S-nitrosothiols signal the ventilatory response to hypoxia

Increased ventilation in response to hypoxia has been appreciated for over a century, but the biochemistry underlying this response remains poorly understood. Here we define a pathway in which increased minute ventilation (&Vdot;E ) is signalled by deoxyhaemoglobin-derived S-nitrosothiols (SNOs). Specifically, we demonstrate that S-nitrosocysteinyl glycine (CGSNO) and S-nitroso-l-cysteine (l-CSNO)-but not S-nitroso-d-cysteine (d-CSNO)-reproduce the ventilatory effects of hypoxia at the level of the nucleus tractus solitarius (NTS). We show that plasma from deoxygenated, but not from oxygenated, blood produces the ventilatory effect of both SNOs and hypoxia. Further, this activity is mediated by S-nitrosoglutathione (GSNO), and GSNO activation by gamma-glutamyl transpeptidase (gamma-GT) is required. The normal response to hypoxia is impaired in a knockout mouse lacking gamma-GT. These observations suggest that S-nitrosothiol biochemistry is of central importance to the regulation of breathing.     

含葡聚糖颗粒物暴露致小鼠肺部GSNO还原酶活化的研究

动物实验和体外研究都已表明,空气中有机颗粒物可引起肺部炎症反应,该反应的显著特点为细胞因子表达上调和分泌增多.葡聚糖是霉菌和细菌代谢及裂解的产物,当颗粒物受到霉菌和细菌污染之后,就会含有葡聚糖.最近的研究表明,含葡聚糖颗粒物的暴露会影响鼻腔和肺部的功能,并伴随炎症反应.然而,含葡聚糖颗粒物的暴露对一氧化氮合酶(NOS)和亚硝基谷胱甘肽还原酶(GSNOR)的影响仍不太清楚.本研究旨在检测含葡聚糖颗粒物暴露对呼吸道中NO信号通路的影响.本实验力小鼠被分别暴露于20μL PBS(空白组),20μL浓度为25μg/20μL的OVA和20μL浓度为100μL/20μL的含葡聚糖颗粒物环境中,暴露持续12d.暴露结束后在肺组织匀浆中检测NOS和GSNOR的活性.同时测定肺组织中谷胱甘肽(GSH)浓度和SOD活性用以评估氧化应激水平.另外,检测肺组织中的IL–6浓度确定炎症反应的发生与否.结果显示,12天OVA和葡聚糖颗粒物暴露并未明显影响NOS活性、GSH含量、SOD活性和IL-6浓度.然而,含葡聚糖颗粒物12d的暴露却明显增加GSNOR的活性和表达.我们的研究结果表明,含葡聚糖颗粒物暴露会激活呼吸道中的GSNOR,但不激活NOS.由于GSNOR在NO信号通路中起着举足轻重的作用,我们的研究结果具有一定的临床重要性.     

Identification of the haematopoietic stem cell niche and control of the niche size

Haematopoietic stem cells (HSCs) are a subset of bone marrow cells that are capable of self-renewal and of forming all types of blood cells (multi-potential). However, the HSC 'niche'--the in vivo regulatory microenvironment where HSCs reside--and the mechanisms involved in controlling the number of adult HSCs remain largely unknown. The bone morphogenetic protein (BMP) signal has an essential role in inducing haematopoietic tissue during embryogenesis. We investigated the roles of the BMP signalling pathway in regulating adult HSC development in vivo by analysing mutant mice with conditional inactivation of BMP receptor type IA (BMPRIA). Here we show that an increase in the number of spindle-shaped N-cadherin+CD45- osteoblastic (SNO) cells correlates with an increase in the number of HSCs. The long-term HSCs are found attached to SNO cells. Two adherens junction molecules, N-cadherin and beta-catenin, are asymmetrically localized between the SNO cells and the long-term HSCs. We conclude that SNO cells lining the bone surface function as a key component of the niche to support HSCs, and that BMP signalling through BMPRIA controls the number of HSCs by regulating niche size.     

