Characterization of an essential histidine residue in thermophilic malate dehydrogenase

Heat-stable malate dehydrogenase isolated from Thermus flavus AT62 was completely inactivated by treatment with diethylpyrocarbonate. The inactivation was accompanied by the loss of 1.2 histidine residues per subunit of the enzyme. The enzyme was protected from inactivation by NADH. The enzyme was also inactivated by dye-sensitized photooxidation. Methionine residues, in addition to histidine residues, were destroyed in the inactivated enzyme. Kinetic analyses of the inactivation indicated that the pK value of the residue involved in the inactivation was 8.20 at 25.0 degrees C and 7.52 at 60.0 degrees C. From the pK values and the heat of ionization calculated from the van't Hoff plot of pKs, a histidine residue was identified to be primarily involved in the inactivation. The effect of temperature on the pK value of the essential group in this enzyme from a thermophilic organism is discussed.     

The telomerase activities in several organs and strains of rats with ageing

Telomerase activity is known to be implicated both in cell immortalization and carcinogenesis. Telomerase activity has not been detected in most human somatic tissues. However, we previously confirmed that the activity is present both in methylazoxymethanol acetate-induced rat colonic adenocarcinoma and non-treated colonic mucosa, presumably indicating the tissue-specific activity of the enzyme in rats. To determine the standard activity of rat telomerase in various organs in relation to differences in sex, age and strain, we examined the activity by using the telomeric repeat amplification protocol (TRAP) assay. The testis, liver, and colon mucosa showed the activity. The brain had very low or negative activity in 5-week-old male rats of the F344, SD, Wistar, Donryu or ACI strains. Age (5-week-old and 9-month-old) or sex difference for the activity was not apparent in rats of these strains. In general, telomerase activity in the fetal brain, liver and kidney was stronger than in the adult organ. The telomerase activity of each organ was different from that of human. This difference may indicate that the rat has a specific mechanism for maintaining the telomeric repeats of the chromosome even in somatic tissues. The basic information resulting from this study may be useful for the study of the role of telomerase in tumorigenesis in animal experiment models.     

Estrogenic antagonists bearing dicarba-closo-dodecaborane as a hydrophobic pharmacophore

Dicarba-closo-dodecaboranes (carboranes), which have spherical geometry and hydrophobicity, are applicable as a hydrophobic pharmacophore of biologically active molecules. We have designed and synthesized estrogenic antagonists based on the structure of the potent agonist 1-hydroxymethyl-12-(4-hydroxyphenyl)-1,12-dicarba-closo-dodecaborane, which we have developed. The compounds showed potent antagonistic activity in luciferase reporter gene assay using COS-1 cells transfected with rat ERα-expression plasmid and an appropriate reporter plasmid.     

Loss of Axonal Mitochondria Promotes Tau-Mediated Neurodegeneration and Alzheimer's Disease-Related Tau Phosphorylation Via PAR-1

Abnormal phosphorylation and toxicity of a microtubule-associated protein tau are involved in the pathogenesis of Alzheimer's disease (AD); however, what pathological conditions trigger tau abnormality in AD is not fully understood. A reduction in the number of mitochondria in the axon has been implicated in AD. In this study, we investigated whether and how loss of axonal mitochondria promotes tau phosphorylation and toxicity in vivo. Using transgenic Drosophila expressing human tau, we found that RNAi-mediated knockdown of milton or Miro, an adaptor protein essential for axonal transport of mitochondria, enhanced human tau-induced neurodegeneration. Tau phosphorylation at an AD-related site Ser262 increased with knockdown of milton or Miro; and partitioning defective-1 (PAR-1), the Drosophila homolog of mammalian microtubule affinity-regulating kinase, mediated this increase of tau phosphorylation. Tau phosphorylation at Ser262 has been reported to promote tau detachment from microtubules, and we found that the levels of microtubule-unbound free tau increased by milton knockdown. Blocking tau phosphorylation at Ser262 site by PAR-1 knockdown or by mutating the Ser262 site to unphosphorylatable alanine suppressed the enhancement of tau-induced neurodegeneration caused by milton knockdown. Furthermore, knockdown of milton or Miro increased the levels of active PAR-1. These results suggest that an increase in tau phosphorylation at Ser262 through PAR-1 contributes to tau-mediated neurodegeneration under a pathological condition in which axonal mitochondria is depleted. Intriguingly, we found that knockdown of milton or Miro alone caused late-onset neurodegeneration in the fly brain, and this neurodegeneration could be suppressed by knockdown of Drosophila tau or PAR-1. Our results suggest that loss of axonal mitochondria may play an important role in tau phosphorylation and toxicity in the pathogenesis of AD.     

