HSPC117 deficiency in cloned embryos causes placental abnormality and fetal death

Somatic cell nuclear transfer (SCNT) has been successfully used in many species to produce live cloned offspring, albeit with low efficiency. The low frequency of successful development has usually been ascribed to incomplete or inappropriate reprogramming of the transferred nuclear genome. Elucidating the genetic differences between normal fertilized and cloned embryos is key to understand the low efficiency of SCNT. Here, we show that expression of HSPC117, which encodes a hypothetical protein of unknown function, was absent or very low in cloned mouse blastocysts. To investigate the role of HSPC117 in embryo development, we knocked-down this gene in normal fertilized embryos using RNA interference. We assessed the post-implantation survival of HSPC117 knock-down embryos at 3 stages: E9 (prior to placenta formation); E12 (after the placenta was fully functional) and E19 (post-natal). Our results show that, although siRNA-treated in vivo fertilized/produced (IVP) embryos could develop to the blastocyst stage and implanted without any difference from control embryos, the knock-down embryos showed substantial fetal death, accompanied by placental blood clotting, at E12. Furthermore, comparison of HSPC117 expression in placentas of nuclear transfer (NT), intracytoplasmic sperm injection (ICSI) and IVP embryos confirmed that HSPC117 deficiency correlates well with failures in embryo development: all NT embryos with a fetus, as well as IVP and ICSI embryos, had normal placental HSPC117 expression while those NT embryos showing reduced or no expression of HSPC117 failed to form a fetus. In conclusion, we show that HSPC117 is an important gene for post-implantation development of embryos, and that HSPC117 deficiency leads to fetal abnormalities after implantation, especially following placental formation. We suggest that defects in HSPC117 expression may be an important contributing factor to loss of cloned NT embryos in vivo. (C) 2010 Elsevier Inc. All rights reserved.

Coupling between NMDA receptor and acid-sensing ion channel contributes to ischemic neuronal death

Acid-sensing ion channels (ASICs) composed of ASIC1a subunit exhibit a high Ca2+ permeability and play important roles in synaptic plasticity and acid-induced cell death. Here, we show that ischemia enhances ASIC currents through the phosphorylation at Ser478 and Ser479 of ASIC1a, leading to exacerbated ischemic cell death. The phosphorylation is catalyzed by Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, as a result of activation of NR2B-containing N-methyl-D-aspartate subtype of glutamate receptors (NMDARs) during ischemia. Furthermore, NR2B-specific antagonist, CaMKII inhibitor, or overexpression of mutated form of ASIC1a with Ser478 or Ser479 replaced by alanine (ASICla-S478A, ASIC1a-S479A) in cultured hippocampal neurons prevented ischemia-induced enhancement of ASIC currents, cytoplasmic Ca2+ elevation, as well as neuronal death. Thus, NMDAR-CaMKII cascade is functionally coupled to ASICs and contributes to acidotoxicity during ischemia. Specific blockade of NMDAR/CaMKII-ASIC coupling may reduce neuronal death after ischemia and other pathological conditions involving excessive glutamate release and acidosis.

Microcystin-RR induces physiological stress and cell death in the cyanobacterium Aphanizomenon sp DC01 isolated from Lake Dianchi, China

The phytoplankton community in Lake Dianchi (Yunnan Province, Southwestern China) is dominated in April by a bloom of Aphanizomenon, that disappears Suddenly and is displaced by a Microcystis bloom in May. The reasons for the rapid bloom disappearance phenomenon and the temporal variability in the composition of phytoplankton assemblages are poorly understood. Cell growth, ultrastructure and physiological changes were examined in cultures of Aphanizomenon sp. DC01 isolated from Lake Dianchi exposed to different closes of rnicrocystin-RR (MC-RR) produced by the Microcystis bloom. MC-RR concentrations above 100 mu g L-1 markedly inhibited the pigment (chlorophyll-a, phycocyanin) synthesis and caused an increase of soluble carbohydrate and protein contents and nitrate reductase activity of toxin-treated blue-green algae. A drastic. reduction in photochemical efficiency of PSII (Fv/Fm) was also found. Morphological examinationn showed that the Aphanizomenon filaments disintegrated and file cells lysed gradually after 48 h Of toxin exposure. Transmission electron microscopy revealed that cellular inclusions of stressed cells almost leaked out completely and the cell membranes were grossly damaged. These findings demonstrate the allelopathic activity of Microcystis aeruginosa inducing physiological stress and cell death of Aphanizomenon sp. DC01 Although the active concentrations of microcystin were rather high, we propose that microcystin may function as allelopathic Substance due to inhomogeneous toxin concentrations close to Microcystis cells. Hence, it may play a role in species Succession of Aphanizomenon and Microcystis in Lake Dianchi.

