A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter

Mitochondrial Ca(2+) homeostasis has a key role in the regulation of aerobic metabolism and cell survival, but the molecular identity of the Ca(2+) channel, the mitochondrial calcium uniporter, is still unknown. Here we have identified in silico a protein (named MCU) that shares tissue distribution with MICU1 (also known as CBARA1), a recently characterized uniporter regulator, is present in organisms in which mitochondrial Ca(2+) uptake was demonstrated and whose sequence includes two transmembrane domains. Short interfering RNA (siRNA) silencing of MCU in HeLa cells markedly reduced mitochondrial Ca(2+) uptake. MCU overexpression doubled the matrix Ca(2+) concentration increase evoked by inositol 1,4,5-trisphosphate-generating agonists, thus significantly buffering the cytosolic elevation. The purified MCU protein showed channel activity in planar lipid bilayers, with electrophysiological properties and inhibitor sensitivity of the uniporter. A mutant MCU, in which two negatively charged residues of the putative pore-forming region were replaced, had no channel activity and reduced agonist-dependent matrix Ca(2+) concentration transients when overexpressed in HeLa cells. Overall, these data demonstrate that the 40-kDa protein identified is the channel responsible for ruthenium-red-sensitive mitochondrial Ca(2+) uptake, thus providing a molecular basis for this process of utmost physiological and pathological relevance.     

The mitochondrial calcium uniporter is a highly selective ion channel

During intracellular Ca2+ signalling mitochondria accumulate significant amounts of Ca2+ from the cytosol. Mitochondrial Ca2+ uptake controls the rate of energy production, shapes the amplitude and spatio-temporal patterns of intracellular Ca2+ signals, and is instrumental to cell death. This Ca2+ uptake is undertaken by the mitochondrial Ca2+ uniporter (MCU) located in the organelle's inner membrane. The uniporter passes Ca2+ down the electrochemical gradient maintained across this membrane without direct coupling to ATP hydrolysis or transport of other ions. Carriers are characterized by turnover numbers that are typically 1,000-fold lower than ion channels, and until now it has been unclear whether the MCU is a carrier or a channel. By patch-clamping the inner mitochondrial membrane, we identified a previously unknown Ca2+-selective ion channel sensitive to inhibitors of mitochondrial Ca2+ uptake. Our data indicate that this unique channel binds Ca2+ with extremely high affinity (dissociation constant < or =2 nM), enabling high Ca2+ selectivity despite relatively low cytoplasmic Ca2+ concentrations. The channel is inwardly rectifying, making it especially effective for Ca2+ uptake into energized mitochondria. Thus, we conclude that the properties of the current mediated by this novel channel are those of the MCU.     

A proteome-wide screen identifies valosin-containing protein as an essential regulator of podocyte endoplasmic reticulum stress

To investigate proteins expressed in the renal tissue of the passive Heymann nephritis (pHN) rat model,we prepared pHN rat models with anti-FxA1 serum and analyzed the proteins differentially expressed in the kidney tissue with label-free liquid chromatography-tandem mass spectrometry.We then analyzed in depth the endoplasmic reticulum stress (ERS)-related protein using an online bioinformatics platform.Forty-one differential proteins and their annotations were obtained.Gene Ontology (GO) function analysis showed that 16 proteins were involved in cellular metabolism and 22 were proteins related to catalytic activity,including protein folding or ATPase.Protein-GO networks indicated that VCP could interact with the ERS marker HSPa5,with both involved in a single pathway.On inhibition of podocyte VCP by RNAi under normal conditions,the HSPa5 expression level did not change,but when the cell was subjected to ERS by tunicamycin,HSPa5 expression significantly increased with RNAi of VCP when compared with the tunicamycin-treated group.Our results showed that ERS plays an important role in podocyte injury of membranous nephropathy and is mediated by an HSPa5-VCP signaling pathway,in which the most predominant proteins are those related to cellular metabolism and catalytic activity.     

