Role of Smac/DIABLO in hydrogen peroxide-induced apoptosis in C2C12 myogenic cells

Smac/DIABLO was recently identified as a protein released from mitochondria in response to apoptotic stimuli which promotes apoptosis by antagonizing inhibitors of apoptosis proteins. Furthermore, Smac/DIABLO plays an important regulatory role in the sensitization of cancer cells to both immune-and drug-induced apoptosis. However, little is known about the role of Smac/DIABLO in hydrogen peroxide (H(2)O(2))-induced apoptosis of C2C12 myogenic cells. In this study, Hoechst 33258 staining was used to examine cell morphological changes and to quantitate apoptotic nuclei. DNA fragmentation was observed by agarose gel electrophoresis. Intracellular translocation of Smac/DIABLO from mitochondria to the cytoplasm was observed by Western blotting. Activities of caspase-3 and caspase-9 were assayed by colorimetry and Western blotting. Full-length Smac/DIABLO cDNA and antisense phosphorothioate oligonucleotides against Smac/DIABLO were transiently transfected into C2C12 myogenic cells and Smac/DIABLO protein levels were analyzed by Western blotting. The results showed that: (1) H(2)O(2) (0.5 mmol/L) resulted in a marked release of Smac/DIABLO from mitochondria to cytoplasm 1 h after treatment, activation of caspase-3 and caspase-9 4 h after treatment, and specific morphological changes of apoptosis 24 h after treatment; (2) overexpression of Smac/DIABLO in C2C12 cells significantly enhanced H(2)O(2)-induced apoptosis and the activation of caspase-3 and caspase-9 (P<0.05). (3) Antisense phosphorothioate oligonucleotides against Smac/DIABLO markedly inhibited de novo synthesis of Smac/DIABLO and this effect was accompanied by decreased apoptosis and activation of caspase-3 and caspase-9 induced by H(2)O(2) (P<0.05). These data demonstrate that H(2)O(2) could result in apoptosis of C2C12 myogenic cells, and that release of Smac/DIABLO from mitochondria to cytoplasm and the subsequent activation of caspase-9 and caspase-3 played important roles in H(2)O(2)-induced apoptosis in C2C12 myogenic cells.     

Vacuolar ATPase Regulates Surfactant Secretion in Rat Alveolar Type II Cells by Modulating Lamellar Body Calcium

Lung surfactant reduces surface tension and maintains the stability of alveoli. How surfactant is released from alveolar epithelial type II cells is not fully understood. Vacuolar ATPase (V-ATPase) is the enzyme responsible for pumping H⁺ into lamellar bodies and is required for the processing of surfactant proteins and the packaging of surfactant lipids. However, its role in lung surfactant secretion is unknown. Proteomic analysis revealed that vacuolar ATPase (V-ATPase) dominated the alveolar type II cell lipid raft proteome. Western blotting confirmed the association of V-ATPase a1 and B1/2 subunits with lipid rafts and their enrichment in lamellar bodies. The dissipation of lamellar body pH gradient by Bafilomycin A1 (Baf A1), an inhibitor of V-ATPase, increased surfactant secretion. Baf A1-stimulated secretion was blocked by the intracellular Ca²⁺ chelator, BAPTA-AM, the protein kinase C (PKC) inhibitor, staurosporine, and the Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), KN-62. Baf A1 induced Ca²⁺ release from isolated lamellar bodies. Thapsigargin reduced the Baf A1-induced secretion, indicating cross-talk between lamellar body and endoplasmic reticulum Ca²⁺ pools. Stimulation of type II cells with surfactant secretagogues dissipated the pH gradient across lamellar bodies and disassembled the V-ATPase complex, indicating the physiological relevance of the V-ATPase-mediated surfactant secretion. Finally, silencing of V-ATPase a1 and B2 subunits decreased stimulated surfactant secretion, indicating that these subunits were crucial for surfactant secretion. We conclude that V-ATPase regulates surfactant secretion via an increased Ca²⁺ mobilization from lamellar bodies and endoplasmic reticulum, and the activation of PKC and CaMKII. Our finding revealed a previously unrealized role of V-ATPase in surfactant secretion.     

