Mice overexpressing human uncoupling protein-3 in skeletal muscle are hyperphagic and lean

Uncoupling protein-3 (UCP-3) is a recently identified member of the mitochondrial transporter superfamily that is expressed predominantly in skeletal muscle. However, its close relative UCP-1 is expressed exclusively in brown adipose tissue, a tissue whose main function is fat combustion and thermogenesis. Studies on the expression of UCP-3 in animals and humans in different physiological situations support a role for UCP-3 in energy balance and lipid metabolism. However, direct evidence for these roles is lacking. Here we describe the creation of transgenic mice that overexpress human UCP-3 in skeletal muscle. These mice are hyperphagic but weigh less than their wild-type littermates. Magnetic resonance imaging shows a striking reduction in adipose tissue mass. The mice also exhibit lower fasting plasma glucose and insulin levels and an increased glucose clearance rate. This provides evidence that skeletal muscle UCP-3 has the potential to influence metabolic rate and glucose homeostasis in the whole animal.     

急性运动中骨骼肌线粒体氧化应激机制研究

以SD大鼠3级递增负荷跑台运动为实验模型,分别选取安静态和运动45,90,120,150 min为实验观察点,测定其骨骼肌线粒体活性氧(ROS)生成、脂质过氧化水平(MDA)和UCP-3mRNA及蛋白表达.实验结果表明:运动过程中ROS生成呈先上升后下降的趋势,运动120 min时达到峰值,运动150 min时下降并具有显著性,其中运动45,90,120,150 min时均较安静时显著性升高;线粒体MDA含量总体呈上升趋势,但变化无显著性;运动过程中UCP-3mRNA和蛋白表达水平总体呈上升趋势,其中UCP-3mRNA在运动90,120,150 min时均较安静时呈显著性升高,而蛋白表达水平相对滞后一个时间段,在运动120,150 min时较安静时呈显著性增高.运动中线粒体ROS生成显著增加,但MDA水平无明显变化,这可能是运动中抗氧化能力提高,足以清除线粒体产生的过多ROS所致.运动中UCP-3表达的增加减少了线粒体ROS生成及其引发的氧化损伤.在ROS大量生成的情况下未发现线粒体有明显的脂质过氧化损伤,提示ROS在运动中可能具有重要的生理意义,而不仅仅是造成损伤.     

Purified inositol 1,4,5-trisphosphate receptor mediates calcium flux in reconstituted lipid vesicles

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), a second messenger molecule involved in actions of neurotransmitters, hormones and growth factors, releases calcium from vesicular non-mitochondrial intracellular stores. An Ins(1,4,5)P3 binding protein, purified from brain membranes, has been shown to be phosphorylated by cyclic-AMP-dependent protein kinase and localized by immunohistochemical techniques to intracellular particles associated with the endoplasmic reticulum. Although the specificity of the Ins(1,4,5)P3 binding protein for inositol phosphates and the high affinity of the protein for Ins(1,4,5)P3 indicate that it is a physiological Ins(1,4,5)P3 receptor mediating calcium release, direct evidence for this has been difficult to obtain. Also, it is unclear whether a single protein mediates both the recognition of Ins(1,4,5)P3 and calcium transport or whether these two functions involve two or more distinct proteins. In the present study we report reconstitution of the purified Ins(1,4,5)P3 binding protein into lipid vesicles. We show that Ins(1,4,5)P3 and other inositol phosphates stimulate calcium flux in the reconstituted vesicles with potencies and specificities that match the calcium releasing actions of Ins(1,4,5)P3. These results indicate that the purified Ins(1,4,5)P3 binding protein is a physiological receptor responsible for calcium release.     

A saturable receptor for 32P-inositol-1,4,5-triphosphate in hepatocytes and neutrophils

Several receptors for neurotransmitters, hormones and growth factors cause accelerated phosphodiesteratic breakdown of polyphosphoinositides when activated. One of the soluble products of this reaction, inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) is thought to act as a second messenger signalling the release of Ca2+ from intracellular stores. In support of this hypothesis, several studies have shown that Ins(1,4,5)P3 releases sequestered Ca2+ from permeable cells and microsomes. On the basis of certain structural requirements for Ca2+-releasing activity by inositol phosphates, it has been postulated that Ins(1,4,5)P3 acts by binding to a specific intracellular receptor, probably on a component of the endoplasmic reticulum. Here we report that 32P-Ins(1,4,5)P3 binds to a specific saturable site in permeabilized guinea pig hepatocytes and rabbit neutrophils, and that the properties of this binding site suggest that it is the physiological receptor for Ins(1,4,5)P3.     

