Transcription Of Pathway Genes Research Articles

Cucurbitacin IIa interferes with EGFR-MAPK signaling pathway leads to proliferation inhibition in A549 cells

Cucurbitacin IIa (CuIIa), a tetracyclic triterpenoid harboring anticancer activity, was investigated in A549 cells to reveal its mechanism of targeting on epidermal growth factor receptor (EGFR) signaling pathway. Results showed that CuIIa was capable of inducing apoptosis and cell cycle arrest at G2/M phase. The transcription of EGFR pathway genes and their proteins accumulation was inconsistently influenced by CuIIa. Notably, transcription of Raf1 was significantly upregulated, nevertheless, MEK1 and ERK1 were significantly downregulated. On the other hand, the accumulation of the total and phosphorylated proteins of the most members in EGFR-mitogen-activated protein kinase (MAPK) pathway, as well as CylclinB1 and survivin were also shifted by CuIIa treatment. Remarkably, total MEK remained constant but survivin completely degraded. Moreover, phosphorylated BRAF continuously increased while Raf1 and MEK decreased continuously. CuIIa was further confirmed to be a tyrosine kinase inhibitor (TKI) of EGFR by kinase inhibition assay. The results of molecular simulation showed that the long side chain of CuIIa occupied the binding pocket of EGFR and the ligand was stabilized at the active site of EGFR. In view of the results above, it is suggested that CuIIa inhibits cell proliferation by interfering the EGFR-MAPK signaling pathway.

Novel Protein Mg2046 Regulates Magnetosome Synthesis in Magnetospirillum gryphiswaldense MSR-1 by Modulating a Proper Redox Status.

Magnetotactic bacteria (MTB) are a large, polyphyletic group of aquatic microorganisms capable of absorbing large amounts of iron and synthesizing intercellular nano-scaled nanoparticles termed magnetosomes. In our previous transcriptomic studies, we discovered that a novel gene (MGMSRv2_2046, termed as mg2046) in Magnetospirillum gryphiswaldense strain MSR-1 was significantly up-regulated during the period of magnetosome synthesis. In the present study, we constructed a MSR-1 mutant strain with deletion of mg2046 (termed Δmg2046) in order to evaluate the role of this gene in cell physiological status and magnetosome formation process. In comparison with wild-type MSR-1, Δmg2046 showed similar cell growth, but much lower cell magnetic response, smaller number and size of magnetosomes, and reduced iron absorption ability. mg2046 deletion evidently disrupted iron uptake, and redox equilibrium, and strongly inhibited transcription of dissimilatory denitrification pathway genes. Our experimental findings, taken together with results of gene homology analysis, indicate that Mg2046 acts as a positive regulator in MSR-1 under microaerobic conditions, responding to hypoxia signals and participating in regulation of oxygen metabolism, in part as a co-regulator of dissimilatory denitrification pathway with oxygen sensor MgFnr (MGMSRv2_2946, termed as Mg2946). Mg2046 is clearly involved in coupled regulation of cellular oxygen, iron and nitrogen metabolism under micro-aerobic or anaerobic conditions. Our findings help explain how MSR-1 cells initiate dissimilatory denitrification pathway and overcome energy deficiency under microaerobic conditions, and have broader implications regarding bacterial survival and energy metabolism strategies under hypoxia.

Induction of inflammatory responses in splenocytes by exosomes released from intestinal epithelial cells following Cryptosporidium parvum infection