Expression of the SOD gene from Trichoderma harzianum in Saccharomyces cerevisiae

Superoxide dismutases are metalloproteins which play a major role in defense against oxygen radicalmediated toxicity in aerobic organisms. Such proteins are important endogeneity cytoprotection factor involving defence. A 751-bp full-length cDNA sequence of an SOD gene was isolated from the Trichoderma harzianum. The full-length cDNA of the SOD gene consists of one 465-bp open reading frame nucleotide, which encodes a 15.7-kDa polypeptide consisting of 154 amino acid residues. Sequence analysis revealed that SOD gene has more than 72%-86% amino acid sequence homology with those of other fungi. The SOD gene was integrated into the genomic DNA of pYES2 by insertion into a single site for recombination, yielding the recombinant pYES2-SOD. SOD expressed by pYES2-SOD was induced by galactose. We test whether SOD could offer abiotic stress resistance when it was introduced into yeast ceils. A transgenic yeast harboring T. harzianum SOD was generated under the control of a constitutively expressed GAL promoter. The results indicated that SOD yeast transformants had significantly higher resistance to salt and drought stress.     

Export by red blood cells of nitric oxide bioactivity

Previous studies support a model in which the physiological O2 gradient is transduced by haemoglobin into the coordinate release from red blood cells of O2 and nitric oxide (NO)-derived vasoactivity to optimize oxygen delivery in the arterial periphery. But whereas both O2 and NO diffuse into red blood cells, only O2 can diffuse out. Thus, for the dilation of blood vessels by red blood cells, there must be a mechanism to export NO-related vasoactivity, and current models of NO-mediated intercellular communication should be revised. Here we show that in human erythrocytes haemoglobin-derived S-nitrosothiol (SNO), generated from imported NO, is associated predominantly with the red blood cell membrane, and principally with cysteine residues in the haemoglobin-binding cytoplasmic domain of the anion exchanger AE1. Interaction with AE1 promotes the deoxygenated structure in SNO-haemoglobin, which subserves NO group transfer to the membrane. Furthermore, we show that vasodilatory activity is released from this membrane precinct by deoxygenation. Thus, the oxygen-regulated cellular mechanism that couples the synthesis and export of haemoglobin-derived NO bioactivity operates, at least in part, through formation of AE1-SNO at the membrane-cytosol interface.     

层流剪切力对悬浮培养红豆杉细胞HMGR酶基因转录的影响

以HMGR酶基因为研究对象,采用实时荧光RT-PCR技术,研究了Couette式反应器中0．3 Pa层流剪切力作用后悬浮培养的对数期东北红豆杉细胞HMGR酶基因转录情况．结果显示,在对数生长时期,未经剪切处理的悬浮培养虹豆杉细胞HMGR基因转录水平呈增长趋势,而剪切处理后的悬浮培养红豆杉细胞HMGR基因转录水平则基本保持不变．同时,对相应细胞生物量的测定结果也显示出类似的变化．以上结果说明在对数生长时期施加一定强度的剪切力,将使细胞生长发生停滞,谊生长停滞现象可能与因剪切所引起的HMGR基因转录水平下降有一定相关性．     

Is ribonucleotide reductase the transforming function of herpes simplex virus 2?

Transformation of cells by herpes simplex virus 2 (HSV-2) can be induced by the BglII C (0.43-0.58 map units) or N (0.58-0.625) fragments of the viral genome. Sequences partially overlapping both fragments (0.566-0.602) encode two 3' coterminal mRNAs; these in turn direct the synthesis of two related polypeptides of molecular weight 140,000 (140K) and 35K (refs 4, 7), which may be involved in transformation. Recently, a temperature-sensitive (ts) mutation affecting HSV-induced ribonucleotide reductase has been mapped within this common region (B.M. Dutia, personal communication). We have partially purified the induced reductase and raised a rabbit antiserum to it which inhibits the enzyme activity and immunoprecipitates from infected cells a 144K polypeptide and minor species including a 38K polypeptide. Here we show that a monoclonal antibody to the putative transforming proteins competes with the rabbit serum for the 144K and 38K antigens and also immunoprecipitates specifically the induced reductase activity. These results suggest a possible role for ribonucleotide reductase in HSV-2-induced transformation.     

