The kinetic model of the shikimate pathway as a tool to optimize enzyme assays for high-throughput screening

Four-enzyme section of the shikimate pathway (Aro B, D, E, and K) of Streptococcus pneumoniae has been studied. Kinetic properties of the individual enzymes and three- and four-enzyme linked reactions have been characterized in vitro. On the basis of the data measured in spectrophotometric and LC-MS experiments, kinetic mechanisms of the enzymes have been suggested and all kinetic parameters have been identified. Kinetic models for these three- and four-enzyme sections of the shikimate pathway have been constructed and validated. The model of the four-enzyme section of shikimate pathway has been employed to design an inhibition-sensitive reconstituted pathway for a high-throughput screening effort on the shikimate pathway. It was demonstrated that using the model it was possible to optimize this reconstituted pathway in such a way to provide equal sensitivity of the enzymes to inhibition.     

The ubiquitin-proteasome pathway in viral infections

The cellular biological function of the ubiquitin-proteasome pathway as a major intracellular protein degradation pathway, and as an important modulator for the regulation of many fundamental cellular processes has been greatly appreciated over the last decade. The critical role of the ubiquitin-proteasome pathway in viral pathogenesis has become increasingly apparent. Many viruses have been reported to evolve different strategies to utilize the ubiquitin-proteasome pathway for their own benefits. Here, we review the general background and function of the ubiquitin-proteasome pathway, summarize our current understanding of how viruses use this pathway to target cellular proteins, and finally, discuss the roles of this pathway in enteroviral infection, and the potential therapeutic application of proteasome inhibition in myocarditis.     

哺乳动物Hippo信号通路:肿瘤治疗的新标靶

Hippo信号通路是首次在果蝇中发现具有调节细胞增殖与凋亡作用的信号通路。最近发现果蝇Hippo信号通路的组成、分子作用机制和生物学功能在进化过程中高度保守。Hippo信号通路在胚胎发育中对细胞的生长分化、组织器官形成以及成体干细胞的维持和自稳态的保持等方面具有重要作用。同时,Hippo信号通路与Wnt信号通路、Notch信号通路等相互作用、密切联系,在肿瘤的发生、发展过程中也起到关键作用。文章综述了哺乳动物Hippo信号通路的作用机理、与其他信号通路和蛋白质因子的相互联系及与肿瘤的关系,对于肿瘤的诊断、预防和治疗具有一定的参考价值。     

Engineering a functional 1-deoxy-D-xylulose 5-phosphate (DXP) pathway in Saccharomyces cerevisiae

Isoprenoids are used in many commercial applications and much work has gone into engineering microbial hosts for their production. Isoprenoids are produced either from acetyl-CoA via the mevalonate pathway or from pyruvate and glyceraldehyde 3-phosphate via the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway. Saccharomyces cerevisiae exclusively utilizes the mevalonate pathway to synthesize native isoprenoids and in fact the alternative DXP pathway has never been found or successfully reconstructed in the eukaryotic cytosol. There are, however, several advantages to isoprenoid synthesis via the DXP pathway, such as a higher theoretical yield, and it has long been a goal to transplant the pathway into yeast. In this work, we investigate and address barriers to DXP pathway functionality in S. cerevisiae using a combination of synthetic biology, biochemistry and metabolomics. We report, for the first time, functional expression of the DXP pathway in S. cerevisiae. Under low aeration conditions, an engineered strain relying solely on the DXP pathway for isoprenoid biosynthesis achieved an endpoint biomass 80% of that of the same strain using the mevalonate pathway.     

Wnt Signaling in Remyelination in Multiple Sclerosis: Friend or Foe?

