.     

Altering the substrate chain-length specificity of an alpha-glucosidase

Dextran glucosidases show high sequence identity (50%) to Bacillus sp. SAM1606 alpha-glucosidase, which is more specific for short-chain substrates. Sequence comparison of these enzymes as well as molecular modeling studies predicted that the extension of loop 4 of the (beta/alpha)(8)-barrel fold may be responsible for the narrower specificity of SAM1606 alpha-glucosidase with respect to substrate chain length. Indeed, deletion mutants of SAM1606 alpha-glucosidase that lack this extension showed higher relative activities toward dextran and long-chain isomaltooligosaccharides. Kinetic and thermodynamic analyses of oligosaccharide hydrolysis catalyzed by SAM1606 alpha-glucosidase and its deletion mutants suggested that the loss of such extension(s) in loop 4 should energetically destabilize the Michaelis complexes with long-chain substrates to result in smaller differences between the activation free energies for the enzymatic hydrolyses of isomaltoheptaose and isomaltose than those observed for the wild-type enzyme. This is the reason that dextran glucosidase, whose loop 4 is shorter in length, shows broader substrate chain-length specificity than does SAM1606 alpha-glucosidase.     

The crystal structure of a cockroach pheromone-binding protein suggests a new ligand binding and release mechanism

Pheromone-binding proteins (PBPs) are small helical proteins found in sensorial organs, particularly in the antennae, of moth and other insect species. They were proposed to solubilize and carry the hydrophobic pheromonal compounds through the antennal lymph to receptors, participating thus in the peri-receptor events of signal transduction. The x-ray structure of Bombyx mori PBP (BmorPBP), from male antennae, revealed a six-helix fold forming a cavity that contains the pheromone bombykol. We have identified a PBP (LmaPBP) from the cockroach Leucophaea maderae in the antennae of the females, the gender attracted by pheromones in this species. Here we report the crystal structure of LmaPBP alone or in complex with a fluorescent reporter (amino-naphthalen sulfonate, ANS) or with a component of the pheromonal blend, 3-hydroxy-butan-2-one. Both compounds bind in the internal cavity of LmaPBP, which is more hydrophilic than BmorPBP cavity. LmaPBP structure ends just after the sixth helix (helix F). BmorPBP structure extends beyond the sixth helix with a stretch of residues elongated at neutral pH and folding as a seventh internalized helix at low pH. These differences between LmaPBP and BmorPBP structures suggest that different binding and release mechanism may be adapted to the hydrophilicity or hydrophobicity of the pheromonal ligand.     

Synthesis and structure-activity relationships of 5-phenylthiophenecarboxylic acid derivatives as antirheumatic agents

5-(Phenylthiophene)-3-carboxylic acid (2a), a metabolite of esonarimod (1), which was developed as a new antirheumatic drug, was considered as a lead compound for new antirheumatic drugs. A new series of 2a derivatives were synthesized and their characteristic pharmacological effects, that is their antagonistic effect toward interleukin (IL)-1 in mice and the suppressive effect against adjuvant-induced arthritis (AIA) in rats, were evaluated and compared with those of 1. The structure-activity relationships indicated that [5-(4-bromophenyl)-thiophen-3-yl]acetic acid (5d), methyl [5-(4-chlorophenyl)-thiophen-3-yl]acetate (5h), and methyl [5-(4-bromophenyl)-thiophen-3-yl]acetate (5i) suppressed AIA more potently than 1 and all of the other synthesized compounds.     

The endogenous Mus81-Eme1 complex resolves Holliday junctions by a nick and counternick mechanism

Functional studies strongly suggest that the Mus81-Eme1 complex resolves Holliday junctions (HJs) in fission yeast, but in vitro it preferentially cleaves flexible three-way branched structures that model replication forks or 3' flaps. Here we report that a nicked HJ is the preferred substrate of endogenous and recombinant Mus81-Eme1. Cleavage occurs specifically on the strand that opposes the nick, resulting in resolution of the structure into linear duplex products. Resolving cuts made by the endogenous Mus81-Eme1 complex on an intact HJ are quasi-simultaneous, indicating that Mus81-Eme1 resolves HJs by a nick and counternick mechanism, with a large rate enhancement of the second cut arising from the flexible nature of the nicked HJ intermediate. Recombinant Mus81-Eme1 is ineffective at making the first cut. We also report that HJs accumulate in a DNA polymerase alpha mutant that lacks Mus81, providing further evidence that the Mus81-Eme1 complex targets HJs in vivo.     

PAK5, a New Brain-Specific Kinase, Promotes Neurite Outgrowth in N1E-115 Cells

We have characterized a new member of the mammalian PAK family of serine/threonine kinases, PAK5, which is a novel target of the Rho GTPases Cdc42 and Rac. The kinase domain and GTPase-binding domain (GBD) of PAK5 are most closely related in sequence to those of mammalian PAK4. Outside of these domains, however, PAK5 is completely different in sequence from any known mammalian proteins. PAK5 does share considerable sequence homology with the Drosophila MBT protein (for "mushroom body tiny"), however, which is thought to play a role in development of cells in Drosophila brain. Interestingly, PAK5 is highly expressed in mammalian brain and is not expressed in most other tissues. We have found that PAK5, like Cdc42, promotes the induction of filopodia. In N1E-115 neuroblastoma cells, expression of PAK5 also triggered the induction of neurite-like processes, and a dominant-negative PAK5 mutant inhibited neurite outgrowth. Expression of activated PAK1 caused no noticeable changes in these cells. An activated mutant of PAK5 had an even more dramatic effect than wild-type PAK5, indicating that the morphologic changes induced by PAK5 are directly related to its kinase activity. Although PAK5 activates the JNK pathway, dominant-negative JNK did not inhibit neurite outgrowth. In contrast, the induction of neurites by PAK5 was abolished by expression of activated RhoA. Previous work has shown that Cdc42 and Rac promote neurite outgrowth by a pathway that is antagonistic to Rho. Our results suggest, therefore, that PAK5 operates downstream to Cdc42 and Rac and antagonizes Rho in the pathway, leading to neurite development.     

