The alternative oxidase of Yarrowia lipolytica mitochondria is unable to compete with the cytochrome pathway for electrons

The activity of the cyanide-resistant alternative oxidase (pathway) of Y. lipolytica mitochondria was studied as a function of the activity of the major, cyanide-sensitive, cytochrome pathway. The contribution of the alternative oxidase to the total respiration of mitochondria was evaluated by measuring the rate of oxygen consumption in the presence of cyanide (an inhibitor of the cytochrome pathway). The potential activity of the cytochrome pathway was evaluated spectrophotometrically, by measuring the oxidation rate of cytochrome c by ferricyanide, which accepts electrons from complex III (cytochrome c) of this pathway. The oxidation of succinate by mitochondria in the presence of ferricyanide and cyanide was accompanied by oxygen consumption due to the transfer of electrons through the alternative pathway. The subsequent addition of ADP or FCCP (an uncoupler of oxidative phosphorylation in the cytochrome pathway) completely inhibited the consumption of oxygen by the mitochondria. Under these conditions, the inhibition of the alternative pathway by benzohydroxamic acid failed to affect the transfer of electrons from cytochrome c to ferricyanide. Benzohydroxamic acid did not influence the rate of ferricyanide reduction by the cytochrome pathway occurring in controlled state 4, nor could it change the phosphorylation quotient ATP/O upon the oxidation of various substrates. These findings indicate that the alternative pathway is unable to compete with the cytochrome respiratory chain for electrons. The alternative pathway transfers only electrons that are superfluous for the cytochrome chain.     

The outcomes of pathway database computations depend on pathway ontology

Different biological notions of pathways are used in different pathway databases. Those pathway ontologies significantly impact pathway computations. Computational users of pathway databases will obtain different results depending on the pathway ontology used by the databases they employ, and different pathway ontologies are preferable for different end uses. We explore differences in pathway ontologies by comparing the BioCyc and KEGG ontologies. The BioCyc ontology defines a pathway as a conserved, atomic module of the metabolic network of a single organism, i.e. often regulated as a unit, whose boundaries are defined at high-connectivity stable metabolites. KEGG pathways are on average 4.2 times larger than BioCyc pathways, and combine multiple biological processes from different organisms to produce a substrate-centered reaction mosaic. We compared KEGG and BioCyc pathways using genome context methods, which determine the functional relatedness of pairs of genes. For each method we employed, a pair of genes randomly selected from a BioCyc pathway is more likely to be related by that method than is a pair of genes randomly selected from a KEGG pathway, supporting the conclusion that the BioCyc pathway conceptualization is closer to a single conserved biological process than is that of KEGG.     

Effects of KCN and NaN3 Pretreatment on the Cyanide-Resistant Respiration in Tobacco Callus

The effects of KCN (0.5mmol/L) and NaN3 (0.01 mmol/L) pretreatment on the operation of the alternative pathway in subcultured tobacco (Nicotiana rustica L. cv. Gansu yellow flower) callus were analyzed. After treatment with KCN and NaN3 for 12 h, the total respiration rate (Vt) decreased by 12% and 17%, whereas oxygen consuption by the cytochrome pathway decreased by 22% and 28% respectively. The capacity of the alternative pathway (Valt) remained constant, while the activity of the alternative pathway (ρ· Valt ) inreased slightly. This changing pattern led to a declined contribution of the cytochrome pathway to the total respiration rate and an increased activity of the alternative pathway. Treatment with KCN for 24 h brought about a slight rise of oxygen consumption by the cytochrome pathway as compared with that in callus treated for 12 h, but the oxygen consumption was still lower than that in the untreated callus. Treatment with NaN3 for 24 h resulted in a profound decrease of the cytochrome pathway operation and a continuing increase of the alternative pathway operation. These data indicated that the enhanced operation of the alternative pathway played a compensatory role to the total respiration when the cytochrome pathway was partially inhibited in tobacco callus.     

