Dysregulation of hepatic fatty acid metabolism in chronic kidney disease.

PubMed

Jin, Kyubok; Norris, Keith; Vaziri, Nosratola D

2013-02-01

Chronic kidney disease (CKD) results in hypertriglyceridemia which is largely due to impaired clearance of triglyceride-rich lipoproteins occasioned by downregulation of lipoprotein lipase and very low-density lipoprotein (LDL) receptor in the skeletal muscle and adipose tissue and of hepatic lipase and LDL receptor-related protein in the liver. However, data on the effect of CKD on fatty acid metabolism in the liver is limited and was investigated here. Male Sprague-Dawley rats were randomized to undergo 5/6 nephrectomy (CRF) or sham operation (control) and observed for 12 weeks. The animals were then euthanized and their liver tissue tested for nuclear translocation (activation) of carbohydrate-responsive element binding protein (ChREBP) and sterol-responsive element binding protein-1 (SREBP-1) which independently regulate the expression of key enzyme in fatty acid synthesis, i.e. fatty acid synthase (FAS) and acyl-CoA carboxylase (ACC) as well as nuclear Peroxisome proliferator-activated receptor alpha (PPARα) which regulates the expression of enzymes involved in fatty acid oxidation and transport, i.e. L-FABP and CPT1A. In addition, the expression of ATP synthase α, ATP synthase β, glycogen synthase and diglyceride acyltransferase 1 (DGAT1) and DGAT2 were determined. Compared with controls, the CKD rats exhibited hypertriglyceridemia, elevated plasma and liver tissue free fatty acids, increased nuclear ChREBP and reduced nuclear SREBP-1 and PPARα, upregulation of ACC and FAS and downregulation of L-FABP, CPT1A, ATP synthase α, glycogen synthase and DGAT in the liver tissue. Liver in animals with advanced CKD exhibits ChREBP-mediated upregulation of enzymes involved in fatty acid synthesis, downregulation of PPARα-regulated fatty acid oxidation system and reduction of DGAT resulting in reduced fatty acid incorporation in triglyceride.

Transmembrane transporter expression regulated by the glucosylceramide pathway in Cryptococcus neoformans.

PubMed

Singh, Arpita; Rella, Antonella; Schwacke, John; Vacchi-Suzzi, Caterina; Luberto, Chiara; Del Poeta, Maurizio

2015-11-16

The sphingolipid glucosylceramide (GlcCer) and factors involved in the fungal GlcCer pathways were shown earlier to be an integral part of fungal virulence, especially in fungal replication at 37 °C, in neutral/alkaline pH and 5 % CO2 environments (e.g. alveolar spaces). Two mutants, ∆gcs 1 lacking glucosylceramide synthase 1 gene (GCS1) which catalyzes the formation of sphingolipid GlcCer from the C9-methyl ceramide and ∆smt1 lacking sphingolipid C9 methyltransferase gene (SMT1), which adds a methyl group to position nine of the sphingosine backbone of ceramide, of this pathway were attenuated in virulence and have a growth defect at the above-mentioned conditions. These mutants with either no or structurally modified GlcCer located on the cell-membrane have reduced membrane rigidity, which may have altered not only the physical location of membrane proteins but also their expression, as the pathogen's mode of adaptation to changing need. Importantly, pathogens are known to adapt themselves to the changing host environments by altering their patterns of gene expression. By transcriptional analysis of gene expression, we identified six genes whose expression was changed from their wild-type counterpart grown in the same conditions, i.e. they became either down regulated or up regulated in these two mutants. The microarray data was validated by real-time PCR, which confirmed their fold change in gene expression. All the six genes we identified, viz siderochrome-iron transporter (CNAG_02083), monosaccharide transporter (CNAG_05340), glucose transporter (CNAG_03772), membrane protein (CNAG_03912), membrane transport protein (CNAG_00539), and sugar transporter (CNAG_06963), are membrane-localized and have significantly altered gene expression levels. Therefore, we hypothesize that these genes function either independently or in tandem with a structurally modified cell wall/plasma membrane resulting from the modifications of the GlcCer pathway and thus possibly disrupt transmembrane signaling complex, which in turn contributes to cryptococcal osmotic, pH, ion homeostasis and its pathobiology. Six genes identified from gene expression microarrays by gene set enrichment analysis and validated by RT-PCR, are membrane located and associated with the growth defect at neutral-alkaline pH due to the absence and or presence of a structurally modified GlcCer. They may be involved in the transmembrane signaling network in Cryptococcus neoformans, and therefore the pathobiology of the fungus in these conditions.

Excessive interferon-α signaling in autoimmunity alters glycosphingolipid processing in B cells.

PubMed

Tan, Andy Hee-Meng; Sanny, Arleen; Ng, Sze-Wai; Ho, Ying-Swan; Basri, Nurhidayah; Lee, Alison Ping; Lam, Kong-Peng

2018-05-01

Excessive interferon-α (IFN-α) production by innate immune cells is a hallmark of autoimmune diseases. What other cell type secretes IFN-α and how IFN-α affects immune cell metabolism and homeostasis in autoimmunity are largely unclear. Here, we report that autoimmune B cells, arising from two different B cell-specific genetic lesions in mice, secrete IFN-α. In addition, IFN-α, found in abundance in autoimmunity, elicited profound changes in the B cell lipidome, increasing their expression of glycosphingolipids (GSLs) and leading to their CD1d-mediated depletion of iNKT cells in vitro and in vivo. IFN-α receptor blockade could reverse the loss of iNKT cells. Excessive stimulation of B cells with IFN-α altered the expression of enzymes that catalyze critical steps in GSL processing, increasing the expressions of glucosylceramide synthase (GCS) and globotrihexosylceramide synthase (Gb3S) but decreasing that of α-galactosidase A (α-galA). Inhibiting GCS or restoring α-galA expression prevented iNKT depletion by IFN-α-activated B cells. Taken together, our work indicated that excessive IFN-α perturbs GSL metabolism in B cells which in turn adversely affects iNKT homeostasis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Reduction of nuclear encoded enzymes of mitochondrial energy metabolism in cells devoid of mitochondrial DNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mueller, Edith E., E-mail: ed.mueller@salk.at; Mayr, Johannes A., E-mail: h.mayr@salk.at; Zimmermann, Franz A., E-mail: f.zimmermann@salk.at

2012-01-20

Highlights: Black-Right-Pointing-Pointer We examined OXPHOS and citrate synthase enzyme activities in HEK293 cells devoid of mtDNA. Black-Right-Pointing-Pointer Enzymes partially encoded by mtDNA show reduced activities. Black-Right-Pointing-Pointer Also the entirely nuclear encoded complex II and citrate synthase exhibit reduced activities. Black-Right-Pointing-Pointer Loss of mtDNA induces a feedback mechanism that downregulates complex II and citrate synthase. -- Abstract: Mitochondrial DNA (mtDNA) depletion syndromes are generally associated with reduced activities of oxidative phosphorylation (OXPHOS) enzymes that contain subunits encoded by mtDNA. Conversely, entirely nuclear encoded mitochondrial enzymes in these syndromes, such as the tricarboxylic acid cycle enzyme citrate synthase (CS) and OXPHOS complexmore » II, usually exhibit normal or compensatory enhanced activities. Here we report that a human cell line devoid of mtDNA (HEK293 {rho}{sup 0} cells) has diminished activities of both complex II and CS. This finding indicates the existence of a feedback mechanism in {rho}{sup 0} cells that downregulates the expression of entirely nuclear encoded components of mitochondrial energy metabolism.« less

Accumulation and distribution of α-synuclein and ubiquitin in the CNS of Gaucher disease mouse models