ClpS is an essential component of the N-end rule pathway in Escherichia coli

The N-end rule states that the half-life of a protein is determined by the nature of its amino-terminal residue. Eukaryotes and prokaryotes use N-terminal destabilizing residues as a signal to target proteins for degradation by the N-end rule pathway. In eukaryotes an E3 ligase, N-recognin, recognizes N-end rule substrates and mediates their ubiquitination and degradation by the proteasome. In Escherichia coli, N-end rule substrates are degraded by the AAA + chaperone ClpA in complex with the ClpP peptidase (ClpAP). Little is known of the molecular mechanism by which N-end rule substrates are initially selected for proteolysis. Here we report that the ClpAP-specific adaptor, ClpS, is essential for degradation of N-end rule substrates by ClpAP in bacteria. ClpS binds directly to N-terminal destabilizing residues through its substrate-binding site distal to the ClpS-ClpA interface, and targets these substrates to ClpAP for degradation. Degradation by the N-end rule pathway is more complex than anticipated and several other features are involved, including a net positive charge near the N terminus and an unstructured region between the N-terminal signal and the folded protein substrate. Through interaction with this signal, ClpS converts the ClpAP machine into a protease with exquisitely defined specificity, ideally suited to regulatory proteolysis.     

Structure of the zinc-binding domain of an essential component of the hepatitis C virus replicase

Hepatitis C virus (HCV) is a human pathogen affecting nearly 3% of the world's population. Chronic infections can lead to cirrhosis and liver cancer. The RNA replication machine of HCV is a multi-subunit membrane-associated complex. The non-structural protein NS5A is an active component of HCV replicase, as well as a pivotal regulator of replication and a modulator of cellular processes ranging from innate immunity to dysregulated cell growth. NS5A is a large phosphoprotein (56-58 kDa) with an amphipathic alpha-helix at its amino terminus that promotes membrane association. After this helix region, NS5A is organized into three domains. The N-terminal domain (domain I) coordinates a single zinc atom per protein molecule. Mutations disrupting either the membrane anchor or zinc binding of NS5A are lethal for RNA replication. However, probing the role of NS5A in replication has been hampered by a lack of structural information about this multifunctional protein. Here we report the structure of NS5A domain I at 2.5-A resolution, which contains a novel fold, a new zinc-coordination motif and a disulphide bond. We use molecular surface analysis to suggest the location of protein-, RNA- and membrane-interaction sites.     

Imaging of Rab5 activity identifies essential regulators for phagosome maturation

Efficient phagocytosis of apoptotic cells is crucial for tissue homeostasis and the immune response. Rab5 is known as a key regulator of the early endocytic pathway and we have recently shown that Rab5 is also implicated in apoptotic cell engulfment; however, the precise spatio-temporal dynamics of Rab5 activity remain unknown. Here, using a newly developed fluorescence resonance energy transfer biosensor, we describe a change in Rab5 activity during the engulfment of apoptotic thymocytes. Rab5 activity on phagosome membranes began to increase on disassembly of the actin coat encapsulating phagosomes. Rab5 activation was either continuous or repetitive for up to 10 min, but it ended before the collapse of engulfed apoptotic cells. Expression of a dominant-negative mutant of Rab5 delayed this collapse of apoptotic thymocytes, showing a role for Rab5 in phagosome maturation. Disruption of microtubules with nocodazole inhibited Rab5 activation on the phagosome membrane without perturbing the engulfment of apoptotic cells. Furthermore, we found that Gapex-5 is the guanine nucleotide exchange factor essential for Rab5 activation during the engulfment of apoptotic cells. Gapex-5 was bound to a microtubule-tip-associating protein, EB1, whose depletion inhibited Rab5 activation during phagocytosis. We therefore propose a mechanistic model in which the recruitment of Gapex-5 to phagosomes through the microtubule network induces the transient Rab5 activation.     