Abstract Cryptosporidium, a protozoan parasite that infects the gastrointestinal epithelium and other mucosal surfaces in humans and animals, is an important opportunistic pathogen in AIDS patients and one of the most common enteric pathogens affecting young children in developing regions. We previously demonstrated that C. parvum infection stimulates host epithelial cells to release exosomes and these released exosomes shuttle several antimicrobial peptides to carry out anti-C. parvum activity. Murine intestinal epithelial (IEC4.1) cells and neonatal mice were applied for infection. Primary splenocytes were exposed to exosomes isolated from infected cells or un-infected controls. Inflammatory response in splenocytes were measured. We detected upregulation of inflammatory genes in the liver and spleen following C. parvum intestinal infection in neonatal mice. Interestingly, exosomes released from intestinal epithelial cells following C. parvum infection could activate the nuclear factor kappa B signaling pathway and trigger inflammatory gene transcription in isolated primary splenocytes. Several epithelial cell-derived proteins and a subset of parasite RNAs were detected in the exosomes released from C. parvum-infected intestinal epithelial cells. Shuttling of these effector molecules, including the high mobility group box 1 protein, was involved in the induction of inflammatory responses in splenocytes induced by the released exosomes from infected cells. Our data indicate that exosomes released from intestinal epithelial cells upon C. parvum infection can activate immune cells through shuttling various effector molecules, a process that may be relevant to host systemic responses to Cryptosporidium infection.

Overexpression of endogenous stress-tolerance related genes in Saccharomyces cerevisiae improved strain robustness and production of heterologous cellobiohydrolase.

To enable Saccharomyces cerevisiae to produce renewable fuels from lignocellulose in a consolidated bioprocess, a heterologous cellulase system must be engineered into this yeast. In addition, inherently low secretion titers and sensitivity to adverse environmental conditions must be overcome. Here, two native S. cerevisiae genes related to yeast stress tolerance, YHB1 and SET5, were overexpressed under transcriptional control of the constitutive PGK1 promoter and their effects on heterologous secretion of Talaromyces emersonii cel7A cellobiohydrolase was investigated. Transformants showed increased secreted enzyme activity that ranged from 22% to 55% higher compared to the parental strains and this did not lead to deleterious growth effects. The recombinant strains overexpressing either YHB1 or SET5 also demonstrated multi-tolerant characteristics desirable in bioethanol production, i.e. improved tolerance to osmotic and heat stress. Quantitative reverse transcriptase PCR analysis in these strains showed decreased transcription of secretion pathway genes. However, decreased unfolded protein response was also observed, suggesting novel mechanisms for enhancing enzyme production through stress modulation. Overexpression of YHB1 in an unrelated diploid strain also enhanced stress tolerance and improved ethanol productivity in medium containing acetic acid. To our knowledge, this is the first demonstration that improved heterologous secretion and environmental stress tolerance could be engineered into yeast simultaneously.

Induction of Inflammatory Responses in Splenocytes by Exosomes Released from Intestinal Epithelial Cells following Cryptosporidium parvum Infection.

Cryptosporidium, a protozoan parasite that infects the gastrointestinal epithelium and other mucosal surfaces in humans and animals, is an important opportunistic pathogen in AIDS patients and one of the most common enteric pathogens affecting young children in developing regions. This parasite is referred to as a "minimally invasive" mucosal pathogen, and epithelial cells play a central role in activating and orchestrating host immune responses. We previously demonstrated that Cryptosporidium parvum infection stimulates host epithelial cells to release exosomes, and these released exosomes shuttle several antimicrobial peptides to carry out anti-C. parvum activity. In this study, we detected the upregulation of inflammatory genes in the liver and spleen following C. parvum intestinal infection in neonatal mice. Interestingly, exosomes released from intestinal epithelial cells following C. parvum infection could activate the nuclear factor kappa B signaling pathway and trigger inflammatory gene transcription in isolated primary splenocytes. Several epithelial cell-derived proteins and a subset of parasite RNAs were detected in the exosomes released from C. parvum-infected intestinal epithelial cells. Shuttling of these effector molecules, including the high mobility group box 1 protein, was involved in the induction of inflammatory responses in splenocytes induced by the exosomes released from infected cells. Our data indicate that exosomes released from intestinal epithelial cells upon C. parvum infection can activate immune cells by shuttling various effector molecules, a process that may be relevant to host systemic responses to Cryptosporidium infection.

Variation in ICE1 Methylation Primarily Determines Phenotypic Variation in Freezing Tolerance in Arabidopsis thaliana.