The Alternative Oxidase of Yarrowia lipolytica Mitochondria Is Unable To Compete with the Cytochrome Pathway for Electrons

The activity of the cyanide-resistant alternative oxidase (pathway) of Yarrowia lipolytica mitochondria was studied as a function of the activity of the major, cyanide-sensitive, cytochrome pathway. The contribution of the alternative oxidase to the total respiration of mitochondria was evaluated by measuring the rate of oxygen consumption in the presence of cyanide (an inhibitor of the cytochrome pathway). The potential activity of the cytochrome pathway was evaluated spectrophotometrically, by measuring the oxidation rate of cytochrome c by ferricyanide, which accepts electrons from complex III (cytochrome c) of this pathway. The oxidation of succinate by mitochondria in the presence of ferricyanide and cyanide was accompanied by oxygen consumption due to the transfer of electrons through the alternative pathway. The subsequent addition of ADP or FCCP (an uncoupler of oxidative phosphorylation in the cytochrome pathway) completely inhibited the consumption of oxygen by the mitochondria. Under these conditions, the inhibition of the alternative pathway by benzohydroxamic acid failed to affect the transfer of electrons from cytochrome c to ferricyanide. Benzohydroxamic acid did not influence the rate of ferricyanide reduction by the cytochrome pathway occurring in controlled state 4, nor could it change the phosphorylation quotient ATP/O upon the oxidation of various substrates. These findings indicate that the alternative pathway is unable to compete with the cytochrome respiratory chain for electrons. The alternative pathway transfers only electrons that are superfluous for the cytochrome chain.     

Response of the ubiquitin-proteasome pathway to changes in muscle activity

The ubiquitin-proteasome pathway plays a critical role in the adaptation of skeletal muscle to persistent decreases or increases in muscle activity. This article outlines the basics of pathway function and reviews what we know about pathway responses to altered muscle use. The ubiquitin-proteasome pathway regulates proteolysis in mammalian cells by attaching ubiquitin polymers to damaged proteins; this targets the protein for degradation via the 26S proteasome. The pathway is constitutively active in muscle and continually regulates protein turnover. Conditions of decreased muscle use, e.g., unloading, denervation, or immobilization, stimulate general pathway activity. This activity increase is caused by upregulation of regulatory components in the pathway and leads to accelerated proteolysis, resulting in net loss of muscle protein. Pathway activity is also increased in response to exercise, a two-phase response. An immediate increase in selective ubiquitin conjugation by constitutive pathway components contributes to exercise-stimulated signal transduction. Over hours-to-days, exercise also stimulates a delayed increase in general ubiquitin conjugating activity by inducing expression of key components in the pathway. This increase mediates a late-phase rise in protein degradation that is required for muscle adaptation to exercise. Thus the ubiquitin-proteasome pathway functions as an essential mediator of muscle remodeling, both in atrophic states and exercise training.     

烟草叶片中呼吸电子传递途径在缓解叶绿体PSⅡ光抑制中的作用

前期研究发现线粒体交替氧化酶(AOX)呼吸途径对叶绿体光系统II(PSII)的光抑制有明显的缓解作用。线粒体内另一条呼吸途径——细胞色素氧化酶(COX)呼吸途径是否也具有光保护作用尚不清楚。该文通过荧光快速诱导动力学和荧光淬灭分析,解析了烟草(Nicotiana tabacum)叶片中COX途径对PSII光保护的贡献及其与AOX途径的关系。结果表明,强光处理后PSII活性在所有叶片中均下降。AOX途径受抑明显加速了叶片PSII活性的下降。而当COX途径受抑后,叶片PSII活性的下降与水处理的对照叶片无明显差异。当AOX途径与COX途径同时受抑时,叶片PSII活性的下降比单独抑制AOX途径时更严重。此外,呼吸电子传递受抑均导致叶片非光化学淬灭(NPQ)增加,AOX途径受抑导致的NPQ上调比COX途径受抑时更明显,AOX和COX途径同时受抑时NPQ的增幅最大。上述结果表明,烟草叶片中COX途径和AOX途径均参与PSⅡ的光保护。当COX途径受抑时,其光保护功能可以被AOX途径和NPQ补偿,而AOX途径的光保护作用不能被COX途径和NPQ完全补偿。     

The role of extra-hepatic tissues in the receptor-mediated plasma clearance of glycoproteins terminated by mannose or N-acetylglucosamine.