Functional genomics reveal that the serine synthesis pathway is essential in breast cancer

Cancer cells adapt their metabolic processes to drive macromolecular biosynthesis for rapid cell growth and proliferation. RNA interference (RNAi)-based loss-of-function screening has proven powerful for the identification of new and interesting cancer targets, and recent studies have used this technology in vivo to identify novel tumour suppressor genes. Here we developed a method for identifying novel cancer targets via negative-selection RNAi screening using a human breast cancer xenograft model at an orthotopic site in the mouse. Using this method, we screened a set of metabolic genes associated with aggressive breast cancer and stemness to identify those required for in vivo tumorigenesis. Among the genes identified, phosphoglycerate dehydrogenase (PHGDH) is in a genomic region of recurrent copy number gain in breast cancer and PHGDH protein levels are elevated in 70% of oestrogen receptor (ER)-negative breast cancers. PHGDH catalyses the first step in the serine biosynthesis pathway, and breast cancer cells with high PHGDH expression have increased serine synthesis flux. Suppression of PHGDH in cell lines with elevated PHGDH expression, but not in those without, causes a strong decrease in cell proliferation and a reduction in serine synthesis. We find that PHGDH suppression does not affect intracellular serine levels, but causes a drop in the levels of α-ketoglutarate, another output of the pathway and a tricarboxylic acid (TCA) cycle intermediate. In cells with high PHGDH expression, the serine synthesis pathway contributes approximately 50% of the total anaplerotic flux of glutamine into the TCA cycle. These results reveal that certain breast cancers are dependent upon increased serine pathway flux caused by PHGDH overexpression and demonstrate the utility of in vivo negative-selection RNAi screens for finding potential anticancer targets.     

I-J as an idiotype of the recognition component of antigen-specific suppressor T-cell factor

The I-J determinant of membrane glycoprotein is known to be expressed exclusively on suppressor T cells (TS), which have a crucial role in the regulation of immune responses. I-J also comprises part of the soluble factor (TSF) with suppressor activity which is secreted from TS. Gene-mapping experiments have indicated that the I-J gene lies between the I-A and I-E subregions of the mouse major histocompatibility complex (MHC) and is defined by the H-2 congeneic pair, that is, B10.A(3R) and B10.A(5R). In fact, antibodies raised in the reciprocal combinations of B10.A(3R) and B10.A(5R) define the I-Jb and I-Jk alleles, and are able to detect the I-J determinants on TS and TSF. Biochemical and functional analyses, using I-J-positive TS clones and hybridomas, have demonstrated that monoclonal anti-I-J antibodies precipitate I-Jk or I-Jb with a relative molecular mass of 25,000-28,000 (25-28K) and that the I-J+ molecule mediates the restriction specificity of TSF in association with an antigen-binding protein (45K). However, molecular genetic studies on the I-J gene reveal no genetic difference between B10.A(3R) and B10.A(5R) and also that there is no room to accommodate a gene encoding I-J in the expected I region. These discrepancies between the molecular genetic and serological/functional data require explanation. Here we demonstrate that TS and TSF expressing I-J of the host type were produced by fully allogeneic bone marrow cells of donor origin in chimaeric mice, when the chimaeras received the host antigen-presenting cells (APC) at the time of immunization. The results show that APC are necessary for the activation and clonal expansion of TS and also support the notion that I-J is an idiotypic determinant of the recognition component of TS and TSF.     

The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus

Acquired immune deficiency syndrome (AIDS) is characterized by opportunistic infections and by 'opportunistic neoplasms' (for example, Kaposi's sarcoma). Persistent generalized lymphadenopathy (PGL) is epidemiologically associated with AIDS, especially in male homosexuals. A subset of T lymphocytes positive for the CD4 antigen (also termed T4 antigen), is depleted in AIDS and PGL patients. A retrovirus found in T-cell cultures from these patients is strongly implicated in the aetiology of AIDS because of the high frequency of isolation and the prevalence of specific antibodies in the patients. Here we have detected cell-surface receptors for the AIDS retrovirus (human T-cell leukaemia virus-III (HTLV-III) and lymphadenopathy-associated virus-1 (LAV-1) isolates) by testing the susceptibility of cells to infection with pseudotypes of vesicular stomatitis virus bearing retroviral envelope antigens, and by the formation of multinucleated syncytia on mixing virus-producing cells with receptor-bearing cells. Receptors were present only on cells expressing CD4 antigen; among 155 monoclonal antibodies tested, each of the 14 anti-CD4 antibodies inhibited formation of syncytia and blocked pseudotypes. Productive infection of CD4+ cells with HTLV-III or LAV-1 markedly reduced cell-surface expression of CD4. In contrast, receptors for HTLV-I and HTLV-II were not restricted to CD4+ cells, were not blocked by anti-CD4 antibodies; cells productively infected with HTLV-I and HTLV-II expressed surface CD4. Hence, we conclude that the CD4 antigen is an essential and specific component of the receptor for the causative agent of AIDS.     

