Silencing of cytosolic NADP+-dependent isocitrate dehydrogenase by small interfering RNA enhances the sensitivity of HeLa cells toward staurosporine

Staurosporine induces the production of reactive oxygen species, which play an important causative role in apoptotic cell death. Recently, it was demonstrated that the control of cellular redox balance and the defense against oxidative damage is one of the primary functions of cytosolic NADP⁺-dependent isocitrate dehydrogenase (IDPc) by supplying NADPH for antioxidant systems. The present report shows that silencing of IDPc expression in HeLa cells greatly enhances apoptosis induced by staurosporine. Transfection of HeLa cells with an IDPc small interfering RNA (siRNA) markedly decreased activity of IDPc, enhancing the susceptibility of staurosporine-induced apoptosis reflected by DNA fragmentation, cellular redox status and the modulation of apoptotic marker proteins. These results indicate that IDPc may play an important role in regulating the apoptosis induced by staurosporine and the sensitizing effect of IDPc siRNA on the apoptotic cell death of HeLa cells offers the possibility of developing a modifier of cancer chemotherapy.     

Phylogenetic analyses of anatomical and nuclear SSU rDNA sequence data indicate that the Dasyaceae and Delesseriaceae (Ceramiales,Rhodophyta) are polyphyletic

The aim of the current investigation was to test the general convention that the Dasyaceae, Delesseriaceae and Rhodomelaceae are all monophyletic families of the red algal order Ceramiales. Phylogenetic relationships among 45 ceramialean taxa were determined, including eight ceramiacean, 18 dasyacean, nine delesseriacean and eight rhodomelacean species, plus two of uncertain ceramialean affinities, based on 34 anatomical characters and nuclear small subunit (SSU) rDNA sequences. Results from our ‘total-evidence’ approach were consistent with the notion that the Dasyaceae, Delesseriaceae and Rhodomelaceae have evolved from a common ancestor within a paraphyletic Ceramiaceae. Our data indicate, however, that the Rhodomelaceae alone is monophyletic at the family level, with both the Dasyaceae and Delesseriaceae polyphyletic. In particular we resolved two independent and well-supported lineages for the included Dasyaceae, viz., a Dasya group and a Heterosiphonia group, which were as distinct from one another as they were from the Rhodomelaceae and the various lineages of Delesseriaceae. The molecular data alone were equivocal on the issue of monophyly of the Dasyaceae. We therefore advocate conservative taxonomic revisions as an interim step towards eventual resolution of familial-level taxonomy in the Ceramiales. Based on our results, the Heterosiphonioideae H.-G. Choi, Kraft, I.K. Lee et G.W. Saunders subfam. nov. is proposed for Heterosiphonia and five closely related genera, and the Dasyoideae Schmitz et Falkenberg is emended for the remaining taxa. Although the Dasyoideae is a natural group, it is in need of a thorough systematic reinvestigation at the generic level. Our analyses indicate that the genus Dasya is polyphyletic or paraphyletic in excluding Dasysiphonia, Eupogodon and Rhodoptilum and that Heterosiphonia japonica also has affinities to this group, taxonomic issues that will be addressed in detail elsewhere.     

EFFECTS OF THE PHYSICAL ENVIRONMENT ON SOME LIPOPROTEIN LAYER SYSTEMS AND OBSERVATIONS ON THEIR MORPHOGENESIS

Lipoprotein membrane systems such as chloroplasts and the endoplasmic reticulum exhibit a generalized swelling response. The initial effect is an increase in interlamellar spacing, but as swelling proceeds, the membranes are transformed into closed thin-walled spherical vesicles. Available evidence suggests that morphogenesis of the endoplasmic reticulum of Nitella and the lamellar system of the Zea chloroplasts involves fusion of small spherical vesicles to yield closed double membrane structures, which subsequently undergo further differentiation. It is suggested that the vesicles comprise a convenient "micellar" form by which lipides may be transported within the cell from the sites of lipide synthesis to regions of lamellar growth. The characteristic formation of vesicles in swelling and the apparent fusion of vesicles in morphogenesis appear to represent two aspects of a fundamental plasticity of lipoprotein layer systems.     

Hyphantria cunea ferritin heavy chain homologue: cDNA sequence and mRNA expression

We have sequenced a cDNA clone encoding a 26-kDa ferritin subunit, which was heavy chain homologue (HCH), in fall webworm, Hyphantria cunea. The HCH cDNA was obtained from the screening of a cDNA library using a PCR product. H. cunea ferritin is composed of 221 amino acid residues and their calculated mass is 26,160 Da. The protein contains the conserved motifs for the ferroxidase center typical for heavy chains of vertebrate ferritin. The iron-responsive element sequence with a predicted stem-loop structure is present in the 5'-untranslated region of ferritin HCH mRNA. The sequence alignment of ferritin HCH shows 68.9 and 68.7% identity with Galleria mellonella HCH (26 kDa ferritin) and Manduca sexta HCH, respectively. While G type insect ferritin vertebrate light chain homologue (LCH) is distantly related to H. cunea ferritin HCH (17.2-20.8%), the Northern blot analysis revealed that H. cunea ferritin HCH was ubiquitously expressed in various tissues and all developmental stages. The ferritin expression of midgut is more responsive to iron-fed, compared to fat body in H. cunea.     