The mannose- and N-acetylglucosamine-specific pathway for the clearance of mammalian glycoproteins has been characterized by using 125I-labelled neoglycoproteins, glycosidase-treated orosomucoid and lysosomal glycosidases (beta-glucuronidase and beta-N-acetylglucosaminidase) as probes. There are two components to this pathway in vivo; one liver-dependent and the other extrahepatic or liver-independent. Cells that mediate clearance by the latter component of the pathway are present in spleen, bone and in elements of the reticuloendothelial system, but not in the kidney. Glycoproteins that possess terminal mannose, glucose or N-acetylglucosamine residues, including various lysosomal enzymes, are rapidly cleared from plasma via this pathway. Glucose-terminated glycoproteins are recognized by two pathways in the intact animal; the hepatic galactose-specific pathway and the mannose/N-acetylglycosamine-specific pathway, which is present in liver and in peripheral tissues. Following removal of the liver by surgical evisceration, glucose-terminated glycoproteins are cleared whereas glycoproteins bearing galactose are not cleared. Uptake of 125I-labelled neoglycoproteins and agalacto-orosomucoid by isolated alveolar macrophages closely mimics clearance in vivo by the mannose/N-acetylglucosamine pathway. Neoglycoproteins terminated by mannose, glucose or N-acetylglucosamine all compete with 125I-labelled agalacto-orosomucoid for uptake by receptor-mediated pinocytosis. The extent of substitution of the neoglycoproteins is a critical determinant of their inhibitory potency. It is proposed that mononuclear phagocytes are in important component of the clearance pathway in vivo. The mannose/N-acetylglucosamine pathway may be important in the regulation of extracellular levels of various glycosylated macromolecules, including lysosomal hydrolases.     

水分胁迫的基因表达和信号转导(综述)

植物在水分胁迫条件下的依赖ABA和不依赖ABA的基因表达途径来调节对逆境的适应。植物通过渗透感受器感知胁迫信号,以MAPK和CDPK等途径传递信号,最终引起基因表达。     

细菌通过聚酮合成酶途径合成多不饱和脂肪酸的研究进展

细菌利用聚酮合成酶途径合成多不饱和脂肪酸是近年发现的新的脂肪酸合成途径。这种途径与常规的由脂肪酸去饱和酶和脂肪酸延长酶引导的脂肪酸合成途径有着本质上的差别。总结了近些年细菌利用聚酮合成酶合成多不饱和脂肪酸这一新途径的研究状况,重点阐明其分子机制,并对其研究趋势及应用前景进行了展望。     

外源~1O_2 和·OH及其清除剂对烟草愈伤组织交替途径发生与运行的影响(英文)

外源1 O2 和·OH处理继代培养 1 4d的烟草愈伤组织 2 4h后 ,愈伤组织内的交替途径的实际运行显著上升 ,但对交替途径容量影响不大 ;而经·OH处理后的愈伤组织交替途径容量和实际运行均明显地受到抑制 ,但交替途径的实际运行对·OH更加敏感。活性氧产生系统中加入相关清除剂进行实验 ,得到与此相对应的结果 :1 O2 的清除剂His处理能明显地降低或抑制1 O2 所增加的交替途径实际运行量 ,但对交替途径容量则几乎均无影响 ,而用·OH的清除剂DMSO和MAN分别处理愈伤组织后 ,DMSO和MAN均能解除·OH对交替途径容量和实际运行的抑制。这些结果表明 ,1 O2 诱导烟草愈伤组织交替途径的实际运行 ,但对其容量的作用不大 ,而·OH则明显地抑制交替途径容量和实际运行。推测1 O2 和·OH对抗氰途径的影响可能是通过对AOX活性的调节     

Glucose Utilization by Chick Embryo Heart Homogenates

Homogenates of early chick embryos and homogenates of early chick embryonic hearts utilized the phosphogluconate pathway of glucose catabolism to a greater extent, relative to the glycolytic-Krebs cycle pathway, than did homogenates of hearts from older chick embryos or adult chicks. An abrupt drop in the relative participation by the phosphogluconate pathway in embryo heart homogenates occurs at about 5 to 7 days of incubation. Heart homogenates from adult chicks catabolize glucose almost entirely by the glycolytic-Krebs cycle pathway, with negligible participation by the phosphogluconate pathway.     