Myelination is critical to normal functioning of the vertebrate nervous system. In demyelinating diseases such as multiple sclerosis, oligodendrocytes, the myelinating cells in the central nervous system, are targeted, resulting in myelin loss, axonal damage, and severe functional impairment. While spontaneous remyelination has been proven a failure in multiple sclerosis, understanding the molecular mechanism underlying oligodendrocyte biology, myelination, and remyelination becomes crucial. To date, a series of signaling pathways in regulating oligodendrocyte development and remyelination have been suggested and, among them, the Wnt/β-catenin/Tcf pathway has been considered a negative factor in the myelinating process. However, this notion has been challenged by recent studies, which showed a pro-myelinating effect of this pathway. This review summarizes the current contradictory concepts concerning the role of the Wnt pathway in the oligodendrocyte development and remyelination process, attempts to address the potential mechanism underlying this controversy, and recommends caution in targeting the Wnt pathway as a potential demyelinating therapy.     

Dishevelled activates Ca2+ flux,PKC, and CamKII in vertebrate embryos

Wnt ligands and Frizzled (Fz) receptors have been shown to activate multiple intracellular signaling pathways. Activation of the Wnt-beta-catenin pathway has been described in greatest detail, but it has been reported that Wnts and Fzs also activate vertebrate planar cell polarity (PCP) and Wnt-Ca2+ pathways. Although the intracellular protein Dishevelled (Dsh) plays a dual role in both the Wnt-beta-catenin and the PCP pathways, its potential involvement in the Wnt-Ca2+ pathway has not been investigated. Here we show that a Dsh deletion construct, XDshDeltaDIX, which is sufficient for activation of the PCP pathway, is also sufficient for activation of three effectors of the Wnt-Ca2+ pathway: Ca2+ flux, PKC, and calcium/calmodulin-dependent protein kinase II (CamKII). Furthermore, we find that interfering with endogenous Dsh function reduces the activation of PKC by Xfz7 and interferes with normal heart development. These data suggest that the Wnt-Ca2+ pathway utilizes Dsh, thereby implicating Dsh as a component of all reported Fz signaling pathways.     

Identification of Drosophila genes modulating Janus kinase/signal transducer and activator of transcription signal transduction

The JAK/STAT pathway was first identified in mammals as a signaling mechanism central to hematopoiesis and has since been shown to exert a wide range of pleiotropic effects on multiple developmental processes. Its inappropriate activation is also implicated in the development of numerous human malignancies, especially those derived from hematopoietic lineages. The JAK/STAT signaling cascade has been conserved through evolution and although the pathway identified in Drosophila has been closely examined, the full complement of genes required to correctly transduce signaling in vivo remains to be identified. We have used a dosage-sensitive dominant eye overgrowth phenotype caused by ectopic activation of the JAK/STAT pathway to screen 2267 independent, newly generated mutagenic P-element insertions. After multiple rounds of retesting, 23 interacting loci that represent genes not previously known to interact with JAK/STAT signaling have been identified. Analysis of these genes has identified three signal transduction pathways, seven potential components of the pathway itself, and six putative downstream pathway target genes. The use of forward genetics to identify loci and reverse genetic approaches to characterize them has allowed us to assemble a collection of genes whose products represent novel components and regulators of this important signal transduction cascade.     

运动对线粒体介导骨骼肌细胞凋亡信号通路的影响

细胞凋亡是一种程序化的细胞死亡方式,其信号传导通路分为外源性和内源性两条主要途径,线粒体在内源性细胞凋亡途径中扮演着重要的角色.研究表明,运动可通过调节线粒体介导骨骼肌细胞凋亡的进程,而运动调节线粒体介导骨骼肌细胞凋亡信号通路影响机体细胞生物进程的机制仍有待研究.该文主要阐述了线粒体介导细胞凋亡信号传导通路及运动对其的...     

Rewriting the lignin roadmap

Considerable interest in lignin biosynthesis has been fueled by the many roles that lignin plays in development and in resistance to biotic and abiotic stress, as well as its importance to industry and agriculture. Although the pathway leading to the lignin polymer has been studied for decades, new insights into the enzymes of the pathway have required a complete re-evaluation of how we think lignin precursors are synthesized. Although free hydroxycinnamic acids have long been thought to be key intermediates, it has become apparent that many of the hydroxylation and methylation steps in the pathway occur instead at the level of hydroxycinnamic acid esters, and their corresponding aldehydes and alcohols.     