Control of the sperm-oocyte switch in Caenorhabditis elegans hermaphrodites by the fem-3 3' untranslated region.

In the Caenorhabditis elegans hermaphrodite germ line, sperm and then oocytes are made from a common pool of germ-cell precursors. The decision to differentiate as a sperm or an oocyte is regulated by the sex-determining gene, fem-3. Expression of fem-3 in the hermaphrodite germ line directs spermatogenesis and must be negatively regulated to allow the switch to oogenesis. In adult hermaphrodites (which are producing oocytes), most fem-3 RNA is found in the germ line, consistent with both the requirement for fem-3 in hermaphrodite spermatogenesis and the maternal effects of fem-3 on embryonic sex determination. Whereas loss-of-function mutants in fem-3 produce only oocytes, hermaphrodites carrying any of nine fem-3 gain-of-function (gf) mutations make none; instead sperm are produced continuously and in vast excess over wild-type amounts. Genetic analyses suggest that fem-3(gf) mutations have escaped a negative control required for the switch to oogenesis. Here we report that all nine fem-3(gf) mutants carry sequence alterations in the fem-3 3' untranslated region (3' UTR). There is no increase in the steady-state level of fem-3(gf) RNA over wild-type, but there is an increase in the polyadenylation of fem-3(gf) RNA that is coincident with the unregulated fem-3 activity. Results of a titration experiment support the hypothesis that a regulatory factor may bind the fem-3 3' UTR. We speculate that fem-3 RNA is regulated through its 3' UTR by binding a factor that inhibits translation, and discuss the idea that this control may be part of a more general regulation of maternal RNAs.     

Platelet-derived growth factor stimulates synthesis of PtdIns(3,4,5)P3 by activating a PtdIns(4,5)P2 3-OH kinase.

Although the hormone-stimulated synthesis of 3-phosphorylated inositol lipids is known to form an intracellular signalling system, there is no consensus on the crucial receptor-regulated event in this pathway and it is still not clear which of the intermediates represent potential output signals. We show here that the key step in the synthesis of 3-phosphorylated inositol lipids in 3T3 cells stimulated by platelet-derived growth factor is the activation of a phosphatidylinositol(4,5)-bisphosphate (3)-hydroxy (PtdIns(4,5)P2 3-OH) kinase. A similar conclusion has been applied to explain the actions of formyl-Met-Leu-Phe on neutrophils, and it may be that receptors that couple through intrinsic tyrosine kinases or through G proteins stimulate the same step in 3-phosphorylated inositol lipid metabolism. The close parallel between these two mechanisms for the activation of PtdIns(4,5)P2 3-OH kinase and those described for the activation of another key signalling enzyme, phospholipase C (ref. 7), focuses attention on the product of the PtdIns(4,5)P2 3-OH kinase, PtdIns(3,4,5)P3, as a possible new second messenger.     

Crucial role for DNA ligase III in mitochondria but not in Xrcc1-dependent repair

Mammalian cells have three ATP-dependent DNA ligases, which are required for DNA replication and repair. Homologues of ligase I (Lig1) and ligase IV (Lig4) are ubiquitous in Eukarya, whereas ligase III (Lig3), which has nuclear and mitochondrial forms, appears to be restricted to vertebrates. Lig3 is implicated in various DNA repair pathways with its partner protein Xrcc1 (ref. 1). Deletion of Lig3 results in early embryonic lethality in mice, as well as apparent cellular lethality, which has precluded definitive characterization of Lig3 function. Here we used pre-emptive complementation to determine the viability requirement for Lig3 in mammalian cells and its requirement in DNA repair. Various forms of Lig3 were introduced stably into mouse embryonic stem (mES) cells containing a conditional allele of Lig3 that could be deleted with Cre recombinase. With this approach, we find that the mitochondrial, but not nuclear, Lig3 is required for cellular viability. Although the catalytic function of Lig3 is required, the zinc finger (ZnF) and BRCA1 carboxy (C)-terminal-related (BRCT) domains of Lig3 are not. Remarkably, the viability requirement for Lig3 can be circumvented by targeting Lig1 to the mitochondria or expressing Chlorella virus DNA ligase, the minimal eukaryal nick-sealing enzyme, or Escherichia coli LigA, an NAD(+)-dependent ligase. Lig3-null cells are not sensitive to several DNA-damaging agents that sensitize Xrcc1-deficient cells. Our results establish a role for Lig3 in mitochondria, but distinguish it from its interacting protein Xrcc1.     