木香薷挥发油主要化学成分的研究

应用ＧＣ、ＧＣＭＳＣＯＭＰ法从采自甘肃天水地区的木香薷（ＥｌｓｈｏｌｔｚｉａｓｔａｕｎｔｏｎｉＢｅｎｔｈ）挥发油中分离出６９个化学成分．鉴定了以１８桉叶油素（２２３５％）、反式石竹烯（１３８６％）、莰醌（４６％）、反式β罗勒烯（３２１％）、苯甲醇（３７１％）、２（２′３′环氧３′甲基丁基）３甲基呋喃（１０６７％）等为主的５５个成分．其中有２６个成分为首次从木香薷中检出．其主要成分占挥发油总量的９４％以上     

单片机应用系统的可靠性设计

可靠性设计是单片机应用系统设计必不可少的设计内容。本文从现代电子系统的可靠性出发,详细论述了单片机应用系统的可靠性特点,提出了芯片选择、电源设计、PCB制作、噪声失敏控制、程序失控回复等集合硬件系统的可靠性设计技术和软件系统的可靠性设计技术的解决方法,可供单片机应用系统的开发人员借鉴与参考。     

Mycorrhizal weathering of apatite as an important calcium source in base-poor forest ecosystems

The depletion of calcium in forest ecosystems of the northeastern USA is thought to be a consequence of acidic deposition and to be at present restricting the recovery of forest and aquatic systems now that acidic deposition itself is declining. This depletion of calcium has been inferred from studies showing that sources of calcium in forest ecosystems namely, atmospheric deposition and mineral weathering of silicate rocks such as plagioclase, a calcium-sodium silicate do not match calcium outputs observed in forest streams. It is therefore thought that calcium is being lost from exchangeable and organically bound calcium in forest soils. Here we investigate the sources of calcium in the Hubbard Brook experimental forest, through analysis of calcium and strontium abundances and strontium isotope ratios within various soil, vegetation and hydrological pools. We show that the dissolution of apatite (calcium phosphate) represents a source of calcium that is comparable in size to known inputs from atmospheric sources and silicate weathering. Moreover, apatite-derived calcium was utilized largely by ectomycorrhizal tree species, suggesting that mycorrhizae may weather apatite and absorb the released ions directly, without the ions entering the exchangeable soil pool. Therefore, it seems that apatite weathering can compensate for some of the calcium lost from base-poor ecosystems, and should be considered when estimating soil acidification impacts and calcium cycling.     

基于单片机的智能防火报警装置设计

针对用电器电流过大导致短路而造成的火灾突发状况,本设计是一款基于单片机的智能防火报警装置。装置以STC89C51单片机为主控制器,采用DS18B20数字温度传感器实时测量插座温度,并配合数码管显示实测温度值。通过实测温度值与温度阈值的比较,装置可实现声光报警和排查断电功能。实验结果表明:当实测温度超过低温度阈值时,装置产生报警功能;当实测温度超过高温度阈值时,实现用电器的断电处理,该装置对火灾预警具有一定功效。     

A 42K outer-membrane protein is a component of the yeast mitochondrial protein import site

An engineered precursor protein that sticks in the import site of isolated yeast mitochondria can be specifically photo-crosslinked to a mitochondrial outer-membrane protein of relative molecular mass 42,000 (42K). This protein (termed import-site protein 42 or ISP 42) is exposed on the mitochondrial surface; antibodies against it block protein import into mitochondria. ISP 42 is the first identified component of the putative transmembrane machinery that imports proteins into mitochondria.     

一种单片机在线仿真方法的实现

单片机系统的仿真调试是单片机系统开发工作的重要环节,也是最困难的步骤。文章介绍了基于KEIL单片机开发软件,实现MCS51单片机系统在线仿真调试的关键技术。文章分析了实现该技术所需的单片机系统的硬件体系结构、要求,分析了使用汇编或C语言编写单片机程序,以实现在线仿真的程序结构及要求。