Cutting Edge: B cell receptor (BCR) cross-talk: the IL-4-induced alternate pathway for BCR signaling operates in parallel with the classical pathway, is sensitive to Rottlerin, and depends on Lyn

B cell exposure to IL-4 alters subsequent BCR signaling such that ERK phosphorylation becomes signalosome-independent; however, the nature of this new, alternate signaling pathway and its relationship to the classical, signalosome-dependent signaling pathway are not known. In this study, we report that the alternate and classical pathways for BCR signaling are differentially affected by rottlerin, and by Go6976 or LY294002, respectively. Furthermore, in B cells lacking protein kinase C (PKC)beta, the classical pathway for BCR signaling is blocked, whereas the alternate pathway is little affected. Conversely, in B cells lacking Lyn, the alternate pathway for BCR signaling is blocked, whereas the classical pathway is little affected. The rottlerin-sensitive element is not PKCdelta, inasmuch as the alternate pathway is not blocked in PKCdelta-deficient B cells. These results indicate that the rottlerin-sensitive, Lyn-dependent alternate pathway, and the classical pathway, for BCR signaling operate in parallel when BCR engagement follows IL-4 exposure.     

Characterization of the TGN exit routes in AtT20 cells using pancreatic amylase and serum albumin

The AtT20 pituitary cell is the one that was originally used to define the pathways taken by secretory proteins in mammalian cells. It possesses two secretory pathways, the constitutive for immediate secretion and the regulated for accumulation and release under hormonal stimulation. It is in the regulated pathway, most precisely in the immature granule of the regulated pathway, that proteolytic maturation takes place. A pathway that stems from the regulated one, namely the constitutive-like pathway releases proteins present in immature granules that are not destined for accumulation in mature granules. In AtT20 cells proopiomelanocortin the endogenous precursor of the accumulated adrenocorticotropic hormone, is predominantly secreted in a constitutive manner without proteolytic maturation. In order to better understand by which secretory pathway intact proopiomelanocortin is secreted by a cell line possessing a regulated secretory pathway, it was transfected with rat serum albumin (a marker of constitutive secretory proteins), and pancreatic amylase (a marker of regulated proteins). COS cells were also transfected in order to serve as control of release by the constitutive pathway. It was observed that both the basal and stimulated secretions of albumin and proopiomelanocortin from AtT20 cells are identical. In addition, secretagogue stimulation when POMC is in transit in the trans-Golgi network decreases its constitutive secretion by 50%. It was also observed using cell fractionation and 20 degrees C secretion blocks that albumin and proopiomelanocortin are present in the regulated pathway, presumably in the immature granules, and are secreted by the constitutive-like secretory pathway. These observations show that stimulation can increase sorting into the regulated pathway, and confirm the importance of the constitutive-like secretory pathway in the model AtT20 cell line.     

Ionizing radiation-induced mitogen-activated protein (MAP) kinase activation in DU145 prostate carcinoma cells: MAP kinase inhibition enhances radiation-induced cell killing and G2/M-phase arrest