PubMed Central

Xu, YH; Sun, Y; Ran, H; Quinn, B; Witte, D; Grabowski, GA

2011-01-01

Gaucher disease, a prevalent lysosomal storage disease, is caused by insufficient activity of acid β-glucosidase (GCase) and resultant glucosylceramide accumulation. Recently in Parkinson disease (PD) patients, heterozygous mutations in GCase have been associated with earlier onset and more progressive PD. To understand the pathogenic relationships between GCase variants and Parkinsonism, α-synuclein and ubiquitin distributions and levels in the brains of several mouse models containing GCase variants were evaluated by immunohistochemistry. Progressive α-synuclein and ubiquitin aggregate accumulations were observed in the cortex, hippocampus, basal ganglia, brainstem, and some cerebellar regions between 4-24 wks in mice that were homozygous for GCase [D409H (9H) or V394L (4L)] variants and also had a prosaposin hypomorphic (PS-NA) transgene. In 4L/PS-NA and 9H/PS-NA mice, this was coincident with progressive neurological manifestations and brain glucosylceramide accumulation. Ultrastructural studies showed electron dense inclusion bodies in neurons and axons of 9H/PS-NA brains. α-Synuclein aggregates were also observed in ventricular, brainstem, and cerebellar regions of older mice (>42-wk) with the GCase variant (D409H/D409H) without overt neurological disease. In a chemically induced GCase deficiency, α-synuclein aggregates and glucosylceramide accumulation also occurred. These studies demonstrate a relationship between glucosylceramide accumulation and α-synuclein aggregates, and implicate glucosylceramide accumulation as risk factor for the α-synucleinopathies. PMID:21257328

Glucosylceramide is Critical for Cell-Type Differentiation and Organogenesis, but not for Cell Viability in Arabidopsis

PubMed Central

Msanne, Joseph; Chen, Ming; Luttgeharm, Kyle D.; Bradley, Amanda M.; Mays, Elizabeth S.; Paper, Janet M.; Boyle, Daniel L.; Cahoon, Rebecca E.; Schrick, Kathrin; Cahoon, Edgar B.

2015-01-01

Summary Glucosylceramides (GlcCer), glucose-conjugated sphingolipids, are major components of the endomembrane system and plasma membrane in most eukaryote cells. Yet, the quantitative significance and cellular functions of GlcCer are not well characterized in plants and other multi-organ eukaryotes. To address this, we examined Arabidopsis lines lacking or deficient in GlcCer by insertional disruption or by RNAi suppression of the single gene for GlcCer synthase (GCS, At2g19880), the enzyme that catalyzes GlcCer synthesis. Null mutants for GCS (designated “gcs-1”) were viable as seedlings, albeit strongly reduced in size, and failed to develop beyond the seedling stage. Heterozygous plants harboring the insertion allele exhibited reduced transmission through the male gametophyte. Undifferentiated calli generated from gcs-1 seedlings and lacking GlcCer proliferated in a manner similar to calli from wild-type plants. However, gcs-1 calli, in contrast to wild-type calli, were unable to develop organs on differentiation media. Consistent with a role for GlcCer in organ-specific cell differentiation, calli from gcs-1 mutants formed roots and leaves on media supplemented with the glucosylated sphingosine glucopsychosine, which was readily converted to GlcCer independent of GCS. Underlying these phenotypes, gcs-1 cells had altered Golgi morphology and fewer cisternae per Golgi apparatus relative to wild-type cells, indicative of protein trafficking defects. Despite seedling lethality in the null mutant, GCS RNAi suppression lines with ≤2% of wild-type GlcCer levels were viable and fertile. Collectively, these results indicate that GlcCer are essential for cell-type differentiation and organogenesis, and plant cells produce GlcCer amounts in excess of that required for normal development. PMID:26313010

A novel approach for quantitation of glucosylceramide in human dried blood spot using LC-MS/MS.

PubMed

Ji, Allena Ji; Wang, Haixing; Ziso-Qejvanaj, Enida; Zheng, Kefei; Chung, Lee Lee; Foley, Timothy; Chuang, Wei-Lien; Richards, Susan; Sung, Crystal

2015-01-01

Glucosylceramide, an efficacy biomarker for Gaucher Type 1 disease, exhibits poor solubility in polar solvents and whole blood which makes it difficult to prepare a homogenous blood standard. We developed a novel method using standard addition approach by spiking a small volume of analyte solution on the surface of prespotted dried blood spot. The whole spots were punched out for subsequent extraction and LC-MS/MS analysis. The assay performance met all validation acceptance criteria. Glucosylceramide concentrations in 50 paired plasma and dry blood spot samples obtained from Gaucher Type 1 patients were tested and the results demonstrated the feasibility of using the DBS method for clinical biomarker monitoring. The new approach greatly improves assay precision and accuracy.

The Role of Light–Dark Regulation of the Chloroplast ATP Synthase

DOE PAGES

Kohzuma, Kaori; Froehlich, John E.; Davis, Geoffry A.; ...

2017-07-24

The chloroplast ATP synthase catalyzes the light-driven synthesis of ATP and is activated in the light and inactivated in the dark by redox-modulation through the thioredoxin system. It has been proposed that this down-regulation is important for preventing wasteful hydrolysis of ATP in the dark. To test this proposal, we compared the effects of extended dark exposure in Arabidopsis lines expressing the wild-type and mutant forms of ATP synthase that are redox regulated or constitutively active. In contrast to the predictions of the model, we observed that plants with wild-type redox regulation lost photosynthetic capacity rapidly in darkness, whereas thosemore » expressing redox-insensitive form were far more stable. To explain these results, we propose that in wild-type plants, down-regulation of ATP synthase inhibits ATP hydrolysis, leading to dissipation of thylakoid proton motive force (pmf) and subsequent inhibition of protein transport across the thylakoid through the twin arginine transporter (Tat)-dependent and Secdependent import pathways, resulting in the selective loss of specific protein complexes. By contrast, in mutants with a redox-insensitive ATP synthase, pmf is maintained by ATP hydrolysis, thus allowing protein transport to maintain photosynthetic activities for extended periods in the dark. Hence, a basal level of Tat-dependent, as well as, Sec-dependent import activity, in the dark helps replenishes certain components of the photosynthetic complexes and thereby aids in maintaining overall complex activity. But, the influence of a dark pmf on thylakoid protein import, by itself, could not explain all the effects we observed in this study. For example, we also observed in wild type plants a large transient buildup of thylakoid pmf and nonphotochemical exciton quenching upon sudden illumination of dark adapted plants. Thus, we conclude that down-regulation of the ATP synthase is probably not related to preventing loss of ATP per se. Instead, ATP synthase redox regulation may be impacting a number of cellular processes such as (1) the accumulation of chloroplast proteins and/or ions or (2) the responses of photosynthesis to rapid changes in light intensity. A model highlighting the complex interplay between ATP synthase regulation and pmf in maintaining various chloroplast functions in the dark is presented.« less

The Role of Light–Dark Regulation of the Chloroplast ATP Synthase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kohzuma, Kaori; Froehlich, John E.; Davis, Geoffry A.

The chloroplast ATP synthase catalyzes the light-driven synthesis of ATP and is activated in the light and inactivated in the dark by redox-modulation through the thioredoxin system. It has been proposed that this down-regulation is important for preventing wasteful hydrolysis of ATP in the dark. To test this proposal, we compared the effects of extended dark exposure in Arabidopsis lines expressing the wild-type and mutant forms of ATP synthase that are redox regulated or constitutively active. In contrast to the predictions of the model, we observed that plants with wild-type redox regulation lost photosynthetic capacity rapidly in darkness, whereas thosemore » expressing redox-insensitive form were far more stable. To explain these results, we propose that in wild-type plants, down-regulation of ATP synthase inhibits ATP hydrolysis, leading to dissipation of thylakoid proton motive force (pmf) and subsequent inhibition of protein transport across the thylakoid through the twin arginine transporter (Tat)-dependent and Secdependent import pathways, resulting in the selective loss of specific protein complexes. By contrast, in mutants with a redox-insensitive ATP synthase, pmf is maintained by ATP hydrolysis, thus allowing protein transport to maintain photosynthetic activities for extended periods in the dark. Hence, a basal level of Tat-dependent, as well as, Sec-dependent import activity, in the dark helps replenishes certain components of the photosynthetic complexes and thereby aids in maintaining overall complex activity. But, the influence of a dark pmf on thylakoid protein import, by itself, could not explain all the effects we observed in this study. For example, we also observed in wild type plants a large transient buildup of thylakoid pmf and nonphotochemical exciton quenching upon sudden illumination of dark adapted plants. Thus, we conclude that down-regulation of the ATP synthase is probably not related to preventing loss of ATP per se. Instead, ATP synthase redox regulation may be impacting a number of cellular processes such as (1) the accumulation of chloroplast proteins and/or ions or (2) the responses of photosynthesis to rapid changes in light intensity. A model highlighting the complex interplay between ATP synthase regulation and pmf in maintaining various chloroplast functions in the dark is presented.« less

The Role of Light-Dark Regulation of the Chloroplast ATP Synthase.