Cold stress is a major abiotic factor plants face during their life cycle. Although plants often exhibit phenotypic variation in cold tolerance, the underlying mechanism remains poorly understood. In the present study, the 50% lethal temperature (LT50) values of 37 Arabidopsis thaliana accessions at latitudes from 15° to 58° ranged from -13.2°C to -4.9°C and were closely correlated with the cold climates of the collection sites. According to a methylation analysis of all C-repeat (CRT)-binding factor (CBF) pathway genes, the coding and promoter regions of AtICE1, a regulator of CBF genes, exhibited the greatest variability in methylation levels among the accessions and included 5-122 methylated cytosine residues. In contrast, unmethylated or only slightly methylated genes in the CBF pathway showed little variation among the accessions. According to a gene expression analysis of four selected A. thaliana populations with distinct methylation patterns, except for the down-regulated gene AtCBF2, the expression levels of all members of the CBF pathway were negatively correlated with AtICE1 gene methylation levels. Treatment of the four A. thaliana populations with the DNA methylation inhibitory reagent 5-azacytidine resulted in a 30.0-78.3% enhancement of freezing tolerance and decreases in LT50 values of approximately 1.9-3.6°C. Similar effects were observed in drm2 mutants, including 30.0-48.3% increases in freezing tolerance and decreases in LT50 values of approximately 0.7-3.4°C. Thus, the AtICE1 methylation-regulated transcription of CBF pathway genes is responsible for the phenotypic variation in the freezing tolerance observed in A. thaliana.

Sialic Acid-Like Sugars in Archaea: Legionaminic Acid Biosynthesis in the Halophile Halorubrum sp. PV6

N-glycosylation is a post-translational modification that occurs in all three domains. In Archaea, however, N-linked glycans present a degree of compositional diversity not observed in either Eukarya or Bacteria. As such, it is surprising that nonulosonic acids (NulOs), nine-carbon sugars that include sialic acids, pseudaminic acids, and legionaminic acids, are routinely detected as components of protein-linked glycans in Eukarya and Bacteria but not in Archaea. In the following, we report that the N-linked glycan attached to the S-layer glycoprotein of the haloarchaea Halorubrum sp. PV6 includes an N-formylated legionaminic acid. Analysis of the Halorubrum sp. PV6 genome led to the identification of sequences predicted to comprise the legionaminic acid biosynthesis pathway. The transcription of pathway genes was confirmed, as was the co-transcription of several of these genes. In addition, the activities of LegI, which catalyzes the condensation of 2,4-di-N-acetyl-6-deoxymannose and phosphoenolpyruvate to generate legionaminic acid, and LegF, which catalyzes the addition of cytidine monophosphate (CMP) to legionaminic acid, both heterologously expressed in Haloferax volcanii, were demonstrated. Further genome analysis predicts that the genes encoding enzymes of the legionaminic acid biosynthetic pathway are clustered together with sequences seemingly encoding components of the N-glycosylation pathway in this organism. In defining the first example of a legionaminic acid biosynthesis pathway in Archaea, the findings reported here expand our insight into archaeal N-glycosylation, an almost universal post-translational modification in this domain of life.

Overexpression of SmbHLH10 enhances tanshinones biosynthesis in Salvia miltiorrhiza hairy roots