These studies examine the role(s) played by the mitogen-activated protein kinase (MAPK) pathway after exposure of DU145 prostate carcinoma cells to radiation. Radiation (2 Gy) was found to cause both immediate primary (0-30 min) and prolonged secondary activations (90-1440 min) of the MAPK pathway. These activations of the MAPK pathway were abolished by inhibition of epidermal growth factor receptor (EGFR) function. The secondary activation was also abolished by addition of a neutralizing monoclonal antibody against transforming growth factor alpha (TGFA). Activation of the MAPK pathway could be induced in nonirradiated cells by the transfer of medium from irradiated cultures. Neutralizing antibody to TGFA blocked this effect, indicating that radiation causes secondary activation of the MAPK pathway by release of TGFA in DU145 cells. Radiation induced a transient G(2)/M-phase growth arrest that was prolonged for up to 24 h by inhibition of the MAPK pathway. Inhibition of the MAPK pathway significantly increased the ability of radiation to cause apoptosis 24 h after exposure. The ability of DU145 cells to proliferate after irradiation became dependent on MAPK signaling. When cells were subjected to single doses or fractionated radiation exposure, continuous inhibition of the MAPK pathway significantly decreased clonogenic survival. Only a small fraction of this cell killing could be accounted for by apoptosis within the first 96 h. Thus inhibition of the MAPK pathway increased radiation-induced cell killing likely by both apoptotic and nonapoptotic mechanisms. Collectively, our findings indicate that disruption of the TGFA/EGFR/MAPK pathway may represent a strategy that could be exploited to manipulate prostate carcinoma growth and cell survival after irradiation.     

Casein kinase Iepsilon modulates the signaling specificities of dishevelled

Wnt signaling is critical to many aspects of development, and aberrant activation of the Wnt signaling pathway can cause cancer. Dishevelled (Dvl) protein plays a central role in this pathway by transducing the signal from the Wnt receptor complex to the beta-catenin destruction complex. Dvl also plays a pivotal role in the planar cell polarity pathway that involves the c-Jun N-terminal kinase (JNK). How functions of Dvl are regulated in these two distinct pathways is not clear. We show that deleting the C-terminal two-thirds of Dvl, which includes the PDZ and DEP domains and is essential for Dvl-induced JNK activation, rendered the molecule a much more potent activator of the beta-catenin pathway. We also found that casein kinase Iepsilon (CKIepsilon), a previously identified positive regulator of Wnt signaling, stimulated Dvl activity in the Wnt pathway, but dramatically inhibited Dvl activity in the JNK pathway. Consistent with this, overexpression of CKIepsilon in Drosophila melanogaster stimulated Wnt signaling and disrupted planar cell polarity. We also observed a correlation between the localization and the signaling activity of Dvl in the beta-catenin pathway and the JNK pathway. Furthermore, by using RNA interference, we demonstrate that the Drosophila CKIepsilon homologue Double time positively regulates the beta-catenin pathway through Dvl and negatively regulates the Dvl-induced JNK pathway. We suggest that CKIepsilon functions as a molecular switch to direct Dvl from the JNK pathway to the beta-catenin pathway, possibly by altering the conformation of the C terminus of Dvl.     

EGF receptor is involved in WNT3a-mediated proliferation and motility of NIH3T3 cells via ERK pathway activation

WNT3a stimulates proliferation of NIH3T3 cells via activation of the extracellular signal-regulated kinase (ERK) pathway. The RAF-1-->MEK-->ERK cascade was immediately increased by WNT3a treatment, however, the upstream event triggering ERK pathway activation by WNT3a is not clear. WNT3a activated RAS and WNT3a-induced ERK activation was blocked by dominant-negative RAS, indicating that WNT3a might act upstream of RAS. WNT3a-induced ERK pathway activations were blocked by AG1478, the epidermal growth factor receptor (EGFR) inhibitor, and EGFR siRNA. The WNT3a-induced ERK pathway activation was not observed in fibroblasts retaining defective EGFR, but the WNT3a effect was restored by EGFR reconstitution. These results indicate involvement of EGFR in the WNT3a-induced ERK pathway activation. WNT3a-induced motility and cytoskeletal rearrangement as well as proliferation of NIH3T3 cells were blocked by AG1478 and EGFR siRNA or abolished in EGFR knock-out fibroblasts, indicating involvement of EGFR in those cellular processes. WNT3a-induced ERK pathway activation was not affected by Dickkoff-1 (DKK-1), although WNT3a-induced activations of the WNT/beta-catenin pathway and proliferation were reduced by DKK-1. EGFR is involved in WNT3a-induced proliferation via both routes dependent on and independent of the WNT/beta-catenin pathway. These results indicate that WNT3a stimulates proliferation and motility of NIH3T3 fibroblasts via EGFR-mediated ERK pathway activation.     