PubMed

Kohzuma, Kaori; Froehlich, John E; Davis, Geoffry A; Temple, Joshua A; Minhas, Deepika; Dhingra, Amit; Cruz, Jeffrey A; Kramer, David M

2017-01-01

The chloroplast ATP synthase catalyzes the light-driven synthesis of ATP and is activated in the light and inactivated in the dark by redox-modulation through the thioredoxin system. It has been proposed that this down-regulation is important for preventing wasteful hydrolysis of ATP in the dark. To test this proposal, we compared the effects of extended dark exposure in Arabidopsis lines expressing the wild-type and mutant forms of ATP synthase that are redox regulated or constitutively active. In contrast to the predictions of the model, we observed that plants with wild-type redox regulation lost photosynthetic capacity rapidly in darkness, whereas those expressing redox-insensitive form were far more stable. To explain these results, we propose that in wild-type plants, down-regulation of ATP synthase inhibits ATP hydrolysis, leading to dissipation of thylakoid proton motive force (pmf) and subsequent inhibition of protein transport across the thylakoid through the twin arginine transporter (Tat)-dependent and Sec-dependent import pathways, resulting in the selective loss of specific protein complexes. By contrast, in mutants with a redox-insensitive ATP synthase, pmf is maintained by ATP hydrolysis, thus allowing protein transport to maintain photosynthetic activities for extended periods in the dark. Hence, a basal level of Tat-dependent, as well as, Sec-dependent import activity, in the dark helps replenishes certain components of the photosynthetic complexes and thereby aids in maintaining overall complex activity. However, the influence of a dark pmf on thylakoid protein import, by itself, could not explain all the effects we observed in this study. For example, we also observed in wild type plants a large transient buildup of thylakoid pmf and nonphotochemical exciton quenching upon sudden illumination of dark adapted plants. Therefore, we conclude that down-regulation of the ATP synthase is probably not related to preventing loss of ATP per se . Instead, ATP synthase redox regulation may be impacting a number of cellular processes such as (1) the accumulation of chloroplast proteins and/or ions or (2) the responses of photosynthesis to rapid changes in light intensity. A model highlighting the complex interplay between ATP synthase regulation and pmf in maintaining various chloroplast functions in the dark is presented. Significance Statement: We uncover an unexpected role for thioredoxin modulation of the chloroplast ATP synthase in regulating the dark-stability of the photosynthetic apparatus, most likely by controlling thylakoid membrane transport of proteins and ions.

The Role of Light–Dark Regulation of the Chloroplast ATP Synthase

PubMed Central

Kohzuma, Kaori; Froehlich, John E.; Davis, Geoffry A.; Temple, Joshua A.; Minhas, Deepika; Dhingra, Amit; Cruz, Jeffrey A.; Kramer, David M.

2017-01-01

The chloroplast ATP synthase catalyzes the light-driven synthesis of ATP and is activated in the light and inactivated in the dark by redox-modulation through the thioredoxin system. It has been proposed that this down-regulation is important for preventing wasteful hydrolysis of ATP in the dark. To test this proposal, we compared the effects of extended dark exposure in Arabidopsis lines expressing the wild-type and mutant forms of ATP synthase that are redox regulated or constitutively active. In contrast to the predictions of the model, we observed that plants with wild-type redox regulation lost photosynthetic capacity rapidly in darkness, whereas those expressing redox-insensitive form were far more stable. To explain these results, we propose that in wild-type plants, down-regulation of ATP synthase inhibits ATP hydrolysis, leading to dissipation of thylakoid proton motive force (pmf) and subsequent inhibition of protein transport across the thylakoid through the twin arginine transporter (Tat)-dependent and Sec-dependent import pathways, resulting in the selective loss of specific protein complexes. By contrast, in mutants with a redox-insensitive ATP synthase, pmf is maintained by ATP hydrolysis, thus allowing protein transport to maintain photosynthetic activities for extended periods in the dark. Hence, a basal level of Tat-dependent, as well as, Sec-dependent import activity, in the dark helps replenishes certain components of the photosynthetic complexes and thereby aids in maintaining overall complex activity. However, the influence of a dark pmf on thylakoid protein import, by itself, could not explain all the effects we observed in this study. For example, we also observed in wild type plants a large transient buildup of thylakoid pmf and nonphotochemical exciton quenching upon sudden illumination of dark adapted plants. Therefore, we conclude that down-regulation of the ATP synthase is probably not related to preventing loss of ATP per se. Instead, ATP synthase redox regulation may be impacting a number of cellular processes such as (1) the accumulation of chloroplast proteins and/or ions or (2) the responses of photosynthesis to rapid changes in light intensity. A model highlighting the complex interplay between ATP synthase regulation and pmf in maintaining various chloroplast functions in the dark is presented. Significance Statement: We uncover an unexpected role for thioredoxin modulation of the chloroplast ATP synthase in regulating the dark-stability of the photosynthetic apparatus, most likely by controlling thylakoid membrane transport of proteins and ions. PMID:28791032

Ceramide-mediated macroautophagy involves inhibition of protein kinase B and up-regulation of beclin 1.

PubMed

Scarlatti, Francesca; Bauvy, Chantal; Ventruti, Annamaria; Sala, Giusy; Cluzeaud, Françoise; Vandewalle, Alain; Ghidoni, Riccardo; Codogno, Patrice

2004-04-30

The sphingolipid ceramide is involved in the cellular stress response. Here we demonstrate that ceramide controls macroautophagy, a major lysosomal catabolic pathway. Exogenous C(2)-ceramide stimulates macroautophagy (proteolysis and accumulation of autophagic vacuoles) in the human colon cancer HT-29 cells by increasing the endogenous pool of long chain ceramides as demonstrated by the use of the ceramide synthase inhibitor fumonisin B(1). Ceramide reverted the interleukin 13-dependent inhibition of macroautophagy by interfering with the activation of protein kinase B. In addition, C(2)-ceramide stimulated the expression of the autophagy gene product beclin 1. Ceramide is also the mediator of the tamoxifen-dependent accumulation of autophagic vacuoles in the human breast cancer MCF-7 cells. Monodansylcadaverine staining and electron microscopy showed that this accumulation was abrogated by myriocin, an inhibitor of de novo synthesis ceramide. The tamoxifen-dependent accumulation of vacuoles was mimicked by 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of glucosylceramide synthase. 1-Phenyl-2-decanoylamino-3-morpholino-1-propanol, tamoxifen, and C(2)-ceramide stimulated the expression of beclin 1, whereas myriocin antagonized the tamoxifen-dependent up-regulation. Tamoxifen and C(2)-ceramide interfere with the activation of protein kinase B, whereas myriocin relieved the inhibitory effect of tamoxifen. In conclusion, the control of macroautophagy by ceramide provides a novel function for this lipid mediator in a cell process with major biological outcomes.

Principal component analysis (PCA) of volatile terpene compounds dataset emitted by genetically modified sweet orange fruits and juices in which a D-limonene synthase was either up- or down-regulated vs. empty vector controls.

PubMed

Rodríguez, Ana; Peris, Josep E; Redondo, Ana; Shimada, Takehiko; Peña, Leandro

2016-12-01

We have categorized the dataset from content and emission of terpene volatiles of peel and juice in both Navelina and Pineapple sweet orange cultivars in which D-limonene was either up- (S), down-regulated (AS) or non-altered (EV; control) ("Impact of D-limonene synthase up- or down-regulation on sweet orange fruit and juice odor perception"(A. Rodríguez, J.E. Peris, A. Redondo, T. Shimada, E. Costell, I. Carbonell, C. Rojas, L. Peña, (2016)) [1]). Data from volatile identification and quantification by HS-SPME and GC-MS were classified by Principal Component Analysis (PCA) individually or as chemical groups. AS juice was characterized by the higher influence of the oxygen fraction, and S juice by the major influence of ethyl esters. S juices emitted less linalool compared to AS and EV juices.

Inhibition of β-Glucocerebrosidase Activity Preserves Motor Unit Integrity in a Mouse Model of Amyotrophic Lateral Sclerosis.