S-glutathionylation uncouples eNOS and regulates its cellular and vascular function

Endothelial nitric oxide synthase (eNOS) is critical in the regulation of vascular function, and can generate both nitric oxide (NO) and superoxide (O(2)(?-)), which are key mediators of cellular signalling. In the presence of Ca(2+)/calmodulin, eNOS produces NO, endothelial-derived relaxing factor, from l-arginine (l-Arg) by means of electron transfer from NADPH through a flavin containing reductase domain to oxygen bound at the haem of an oxygenase domain, which also contains binding sites for tetrahydrobiopterin (BH(4)) and l-Arg. In the absence of BH(4), NO synthesis is abrogated and instead O(2)(?-) is generated. While NOS dysfunction occurs in diseases with redox stress, BH(4) repletion only partly restores NOS activity and NOS-dependent vasodilation. This suggests that there is an as yet unidentified redox-regulated mechanism controlling NOS function. Protein thiols can undergo S-glutathionylation, a reversible protein modification involved in cellular signalling and adaptation. Under oxidative stress, S-glutathionylation occurs through thiol-disulphide exchange with oxidized glutathione or reaction of oxidant-induced protein thiyl radicals with reduced glutathione. Cysteine residues are critical for the maintenance of eNOS function; we therefore speculated that oxidative stress could alter eNOS activity through S-glutathionylation. Here we show that S-glutathionylation of eNOS reversibly decreases NOS activity with an increase in O(2)(?-) generation primarily from the reductase, in which two highly conserved cysteine residues are identified as sites of S-glutathionylation and found to be critical for redox-regulation of eNOS function. We show that eNOS S-glutathionylation in endothelial cells, with loss of NO and gain of O(2)(?-) generation, is associated with impaired endothelium-dependent vasodilation. In hypertensive vessels, eNOS S-glutathionylation is increased with impaired endothelium-dependent vasodilation that is restored by thiol-specific reducing agents, which reverse this S-glutathionylation. Thus, S-glutathionylation of eNOS is a pivotal switch providing redox regulation of cellular signalling, endothelial function and vascular tone.     

Validation of AMSU-A measurements from two different calibrations in the lower stratosphere using COSMIC radio occultation data

GPS radio occultation data from the Constellation Observing System for Meteorology, Ionosphere, and Climate(COSMIC) mission were used to validate the measurements of the advanced microwave sounding unit-A(AMSU-A) in the lower stratosphere from different satellites. AMSU-A observations from two different calibrations—the pre-launch operational and post-launch simultaneous nadir overpass(SNO) calibrations—were compared to microwave brightness temperatures(Tb)simulated from COSMIC data. Observations from three satellites(NOAA-15,-16, and-18) were used in the comparison. The results showed that AMSU-A Tb measurements from both calibrations and from all three NOAA satellites were underestimated in the lower stratosphere,and that the biases were larger in polar winters, especially over the southern high latitudes. In comparison to operational calibration, the SNO-calibrated AMSU-A data produced much smaller biases relative to the COSMIC data.The improvement due to SNO calibration was quantified by a Ratio index, which measured the bias changes from operational to SNO calibrations relative to the biases between the operational-calibrated AMSU-A data and the COSMIC data. The Ratio values were 70 % for NOAA-15and [80 % for NOAA-18 and-16, indicating that the SNO calibration method significantly reduced AMSU-A biases and effectively improved AMSU-A data quality.     

The Toll-like receptor 2 is recruited to macrophage phagosomes and discriminates between pathogens.

Macrophages orchestrate innate immunity by phagocytosing pathogens and coordinating inflammatory responses. Effective defence requires the host to discriminate between different pathogens. The specificity of innate immune recognition in Drosophila is mediated by the Toll family of receptors; Toll mediates anti-fungal responses, whereas 18-wheeler mediates anti-bacterial defence. A large number of Toll homologues have been identified in mammals, and Toll-like receptor 4 is critical in responses to Gram-negative bacteria. Here we show that Toll-like receptor 2 is recruited specifically to macrophage phagosomes containing yeast, and that a point mutation in the receptor abrogates inflammatory responses to yeast and Gram-positive bacteria, but not to Gram-negative bacteria. Thus, during the phagocytosis of pathogens, two classes of innate immune receptors cooperate to mediate host defence: phagocytic receptors, such as the mannose receptor, signal particle internalization, and the Toll-like receptors sample the contents of the vacuole and trigger an inflammatory response appropriate to defence against the specific organism.     