Synthesis of L-fucose in thyroid tissue

A de novo pathway for L-fucose synthesis has been detected in porcine thyroid tissue. This system uses guanosine diphospho-alpha-D mannose as a precursor and forms guanosine diphospho-beta-L-fucose as product. The system seems similar to those reported by others to exist in microorganisms and plants in that the first step of the pathway involves a 4-keto sugar nucleotide intermediate. The first enzyme of the pathway, guanosine diphospho-alpha-D-mannose oxidoreductase has been purified 57-fold from crude extracts by virtue of its affinity for Blue Sepharose.     

A novel series of potent and selective small molecule inhibitors of the complement component C1s

Activation of the classical pathway of complement has been implicated in disease states such as hereditary angioedema, ischemia-reperfusion injury and acute transplant rejection. The trypsin-like serine protease C1s represents a pivotal upstream point of control in the classical pathway of complement activation and is therefore likely to be a useful target in the therapeutic intervention of these disease states. A series of thiopheneamidine-based inhibitors of C1s has been optimized to give a 70 nM inhibitor that inhibits the classical pathway of complement activation in vitro.     

Mercury methylation independent of the acetyl-coenzyme A pathway in sulfate-reducing bacteria

Sulfate-reducing bacteria (SRB) in anoxic waters and sediments are the major producers of methylmercury in aquatic systems. Although a considerable amount of work has addressed the environmental factors that control methylmercury formation and the conditions that control bioavailability of inorganic mercury to SRB, little work has been undertaken analyzing the biochemical mechanism of methylmercury production. The acetyl-coenzyme A (CoA) pathway has been implicated as being key to mercury methylation in one SRB strain, Desulfovibrio desulfuricans LS, but this result has not been extended to other SRB species. To probe whether the acetyl-CoA pathway is the controlling biochemical process for methylmercury production in SRB, five incomplete-oxidizing SRB strains and two Desulfobacter strains that do not use the acetyl-CoA pathway for major carbon metabolism were assayed for methylmercury formation and acetyl-CoA pathway enzyme activities. Three of the SRB strains were also incubated with chloroform to inhibit the acetyl-CoA pathway. So far, all species that have been found to have acetyl-CoA activity are complete oxidizers that require the acetyl-CoA pathway for basic metabolism, as well as methylate mercury. Chloroform inhibits Hg methylation in these species either by blocking the methylating enzyme or by indirect effects on metabolism and growth. However, we have identified four incomplete-oxidizing strains that clearly do not utilize the acetyl-CoA pathway either for metabolism or mercury methylation (as confirmed by the absence of chloroform inhibition). Hg methylation is thus independent of the acetyl-CoA pathway and may not require vitamin B(12) in some and perhaps many incomplete-oxidizing SRB strains.     

TOR pathway activation in Zea mays L. tissues: Conserved function between animal and plant kingdoms

In most non-photosynthetic eukaryotes it has been demonstrated a conserved signal transduction pathway, namely TOR-S6K, that coordinates growth and cell proliferation. This pathway targets the translational apparatus to induce selective translation of ribosomal mRNAs as well as stimulate the cell cycle transition through the G₁/S phase. Thus, by activation of this pathway through environmental signals, nutrients, stress, or specific growth factors, such as insulin or insulin-like growth factors (IGF), this pathway allows organisms to regulate growth and cell division. In plants, evidence has shown that TOR protein has been highly conserved through evolution, being involved in growth and cell proliferation control as well. Particularly in maize, a peptide named ZmIGF has been found in actively growing tissues. It targets the maize TOR pathway at the same extent as insulin and, by doing so it induces growth, as well as ribosomal proteins and DNA synthesis. Thus, higher metazoans and plants seem to conserve similar biochemical paths to regulate cell growth through equivalent targets that conduce to activation of the TOR-S6K pathway. Recent research shows evidence that supports this proposal by uncovering the ZmIGF receptor in maize, providing further means for analyzing the role of the conserved TOR signaling pathway in this plant.     