PubMed

Henriques, Alexandre; Huebecker, Mylene; Blasco, Hélène; Keime, Céline; Andres, Christian R; Corcia, Philippe; Priestman, David A; Platt, Frances M; Spedding, Michael; Loeffler, Jean-Philippe

2017-07-12

Recent metabolomic reports connect dysregulation of glycosphingolipids, particularly ceramide and glucosylceramide, to neurodegeneration and to motor unit dismantling in amyotrophic lateral sclerosis at late disease stage. We report here altered levels of gangliosides in the cerebrospinal fluid of amyotrophic lateral sclerosis patients in early disease stage. Conduritol B epoxide is an inhibitor of acid beta-glucosidase, and lowers glucosylceramide degradation. Glucosylceramide is the precursor for all of the more complex glycosphingolipids. In SOD1 G86R mice, an animal model of amyotrophic lateral sclerosis, conduritol B epoxide preserved ganglioside distribution at the neuromuscular junction, delayed disease onset, improved motor function and preserved motor neurons as well as neuromuscular junctions from degeneration. Conduritol B epoxide mitigated gene dysregulation in the spinal cord and restored the expression of genes involved in signal transduction and axonal elongation. Inhibition of acid beta-glucosidase promoted faster axonal elongation in an in vitro model of neuromuscular junctions and hastened recovery after peripheral nerve injury in wild type mice. Here, we provide evidence that glycosphingolipids play an important role in muscle innervation, which degenerates in amyotrophic lateral sclerosis from the early disease stage. This is a first proof of concept study showing that modulating the catabolism of glucosylceramide may be a therapeutic target for this devastating disease.

Inhibition of invasion by glycogen synthase kinase-3 beta inhibitors through dysregulation of actin re-organisation via down-regulation of WAVE2.

PubMed

Yoshino, Yuki; Suzuki, Manami; Takahashi, Hidekazu; Ishioka, Chikashi

2015-08-14

Cancer cell invasion is a critical phenomenon in cancer pathogenesis. Glycogen synthase kinase-3β (GSK-3β) has been reported to regulate cancer cell invasion both negatively and positively. Thus, the net effect of GSK-3β on invasion is unclear. In this report, we showed that GSK-3β inhibitors induced dysregulation of the actin cytoskeleton and functional insufficiency of focal adhesion, which resulted in suppressed invasion. In addition, WAVE2, an essential molecule for actin fibre branching, was down-regulated after GSK-3β inhibition. Collectively, we propose that the WAVE2-actin cytoskeleton axis is an important target of GSK-3β inhibitors in cancer cell invasion. Copyright © 2015 Elsevier Inc. All rights reserved.

Diastereomer-specific quantification of bioactive hexosylceramides from bacteria and mammals.

PubMed

von Gerichten, Johanna; Schlosser, Kerstin; Lamprecht, Dominic; Morace, Ivan; Eckhardt, Matthias; Wachten, Dagmar; Jennemann, Richard; Gröne, Hermann-Josef; Mack, Matthias; Sandhoff, Roger

2017-06-01

Mammals synthesize, cell-type specifically, the diastereomeric hexosylceramides, β-galactosylceramide (GalCer) and β-glucosylceramide (GlcCer), which are involved in several diseases, such as sphingolipidosis, diabetes, chronic kidney diseases, or cancer. In contrast, Bacteroides fragilis , a member of the human gut microbiome, and the marine sponge, Agelas mauritianus , produce α-GalCer, one of the most potent stimulators for invariant natural killer T cells. To dissect the contribution of these individual stereoisomers to pathologies, we established a novel hydrophilic interaction chromatography-based LC-MS 2 method and separated ( R > 1.5) corresponding diastereomers from each other, independent of their lipid anchors. Testing various bacterial and mammalian samples, we could separate, identify (including the lipid anchor composition), and quantify endogenous β-GlcCer, β-GalCer, and α-GalCer isomers without additional derivatization steps. Thereby, we show a selective decrease of β-GlcCers versus β-GalCers in cell-specific models of GlcCer synthase-deficiency and an increase of specific β-GlcCers due to loss of β-glucoceramidase 2 activity. Vice versa, β-GalCer increased specifically when cerebroside sulfotransferase ( Gal3st1 ) was deleted. We further confirm β-GalCer as substrate of globotriaosylceramide synthase for galabiaosylceramide synthesis and identify additional members of the human gut microbiome to contain immunogenic α-GalCers. Finally, this method is shown to separate corresponding hexosylsphingosine standards, promoting its applicability in further investigations. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

Hypoxic HepG2 cell adaptation decreases ATP synthase dimers and ATP production in inflated cristae by mitofilin down-regulation concomitant to MICOS clustering.

PubMed

Plecitá-Hlavatá, Lydie; Engstová, Hana; Alán, Lukáš; Špaček, Tomáš; Dlasková, Andrea; Smolková, Katarína; Špačková, Jitka; Tauber, Jan; Strádalová, Vendula; Malínský, Jan; Lessard, Mark; Bewersdorf, Joerg; Ježek, Petr

2016-05-01

The relationship of the inner mitochondrial membrane (IMM) cristae structure and intracristal space (ICS) to oxidative phosphorylation (oxphos) is not well understood. Mitofilin (subunit Mic60) of the mitochondrial contact site and cristae organizing system (MICOS) IMM complex is attached to the outer membrane (OMM) via the sorting and assembly machinery/topogenesis of mitochondrial outer membrane β-barrel proteins (SAM/TOB) complex and controls the shape of the cristae. ATP synthase dimers determine sharp cristae edges, whereas trimeric OPA1 tightens ICS outlets. Metabolism is altered during hypoxia, and we therefore studied cristae morphology in HepG2 cells adapted to 5% oxygen for 72 h. Three dimensional (3D), super-resolution biplane fluorescence photoactivation localization microscopy with Eos-conjugated, ICS-located lactamase-β indicated hypoxic ICS expansion with an unchanged OMM (visualized by Eos-mitochondrial fission protein-1). 3D direct stochastic optical reconstruction microscopy immunocytochemistry revealed foci of clustered mitofilin (but not MICOS subunit Mic19) in contrast to its even normoxic distribution. Mitofilin mRNA and protein decreased by ∼20%. ATP synthase dimers vs monomers and state-3/state-4 respiration ratios were lower during hypoxia. Electron microscopy confirmed ICS expansion (maximum in glycolytic cells), which was absent in reduced or OMM-detached cristae of OPA1- and mitofilin-silenced cells, respectively. Hypoxic adaptation is reported as rounding sharp cristae edges and expanding cristae width (ICS) by partial mitofilin/Mic60 down-regulation. Mitofilin-depleted MICOS detaches from SAM while remaining MICOS with mitofilin redistributes toward higher interdistances. This phenomenon causes partial oxphos dormancy in glycolytic cells via disruption of ATP synthase dimers.-Plecitá-Hlavatá, L., Engstová, H., Alán, L., Špaček, T., Dlasková, A., Smolková, K., Špačková, J., Tauber, J., Strádalová, V., Malínský, J., Lessard, M., Bewersdorf, J., Ježek, P. Hypoxic HepG2 cell adaptation decreases ATP synthase dimers and ATP production in inflated cristae by mitofilin down-regulation concomitant to MICOS clustering. © FASEB.

Recovery Effects of Oral Administration of Glucosylceramide and Beet Extract on Skin Barrier Destruction by UVB in Hairless Mice.

PubMed

Tokudome, Yoshihiro; Masutani, Noriomi; Uchino, Shohei; Fukai, Hisano

2017-10-27

Purified glucosylceramide from beet extract (beet GlcCer) and beet extract containing an equal amount of GlcCer were administered orally to ultra violet B (UVB)-irradiated mice, and differences in the protective effects against skin barrier dysfunction caused by UVB irradiation were compared. In the beet GlcCer group, epidermal thickening and the decrease in stratum corneum (SC) ceramide content caused by UVB irradiation were reduced. In the group that was orally administered beet extract containing glucosylceramide, effects similar to those in the beet GlcCer group were observed. Oral administration of beet GlcCer had no obvious effects against an increase in TEWL or decrease in SC water content after UVB irradiation, but there was improvement in the beet extract group. Oral administration of beet GlcCer is effective in improving skin barrier function in UVB-irradiated mice. Beet extract contains constituents other than GlcCer that are also effective in improving skin barrier function.