Transcriptional Regulation of Cannabinoid Receptor-1 Expression in the Liver by Retinoic Acid Acting via Retinoic Acid Receptor-γ

Alcoholism can result in fatty liver that can progress to steatohepatitis, cirrhosis, and liver cancer. Mice fed alcohol develop fatty liver through endocannabinoid activation of hepatic CB₁ cannabinoid receptors (CB₁R), which increases lipogenesis and decreases fatty acid oxidation. Chronic alcohol feeding also up-regulates CB₁R in hepatocytes in vivo, which could be replicated in vitro by co-culturing control hepatocytes with hepatic stellate cells (HSC) isolated from ethanol-fed mice, implicating HSC-derived mediator(s) in the regulation of hepatic CB₁R (Jeong, W. I., Osei-Hyiaman, D., Park, O., Liu, J., Bátkai, S., Mukhopadhyay, P., Horiguchi, N., Harvey-White, J., Marsicano, G., Lutz, B., Gao, B., and Kunos, G. (2008) Cell Metab. 7, 227–235). HSC being a rich source of retinoic acid (RA), we tested whether RA and its receptors may regulate CB₁R expression in cultured mouse hepatocytes. Incubation of hepatocytes with RA or RA receptor (RAR) agonists increased CB₁R mRNA and protein, the most efficacious being the RARγ agonist CD437 and the pan-RAR agonist TTNPB. The endocannabinoid 2-arachidonoylglycerol (2-AG) also increased hepatic CB₁R expression, which was mediated indirectly via RA, because it was absent in hepatocytes from mice lacking retinaldehyde dehydrogenase 1, the enzyme catalyzing the generation of RA from retinaldehyde. The binding of RARγ to the CB₁R gene 5′ upstream domain in hepatocytes treated with RAR agonists or 2-AG was confirmed by chromatin immunoprecipitation and electrophoretic mobility shift and antibody supershift assays. Finally, TTNPB-induced CB₁R expression was attenuated by small interfering RNA knockdown of RARγ in hepatocytes. We conclude that RARγ regulates CB₁R expression and is thus involved in the control of hepatic fat metabolism by endocannabinoids.     

The naphthylamine palladium(II) template induced E-Z isomerism of exocyclic vinyl group in five-membered P-N chelates: detailed spectroscopic studies

The exocyclic CC bond E-Z isomerism of chelating Ph₂PC(CHPh)-CHNAr in organopalladium complexes containing orthometallated [(S)-1-(dimethylamino)ethyl]naphthalene is reported. In dilute solutions of non-coordinating CH₂Cl₂ or CHCl₃, all the original E-isomers, in which the CHPh phenyl rings are located trans to PPh₂ moieties were partly converted to their Z-isomers. The isomerism was found to be dependent on temperature, concentration and solvent. At higher temperature, the Z-isomers were transformed completely back to their original E-isomers. Removal of the chiral auxiliaries of the E-Z mixtures by concentrated HCl, gave only the dichloro complexes of the E-isomers. The E-Z isomerization processes were well established by detailed spectroscopic studies, including ³¹P NMR, ¹H NMR and 2D ¹H-¹H ROESY NMR studies. It is noteworthy that the dichloro complexes and free P-N ligands did not show such isomerization processes, indicating that the isomerization processes were triggered by the orthopalladated naphthylamine moiety.     

Acute thoracic aortic dissection presenting as sore throat: report of a case

Acute dissection of the aorta can be one of the most dramatic of cardiovascular emergencies. Its symptoms can occur abruptly and progress rapidly. Prompt recognition and appropriate intervention is crucial. However, not all aortic dissections present with classic symptoms of abrupt chest, back, or abdominal pain, and the diagnosis may be missed. Aortic dissection presenting as a sore throat is quite unusual. A 53-year-old man presented with sore throat as the early symptom of an acute thoracic aortic dissection. Unfortunately, the diagnosis was delayed, and the patient died. Given the high morbidity and mortality after delayed recognition or misdiagnosis, aortic dissection should be considered in the differential diagnosis of a patient presenting with sore throat and normal findings of neck and throat, even when there is no classic symptoms.     

