Inositol hexakisphosphate kinase-2, a physiologic mediator of cell death

Diphosphoinositol pentakisphosphate (InsP7) and bis-diphosphoinositol tetrakisphosphate contain pyrophosphate bonds. InsP7 is formed from inositol hexakisphosphate (InsP6) by a family of three inositol hexakisphosphate kinases (InsP6K). In this study we establish one of the InsP6Ks, InsP6K2, as a physiologic mediator of cell death. Overexpression of wild-type InsP6K2 augments the cytotoxic actions of multiple cell stressors in diverse cell lines, whereas transfection with a dominant negative InsP6K2 decreases cell death. During cell death, InsP6 kinase activity is enhanced, and intracellular InsP7 level is augmented. Deletion of InsP6K2 but not the other forms of InsP6K diminishes cell death, suggesting that InsP6K2 is the major InsP6 kinase involved in cell death. Cytotoxicity is associated with a translocation of InsP6K2 from nuclei to mitochondria, whereas the intracellular localization of the other isoforms of the enzyme does not change. The present study provides compelling evidence that endogenous InsP6K2, by generating InsP7, provides physiologic regulation of the apoptotic process.     

Cleavage of carcinoembryonic antigen induces metastatic potential in colorectal carcinoma

Carcinoembryonic antigen (CEA), a widely used tumor marker, is attached by a glycosylphosphatidylinositol (GPI) anchor motif to the cell membrane. Recent study suggested that membrane-bound CEA might be cleaved by glycosylphosphatidylinositol-phospholipase D (GPI-PLD). We studied the effect of GPI-PLD on the cleavage of CEA to elucidate the implication for metastatic potential in colorectal carcinoma cells. CEA amount of conditioned medium was changed by suramin and phenanthroline (activator and inhibitor of GPI-PLD) only in SW620 and SW837 which expressed both CEA and GPI-PLD mRNA. Suramin treatment also augmented migratory activity and decreased cell surface CEA expression in SW620 and SW837. Furthermore, GPI-PLD knockdown cells using GPI-PLD-specific siRNA in SW620 and SW837 showed decreased CEA secretion from cell membrane and the migration activity, increased membrane-bound CEA amount. Splenic injection of SW620 and SW837 induced marked hepatic metastases in nude mice. These results suggest that membrane-bound CEA is cleaved by GPI-PLD and that this cleavage enhances the metastatic potential in colorectal carcinoma cells.     

Alteration of ryanodine receptor in the hippocampus CA1 after hemispheric cerebral ischemia

Alterations in ryanodine binding and local cerebral blood flow (LCBF) were examined at 30 minutes and 2 hours post-ischemia in the gerbil brain in order to evaluate the influence of cerebral ischemia on the intracellular channels of Ca²⁺-induced Ca²⁺ release (CICR). Severe hemispheric cerebral ischemia was induced by occluding the right common carotid artery. LCBF was measured at the end of the experiment using [¹⁴C]iodoantipyrine method, and the ryanodine binding was evaluated in vitro using [³H]ryanodine as a specific ligand for CICR channels. An autoradiographic method developed in our laboratory enabled us to determine both parameters within the same brain. A group of gerbils who underwent a sham procedure served as controls. LCBF was found to be significantly reduced in most of the cerebral regions on the occluded side at both 30 minutes as well as 2 hours post-ischemia. In contrast, a significant reduction in ryanodine binding was noted only in the hippocampus CA1 on the occluded side at 30 minutes and 2 hours after the occlusion. These findings suggest that regionally specific changes of CICR may be the cause of decreased ryanodine binding in the hippocampus CA1, and that these changes may be related to the pathophysiological mechanisms that cause this region to be particularly vulnerable to ischemia.     

Homologous Recombination, but Not DNA Repair, Is Reduced in Vertebrate Cells Deficient in RAD52

Rad52 plays a pivotal role in double-strand break (DSB) repair and genetic recombination in Saccharomyces cerevisiae, where mutation of this gene leads to extreme X-ray sensitivity and defective recombination. Yeast Rad51 and Rad52 interact, as do their human homologues, which stimulates Rad51-mediated DNA strand exchange in vitro, suggesting that Rad51 and Rad52 act cooperatively. To define the role of Rad52 in vertebrates, we generated RAD52^−/− mutants of the chicken B-cell line DT40. Surprisingly, RAD52^−/− cells were not hypersensitive to DNA damages induced by γ-irradiation, methyl methanesulfonate, or cis-platinum(II)diammine dichloride (cisplatin). Intrachromosomal recombination, measured by immunoglobulin gene conversion, and radiation-induced Rad51 nuclear focus formation, which is a putative intermediate step during recombinational repair, occurred as frequently in RAD52^−/− cells as in wild-type cells. Targeted integration frequencies, however, were consistently reduced in RAD52^−/− cells, showing a clear role for Rad52 in genetic recombination. These findings reveal striking differences between S. cerevisiae and vertebrates in the functions of RAD51 and RAD52.     