Phospholipase C-delta1 rescues intracellular Ca2+ overload in ischemic heart and hypoxic neonatal cardiomyocytes

Ischemia and simulated ischemic conditions cause intracellular Ca2+ overload in the myocardium. The relationship between ischemia injury and Ca2+ overload has not been fully characterized. The aim of the present study was to investigate the expression and characteristics of PLC isozymes in myocardial infarction-induced cardiac remodeling and heart failure. In normal rat heart tissue, PLC-delta1 (about 44 ng/mg of heart tissue) was most abundant isozymes compared to PLC-gamma1 (6.8 ng/mg) and PLC-beta1 (0.4 ng/mg). In ischemic heart and hypoxic neonatal cardiomyocytes, PLC-delta1, but not PLC-beta1 and PLC-gamma1, was selectively degraded, a response that could be inhibited by the calpain inhibitor, calpastatin, and by the caspase inhibitor, zVAD-fmk. Overexpression of the PLC-delta1 in hypoxic neonatal cardiomyocytes rescued intracellular Ca2+ overload by ischemic conditions. In the border zone and scar region of infarcted myocardium, and in hypoxic neonatal cardiomyocytes, the selective degradation of PLC-delta1 by the calcium sensitive proteases may play important roles in intracellular Ca2+ regulations under the ischemic conditions. It is suggested that PLC isozyme-changes may contribute to the alterations in calcium homeostasis in myocardial ischemia.     

The alpha-helical domain of Galphat determines specific interaction with regulator of G protein signaling 9

RGS proteins (regulators of G protein signaling) are potent accelerators of the intrinsic GTPase activity of G protein alpha subunits (GAPs), thus controlling the response kinetics of a variety of cell signaling processes. Most RGS domains that have been studied have relatively little GTPase activating specificity especially for G proteins within the Gi subfamily. Retinal RGS9 is unique in its ability to act synergistically with a downstream effector cGMP phosphodiesterase to stimulate the GTPase activity of the alpha subunit of transducin, Galphat. Here we report another unique property of RGS9: high specificity for Galphat. The core (RGS) domain of RGS9 (RGS9) stimulates Galphat GTPase activity by 10-fold and Galphai1 GTPase activity by only 2-fold at a concentration of 10 microM. Using chimeric Galphat/Galphai1 subunits we demonstrated that the alpha-helical domain of Galphat imparts this specificity. The functional effects of RGS9 were well correlated with its affinity for activated Galpha subunits as measured by a change in fluorescence of a mutant Galphat (Chi6b) selectively labeled at Cys-210. Kd values for RGS9 complexes with Galphat and Galphai1 calculated from the direct binding and competition experiments were 185 nM and 2 microM, respectively. The gamma subunit of phosphodiesterase increases the GAP activity of RGS9. We demonstrate that this is because of the ability of Pgamma to increase the affinity of RGS9 for Galphat. A distinct, nonoverlapping pattern of RGS and Pgamma interaction with Galphat suggests a unique mechanism of effector-mediated GAP function of the RGS9.     

Ribose 5-Phosphate Isomerase B Knockdown Compromises Trypanosoma brucei Bloodstream Form Infectivity

Ribose 5-phosphate isomerase is an enzyme involved in the non-oxidative branch of the pentose phosphate pathway, and catalyzes the inter-conversion of D-ribose 5-phosphate and D-ribulose 5-phosphate. Trypanosomatids, including the agent of African sleeping sickness namely Trypanosoma brucei, have a type B ribose-5-phosphate isomerase. This enzyme is absent from humans, which have a structurally unrelated ribose 5-phosphate isomerase type A, and therefore has been proposed as an attractive drug target waiting further characterization. In this study, Trypanosoma brucei ribose 5-phosphate isomerase B showed in vitro isomerase activity. RNAi against this enzyme reduced parasites'' in vitro growth, and more importantly, bloodstream forms infectivity. Mice infected with induced RNAi clones exhibited lower parasitaemia and a prolonged survival compared to control mice. Phenotypic reversion was achieved by complementing induced RNAi clones with an ectopic copy of Trypanosoma cruzi gene. Our results present the first functional characterization of Trypanosoma brucei ribose 5-phosphate isomerase B, and show the relevance of an enzyme belonging to the non-oxidative branch of the pentose phosphate pathway in the context of Trypanosoma brucei infection.     

Production of a value-added hydroxy fatty acid, 7,10-dihydroxy-8(<Emphasis Type="Italic">E</Emphasis>)-octadecenoic acid,from high oleic safflower Oil by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3

Hydroxy fatty acids (HFAs), originally obtained in small amounts from plant systems, are good examples of structurally modified lipids, and they render special properties such as higher viscosity and reactivity compared to normal fatty acids. Based on these properties, HFAs possess high industrial potential in a wide range of applications. Recently, various microbial strains were tested for the production of HFAs from different unsaturated fatty acids since HFA production is limited to plant systems. Among the microbial strains tested, Pseudomonas aeruginosa PR3 has been well studied for the production of 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) from oleic acid. Previously, we reported that strain PR3 could utilize triolein instead of oleic acid as a substrate for the production of DOD (Appl. Microbiol. Biotechnol. 2007, 74: 301–306). In this study, we focused on utilization of vegetable oil as a substrate for DOD production by PR3. Consequently, strain PR3 efficiently utilized high oleic safflower oil as a substrate for DOD production. Optimal initial medium pH and incubation time were pH 8.0 and 72 h, respectively. Optimal carbon and nitrogen sources were fructose and glutamine, respectively. Results from this study demonstrate that normal vegetable oils could be used as efficient substrates for the production of value-added HFAs by microbial bioconversion.