Ocean acidification modulates expression of genes and physiological performance of a marine diatom

NASA Astrophysics Data System (ADS)

Li, Y.; Zhuang, S.; Wu, Y.; Ren, H.; Cheng, F.; Lin, X.; Wang, K.; Beardall, J.; Gao, K.

2015-09-01

Ocean Acidification (OA) is known to affect various aspects of the physiological performance of diatoms, but there is little information on the underlining molecular mechanisms involved. Here, we show that in the model diatom Phaeodactylum tricornutum expression of the genes related to light harvesting, carbon acquisition and carboxylation, nitrite assimilation and ATP synthesis are modulated by OA. Growth and photosynthetic carbon fixation were enhanced by elevated CO2 (1000 μatm) under both constant indoor and fluctuating outdoor light regimes. The genetic expression of nitrite reductase (NiR) was up-regulated by OA regardless of light levels and/or regimes. The transcriptional expression of fucoxanthin chlorophyll a/c protein (lhcf type (FCP)) and mitochondrial ATP synthase (mtATP synthase) genes were also enhanced by OA, but only under high light intensity. OA treatment decreased the expression of β-carbonic anhydrase (β-CA) along with down-regulation of CO2 concentrating mechanisms (CCMs). Additionally, the genes for these proteins (NiR, FCP, mtATP synthase, β-CA) showed diel expressions either under constant indoor light or fluctuating sunlight. Thus, OA enhanced photosynthetic and growth rates by stimulating nitrogen assimilation and indirectly by down-regulating the energy-costly inorganic carbon acquisition process.

Mycobacterial glycolipids di-O-acylated trehalose and tri-O-acylated trehalose downregulate inducible nitric oxide synthase and nitric oxide production in macrophages.

PubMed

Espinosa-Cueto, Patricia; Escalera-Zamudio, Marina; Magallanes-Puebla, Alejandro; López-Marín, Luz María; Segura-Salinas, Erika; Mancilla, Raúl

2015-06-23

Tuberculosis (TB) remains a serious human health problem that affects millions of people in the world. Understanding the biology of Mycobacterium tuberculosis (Mtb) is essential for tackling this devastating disease. Mtb possesses a very complex cell envelope containing a variety of lipid components that participate in the establishment of the infection. We have previously demonstrated that di-O-acylated trehalose (DAT), a non-covalently linked cell wall glycolipid, inhibits the proliferation of T lymphocytes and the production of cytokines. In this work we show that DAT and the closely related tri-O-acylated trehalose (TAT) inhibits nitric oxide (NO) production and the inducible nitric oxide synthase (iNOS) expression in macrophages (MØ). These findings show that DAT and TAT are cell-wall located virulence factors that downregulate an important effector of the immune response against mycobacteria.

SGK2 promotes hepatocellular carcinoma progression and mediates GSK-3β/β-catenin signaling in HCC cells.

PubMed

Liu, Junying; Zhang, Guangdong; Lv, Yanping; Zhang, Xiaoyang; Ying, Cui; Yang, Suocheng; Kong, Xin; Yu, Yanzhang

2017-06-01

The phosphoinositide 3-kinase pathway is one of the most commonly altered pathways in human cancers. The serum/glucocorticoid-regulated kinase (SGK) family of serine/threonine kinases consists of three isoforms, SGK1, SGK2, and SGK3. This family of kinases is highly homologous to the AKT kinase family, sharing similar upstream activators and downstream targets. Few studies have investigated the role of SGK2 in hepatocellular carcinoma. Here, we report that SGK2 expression levels were upregulated in hepatocellular carcinoma tissues and human hepatoma cell lines compared to the adjacent normal liver tissues and a normal hepatocyte line, respectively. We found that downregulated SGK2 inhibits cell migration and invasive potential of hepatocellular carcinoma cell lines (SMMC-7721 and Huh-7).We also found that downregulated SGK2 suppressed the expression level of unphosphorylated (activated) glycogen synthase kinase 3 beta. In addition, SGK2 downregulation decreased the dephosphorylation (activation) of β-catenin by preventing its proteasomal degradation in the hepatocellular carcinoma cell lines. These findings suggest that SGK2 promotes hepatocellular carcinoma progression and mediates glycogen synthase kinase 3 beta/β-catenin signaling in hepatocellular carcinoma cells.

Stilbene synthase gene transfer caused alterations in the phenylpropanoid metabolism of transgenic strawberry (Fragaria×ananassa)

PubMed Central

Hanhineva, Kati; Kokko, Harri; Siljanen, Henri; Rogachev, Ilana; Aharoni, Asaph; Kärenlampi, Sirpa O.

2009-01-01

The gene encoding stilbene synthase is frequently used to modify plant secondary metabolism with the aim of producing the self-defence phytoalexin resveratrol. In this study, strawberry (Fragaria×ananassa) was transformed with the NS-Vitis3 gene encoding stilbene synthase from frost grape (Vitis riparia) under the control of the cauliflower mosaic virus 35S and the floral filament-specific fil1 promoters. Changes in leaf metabolites were investigated with UPLC-qTOF-MS (ultra performance liquid chromatography-quadrupole time of flight mass spectrometry) profiling, and increased accumulation of cinnamate, coumarate, and ferulate derivatives concomitantly with a decrease in the levels of flavonols was observed, while the anticipated resveratrol or its derivatives were not detected. The changed metabolite profile suggested that chalcone synthase was down-regulated by the genetic modification; this was verified by decreased chalcone synthase transcript levels. Changes in the levels of phenolic compounds led to increased susceptibility of the transgenic strawberry to grey mould fungus. PMID:19443619

Targeting glucosylceramide synthase induction of cell surface globotriaosylceramide (Gb3) in acquired cisplatin-resistance of lung cancer and malignant pleural mesothelioma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tyler, Andreas, E-mail: andreas.tyler@medbio.umu.se; Johansson, Anders; Karlsson, Terese

Background: Acquired resistance to cisplatin treatment is a caveat when treating patients with non-small cell lung cancer (NSCLC) and malignant pleural mesothelioma (MPM). Ceramide increases in response to chemotherapy, leading to proliferation arrest and apoptosis. However, a tumour stress activation of glucosylceramide synthase (GCS) follows to eliminate ceramide by formation of glycosphingolipids (GSLs) such as globotriaosylceramide (Gb3), the functional receptor of verotoxin-1. Ceramide elimination enhances cell proliferation and apoptosis blockade, thus stimulating tumor progression. GSLs transactivate multidrug resistance 1/P-glycoprotein (MDR1) and multidrug resistance-associated protein 1 (MRP1) expression which further prevents ceramide accumulation and stimulates drug efflux. We investigated the expressionmore » of Gb3, MDR1 and MRP1 in NSCLC and MPM cells with acquired cisplatin resistance, and if GCS activity or MDR1 pump inhibitors would reduce their expression and reverse cisplatin-resistance. Methods: Cell surface expression of Gb3, MDR1 and MRP1 and intracellular expression of MDR1 and MRP1 was analyzed by flow cytometry and confocal microscopy on P31 MPM and H1299 NSCLC cells and subline cells with acquired cisplatin resistance. The effect of GCS inhibitor PPMP and MDR1 pump inhibitor cyclosporin A for 72 h on expression and cisplatin cytotoxicity was tested. Results: The cisplatin-resistant cells expressed increased cell surface Gb3. Cell surface Gb3 expression of resistant cells was annihilated by PPMP whereas cyclosporin A decreased Gb3 and MDR1 expression in H1299 cells. No decrease of MDR1 by PPMP was noted in using flow cytometry, whereas a decrease of MDR1 in H1299 and H1299res was indicated with confocal microscopy. No certain co-localization of Gb3 and MDR1 was noted. PPMP, but not cyclosporin A, potentiated cisplatin cytotoxicity in all cells. Conclusions: Cell surface Gb3 expression is a likely tumour biomarker for acquired cisplatin resistance of NSCLC and MPM cells. Tumour cell resistance to MDR1 inhibitors of cell surface MDR1 and Gb3 could explain the aggressiveness of NSCLC and MPM. Therapy with GCS activity inhibitors or toxin targeting of the Gb3 receptor may substantially reduce acquired cisplatin drug resistance of NSCLC and MPM cells. - Highlights: • The cisplatin-resistant cells had increased cell surface Gb3 and MDR1. • PPMP decreased extracellular Gb3 in the resistant cell lines. • Cyclosporin A decreased extracellular Gb3 and MDR1 in H1299 cells. • PPMP, but not cyclosporin A, potentiated cisplatin cytotoxicity in all cells. • Resistance to inhibitors of MDR1 and Gb3 could explain aggressiveness of NSCLC and MPM.« less

Ylpex5 mutation partially suppresses the defective hyphal growth of a Yarrowia lipolytica ceramide synthase mutant, Yllac1, by recovering lipid raft polarization and vacuole morphogenesis.