Sex Change and Population Dynamics in Arisaema (Araceae) II. An Examination on the Evolutionary Stability of Sex Changing Schedule of A. serratum (Thunb.) Schott

Abstract The evolutionary stability of the sex-changing habit of Arisaema serratum was examined. To evaluate reproductive success through male functioin properly, the mathematical formulation of Kakehashi and Harada (1987) was employed. Individuals were classified by both their size and sexual state, and the genetic contribution of individuals was estimated by calculating their reproductive value. It was shown that in the size classes where sex change actually occurs, the reproductive values of males and females are basically the same. This means that the genetic contribution of individuals in these size classes is the same whether they are male or female, and no selection pressure is working to change the male ratio in these classes. Thus, it is concluded that the size at which sex change occurs is evolutionarily stable. The adaptive significance of the size of first reproduction was also discussed.     

Drosophila Sirt2/mammalian SIRT3 deacetylates ATP synthase β and regulates complex V activity

Adenosine triphosphate (ATP) synthase β, the catalytic subunit of mitochondrial complex V, synthesizes ATP. We show that ATP synthase β is deacetylated by a human nicotinamide adenine dinucleotide (NAD⁺)–dependent protein deacetylase, sirtuin 3, and its Drosophila melanogaster homologue, dSirt2. dsirt2 mutant flies displayed increased acetylation of specific Lys residues in ATP synthase β and decreased complex V activity. Overexpression of dSirt2 increased complex V activity. Substitution of Lys 259 and Lys 480 with Arg in human ATP synthase β, mimicking deacetylation, increased complex V activity, whereas substitution with Gln, mimicking acetylation, decreased activity. Mass spectrometry and proteomic experiments from wild-type and dsirt2 mitochondria identified the Drosophila mitochondrial acetylome and revealed dSirt2 as an important regulator of mitochondrial energy metabolism. Additionally, we unravel a ceramide–NAD⁺–sirtuin axis wherein increased ceramide, a sphingolipid known to induce stress responses, resulted in depletion of NAD⁺ and consequent decrease in sirtuin activity. These results provide insight into sirtuin-mediated regulation of complex V and reveal a novel link between ceramide and Drosophila acetylome.     

SdrA,a New DeoR Family Regulator Involved in Streptomyces avermitilis Morphological Development and Antibiotic Production

The SAV3339 (SdrA) protein of Streptomyces avermitilis, a member of the DeoR family of regulators, was assessed to determine its in vivo function by gene knockdown through the use of cis-encoded noncoding RNA and knockout of the sdrA gene. These analyses revealed that SdrA represents another class of Streptomyces regulator that controls morphological development and antibiotic production.     

High-resolution spatial and temporal analysis of phytoalexin production in oats

The production of oat (Avena sativa L.) phytoalexins, avenanthramides, occurs in response to elicitor treatment with oligo-N-acetylchitooligosaccharides. In this study, avenanthramides production was investigated by techniques that provide high spatial and temporal resolution in order to clarify the process of phytoalexin production at the cellular level. The amount of avenanthramides accumulation in a single mesophyll cell was quantified by a combination of laser micro-sampling and low-diffuse nanoflow liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) techniques. Avenanthramides, NAD(P)H and chlorophyll were also visualized in elicitor-treated mesophyll cells using line-scanning fluorescence microscopy. We found that elicitor-treated mesophyll cells could be categorized into three characteristic cell phases, which occurred serially over time. Phase 0 indicated the normal cell state before metabolic or morphological change in response to elicitor, in which the cells contained abundant NAD(P)H. In phase 1, rapid NAD(P)H oxidation and marked movement of chloroplasts occurred, and this phase was the early stage of avenanthramides biosynthesis. In phase 2, avenanthramides accumulation was maximized, and chloroplasts were degraded. Avenanthramides appear to be synthesized in the chloroplast, because a fluorescence signal originating from avenanthramides was localized to the chloroplasts. Moreover, our results indicated that avenanthramides biosynthesis and the hypersensitive response (HR) occurred in identical cells. Thus, the avenanthramides production may be one of sequential events programmed in HR leading to cell death. Furthermore, the phase of the defense response was different among mesophyll cells simultaneously treated with elicitor. These results suggest that individual cells may have different susceptibility to the elicitor. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Inositol hexakisphosphate kinases promote autophagy

We and other authors have previously reported that increasing cellular diphosphoinositol pentakisphosphate (InsP(7)) levels increases cell sensitivity to cell death. In the present study, we elucidated the relationship between inositol hexakisphosphate kinases (InsP(6)Ks), which form InsP(7), and autophagy using InsP(6)Ks overexpression and disruption systems. A large number of autophagosomes were induced in cells transfected with InsP(6)Ks, as revealed by the conversion of LC3-I to LC3-II, which was examined using immunoblotting, immunocytochemistry, and immuno-electron microscopy for LC3; consequently, the rate of cell death was higher among these cells than among cells transfected with a control vector, as shown using propidium iodide staining. However, the reduction of InsP(6)Ks levels using RNAi suppressed the formation of autophagosomes. Moreover, the number of autophagosomes and the rate of cell death were significantly higher among cells transfected with InsP(6)Ks subjected to staurosporine-induced stress than among cells transfected with InsP(6)Ks subjected to normal conditions. The cell death induced by InsP(6)Ks was not completely suppressed by z-VAD-fmk, a pan-caspase inhibitor. The phosphorylation of mammalian target of rapamycin (mTOR) was also depressed in cells overexpressing InsP(6)Ks, suggesting that the mTOR pathway regulates autophagosomes generated by InsP(6)Ks. These findings imply that InsP(6)Ks promote autophagy and induce caspase-independent cell death. This phenomenon opens a new pathway of autophagy via InsP(6)Ks.