UBCH7 reactivity profile reveals parkin and HHARI to be RING/HECT hybrids

Although the functional interaction between ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s) is essential in ubiquitin (Ub) signalling, the criteria that define an active E2-E3 pair are not well established. The human E2 UBCH7 (also known as UBE2L3) shows broad specificity for HECT-type E3s, but often fails to function with RING E3s in vitro despite forming specific complexes. Structural comparisons of inactive UBCH7-RING complexes with active UBCH5-RING complexes reveal no defining differences, highlighting a gap in our understanding of Ub transfer. Here we show that, unlike many E2s that transfer Ub with RINGs, UBCH7 lacks intrinsic, E3-independent reactivity with lysine, explaining its preference for HECTs. Despite lacking lysine reactivity, UBCH7 exhibits activity with the RING-in-between-RING (RBR) family of E3s that includes parkin (also known as PARK2) and human homologue of ariadne (HHARI; also known as ARIH1). Found in all eukaryotes, RBRs regulate processes such as translation and immune signalling. RBRs contain a canonical C3HC4-type RING, followed by two conserved Cys/His-rich Zn(2+)-binding domains, in-between-RING (IBR) and RING2 domains, which together define this E3 family. We show that RBRs function like RING/HECT hybrids: they bind E2s via a RING domain, but transfer Ub through an obligate thioester-linked Ub (denoted ～Ub), requiring a conserved cysteine residue in RING2. Our results define the functional cadre of E3s for UBCH7, an E2 involved in cell proliferation and immune function, and indicate a novel mechanism for an entire class of E3s.     

Coupled Pervaporation-Reaction Distillation Process for the Production of n-Bromopropane

IntroductionThere are several industrializable membraneseparation techniques,such as reverse osmosis(RO) ,nanofiltration(NF) ,ultrafiltration(UF) ,dialysis(D) ,electric dialysis(ED) ,gas separation(GS) and pervaporation (PV) [13] . In recentyears,another application of membrane technology tocoupled reaction- separation processes has receivedspecial attention from academic circles andindustrial enterprises[4 7] . When separationmembranes are incorporated into a reversiblereaction system co…     

金红石与石榴石浮选分离及调整剂作用机理

为了实现金红石与主要脉石矿物石榴石在无污染条件下的有效分离,采用浮选方法研究了金红石和石榴石的分离,并采用红外光谱和X光电子能谱分析等手段研究了分离过程中调整剂六偏磷酸钠的作用机理。结果表明,以烷胺双甲基磷酸(ATF1024)为捕收剂,六偏磷酸钠[(NaPO3)6]为调整剂,可以实现金红石与石榴石之间的浮选分离。(NaPO3)6对石榴石的抑制作用存在两种机理:其一,(NaPO3)6与石榴石表面Fe2 发生化学键合导致其牢固吸附而使石榴石表面强烈亲水;其二,(NaPO3)6选择性溶解石榴石表面Ca2 导致石榴石与捕收剂作用的表面活性质点减少。     

Altered cytosol/membrane enzyme redistribution on interleukin-3 activation of protein kinase C

Interleukin-3 (IL-3) is a member of a family of growth and differentiation peptides, collectively referred to as colony-stimulating factors, which regulate haematopoiesis. IL-3 has been highly purified from medium conditioned by WEHI-3B cells, and recently the molecular cloning of complementary DNA for murine IL-3 has been reported. IL-3 seems to stimulate a wide range of colony-forming cells derived from murine bone marrow and has consequently been studied under a variety of names, including burst-promoting activity, mast cell growth factor, P-cell stimulating factor and multi-colony-stimulating factor. Here we present evidence that IL-3-receptor interaction stimulates the rapid and transient redistribution of protein kinase C (PK-C) from cytosol to plasma membrane in FDC-P1 cells. Phorbol myristate acetate (PMA) is shown to have a similar effect in these IL-3-dependent FDC-P1 cells. Our data suggest that IL-3 and phorbol esters share a common feature of transmembrane signalling crucial for growth and differentiation.     