PB2 Protein of a Highly Pathogenic Avian Influenza Virus Strain A/chicken/Yamaguchi/7/2004 (H5N1) Determines Its Replication Potential in Pigs

It has been shown that not all but most of the avian influenza viruses replicate in the upper respiratory tract of pigs (H. Kida et al., J. Gen. Virol. 75:2183-2188, 1994). It was shown that A/chicken/Yamaguchi/7/2004 (H5N1) [Ck/Yamaguchi/04 (H5N1)] did not replicate in pigs (N. Isoda et al., Arch. Virol. 151:1267-1279, 2006). In the present study, the genetic basis for this host range restriction was determined using reassortant viruses generated between Ck/Yamaguchi/04 (H5N1) and A/swine/Hokkaido/2/1981 (H1N1) [Sw/Hokkaido/81 (H1N1)]. Two in vivo-generated single-gene reassortant virus clones of the H5N1 subtype (virus clones 1 and 2), whose PB2 gene was of Sw/Hokkaido/81 (H1N1) origin and whose remaining seven genes were of Ck/Yamaguchi/04 (H5N1) origin, were recovered from the experimentally infected pigs. The replicative potential of virus clones 1 and 2 was further confirmed by using reassortant virus (rg-Ck-Sw/PB2) generated by reverse genetics. Interestingly, the PB2 gene of Ck/Yamaguchi/04 (H5N1) did not restrict the replication of Sw/Hokkaido/81 (H1N1), as determined by using reassortant virus rg-Sw-Ck/PB2. The rg-Sw-Ck/PB2 virus replicated to moderate levels and for a shorter duration than parental Sw/Hokkaido/81 (H1N1). Sequencing of two isolates recovered from the pigs inoculated with rg-Sw-Ck/PB2 revealed either the D256G or the E627K amino acid substitution in the PB2 proteins of the isolates. The D256G and E627K mutations enhanced viral polymerase activity in the mammalian cells, correlating with replication of virus in pigs. These results indicate that the PB2 protein restricts the growth of Ck/Yamaguchi/04 (H5N1) in pigs.     

On the Nature of the Subjectivity of Living Things

A biosemiotic view of living things is presented that supersedes the mechanistic view of life prevalent in biology today. Living things are active agents with autonomous subjectivity, whose structure is triadic, consisting of the individual organism, its Umwelt and the society. Sociality inheres in every living thing since the very origin of life on the earth. The temporality of living things is guided by the purpose to live, which works as the semantic boundary condition for the processes of embodiment of the subjectivity. Freedom at the molecular and cellular levels allows autonomy and spontaneity to emerge even in single cell organisms, and the presence of the dimension of mind in every living thing is deduced. Living things transcend their individualness, as they live in historically formed higher order structure consisting of the lineage-species and the society. They also transcend materiality, having the dimension of mind.     

Stearoyl-CoA desaturase-1 (SCD1) augments saturated fatty acid-induced lipid accumulation and inhibits apoptosis in cardiac myocytes