PubMed

Bal, Jyotiranjan; Lee, Hye-Jeong; Cheon, Seon Ah; Lee, Kyung Jin; Oh, Doo-Byoung; Kim, Jeong-Yoon

2013-01-01

Sphingolipids are involved in cell differentiation and morphogenesis in eukaryotic cells. In this study, YlLac1p, a ceramide synthase required for glucosylceramide (GlcCer) synthesis, was found to be essential for hyphal growth in Yarrowia lipolytica. Y. lipolytica GlcCer was shown to be composed of a C16:0 fatty acid, which is hydroxylated at C2, and a C18:2 long chain base, which is unsaturated at both C4 and C8 and methylated at C9. Domain swapping analysis revealed that the entire TRAM/Lag1/CLN8 (TLC) domain, not the Lag1 motif, is crucial for the function of YlLac1p. YlDes1p, the C4 desaturase of the ceramide synthesized by YlLac1p, was also required for Y. lipolytica morphogenesis. Both Yllac1Δ and Yldes1Δ mutants neither polarize lipid rafts nor form normal vacuoles. Interestingly, mutation in YlPEX5, which encode a peroxisomal targeting signal receptor, partially suppressed the defective hyphal growth of Yllac1Δ. The Yllac1ΔYlpex5Δ mutant restored the ability to polarize lipid rafts and to form normal vacuoles, although it could not synthesize GlcCer. Taken together, our results suggest that GlcCer or GlcCer derivatives may be involved in hyphal morphogenesis in Y. lipolytica, at least in part, by affecting polarization of lipid rafts and vacuole morphogenesis. Copyright © 2012 Elsevier Inc. All rights reserved.

Resistance to pathogens in terpene down-regulated orange fruits inversely correlates with the accumulation of D-limonene in peel oil glands.

PubMed

Rodríguez, Ana; Shimada, Takehiko; Cervera, Magdalena; Redondo, Ana; Alquézar, Berta; Rodrigo, María Jesús; Zacarías, Lorenzo; Palou, Lluís; López, María M; Peña, Leandro

2015-01-01

Volatile organic compounds (VOCs) are secondary metabolites acting as a language for the communication of plants with the environment. In orange fruits, the monoterpene D-limonene accumulates at very high levels in oil glands from the peel. Drastic down-regulation of D-limonene synthase gene expression in the peel of transgenic oranges harboring a D-limonene synthase transgene in antisense (AS) configuration altered the monoterpene profile in oil glands, mainly resulting in reduced accumulation of D-limonene. This led to fruit resistance against Penicillium digitatum (Pd), Xanthomonas citri subsp. citri (Xcc) and other specialized pathogens. Here, we analyze resistance to pathogens in independent AS and empty vector (EV) lines, which have low, medium or high D-limonene concentrations and show that the level of resistance is inversely related to the accumulation of D-limonene in orange peels, thus explaining the need of high D-limonene accumulation in mature oranges in nature for the efficient attraction of specialized microorganism frugivores.

Resistance to pathogens in terpene down-regulated orange fruits inversely correlates with the accumulation of D-limonene in peel oil glands

PubMed Central

Rodríguez, Ana; Shimada, Takehiko; Cervera, Magdalena; Redondo, Ana; Alquézar, Berta; Rodrigo, María Jesús; Zacarías, Lorenzo; Palou, Lluís; López, María M; Peña, Leandro

2015-01-01

Volatile organic compounds (VOCs) are secondary metabolites acting as a language for the communication of plants with the environment. In orange fruits, the monoterpene D-limonene accumulates at very high levels in oil glands from the peel. Drastic down-regulation of D-limonene synthase gene expression in the peel of transgenic oranges harboring a D-limonene synthase transgene in antisense (AS) configuration altered the monoterpene profile in oil glands, mainly resulting in reduced accumulation of D-limonene. This led to fruit resistance against Penicillium digitatum (Pd), Xanthomonas citri subsp. citri (Xcc) and other specialized pathogens. Here, we analyze resistance to pathogens in independent AS and empty vector (EV) lines, which have low, medium or high D-limonene concentrations and show that the level of resistance is inversely related to the accumulation of D-limonene in orange peels, thus explaining the need of high D-limonene accumulation in mature oranges in nature for the efficient attraction of specialized microorganism frugivores. PMID:26023857

Collecting Duct Nitric Oxide Synthase 1ß Activation Maintains Sodium Homeostasis During High Sodium Intake Through Suppression of Aldosterone and Renal Angiotensin II Pathways.

PubMed

Hyndman, Kelly A; Mironova, Elena V; Giani, Jorge F; Dugas, Courtney; Collins, Jessika; McDonough, Alicia A; Stockand, James D; Pollock, Jennifer S

2017-10-24

During high sodium intake, the renin-angiotensin-aldosterone system is downregulated and nitric oxide signaling is upregulated in order to remain in sodium balance. Recently, we showed that collecting duct nitric oxide synthase 1β is critical for fluid-electrolyte balance and subsequently blood pressure regulation during high sodium feeding. The current study tested the hypothesis that high sodium activation of the collecting duct nitric oxide synthase 1β pathway is critical for maintaining sodium homeostasis and for the downregulation of the renin-angiotensin-aldosterone system-epithelial sodium channel axis. Male control and collecting duct nitric oxide synthase 1β knockout (CDNOS1KO) mice were placed on low, normal, and high sodium diets for 1 week. In response to the high sodium diet, plasma sodium was significantly increased in control mice and to a significantly greater level in CDNOS1KO mice. CDNOS1KO mice did not suppress plasma aldosterone in response to the high sodium diet, which may be partially explained by increased adrenal AT1R expression. Plasma renin concentration was appropriately suppressed in both genotypes. Furthermore, CDNOS1KO mice had significantly higher intrarenal angiotensin II with high sodium diet, although intrarenal angiotensinogen levels and angiotensin-converting enzyme activity were similar between knockout mice and controls. In agreement with inappropriate renin-angiotensin-aldosterone system activation in the CDNOS1KO mice on a high sodium diet, epithelial sodium channel activity and sodium transporter abundance were significantly higher compared with controls. These data demonstrate that high sodium activation of collecting duct nitric oxide synthase 1β signaling induces suppression of systemic and intrarenal renin-angiotensin-aldosterone system, thereby modulating epithelial sodium channel and other sodium transporter abundance and activity to maintain sodium homeostasis. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

RNAi-mediated down-regulation of a melanin polyketide synthase (pks1) gene in the fungus Slafractonia leguminicola

USDA-ARS?s Scientific Manuscript database

The fungus Slafractonia leguminicola, the causal agent of blackpatch disease of legumes produces two mycotoxins slaframine and swainsonine, causing slobbers’ symptoms and locoism of grazing animals, respectively. The genetics of this important fungus is poorly understood. This work aimed to develop ...

Artichoke, cynarin and cyanidin downregulate the expression of inducible nitric oxide synthase in human coronary smooth muscle cells.

PubMed

Xia, Ning; Pautz, Andrea; Wollscheid, Ursula; Reifenberg, Gisela; Förstermann, Ulrich; Li, Huige

2014-03-24

Artichoke (Cynara scolymus L.) is one of the world's oldest medicinal plants with multiple health benefits. We have previously shown that artichoke leaf extracts and artichoke flavonoids upregulate the gene expression of endothelial-type nitric oxide synthase (eNOS) in human endothelial cells. Whereas NO produced by the eNOS is a vasoprotective molecule, NO derived from the inducible iNOS plays a pro-inflammatory role in the vasculature. The present study was aimed to investigate the effects of artichoke on iNOS expression in human coronary artery smooth muscle cells (HCASMC). Incubation of HCASMC with a cytokine mixture led to an induction of iNOS mRNA expression. This iNOS induction was concentration- and time-dependently inhibited by an artichoke leaf extract (1-100 µg/mL, 6 h or 24 h). Consistently, the artichoke leaf extract also reduced cytokine-induced iNOS promoter activation and iNOS protein expression. In addition, treatment of HCASMC with four well-known artichoke compounds (cynarin > cyanidin > luteolin ≈ cynaroside) led to a downregulation iNOS mRNA and protein expression, with cynarin being the most potent one. In conclusion, artichoke contains both eNOS-upregulating and iNOS-downregulating compounds. Such compounds may contribute to the beneficial effects of artichoke and may per se have therapeutic potentials.