The Nuclear Factor NF-��B Pathway in Inflammation

The nuclear factor NF-κB pathway has long been considered a prototypical proinflammatory signaling pathway, largely based on the role of NF-κB in the expression of proinflammatory genes including cytokines, chemokines, and adhesion molecules. In this article, we describe how genetic evidence in mice has revealed complex roles for the NF-κB in inflammation that suggest both pro- and anti-inflammatory roles for this pathway. NF-κB has long been considered the “holy grail” as a target for new anti-inflammatory drugs; however, these recent studies suggest this pathway may prove a difficult target in the treatment of chronic disease. In this article, we discuss the role of NF-κB in inflammation in light of these recent studies.NF-κB has long been considered a prototypical proinflammatory signaling pathway, largely based on the activation of NF-κB by proinflammatory cytokines such as interleukin 1 (IL-1) and tumor necrosis factor α (TNFα), and the role of NF-κB in the expression of other proinflammatory genes including cytokines, chemokines, and adhesion molecules, which has been extensively reviewed elsewhere. But inflammation is a complex physiological process and the role of NF-κB in the inflammatory response cannot be extrapolated from in vitro studies. In this article, we describe how genetic evidence in mice has revealed complex roles for the NF-κB pathway in inflammation.     

POSH is involved in Eiger-Basket （TNF-JNK） signaling and embryogenesis in Drosophila

TNFα can trigger different signaling pathways, including the JNK pathway, to regulate various biological functions such as cell death, differentiation and proliferation. The scaffold protein POSH(Plenty of SH3 Domains)has been shown to be an important regulator of the JNK pathway, but whether it is involved in TNF-signaling has not been reported.Although POSH has been implicated to play a role in development in zebrafish,it has not been studied in null mutants and the underlying mechanism of its effects is still not clear.In this study,we provide evidence that the JNK pathway scaffold protein,POSH,is involved in TNF(Eiger)signaling in Drosophila.POSH is likely to act downstream of dTAB2 and upstream of dTAK1 in the TNF-JNK signaling pathway.In addition,we found that POSH is essential during Drosophila embryogenesis,including epidermal dorsal closure,similar to other JNK pathway components such as Slipper,Hemipterous,and Basket. We observed defects in F-actin accumulation and adherens junction formation during dorsal closure in different posh null mutants, suggesting that POSH is required for epidermal cell migration and cell-shape change during epidermal dorsal closure.     

Synthesis of l-fucose in thyroid tissue

A de novo pathway for l-fucose synthesis has been detected in porcine thyroid tissue. This system uses guanosine diphospho-α-D-mannose as a precursor and forms guanosine diphospho-β-l-fucose as product. The system seems similar to those reported by others to exist in microorganisms and plants in that the first step of the pathway involves a 4-keto sugar nucleotide intermediate. The first enzyme of the pathway, guanosine diphospho-α-d-mannose oxidoreductase has been purified 57-fold from crude extracts by virtue of tis affinity for Blue Sepharose.     

Morphogen to mitogen: the multiple roles of hedgehog signalling in vertebrate neural development

Sonic hedgehog has received an enormous amount of attention since its role as a morphogen that directs ventral patterning in the spinal cord was discovered a decade ago. Since that time, a bewildering array of information has been generated concerning both the components of the hedgehog signalling pathway and the remarkable number of contexts in which it functions. Nowhere is this more evident than in the nervous system, where hedgehog signalling has been implicated in events as disparate as axonal guidance and stem cell maintenance. Here we review our present knowledge of the hedgehog signalling pathway and speculate about areas in which further insights into this versatile pathway might be forthcoming.