The bHLH transcription factors have important role in regulation of plant growth, development, and secondary metabolism. Tanshinones are the major pharmaceutical components present in Salvia miltiorrhiza Bunge. It has been reported that bHLHs have functions in terpenoids biosynthesis. Here, we got a bHLH family member named SmbHLH10 which could positively regulate tanshinones biosynthesis in S. miltiorrhiza hairy roots. In the SmbHLH10-overexpressing line 6, four major tanshinones contents were reaching 2.51-fold (dihydrotanshinone I), 2.84-fold (cryptotanshinone), 2.89- fold (tanshinone I), 2.68-fold (tanshinone II A) of WT, respectively. The variation in tanshinones biosynthetic pathway gene transcription was generally consistent with tanshinones content. DXS2, DXS3 and DXR of MEP pathway were induced substantially, reaching 10-fold, 3-fold, 5.74-fold higher of the WT, respectively. The downstream pathway genes CPS1, CPS5 and CYP76AH1 were highest in line OE-SmbHLH10-6, reached 4.93, 16.29 and 3.27-fold of the WT, respectively, while KSL1’s expression was highest in line OE-SmbHLH10-4, 4.64-fold of WT. Yeast one-hybrid assays results showed that SmbHLH10 could binds the predicted G-box motifs within the promoters of DXS2, CPS1 and CPS5. These findings indicated that SmbHLH10 could directly binds to G-box in the pathway genes’ promotor, activate their expression and then upregulate tanshinones biosynthesis.

Novel Intergenically Spliced Chimera, NFATC3-PLA2G15, Is Associated with Aggressive T-ALL Biology and Outcome.

Leukemias are frequently characterized by the expression of oncogenic fusion chimeras that normally arise due to chromosomal rearrangements. Intergenically spliced chimeric RNAs (ISC) are transcribed in the absence of structural genomic changes, and aberrant ISC expression is now recognized as a potential driver of cancer. To better understand these potential oncogenic drivers, high-throughput RNA sequencing was performed on T-acute lymphoblastic leukemia (T-ALL) patient specimens (n = 24), and candidate T-ALL-related ISCs were identified (n = 55; a median of 4/patient). In-depth characterization of the NFATC3-PLA2G15 chimera, which was variably expressed in primary T-ALL, was performed. Functional assessment revealed that the fusion had lower activity than wild-type NFATC3 in vitro, and T-ALLs with elevated NFATC3-PLA2G15 levels had reduced transcription of canonical NFAT pathway genes in vivo Strikingly, high expression of the NFATC3-PLA2G15 chimera correlated with aggressive disease biology in murine patient-derived T-ALL xenografts, and poor prognosis in human T-ALL patients. Mol Cancer Res; 16(3); 470-5. ©2018 AACR.

Altered toll-like receptor responsiveness underlies a dominant heritable defect in Bcell tolerance in autoimmune New Zealand Black mice.

Systemic lupus erythematosus is a debilitating autoimmune disease in which autoantibodies and autoreactive T cells destroy kidneys and other organs. Disease is clinically and genetically heterogeneous, suggesting that underlying mechanisms vary between patients. We previously used an autoantibody transgenic mouse reporter system to examine the effect of different autoimmune backgrounds on B-cell tolerance, failure of which is a fundamental defect in lupus. We identified a defect consistent with reversible anergy induced by endotoxin stimulation of Bcells from Ig transgenic New Zealand Black (NZB) mice. Herein we report that the tolerance defect is revealed by TLR7 and TLR9 as well as TLR4 ligands, with additive effect, and is partially reversed by Mek inhibition. Gene expression analysis reveals significant differences in transcription of multiple TLR pathway genes and ptpn22 in stimulated NZB compared to B6 Bcells. Additionally, the defect is detected in Ig transgenic NZB F1 hybrid strains (NZBxNZW)F1 and (B6xNZB)F1. These results implicate an inherited defect wherein NZB anergic Bcells maintain coordinated TLR/BCR signaling that permits autoantibody production. Agents targeting these pathways may have therapeutic benefit in the subset of lupus patients that manifest similar defects in B-cell regulation.

Induction of Terpene Biosynthesis in Berries of Microvine Transformed with VvDXS1 Alleles.