NADPH oxidase-mediated generation of reactive oxygen species: A new mechanism for X-ray-induced HeLa cell death

Oxidative damage is an important mechanism in X-ray-induced cell death. Radiolysis of water molecules is a source of reactive oxygen species (ROS) that contribute to X-ray-induced cell death. In this study, we showed by ROS detection and a cell survival assay that NADPH oxidase has a very important role in X-ray-induced cell death. Under X-ray irradiation, the upregulation of the expression of NADPH oxidase membrane Subunit gp91(phox) was dose-dependent. Meanwhile, the cytoplasmic subunit p47(phox) was translocated to the cell membrane and localized with p22(phox) and gp91(phox) to form reactive NADPH oxidase. Our data Suggest, for the first time, that NADPH oxidase-mediated generation of ROS is an important contributor to X-ray-induced cell death. This suggests a new target for combined gene transfer and radiotherapy.

Structure of an unexpected trimer from the reaction of ageratochromene II with aluminum chloride

A new trimer from the reaction of ageratochromene [1] (6,7-dimethoxy-2,2-dimethyl-1-benzopyran) with anhydrous aluminum chloride was shown to be 3,4-dihydro-6,7-dimethoxy-2,2-dimethyl-3-(6',7'-dimethoxy-2',2'-di-methyl-2H-1-benzopyran-4'-yl)-4-(3" 4"-dihydro-6", 7"-dimethoxy-2",2"-dimethyl-2H-1-benzopyran-3"-yl)-2H-1-benzopyran. Its structure was confirmed by NMR (H-1, C-13, DEPT-135. COSY, HMBC, HSQC, TOCSY and NOESY), IR, mass spectra and elemental analysis. Copyright (C) 2002 John Wiley Sons, Ltd.

Synthesis and identification of Heterocyclic derivatives of fullerene C-60: Unexpected reaction of anionic C-60 with benzonitrile

During the reaction of reduced C-60 with benzyl bromide in benzonitrile, a novel cis-1 C-60 adduct, 1,4-dibenzyl-2,3-cyclic phenylimidate C-60 (1), Was obtained rather than the expected product of 1,4-dibenzyl C-60. The structure of compound 1 was analyzed by X-ray single-crystal diffraction, identifying the presence of a five-membered heterocycle at a [5,6] bond of C-60. One of the heteroatoms is assigned as a nitrogen atom; however, the identity of the other heteroatom cannot be determined unambiguously by crystallography due to similarity between the nitrogen and oxygen atoms.

Red-Emitting Polyfluorenes Grafted with Quinoline-Based Iridium Complex: "Simple Polymeric Chain, Unexpected High Efficiency"

A series of red-light emitting electrophosphorescent polyfluorenes (PFs) with varying content of a quinoline-based iridium complex, (PPQ)(2)Ir(acac) (bis(2,4-diphenylquinolyl-N,C-2') iridium(acetylacetonate)), in the side chain are synthesized by Suzuki polycondensation. Because of the efficient Forster energy transfer from the PF main chain to (PPQ)(2)Ir(acac) and direct charge trapping on the complex, the electroluminescent emission from PF is nearly completely quenched, even though the amount of iridium complex I incorporated into the polymers is as low as 1 mol %. Based on a single-layer device configuration, a luminous efficiency of up to 5.0 cd A(-1) with a luminance of 2000 cd m(-2) and Commission Internationale de L'Eclairage coordinates of (0.63, 0.35) (x, y) is realized, which is far superior to that of previously reported red-light emitting PFs containing benzothiazole- and isoquinoline-based iridium complexes.