Glycogen synthase kinase-3 negatively regulates anti-inflammatory interleukin-10 for lipopolysaccharide-induced iNOS/NO biosynthesis and RANTES production in microglial cells

PubMed Central

Huang, Wei-Ching; Lin, Yee-Shin; Wang, Chi-Yun; Tsai, Cheng-Chieh; Tseng, Hsiang-Chi; Chen, Chia-Ling; Lu, Pei-Jung; Chen, Po-See; Qian, Li; Hong, Jau-Shyong; Lin, Chiou-Feng

2009-01-01

The inflammatory effects of glycogen synthase kinase-3 (GSK-3) have been identified; however, the potential mechanism is still controversial. In this study, we investigated the effects of GSK-3-mediated interleukin-10 (IL-10) inhibition on lipopolysaccharide (LPS)-induced inflammation. Treatment with GSK-3 inhibitor significantly blocked LPS-induced nitric oxide (NO) production as well as inducible NO synthase (iNOS) expression in BV2 murine microglial cells and primary rat microglia-enriched cultures. Using an antibody array and enzyme-linked immunosorbent assay, we found that GSK-3-inhibitor treatment blocked LPS-induced upregulation of regulated on activation normal T-cell expressed and secreted (RANTES) and increased IL-10 expression. The time kinetics and dose–response relations were confirmed. Reverse transcription–polymerase chain reaction showed changes on the messenger RNA level as well. Inhibiting GSK-3 using short-interference RNA, and transfecting cells with dominant-negative GSK-3β, blocked LPS-elicited NO and RANTES expression but increased IL-10 expression. In contrast, GSK-3β overexpression upregulated NO and RANTES but downregulated IL-10 in LPS-stimulated cells. Treating cells with anti-IL-10 neutralizing antibodies to prevent GSK-3 from downregulating NO and RANTES showed that the anti-inflammatory effects are, at least in part, IL-10-dependent. The involvement of Akt, extracellular signal-regulated kinase, p38 mitogen-activated protein kinase and nuclear factor-κB that positively regulated IL-10 was demonstrated. Furthermore, inhibiting GSK-3 increased the nuclear translocation of transcription factors, that all important for IL-10 expression, including CCAAT/enhancer-binding protein beat (C/EBPβ), C/EBPδ, cAMP response binding element protein and NF-κB. Taken together, these findings reveal that LPS induces iNOS/NO biosynthesis and RANTES production through a mechanism involving GSK-3-mediated IL-10 downregulation. PMID:19175796

Glycogen synthase kinase-3 negatively regulates anti-inflammatory interleukin-10 for lipopolysaccharide-induced iNOS/NO biosynthesis and RANTES production in microglial cells.

PubMed

Huang, Wei-Ching; Lin, Yee-Shin; Wang, Chi-Yun; Tsai, Cheng-Chieh; Tseng, Hsiang-Chi; Chen, Chia-Ling; Lu, Pei-Jung; Chen, Po-See; Qian, Li; Hong, Jau-Shyong; Lin, Chiou-Feng

2009-09-01

The inflammatory effects of glycogen synthase kinase-3 (GSK-3) have been identified; however, the potential mechanism is still controversial. In this study, we investigated the effects of GSK-3-mediated interleukin-10 (IL-10) inhibition on lipopolysaccharide (LPS)-induced inflammation. Treatment with GSK-3 inhibitor significantly blocked LPS-induced nitric oxide (NO) production as well as inducible NO synthase (iNOS) expression in BV2 murine microglial cells and primary rat microglia-enriched cultures. Using an antibody array and enzyme-linked immunosorbent assay, we found that GSK-3-inhibitor treatment blocked LPS-induced upregulation of regulated on activation normal T-cell expressed and secreted (RANTES) and increased IL-10 expression. The time kinetics and dose-response relations were confirmed. Reverse transcription-polymerase chain reaction showed changes on the messenger RNA level as well. Inhibiting GSK-3 using short-interference RNA, and transfecting cells with dominant-negative GSK-3beta, blocked LPS-elicited NO and RANTES expression but increased IL-10 expression. In contrast, GSK-3beta overexpression upregulated NO and RANTES but downregulated IL-10 in LPS-stimulated cells. Treating cells with anti-IL-10 neutralizing antibodies to prevent GSK-3 from downregulating NO and RANTES showed that the anti-inflammatory effects are, at least in part, IL-10-dependent. The involvement of Akt, extracellular signal-regulated kinase, p38 mitogen-activated protein kinase and nuclear factor-kappaB that positively regulated IL-10 was demonstrated. Furthermore, inhibiting GSK-3 increased the nuclear translocation of transcription factors, that all important for IL-10 expression, including CCAAT/enhancer-binding protein beat (C/EBPbeta), C/EBPdelta, cAMP response binding element protein and NF-kappaB. Taken together, these findings reveal that LPS induces iNOS/NO biosynthesis and RANTES production through a mechanism involving GSK-3-mediated IL-10 downregulation.

Structural analysis of cerebrosides from Aspergillus fungi: the existence of galactosylceramide in A. oryzae.

PubMed

Tani, Yasushi; Amaishi, Yasunori; Funatsu, Tori; Ito, Masahiro; Itonori, Saki; Hata, Yoji; Ashida, Hisashi; Yamamoto, Kenji

2014-12-01

Glucosylceramide and galactosylceramide were detected in three Aspergillus species: Aspergillus oryzae, Aspergillus sojae and Aspergillus. awamori, using borate-coated TLC. The cerebrosides from A. oryzae were further purified by ion exchange and iatrobeads column chromatographies with or without borate, and determined the composition of sugar, fatty acid and sphingoid base by GC/MS, MALDI-TOF/MS and (1)H-NMR. We identified them as β-glucosylceramide and β-galactosylceramide. The ceramide moiety of both cerebrosides consisted mainly of 2-hydroxystearic acid and either 9-methyl-octadeca-4, 8-sphingadienine or octadeca-4, 8-sphingadienine. To our knowledge, this is the first study to provide evidence for the presence of β-galactosylceramide in A. oryzae.

14-3-3 protein is a regulator of the mitochondrial and chloroplast ATP synthase.

PubMed

Bunney, T D; van Walraven, H S; de Boer, A H

2001-03-27

Mitochondrial and chloroplast ATP synthases are key enzymes in plant metabolism, providing cells with ATP, the universal energy currency. ATP synthases use a transmembrane electrochemical proton gradient to drive synthesis of ATP. The enzyme complexes function as miniature rotary engines, ensuring energy coupling with very high efficiency. Although our understanding of the structure and functioning of the synthase has made enormous progress in recent years, our understanding of regulatory mechanisms is still rather preliminary. Here we report a role for 14-3-3 proteins in the regulation of ATP synthases. These 14-3-3 proteins are highly conserved phosphoserine/phosphothreonine-binding proteins that regulate a wide range of enzymes in plants, animals, and yeast. Recently, the presence of 14-3-3 proteins in chloroplasts was illustrated, and we show here that plant mitochondria harbor 14-3-3s within the inner mitochondrial-membrane compartment. There, the 14-3-3 proteins were found to be associated with the ATP synthases, in a phosphorylation-dependent manner, through direct interaction with the F(1) beta-subunit. The activity of the ATP synthases in both organelles is drastically reduced by recombinant 14-3-3. The rapid reduction in chloroplast ATPase activity during dark adaptation was prevented by a phosphopeptide containing the 14-3-3 interaction motif, demonstrating a role for endogenous 14-3-3 in the down-regulation of the CF(o)F(1) activity. We conclude that regulation of the ATP synthases by 14-3-3 represents a mechanism for plant adaptation to environmental changes such as light/dark transitions, anoxia in roots, and fluctuations in nutrient supply.