Terpenoids, especially monoterpenes, are major aroma-impact compounds in grape and wine. Previous studies highlighted a key regulatory role for grapevine 1-deoxy-D-xylulose 5-phosphate synthase 1 (VvDXS1), the first enzyme of the methylerythritol phosphate pathway for isoprenoid precursor biosynthesis. Here, the parallel analysis of VvDXS1 genotype and terpene concentration in a germplasm collection demonstrated that VvDXS1 sequence has a very high predictive value for the accumulation of monoterpenes and also has an influence on sesquiterpene levels. A metabolic engineering approach was applied by expressing distinct VvDXS1 alleles in the grapevine model system “microvine” and assessing the effects on downstream pathways at transcriptional and metabolic level in different organs and fruit developmental stages. The underlying goal was to investigate two potential perturbation mechanisms, the former based on a significant over-expression of the wild-type (neutral) VvDXS1 allele and the latter on the ex-novo expression of an enzyme with increased catalytic efficiency from the mutated (muscat) VvDXS1 allele. The integration of the two VvDXS1 alleles in distinct microvine lines was found to alter the expression of several terpenoid biosynthetic genes, as assayed through an ad hoc developed TaqMan array based on cDNA libraries of four aromatic cultivars. In particular, enhanced transcription of monoterpene, sesquiterpene and carotenoid pathway genes was observed. The accumulation of monoterpenes in ripe berries was higher in the transformed microvines compared to control plants. This effect is predominantly attributed to the improved activity of the VvDXS1 enzyme coded by the muscat allele, whereas the up-regulation of VvDXS1 plays a secondary role in the increase of monoterpenes.

Identification and characterization of genes involving the early step of Juvenile Hormone pathway in Helicoverpa armigera

Juvenile hormones (JHs) are crucial regulators for multiple physiological processes in insects. In the current study, 10 genes in mevalonate pathway involved in JH biosynthesis were identified from Helicoverpa armigera. Tissue-specific expression analysis showed that six genes were highly expressed in the head which contained the JH biosynthetic gland (corpora allata). Temporal expression pattern showed that 10 of 12 genes were highly transcribed in the late 2nd-instar when the in vivo JH titer reached the peak, indicating a tight correlation between JH titer and the transcription of JH synthetic pathway genes. Moreover, ingestion of methoprene, a JH analogue, significantly suppressed the transcription of nine JH biosynthetic genes and caused a feedback upregulation of the JH degradation enzyme. Particularly, the Acetoacetyl CoA thiolase (HaAce) and Farnesyl diphosphate synthase gene 4 (HaFpps4) showed high transcript abundance, and their temporal expressions keep pace with JH fluctuations. Further study by RNAi showed that knockdown of HaFpps4 caused the decrease of JH titer, led to a negative effect on the transcript levels of other genes in JH pathway, and resulted in molting disturbance in larvae. Altogether, these results contribute to our understanding of JH biosynthesis in H. armigera and provide target genes for pest control based on JH-dependent regulation.

Peripheral Transcription of NRG-ErbB Pathway Genes Are Upregulated in Treatment-Resistant Schizophrenia.

Investigation of peripheral gene expression patterns of transcripts within the NRG–ErbB signaling pathway, other than neuregulin-1 (NRG1), among patients with schizophrenia and more specifically treatment-resistant schizophrenia (TRS) is limited. The present study built on our previous work demonstrating elevated levels of NRG1 EGFα, EGFβ, and type I(Ig2) containing transcripts in TRS by investigating 11 NRG–ErbB signaling pathway mRNA transcripts (NRG2, ErbB1, ErbB2, ErbB3, ErbB4, PIK3CD, PIK3R3, AKT1, mTOR, P70S6K, eIF4EBP1) in whole blood of TRS patients (N = 71) and healthy controls (N = 57). We also examined the effect of clozapine exposure on transcript levels using cultured peripheral blood mononuclear cells (PBMCs) from 15 healthy individuals. Five transcripts (ErbB3, PIK3CD, AKT1, P70S6K, eIF4EBP1) were significantly elevated in TRS patients compared to healthy controls but only expression of P70S6K (Pcorrected = 0.018), a protein kinase linked to protein synthesis, cell growth, and cell proliferation, survived correction for multiple testing using the Benjamini–Hochberg method. Investigation of clinical factors revealed that ErbB2, PIK3CD, PIK3R3, AKT1, mTOR, and P70S6K expression were negatively correlated with duration of illness. However, no transcript was associated with chlorpromazine equivalent dose or clozapine plasma levels, the latter supported by our in vitro PBMC clozapine exposure experiment. Taken together with previously published NRG1 results, our findings suggest an overall upregulation of transcripts within the NRG–ErbB signaling pathway among individuals with schizophrenia some of which attenuate over duration of illness. Follow-up studies are needed to determine if the observed peripheral upregulation of transcripts within the NRG–ErbB signaling pathway are specific to TRS or are a general blood-based marker of schizophrenia.