Mismatch between the uptake and utilization of long-chain fatty acids in the myocardium leads to abnormally high intracellular fatty acid concentration, which ultimately induces myocardial dysfunction. Stearoyl-Coenzyme A desaturase-1 (SCD1) is a rate-limiting enzyme that converts saturated fatty acids (SFAs) to monounsaturated fatty acids. Previous studies have shown that SCD1-deficinent mice are protected from insulin resistance and diet-induced obesity; however, the role of SCD1 in the heart remains to be determined. We examined the expression of SCD1 in obese rat hearts induced by a sucrose-rich diet for 3 months. We also examined the effect of SCD1 on myocardial energy metabolism and apoptotic cell death in neonatal rat cardiac myocytes in the presence of SFAs. Here we showed that the expression of SCD1 increases 3.6-fold without measurable change in the expression of lipogenic genes in the heart of rats fed a high-sucrose diet. Forced SCD1 expression augmented palmitic acid-induced lipid accumulation, but attenuated excess fatty acid oxidation and restored reduced glucose oxidation. Of importance, SCD1 substantially inhibited SFA-induced caspase 3 activation, ceramide synthesis, diacylglycerol synthesis, apoptotic cell death, and mitochondrial reactive oxygen species (ROS) generation. Experiments using SCD1 siRNA confirmed these observations. Furthermore, we showed that exposure of cardiac myocytes to glucose and insulin induced SCD1 expression. Our results indicate that SCD1 is highly regulated by a metabolic syndrome component in the heart, and such induction of SCD1 serves to alleviate SFA-induced adverse fatty acid catabolism, and eventually to prevent SFAs-induced apoptosis.     

RA-RhoGAP, Rap-activated Rho GTPase-activating protein implicated in neurite outgrowth through Rho

Rap1 and Rho small G proteins have been implicated in the neurite outgrowth, but the functional relationship between Rap1 and Rho in the neurite outgrowth remains to be established. Here we identified a potent Rho GTPase-activating protein (GAP), RA-RhoGAP, as a direct downstream target of Rap1 in the neurite outgrowth. RA-RhoGAP has the RA and GAP domains and showed GAP activity specific for Rho, which was enhanced by the binding of the GTP-bound active form of Rap1 to the RA domain. Overexpression of RA-RhoGAP induced inactivation of Rho for promoting the neurite outgrowth in a Rap1-dependent manner. Knockdown of RA-RhoGAP reduced the Rap1-induced neurite outgrowth. These results indicate that RA-RhoGAP transduces a signal from Rap1 to Rho and regulates the neurite outgrowth.     

Roles and modes of action of nectins in cell-cell adhesion

Nectins are Ca(2+)-independent immunoglobulin (Ig)-like cell-cell adhesion molecules (CAMs), which comprise a family consisting of four members. Each nectin homophilically and heterophilically trans-interacts and causes cell-cell adhesion. Biochemical, cell biological, and knockout mice studies have revealed that nectins play important roles in formation of many types of cell-cell junctions and cell-cell contacts, including cadherin-based adherens junctions (AJs) and synapses. Mode of action of nectins in the formation of AJs has extensively been investigated. Nectins form initial cell-cell adhesion and recruit E-cadherin to the nectin-based cell-cell adhesion sites. In addition, nectins induce activation of Cdc42 and Rac small G proteins, which eventually enhances the formation of cadherin-based AJs through the reorganization of the actin cytoskeleton. Nectins furthermore heterophilically trans-interact with nectin-like molecules (Necls), other Ig-like CAMs, and assist or modify their various functions, such as cell adhesion, migration, and proliferation. We describe here the roles and modes of action of nectins as CAMs.     

The parasitoidOoencyrtus nezarae (Hymenoptera: Encyrtidae) prefers hosts parasitized by conspecifics over unparasitized hosts

Summary Two laboratory experiments were conducted to examine the ovipositional preferences of the egg parasitoidOoencyrtus nezarae Ishii (Hymenoptera: Encyrtidae) for parasitized and unparasitizedMegacopta punctatissimum Montandon (Hemiptera: Plataspidae). Females that had never oviposited or that had not oviposite for 3 days preferred recently parasitized hosts more than unparasitized hosts. The preference for recently parasitized hosts appeared to be mediated by the punctures in already parasitized hosts made by the ovipositor of the first female. Survival of the parasitoid progeny was lower in recently parasitized hosts than in unparasitized hosts. However, handling time of parasitized hosts was extremely short relative to that of unparasitized hosts, because the superparasitizing female could use the punctures made by the previous females. It is concluded that the females preferred the parasitized hosts over unparasitized hosts because the benefit of saving time and energy for drilling was more than the cost of progeny survival.