Crystal structure of the neurotrophin-3 and p75NTR symmetrical complex

Neurotrophins (NTs) are important regulators for the survival, differentiation and maintenance of different peripheral and central neurons. NTs bind to two distinct classes of glycosylated receptor: the p75 neurotrophin receptor (p75(NTR)) and tyrosine kinase receptors (Trks). Whereas p75(NTR) binds to all NTs, the Trk subtypes are specific for each NT. The question of whether NTs stimulate p75(NTR) by inducing receptor homodimerization is still under debate. Here we report the 2.6-A resolution crystal structure of neurotrophin-3 (NT-3) complexed to the ectodomain of glycosylated p75(NTR). In contrast to the previously reported asymmetric complex structure, which contains a dimer of nerve growth factor (NGF) bound to a single ectodomain of deglycosylated p75(NTR) (ref. 3), we show that NT-3 forms a central homodimer around which two glycosylated p75(NTR) molecules bind symmetrically. Symmetrical binding occurs along the NT-3 interfaces, resulting in a 2:2 ligand-receptor cluster. A comparison of the symmetrical and asymmetric structures reveals significant differences in ligand-receptor interactions and p75(NTR) conformations. Biochemical experiments indicate that both NT-3 and NGF bind to p75(NTR) with 2:2 stoichiometry in solution, whereas the 2:1 complexes are the result of artificial deglycosylation. We therefore propose that the symmetrical 2:2 complex reflects a native state of p75(NTR) activation at the cell surface. These results provide a model for NTs-p75(NTR) recognition and signal generation, as well as insights into coordination between p75(NTR) and Trks.     

Stepwise enzymatic dephosphorylation of inositol 1,4,5-trisphosphate to inositol in liver

Many receptors for hormones, neurotransmitters and other signals cause hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and effect a rise in cytosolic Ca2+ concentration. The inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) liberated during PtdIns(4,5)P2 breakdown seems to serve as a second messenger that activates the release of Ca2+ from a nonmitochondrial intracellular compartment. As expected if it is an important intracellular messenger, Ins(1,4,5)P3 is relatively rapidly degraded, both within stimulated cells and when added to homogenates of blowfly salivary gland or to permeabilized, but not intact, hepatocytes. Here we report that the dephosphorylation reactions responsible for the conversion of Ins(1,4,5)P3 to free inositol in rat liver are catalysed by two or more enzymes, and that these reactions are distributed between the plasma membrane and cytosol. The Ins(1,4,5)P3 5-phosphatase and inositol 1-phosphate (Ins(1)P) phosphatase of liver appear similar to enzymes described previously in erythrocytes and brain.     

Electrical conductivity optimization of the Na3AlF6-Al2O3-Sm2O3 molten salts system for Al-Sm intermediate binary ahoy production

Metal Sm has been widely used in making Al-Sm magnet alloy materials.Conventional distillation technology to produce Sm has the disadvantages of low productivity,high costs,and pollution generation.The objective of this study was to develop a molten salt electrolyte system to produce Al-Sm alloy directly,with focus on the electrical conductivity and optimal operating conditions to minimize the energy consumption.The continuously varying cell constant (CVCC) technique was used to measure the conductivity for the Na3AlF6-AlF3-LiF-MgF2-Al2O3-Sm2O3 electrolysis medium in the temperature range from 905 to 1055℃.The temperature (t) and the addition of Al2O3 (W(Al2O3)),Sm2O3 (W(Sm2O3)),and a combination of Al2O3 and Sm2O3 into the basic fluoride system were examined with respect to their effects on the conductivity (κ) and activation energy.The experimental results showed that the molten electrolyte conductivity increases with increasing temperature (t) and decreases with the addition of Al2O3 or Sm2O3 or both.We concluded that the optimal operation conditions for Al-Sm intermediate alloy production in the Na3AlF6-AlF3-LiF-MgF2-Al2O3-Sm2O3 system are W(Al2O3) + W(Sm2O3) =3wt％,W(Al2O3)∶ W(Sm2O3) =7∶3,and a temperature of 965 to 995℃,which results in satisfactory conductivity,low fluoride evaporation losses,and low energy consumption.