Peanuts that keep aflatoxin at bay: a threshold that matters.

SummaryAflatoxin contamination in peanuts poses major challenges for vulnerable populations of sub‐Saharan Africa and South Asia. Developing peanut varieties to combat preharvest Aspergillus flavus infection and resulting aflatoxin contamination has thus far remained a major challenge, confounded by highly complex peanut–Aspergilli pathosystem. Our study reports achieving a high level of resistance in peanut by overexpressing (OE) antifungal plant defensins MsDef1 and MtDef4.2, and through host‐induced gene silencing (HIGS) of aflM and aflP genes from the aflatoxin biosynthetic pathway. While the former improves genetic resistance to A. flavus infection, the latter inhibits aflatoxin production in the event of infection providing durable resistance against different Aspergillus flavus morphotypes and negligible aflatoxin content in several peanut events/lines well. A strong positive correlation was observed between aflatoxin accumulation and decline in transcription of the aflatoxin biosynthetic pathway genes in both OE‐Def and HIGS lines. Transcriptomic signatures in the resistant lines revealed key mechanisms such as regulation of aflatoxin synthesis, its packaging and export control, besides the role of reactive oxygen species‐scavenging enzymes that render enhanced protection in the OE and HIGS lines. This is the first study to demonstrate highly effective biotechnological strategies for successfully generating peanuts that are near‐immune to aflatoxin contamination, offering a panacea for serious food safety, health and trade issues in the semi‐arid regions.

ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways

Stem cell-like brain tumor initiating cells (BTICs) cause recurrence of glioblastomas, with BTIC ‘stemness’ affected by epigenetic mechanisms. The ING family of epigenetic regulators (ING1-5) function by targeting histone acetyltransferase (HAT) or histone deacetylase complexes to the H3K4me3 mark to alter histone acetylation and subsequently, gene expression. Here we find that ectopic expression of ING5, the targeting subunit of HBO1, MOZ and MORF HAT complexes increases expression of the Oct4, Olig2 and Nestin stem cell markers, promotes self-renewal, prevents lineage differentiation and increases stem cell pools in BTIC populations. This activity requires the plant homeodomain region of ING5 that interacts specifically with the H3K4me3 mark. ING5 also enhances PI3K/AKT and MEK/ERK activity to sustain self-renewal of BTICs over serial passage of stem cell-like spheres. ING5 exerts these effects by activating transcription of calcium channel and follicle stimulating hormone pathway genes. In silico analyses of The Cancer Genome Atlas data suggest that ING5 is a positive regulator of BTIC stemness, whose expression negatively correlates with patient prognosis, especially in the Proneural and Classical subtypes, and in tumors with low SOX2 expression. These data suggest that altering histone acetylation status and signaling pathways induced by ING5 may provide useful clinical strategies to target tumor resistance and recurrence in glioblastoma.

Salt-Sensitive Signaling Networks in the Mediation of K+/Na+ Homeostasis Gene Expression in Glycyrrhiza uralensis Roots.