GILT is a critical host factor for Listeria monocytogenes infection

Listeria monocytogenes is a gram-positive, intracellular, food-borne pathogen that can cause severe illness in humans and animals. On infection, it is actively phagocytosed by macrophages; it then escapes from the phagosome, replicates in the cytosol, and subsequently spreads from cell to cell by a non-lytic mechanism driven by actin polymerization. Penetration of the phagosomal membrane is initiated by the secreted haemolysin listeriolysin O (LLO), which is essential for vacuolar escape in vitro and for virulence in animal models of infection. Reduction is required to activate the lytic activity of LLO in vitro, and we show here that reduction by the enzyme gamma-interferon-inducible lysosomal thiol reductase (GILT, also called Ifi30) is responsible for the activation of LLO in vivo. GILT is a soluble thiol reductase expressed constitutively within the lysosomes of antigen-presenting cells, and it accumulates in macrophage phagosomes as they mature into phagolysosomes. The enzyme is delivered by a mannose-6-phosphate receptor-dependent mechanism to the endocytic pathway, where amino- and carboxy-terminal pro-peptides are cleaved to generate a 30-kDa mature enzyme. The active site of GILT contains two cysteine residues in a CXXC motif that catalyses the reduction of disulphide bonds. Mice lacking GILT are deficient in generating major histocompatibility complex class-II-restricted CD4(+) T-cell responses to protein antigens that contain disulphide bonds. Here we show that these mice are resistant to L. monocytogenes infection. Replication of the organism in GILT-negative macrophages, or macrophages expressing an enzymatically inactive GILT mutant, is impaired because of delayed escape from the phagosome. GILT activates LLO within the phagosome by the thiol reductase mechanism shared by members of the thioredoxin family. In addition, purified GILT activates recombinant LLO, facilitating membrane permeabilization and red blood cell lysis. The data show that GILT is a critical host factor that facilitates L. monocytogenes infection.     

Gene targeting in normal and amplified cell lines

Targeted recombination in mammalian cells is rare compared with non-homologous integration. In Saccharomyces cerevisiae the reverse is true. Differences in targeting efficiency could arise because a target of unique DNA is 200 times more dilute in mammalian genomes than it is in yeast. We tested this possibility by measuring gene targeting in normal CHO cells with two copies of the dihydrofolate reductase (DHFR) gene and in amplified CHOC 400 cells, which carry 800 copies. If the concentration of the target gene is critical, amplified cells should show an enhanced frequency of targeted recombination relative to non-homologous integration. Using a positive/negative selection protocol, we demonstrated that the efficiency of targeting into DHFR genes is indistinguishable in normal and amplified CHO cells. As targeting does not depend on the number of targets, the search for homology is not a rate-limiting step in the mammalian pathway of gene targeting. Thus, the difference in genome size is not the basis for the different outcomes of targeting experiments in S. cerevisiae and mammals.     

S-nitrosylated protein-disulphide isomerase links protein misfolding to neurodegeneration

Stress proteins located in the cytosol or endoplasmic reticulum (ER) maintain cell homeostasis and afford tolerance to severe insults. In neurodegenerative diseases, several chaperones ameliorate the accumulation of misfolded proteins triggered by oxidative or nitrosative stress, or of mutated gene products. Although severe ER stress can induce apoptosis, the ER withstands relatively mild insults through the expression of stress proteins or chaperones such as glucose-regulated protein (GRP) and protein-disulphide isomerase (PDI), which assist in the maturation and transport of unfolded secretory proteins. PDI catalyses thiol-disulphide exchange, thus facilitating disulphide bond formation and rearrangement reactions. PDI has two domains that function as independent active sites with homology to the small, redox-active protein thioredoxin. During neurodegenerative disorders and cerebral ischaemia, the accumulation of immature and denatured proteins results in ER dysfunction, but the upregulation of PDI represents an adaptive response to protect neuronal cells. Here we show, in brains manifesting sporadic Parkinson's or Alzheimer's disease, that PDI is S-nitrosylated, a reaction transferring a nitric oxide (NO) group to a critical cysteine thiol to affect protein function. NO-induced S-nitrosylation of PDI inhibits its enzymatic activity, leads to the accumulation of polyubiquitinated proteins, and activates the unfolded protein response. S-nitrosylation also abrogates PDI-mediated attenuation of neuronal cell death triggered by ER stress, misfolded proteins or proteasome inhibition. Thus, PDI prevents neurotoxicity associated with ER stress and protein misfolding, but NO blocks this protective effect in neurodegenerative disorders through the S-nitrosylation of PDI.     