电压激活的钾通道阻断剂抑制山莨菪碱松弛去甲肾上腺素预收缩的兔主动脉平滑肌

研究显示,山莨菪碱预处理不改变高钾引起的兔主动脉环收缩,但可明显减弱去甲肾上腺素(noradrenaline,NA)、组织胺或5-羟色胺引起的收缩,且其减弱作用不受去除血管内皮影响。本实验观察了几种钾通道阻断剂对山良菪碱松弛：NA预收缩的兔主动脉环的影响。结果表明,1、3、10μmol／L山莨菪碱作用8min,可使0．01μmol／L NA预收缩的兔主动脉环松弛(P＜O．01)。10mmol／L,CsCl、1mmol／L 4-氨基吡啶、10μmol／L BaCl2、10μmol/L格列本脲、3μmol／L charybdotoxin和3μmol／L蜂毒明从分别与0．0lμmol／L NA同时加入,可增强后者收缩兔主动脉环的作用(P＜0．01)。10、30mmol／L CsCl或10、30mmol／L 4-氨基吡啶存在时,10μmol／L山茛菪碱对NA预收缩的兔主动脉环的松弛作用减弱,松弛率与对照组比较分别有极显著差异(P＜0．01);10、30μmol／L BaCl2,10、30μmol/L格列本脲,3μmol/L charybdotoxin或3μmol／L蜂毒明肽存在时,山莨菪碱对NA预收缩的兔主动脉环的松弛作用不受影响(P＞O．05)。本研究表明,电压激活的钾通道阻断剂抑制山莨菪碱松弛NA预收缩的兔主动脉平滑肌,初步提示血管平滑肌细胞膜上电压激活的钾通道参与山莨菪碱扩血管作用。     

Comprehensive analysis of pseudogenes in prokaryotes: widespread gene decay and failure of putative horizontally transferred genes

Background  

Pseudogenes often manifest themselves as disabled copies of known genes. In prokaryotes, it was generally believed (with a few well-known exceptions) that they were rare.     

Artemisinin: current state and perspectives for biotechnological production of an antimalarial drug

Artemisinin isolated from the aerial parts of Artemisia annua L. is a promising and potent antimalarial drug which has a remarkable activity against chloroquine-resistant and chloroquine-sensitive strains of Plasmodium falciparum, and is useful in treatment of cerebral malaria. Because the low content (0.01–1 %) of artemisinin in A. annua is a limitation to the commercial production of the drug, many research groups have been focusing their researches on enhancing the production of artemisinin in tissue culture or in the whole plant of A. annua. This review mainly focuses on the progresses made in the production of artemisinin from A. annua by biotechnological strategies including in vitro tissue culture, metabolic regulation of artemisinin biosynthesis, genetic engineering, and bioreactor technology.     

Cytological mechanism of pollen abortion resulting from allelic interaction of F1 pollen sterility locus in rice (Oryza sativa L.)

Pollen abortion is one of the major reasons causing the inter-subspecific F₁ hybrid sterility in rice and is due to allelic interaction of F₁ pollen sterility genes. The microsporogenesis and microgametogenesis of Taichung 65 and its three F₁ hybrids were comparatively studied by using techniques of differential interference contrast microscopy, semi-thin section light microscopy, epifluorescence microscopy and TEM. The results showed that there were differences among the cytological mechanisms of pollen abortion due to allelic interaction at the three F₁ pollen sterility loci. The allelic interaction at S-a locus resulted in microspores unable to extend the protoplasm membrane with the enlargement of the microspore at the middle microspore stage and finally producing empty abortive pollen. The allelic interaction at S-b locus caused asynchronous development of microspores at the middle microspore stage producing stainable abortive pollen. The allelic interaction at S-c locus mainly led to the non-dissolution of the generative cell wall and finally caused the hybrid F₁ mainly producing stainable abortive pollen. Genotypic identification indicated that the abortive pollen were those with S ^j allele.