We investigated the effects of salt-sensitive signaling molecules on ionic fluxes and gene expression related to K+/Na+ homeostasis in a perennial herb, Glycyrrhiza uralensis, during short-term NaCl stress (100 mM, 24 h). Salt treatment caused more pronounced Na+ accumulation in root cells than in leaf cells. Na+ ions were mostly compartmentalized in vacuoles. Roots exposed to NaCl showed increased levels of extracellular ATP (eATP), cytosolic Ca2+, H2O2, and NO. Steady-state flux recordings revealed that these salt-sensitive signaling molecules enhanced NaCl-responsive Na+ efflux, due to the activated Na+/H+ antiport system in the plasma membrane (PM). Moreover, salt-elicited K+ efflux, which was mediated by depolarization-activated cation channels, was reduced with the addition of Ca2+, H2O2, NO, and eATP. The salt-adaptive effects of these molecules (Na+ extrusion and K+ maintenance) were reduced by pharmacological agents, including LaCl3 (a PM Ca2+ channel inhibitor), DMTU (a reactive oxygen species scavenger), cPTIO (an NO scavenger), or PPADS (an antagonist of animal PM purine P2 receptors). RT-qPCR data showed that the activation of the PM Na+/H+ antiport system in salinized roots most likely resulted from the upregulation of two genes, GuSOS1 and GuAHA, which encoded the PM Na+/H+ antiporter, salt overly sensitive 1 (SOS1), and H+-ATPase, respectively. Clear interactions occurred between these salt-sensitive agonists to accelerate transcription of salt-responsive signaling pathway genes in G. uralensis roots. For example, Ca2+, H2O2, NO, and eATP promoted transcription of GuSOS3 (salt overly sensitive 3) and/or GuCIPK (CBL-interacting protein kinase) to activate the predominant Ca2+-SOS signaling pathway in salinized liquorice roots. eATP, a novel player in the salt response of G. uralensis, increased the transcription of GuSOS3, GuCIPK, GuRbohD (respiratory burst oxidase homolog protein D), GuNIR (nitrate reductase), GuMAPK3, and GuMAPK6 (the mitogen-activated protein kinases 3 and 6). Moreover, GuMAPK3 and GuMAPK6 expression levels were enhanced by H2O2 in NaCl-stressed G. uralensis roots. Our results indicated that eATP triggered downstream components and interacted with Ca2+, H2O2, and NO signaling to maintain K+/Na+ homeostasis. We propose that a multiple signaling network regulated K+/Na+ homeostasis in NaCl-stressed G. uralensis roots.

Molecular Basis of Nongenomic Actions of Thyroid Hormone.

Nongenomic actions of thyroid hormone are initiated by the hormone at receptors in the plasma membrane, in cytoplasm, or in mitochondria and do not require the interaction of nuclear thyroid hormone receptors (TRs) with their primary ligand, 3,5,3'-triiodo-l-thyronine (T3). Receptors involved in nongenomic actions may or may not have structural homologies with TRs. Certain nongenomic actions that originate at the plasma membrane may modify the state and function of intranuclear TRs. Reviewed here are nongenomic effects of the hormone-T3 or, in some cases, l-thyroxine (T4)-that are initiated at (a) truncated TRα isoforms, e.g., p30 TRα1, (b) cytoplasmic proteins, or (c) plasma membrane integrin αvβ3. p30 TRα1 is not transcriptionally competent, binds T3 at the cell surface, and consequently expresses a number of important functions in bone cells. Nongenomic hormonal control of mitochondrial respiration involves a TRα isoform, and another truncated TRα isoform nongenomically regulates the state of cellular actin. Cytoplasmic hormone-binding proteins involved in nongenomic actions of thyroid hormone include ketimine reductase, pyruvate kinase, and TRβ that shuttle among intracellular compartments. Functions of the receptor for T4 on integrin αvβ3 include stimulation of proliferation of cancer and endothelial cells (angiogenesis) and regulation of transcription of cancer cell survival pathway genes. T4 serves as a prohormone for T3 in genomic actions of thyroid hormone, but T4 is a hormone at αvβ3 and more important to cancer cell function than is T3. Thus, characterization of nongenomic actions of the hormone has served to broaden our understanding of the cellular roles of T3 and T4.

A WRKY transcription factor from Withania somnifera regulates triterpenoid withanolide accumulation and biotic stress tolerance through modulation of phytosterol and defense pathways.