Synthesis and Characterization of Hyperbranched Poly(ether amide)s with Thermoresponsive Property and Unexpected Strong Blue Photoluminescence

A facile and efficient strategy for the syntheses of novel hyperbranched poly(ether amide)s (HPEA) from multihydroxyl primary amines and (meth)acryloyl chloride has been developed. The chemical structures of the HPEAs were confirmed by IR and NMR spectra. Analyses of SEC (size exclusion chromatography) and viscosity characterizations revealed the highly branched structures of the polymers obtained. The resultant hyperbranched polymers contain abundant hydroxyl groups. The thermoresponsive property was obtained from in situ surface modification of abundant OH end groups with N-isopropylacrylamide (NIPAAm). The study oil temperature-dependent characteristics has revealed that NIPAAm-g-HPEA exhibits an adjustable lower critical solution temperature (LCST) of about 34-42 degrees C depending on the grafting degree. More interestingly, the work provided an interesting phenomenon where the HPEA backbones exhibited strong blue photoluminescence.

Kinetic isotope effects of proton transfer reaction for amines by ultrasonic relaxation methods: Unexpected evidence from the results in ethylamine solutions

Ultrasonic absorption coefficients for ethylamine in heavy water (D2O) and in light water (H2O) have been measured in the frequency range from 0.8 to 220 MHz at 25 degrees C. A single relaxational process has been observed in these two kinds of solutions. From the concentration dependence of the ultrasonic relaxation parameters, and following the reaction mechanism proposed by Eigen et al. for ethylamine in H2O, the causes of the relaxations have been attributed to a perturbation of an equilibrium associated with a deuteron or proton transfer reaction. The rate and equilibrium constants have been estimated from deuterioxide or hydroxide ion concentration dependence of the relaxation frequency, and the kinetic isotope effects have been determined. In addition, the standard volume changes of the reactions have been calculated from the concentration dependence of the maximum absorption per wavelength, and the adiabatic compressibility has also been determined from the density and sound velocity for ethylamine in D2O and in H2O, respectively. These results are compared with those for propylamine and butylamine and are discussed in relation to the different kinetic properties between D2O and H2O, the reaction radii derived by Debye theory, and the structural properties of the reaction intermediate.

A STUDY ON THE INDICATION OF UNEXPECTED COMPONENTS BY KALMAN FILTERING IN FLUOROSPECTROPHOTOMETRY

The potential of Kalman filtering for indication of unexpected components in a mixture was experimentally evaluated by taking the spectrofluorimetric analysis of the tricomponent system;oi phenylalanine, tryhtophen and tyrosine as an example. According to

A novel anti-tumor protein extracted from Meretrix meretrix Linnaeus induces cell death by increasing cell permeability and inhibiting tubulin polymerization

Discovery and development of new pharmaceuticals from marine organisms are attracting increasing interest. Several agents derived from marine organisms are under preclinical and clinical evaluation as potential anticancer drugs. We extracted and purified a novel anti-tumor protein from the coelomic fluid of Meretrix meretrix Linnaeus by ammonium sulphate fractionation, ion exchange and hydrophobic interaction chromatography. The molecular weight of the highly purified protein, designated MML, was 40 kDa as determined by SDS-PAGE analysis. MML exhibited significant cytotoxicity to several cancer cell types, including human hepatoma BEL-7402, human breast cancer MCF-7 and human colon cancer HCT116 cells. However, no inhibitory effect was found when treating murine normal fibroblasts NIH3T3 and benign human breast MCF-10A cells with MML. The cell death induced by MML was characterized by cell morphological changes. The induction of apoptosis of BEL-7402 cells by MML was weak by DNA ladder assay. The possible mechanisms of its anti-tumor effect might be the changes in cell membrane permeability and inhibition of tubulin polymerization. MML may be developed as a novel, highly selective and effective anti-cancer drug.