Induction of apoptosis by stomach cancer-associated protein-tyrosine phosphatase-1

Stomach cancer-associated protein-tyrosine phosphatase-1 (SAP-1), a transmembrane-type protein-tyrosine phosphatase, is thought to inhibit integrin signaling by mediating the dephosphorylation of focal adhesion-associated proteins. Adenovirus-mediated overexpression of wild-type SAP-1, but not that of a catalytically inactive mutant of this enzyme, has now been shown to induce apoptosis in NIH 3T3 fibroblasts. This effect of SAP-1 was dependent on cellular caspase activities and was preceded by inactivation of two serine-threonine protein kinases, Akt and integrin-linked kinase (ILK), both of which function downstream of phosphoinositide (PI) 3-kinase to promote cell survival. Coexpression of constitutively active forms of PI 3-kinase or Akt (which fully restored Akt and ILK activities) resulted in partial inhibition of SAP-1-induced cell death. Furthermore, expression of a dominant negative mutant of PI 3-kinase did not induce cell death as efficiently as did SAP-1, although this mutant inhibited Akt and ILK activities more effectively than did SAP-1. Overexpression of SAP-1 had no substantial effect on Ras activity. These results suggest that SAP-1 induces apoptotic cell death by at least two distinct mechanisms: inhibition of cell survival signaling mediated by PI 3-kinase, Akt, and ILK and activation of a caspase-dependent proapoptotic pathway.     

Molecular and biochemical characterization of two plant inositol polyphosphate 6-/3-/5-kinases

Despite the high deposition of inositol hexakisphosphate (IP(6)), also known as phytate or phytin, in certain plant tissues little is known at the molecular level about the pathway(s) involved in its production. In budding yeast, IP(6) synthesis occurs through the sequential phosphorylation of I(1,4,5)P(3) by two gene products, Ipk2 and Ipk1, a IP(3)/IP(4) dual-specificity 6-/3-kinase and an inositol 1,3,4,5,6-pentakisphosphate 2-kinase, respectively. Here we report the identification and characterization of two inositol polyphosphate kinases from Arabidopsis thaliana, designated AtIpk2alpha and AtIpk2beta that are encoded by distinct genes on chromosome 5 and that are ubiquitously expressed in mature tissue. The primary structures of AtIpk2alpha and AtIpk2beta are 70% identical to each other and 12-18% identical to Ipk2s from yeast and mammals. Similar to yeast Ipk2, purified recombinant AtIpk2alpha and AtIpk2beta have 6-/3-kinase activities that sequentially phosphorylate I(1,4,5)P(3) to generate I(1,3,4,5,6)P(5) predominantly via an I(1,4,5,6)P(4) intermediate. While I(1,3,4,5)P(4) is a substrate for the plant Ipk2s, it does not appear to be a detectable product of the IP(3) reaction. Additionally, we report that the plant and yeast Ipk2 have a novel 5-kinase activity toward I(1,3,4,6)P(4) and I(1,2,3,4,6)P(5), which would allow these proteins to participate in at least two proposed pathways in the synthesis of IP(6). Heterologous expression of either plant isoform in an ipk2 mutant yeast strain restores IP(4) and IP(5) production in vivo and rescues its temperature-sensitive growth defects. Collectively our results provide a molecular basis for the synthesis of higher inositol polyphosphates in plants through multiple routes and indicate that the 6-/3-/5-kinase activities found in plant extracts may be encoded by the IPK2 gene class.     

Crystal structure of rat heme oxygenase-1 in complex with heme bound to azide. Implication for regiospecific hydroxylation of heme at the alpha-meso carbon

Heme oxygenase (HO) catalyzes physiological heme degradation consisting of three sequential oxidation steps that use dioxygen molecules and reducing equivalents. We determined the crystal structure of rat HO-1 in complex with heme and azide (HO-heme-N(3)(-)) at 1.9-A resolution. The azide, whose terminal nitrogen atom is coordinated to the ferric heme iron, is situated nearly parallel to the heme plane, and its other end is directed toward the alpha-meso position of the heme. Based on resonance Raman spectroscopic analysis of HO-heme bound to dioxygen, this parallel coordination mode suggests that the azide is an analog of dioxygen. The azide is surrounded by residues of the distal F-helix with only the direction to the alpha-meso carbon being open. This indicates that regiospecific oxygenation of the heme is primarily caused by the steric constraint between the dioxygen bound to heme and the F-helix. The azide interacts with Asp-140, Arg-136, and Thr-135 through a hydrogen bond network involving five water molecules on the distal side of the heme. This network, also present in HO-heme, may function in dioxygen activation in the first hydroxylation step. From the orientation of azide in HO-heme-N(3)(-), the dioxygen or hydroperoxide bound to HO-heme, the active oxygen species of the first reaction, is inferred to have a similar orientation suitable for a direct attack on the alpha-meso carbon.