Withania somnifera produces pharmacologically important triterpenoid withanolides that are derived via phytosterol pathway; however, their biosynthesis and regulation remain to be elucidated. A jasmonate- and salicin-inducible WRKY transcription factor from W.somnifera (WsWRKY1) exhibiting correlation with withaferin A accumulation was functionally characterized employing virus-induced gene silencing and overexpression studies combined with transcript and metabolite analyses, and chromatin immunoprecipitation assay. WsWRKY1 silencing resulted in stunted plant growth, reduced transcripts of phytosterol pathway genes with corresponding reduction in phytosterols and withanolides in W.somnifera. Its overexpression elevated the biosynthesis of triterpenoids in W.somnifera (phytosterols and withanolides), as well as tobacco and tomato (phytosterols). Moreover, WsWRKY1 binds to W-box sequences in promoters of W.somnifera genes encoding squalene synthase and squalene epoxidase, indicating its direct regulation of triterpenoid pathway. Furthermore, while WsWRKY1 silencing in W.somnifera compromised the tolerance to bacterial growth, fungal infection, and insect feeding, its overexpression in tobacco led to improved biotic stress tolerance. Together these findings demonstrate that WsWRKY1 has a positive regulatory role on phytosterol and withanolides biosynthesis, and defense against biotic stress, highlighting its importance as a metabolic engineering tool for simultaneous improvement of triterpenoid biosynthesis and plant defense.

Molecular Mechanisms Underlying γ-Aminobutyric Acid (GABA) Accumulation in Giant Embryo Rice Seeds.

To uncover the molecular mechanisms underlying GABA accumulation in giant embryo rice seeds, we analyzed the expression levels of GABA metabolism genes and contents of GABA and GABA metabolic intermediates in developing grains and germinated brown rice of giant embryo rice 'Shangshida No. 5' and normal embryo rice 'Chao2-10' respectively. In developing grains, the higher GABA contents in 'Shangshida No. 5' were accompanied with upregulation of gene transcripts and intermediate contents in the polyamine pathway and downregulation of GABA catabolic gene transcripts, as compared with those in 'Chao2-10'. In germinated brown rice, the higher GABA contents in 'Shangshida No. 5' were parallel with upregulation of OsGAD and polyamine pathway gene transcripts and Glu and polyamine pathway intermediate contents and downregulation of GABA catabolic gene transcripts. These results are the first to indicate that polyamine pathway and GABA catabolic genes play a crucial role in GABA accumulation in giant embryo rice seeds.

TIMELESS confers cisplatin resistance in nasopharyngeal carcinoma by activating the Wnt/β-catenin signaling pathway and promoting the epithelial mesenchymal transition

This study investigated the expression, clinicopathological significance and mechanism of action of TIMELESS, a mammalian homolog of a Drosophila circadian rhythm gene, in nasopharyngeal carcinoma. Quantitative real-time PCR, western blotting and immunohistochemistry revealed TIMELESS was upregulated in NPC cell lines (n = 8 vs. NP69 cells), and freshly-frozen (n = 6) and paraffin-embedded human NPC specimens (n = 108 vs. normal samples/non-tumor cells). TIMELESS expression was associated with T category (P = 0.002), N category (P = 0.001), clinical stage (P < 0.001), metastasis (P = 0.047), vital status (P = 0.013) and serum Epstein–Barr DNA (P = 0.005). High TIMELESS expression was associated with poorer overall survival (80.7% vs. 95.9%; P = 0.004) and progression free survival (68.1% vs. 88.0%; P = 0.005). Univariate and multivariate analysis revealed TIMELESS was an independent prognostic factor for overall survival and progression free survival. Stable ectopic overexpression of TIMELESS in NPC cell lines conferred resistance to cisplatin-induced apoptosis in vitro and in vivo, promoted an epithelial-to-mesenchymal transition phenotype, and activated the Wnt/β-catenin pathway and downstream gene transcription; knockdown of TIMELESS had the opposite effects. TIMELESS may play a role in the development of NPC and could represent a valuable prognostic factor and potential therapeutic target.