Programmed cell death in Laminaria japonica (Phaeophyta) tissues infected with alginic acid decomposing bacterium

TdT-mediated dUTP-biotin nick end labeling (TUNEL) is a sensitive and valid method for detecting DNA cleavage in programmed cell death (PCD). Using this method, DNA cleavage was observed in Laminaria japonica sporophytic tissues, which were infected with alginic acid decomposing bacterium. It was found that DNA cleavage occurred 5 min after the infection, the fragments with 3'-OH groups of cleaved nuclear DNA increased with time of infection and spread from the infection site. Although no typical DNA ladder (200 bp/ 180 bp) was detected by routine agarose gel electrophoresis, the cleavage of nuclear DNA fragments of 97 similar to 48.5 kb could be detected by pulsed field gel electrophoresis (PFGE). By using CaspGLOW(TM) fluorescein active caspase-3 staining method, caspase-3 activity has been detected in response to the infection of alginic acid decomposing bacterium. Our results are similar to the observations in hypersensitive response (HR) of higher plant, suggesting that the rapid cell death of L. japonica infected by alginic acid decomposing bacterium might be involved in PCD, and indicating that the occurrence of PCD is an active defense process against the pathogen's infection.

Molecular cloning and responsive expression to injury stimulus of a defender against cell death 1 (DAD1) gene from bay scallops Argopecten irradians

Apoptosis is an active process of cell death, which is an integral part of growth and development in multicellular organisms. The defender against cell death 1 (DAD1), the regulatory protein to inhibit the apoptosis process, was first cloned from the bay scallop Argopecten irradians by randomly sequencing a whole tissue cDNA library and rapid amplification of cDNA end (RACE). The full-length cDNA of the A. irradians DAD1 was 607 bp, consist of a 5'-terminal untranslated region (UTR) of 63 bp, a 3'-terminal UTR of 205 bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 339 bp. The deduced amino acid sequence of the A. irradians DAD1 showed 75.5% identity to Araneus ventricosus, 74.5% to Drosophila melanogaster, and 73.6% to Homo sapiens, Sus scrofa, Mesocricetus auratus, Rattus norvegicus and Mus musculus. Excluding the Saccharomyces cerevisiae DAD1 homologue, all animal DAD1 including A. irradians DAD1 homologue formed a subgroup and all plant DAD1 proteins formed another subgroup in the phylogenetic analysis. The A. irradians DAD1 was expressed in all examined tissues including adductor muscle, mantle, gills, digestive gland, gonad and hemolymph, suggesting that A. irradians DAD1 is expressed in most body tissues. Furthermore, the mRNA expression levels of A. irradians DAD1 gene of hemolymph were particularly high after injury, suggesting that the gene is responsive to injury stimuli.

Fucoidan protects against dopaminergic neuron death in vivo and in vitro

Parkinson's disease is a neurodegenerative disorder of uncertain pathogenesis characterized by a loss of dopaminergic neurons in substantia nigra pars compacta, and can be modeled by the neurotoxin 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP). Oxidative stress may contribute to MPTP- and Parkinson's disease-related neurodegeneration. Fucoidan is a sulfated polysaccharide extracted from brown seaweeds which possesses a wide variety of biological activities including potent antioxidative effects. Here we investigated the effect of fucoidan treatment on locomoter activities of animals, striatal dopamine and its metabolites and survival of nigral dopaminergic neurons in MPTP-induced animal model of Parkinsonism in C57/BL mice in vivo and on the neuronal damage induced by 1-methyl-4-phenylpyridinium (MPP+) in vitro, and to study the possible mechanisms. When administered prior to MPTP, fucoidan reduced behavioral deficits, increased striatal dopamine and its metabolites levels, reduced cell death, and led to a marked increase in tyrosine hydroxylase expression relative to mice treated with MPTP alone. Furthermore, we found that fucoidan inhibited MPTP-induced lipid peroxidation and reduction of antioxidant enzyme activity. In addition, pre-treatment with fucoidan significantly protected against MPP+-induced damage in MN9D cells. Taken together, these findings suggest that fucoidan has protective effect in MPTP-induced neurotoxicity in this model of Parkinson's disease via its antioxidative activity. (C) 2009 Elsevier B.V. All rights reserved.