Synergy between methylerythritol phosphate pathway and mevalonate pathway for isoprene production in Escherichia coli

Isoprene, a key building block of synthetic rubber, is currently produced entirely from petrochemical sources. In this work, we engineered both the methylerythritol phosphate (MEP) pathway and the mevalonate (MVA) pathway for isoprene production in E. coli. The synergy between the MEP pathway and the MVA pathway was demonstrated by the production experiment, in which overexpression of both pathways improved the isoprene yield about 20-fold and 3-fold, respectively, compared to overexpression of the MEP pathway or the MVA pathway alone. The ¹³C metabolic flux analysis revealed that simultaneous utilization of the two pathways resulted in a 4.8-fold increase in the MEP pathway flux and a 1.5-fold increase in the MVA pathway flux. The synergy of the dual pathway was further verified by quantifying intracellular flux responses of the MEP pathway and the MVA pathway to fosmidomycin treatment and mevalonate supplementation. Our results strongly suggest that coupling of the complementary reducing equivalent demand and ATP requirement plays an important role in the synergy of the dual pathway. Fed-batch cultivation of the engineered strain overexpressing the dual pathway resulted in production of 24.0 g/L isoprene with a yield of 0.267 g/g of glucose. The synergy of the MEP pathway and the MVA pathway also successfully increased the lycopene productivity in E. coli, which demonstrates that it can be used to improve the production of a broad range of terpenoids in microorganisms.     

Host immune system strikes back

Autophagy-mediated major histocompatibility complex (MHC) class I presentation can follow either the conventional MHC class I pathway or a recently described vacuolar pathway. In the vacuolar pathway, protein degradation is effected by lysosomal proteases, peptide exchange takes place with recirculating MHC complexes and the newly formed peptide-MHC complexes reach the cell surface by the endocytic pathway. This pathway is independent of the proteasome and the transporter associated with antigen processing (TAP) complex, but generates the same, or a similar, epitope as that from the conventional MHC class I pathway. Here, we discuss different mechanisms by which autophagy mediates MHC class I-restricted antigen presentation, which is crucial to its role in the control of intracellular pathogens.     

Growth of Pseudomonas C on C1 compounds: enzyme activites in extracts of Pseudomonas C cells grown on methanol, formaldehyde, and formate as sole carbon sources.

Pseudomonas C can grow on methanol, formaldehyde, or formate as sole carbon source. It is proposed that the assimilation of carbon by Pseudomonas C grown on different C1 growth substrates proceeds via one of two metabolic pathways, the serine pathway or the allulose pathway (the ribose phosphate cycle of formaldehyde fixation). This contention is based on the distribution of two key enzymes, each of which appears to be specifically involved in one of the assimilation pathways, glycerate dehydrogenase (serine pathway) and hexose phosphate synthetase (allulose pathway). The assimilation of methanol in Pseudomonas C cells appears to occur via the allulose pathway, whereas the utilization of formaldehyde or formate in cells grown on formaldehyde or formate as sole carbon sources appears by the serine pathway. When methanol is present together with formaldehyde or formate in the growth medium, the formaldehyde or formate is utilized by the allulose pathway.     

REGULATION OF ALTERNATIVE PATHWAY RESPIRATION IN CHLAMYDOMONAS REINHARDTH (CHLOROPHYCEAE)1

The inhibitor propyl gallate was used to estimate partitioning of respiratory electron flow between the cytochrome amd alternative pathways in Chlamydomonas reinhardtii Dangeard. Nutrient limitation (nitrogen or phosphorus resulted in a large increase in alternative pathway capacity relative to cytochrome pathway activity, without regulating in engagement of the alternative pathway. High rates of respiration, which could be induced in phosphate-starved cells by a combination of phosphate addition and uncoupler, resulted in alternative pathway activity. Osmotic stress resulted in decreased electron flow through the cytochrome pathway and increased flow through the alternative pathway, while high temperature also resulted in alternative pathway engagement. Incubation with exogenous carbon sources could increase the rate of respiratory O₂ consumption; the increase was mediated entirely by the alternative pathway. We suggest that the alternative pathway functions in these cells both to maintain respiration during environmentally induced stress and as on energy overflow.