Expression and antigen activity determination of human thyroid peroxidase in silkworm and yeast

In this study, we report the expression of human thyroid peroxidase (TPO) in silkworm larvae and Pichia pastoris GS115. Recombinant TPO is sequentially purified from the hemolymph of infected silkworm larvae and yeast using a Ni-NTA resin kit. The concentration of yield of recombinant TPO is 4.87 mg per thousand larvae and 40.83 mg per liter yeast culture. However, the recombinant TPO produced in silkworm show similar binding ability with the specific anti-TPO serum to standard human TPO purified from insect cells. The lower antigen activity indicates the TPO expressed in yeast is not suitable to be used as the coating antigen in enzyme linked immunosorbent assay (ELISA). The cost of TPO expressed in B. mori is about 1/4 that of in insect cells, and the cost of TPO purified from silkworm for ELISA is only 1/8 that of TPO produced from Sf9 cells. It indicates the BmNPV-silkworm expression system is a cost-effective platform for producing TPO with high antigen activity.     

硒对NO诱导的内皮细胞损伤的抑制机制研究

用外源性NO供体S－亚硝基谷胱甘肽（GSNO）处理人脐静脉内皮细胞系ECV－304细胞，研究其对细胞的损伤机制，并探讨硒在这一过程中的保护作用，通过MTT法测定细胞存活率，分光光度法[测定细胞LDH漏出率及细胞脂质过氧化水平，采用荧光标记技术研究细胞膜流动性变化，结果表明，NO可引起细胞脂质过氧化水平升高，细胞膜流动性下降，导致ECV－304细胞损伤，其作用具有浓度效应，细胞内硒可通过抑制细胞脂质过氧化水平及细胞膜流动性变化从而抑制NO诱导的细胞损伤。     

Exogenous and endogenous glycolipid antigens activate NKT cells during microbial infections

CD1d-restricted natural killer T (NKT) cells are innate-like lymphocytes that express a conserved T-cell receptor and contribute to host defence against various microbial pathogens. However, their target lipid antigens have remained elusive. Here we report evidence for microbial, antigen-specific activation of NKT cells against Gram-negative, lipopolysaccharide (LPS)-negative alpha-Proteobacteria such as Ehrlichia muris and Sphingomonas capsulata. We have identified glycosylceramides from the cell wall of Sphingomonas that serve as direct targets for mouse and human NKT cells, controlling both septic shock reaction and bacterial clearance in infected mice. In contrast, Gram-negative, LPS-positive Salmonella typhimurium activates NKT cells through the recognition of an endogenous lysosomal glycosphingolipid, iGb3, presented by LPS-activated dendritic cells. These findings identify two novel antigenic targets of NKT cells in antimicrobial defence, and show that glycosylceramides are an alternative to LPS for innate recognition of the Gram-negative, LPS-negative bacterial cell wall.     

50Hz极低频电磁场对酵母细胞生长和氧化应激的影响初探

探讨50 Hz极低频电磁场(extremely low frequency electromagnetic fields,ELF-EMF)暴露对酵母细胞的生长、细胞内活性氧(ROS)水平,以及超氧化物歧化酶(SOD)和过氧化氢酶(CAT)两种抗氧化酶活性的影响。测定酵母细胞在50 Hz 6 m T ELF-EMF暴露下的菌落形成数目和生长曲线,及ELF-EMF短期暴露(0.5、1、2 h)和长期暴露(6、12、24、96 h)对细胞内ROS水平,以及SOD和CAT活性的影响。实验结果表明,磁感应强度高达6 m T的ELF-EMF暴露下,酵母细胞的菌落形成数目和生长曲线没有发生显著变化(p0.05)。在暴露2 h内,ELF-EMF可显著提高细胞内ROS水平(p0.01);并显著影响酵母细胞内SOD或CAT活性(p0.05)。但暴露时间超过6 h后,酵母细胞内ROS水平、及SOD和CAT活性与对照组大致相同,没有发生显著变化。实验条件下ELF-EMF暴露对酵母的生长没有显著影响,在短期暴露(2 h内)中,可引起酵母细胞内氧化应激反应;但在长期暴露(6 h后)后,对氧化应激反应没有显著影响。