Studies on the Expression of Sesquiterpene Synthases Using Promoter-β-Glucuronidase Fusions in Transgenic Artemisia annua L

PubMed Central

Wang, Hongzhen; Han, Junli; Kanagarajan, Selvaraju; Lundgren, Anneli; Brodelius, Peter E.

2013-01-01

In order to better understand the influence of sesquiterpene synthases on artemisinin yield in Artemisia annua, the expression of some sesquiterpene synthases has been studied using transgenic plants expressing promoter-GUS fusions. The cloned promoter sequences were 923, 1182 and 1510 bp for β-caryophyllene (CPS), epi-cedrol (ECS) and β-farnesene (FS) synthase, respectively. Prediction of cis-acting regulatory elements showed that the promoters are involved in complex regulation of expression. Transgenic A. annua plants carrying promoter-GUS fusions were studied to elucidate the expression pattern of the three sesquiterpene synthases and compared to the previously studied promoter of amorpha-4,11-diene synthase (ADS), a key enzyme of artemisinin biosynthesis. The CPS and ECS promoters were active in T-shaped trichomes of leaves and stems, basal bracts of flower buds and also in some florets cells but not in glandular secretory trichome while FS promoter activity was only observed in leaf cells and trichomes of transgenic shoots. ADS, CPS, ECS and FS transcripts were induced by wounding in a time depended manner. The four sesquiterpene synthases may be involved in responsiveness of A. annua to herbivory. Methyl jasmonate treatment triggered activation of the promoters of all four sesquiterpene synthases in a time depended manner. Southern blot result showed that the GUS gene was inserted into genomic DNA of transgenic lines as a single copy or two copies. The relative amounts of CPS and ECS as well as germacrene A synthase (GAS) transcripts are much lower than that of ADS transcript. Consequently, down-regulation of the expression of the CPS, ECS or GAS gene may not improve artemsinin yield. However, blocking the expression of FS may have effects on artemisinin production. PMID:24278301

The Ubiquitin Ligase SCF(Ucc1) Acts as a Metabolic Switch for the Glyoxylate Cycle.

PubMed

Nakatsukasa, Kunio; Nishimura, Takashi; Byrne, Stuart D; Okamoto, Michiyo; Takahashi-Nakaguchi, Azusa; Chibana, Hiroji; Okumura, Fumihiko; Kamura, Takumi

2015-07-02

Despite the crucial role played by the glyoxylate cycle in the virulence of pathogens, seed germination in plants, and sexual development in fungi, we still have much to learn about its regulation. Here, we show that a previously uncharacterized SCF(Ucc1) ubiquitin ligase mediates proteasomal degradation of citrate synthase in the glyoxylate cycle to maintain metabolic homeostasis in glucose-grown cells. Conversely, transcription of the F box subunit Ucc1 is downregulated in C2-compound-grown cells, which require increased metabolic flux for gluconeogenesis. Moreover, in vitro analysis demonstrates that oxaloacetate regenerated through the glyoxylate cycle induces a conformational change in citrate synthase and inhibits its recognition and ubiquitination by SCF(Ucc1), suggesting the existence of an oxaloacetate-dependent positive feedback loop that stabilizes citrate synthase. We propose that SCF(Ucc1)-mediated regulation of citrate synthase acts as a metabolic switch for the glyoxylate cycle in response to changes in carbon source, thereby ensuring metabolic versatility and flexibility. Copyright © 2015 Elsevier Inc. All rights reserved.

The effect of 24S-hydroxycholesterol on cholesterol homeostasis in neurons: quantitative changes to the cortical neuron proteome.

PubMed

Wang, Yuqin; Muneton, Sabina; Sjövall, Jan; Jovanovic, Jasmina N; Griffiths, William J

2008-04-01

In humans, the brain represents only about 2% of the body's mass but contains about one-quarter of the body's free cholesterol. Cholesterol is synthesized de novo in brain and removed by metabolism to oxysterols. 24S-Hydoxycholesterol represents the major metabolic product of cholesterol in brain, being formed via the cytochrome P450 (CYP) enzyme CYP46A1. CYP46A1 is expressed exclusively in brain, normally by neurons. In this study, we investigated the effect of 24S-hydroxycholesterol on the proteome of rat cortical neurons. With the use of two-dimensional liquid chromatography linked to nanoelectrospray tandem mass spectrometry, over 1040 proteins were identified including members of the cholesterol, isoprenoid and fatty acid synthesis pathways. With the use of stable isotope labeling technology, the protein expression patterns of enzymes in these pathways were investigated. 24S-Hydroxycholesterol was found to down-regulate the expression of members of the cholesterol/isoprenoid synthesis pathways including 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (EC 2.3.3.10), diphosphomevalonate decarboxylase (EC 4.1.1.33), isopentenyl-diphosphate delta isomerase (EC 5.3.3.2), farnesyl-diphosphate synthase (Geranyl trans transferase, EC 2.5.1.10), and dedicated sterol synthesis enzymes, farnesyl-diphosphate farnesyltransferase 1 (squalene synthase, EC 2.5.1.21) and methylsterol monooxygenase (EC 1.14.13.72). The expression of many enzymes in the cholesterol/isoprenoid and fatty acid synthesis pathways are regulated by the membrane-bound transcription factors named sterol regulatory element-binding proteins (SREBPs), which themselves are both transcriptionally and post-transcriptionally regulated. The current proteomic data indicates that 24S-hydroxycholesterol down-regulates cholesterol synthesis in neurons, possibly, in a post-transcriptional manner through SREBP-2. In contrast to cholesterol metabolism, enzymes responsible for the synthesis of fatty acids were not found to be down-regulated in neurons treated with 24S-hydroxycholesterol, while apolipoprotein E (apo E), a cholesterol trafficking protein, was found to be up-regulated. Taken together, this data leads to the hypothesis that, in times of cholesterol excess, 24S-hydroxycholesterols signals down-regulation of cholesterol synthesis enzymes through SREBP-2, but up-regulates apo E synthesis (through the liver X receptor) leading to cholesterol storage and restoration of cholesterol balance.

Structural analysis of glucosylceramides (GlcCer) from species of the Pseudallescheria/Scedosporium complex.

PubMed

Calixto, Renata O R; Rollin-Pinheiro, Rodrigo; da Silva, Mariana I D; Liporagi-Lopes, Livia C; Vieira, Jardel M; Sassaki, Guilherme L; Barreto-Bergter, Eliana

2016-02-01

Glucosylceramides (GlcCer) are the main neutral glycosphingolipids expressed in fungal cells. In this work, glucosylceramides (GlcCer) were extracted from three strains of Scedosporium (Pseudallescheria) boydii, one strain of Pseudallescheria ellipsoidea and one strain of Pseudallescheria angusta and purified by several chromatographic steps. Using high-performance thin layer chromatography (HPTLC), we found a similarity between GlcCer obtained from all of the analysed strains. A detailed structural analysis of the P. ellipsoidea GlcCer was performed via electrospray ionization mass spectrometry (ESI-MS) and confirmed in 1- and 2-D heteronuclear NMR experiments ((1)H-(13) C HSQC). GlcCer species produced by mycelial forms of these strains displayed the same structure previously demonstrated by our group for P. boydii, Cryptococcus neoformans, Pseudallescheria minustipora, Fusarium solani, and Colletotrichum gloesporioides. A monoclonal antibody (mAb) against GlcCer was used for immunofluorescence experiments. Our results revealed that GlcCer is present on the surface of these fungi, and no difference was observed in the GlcCer structure of the present set of strains in terms of geographic or clinical origin, suggesting a conserved GlcCer structure similar to those previously described for Scedosporium apiospermum, Scedosporium aurantiacum, and P. minutispora. The surface distribution of GlcCer in these fungi is suggestive of the involvement of this molecule in fungal growth. Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Cardioprotective Effect of Danshensu against Ischemic/Reperfusion Injury via c-Subunit of ATP Synthase Inhibition

PubMed Central

Zhao, JingYi; Fan, Zixuan; Bao, Jiadi; Sun, Dawei; Sun, Chun

2017-01-01

Mitochondrial permeability transition pore (MPTP) opening is the main culprit of ischemic/reperfusion (IR) injury. It is reported that c-subunit of ATP synthase is the core component of MPTP. Danshensu (DSS), a monomer isolated from the traditional Chinese herb Danshen, has showed cardioprotective effect against IR injury through unknown mechanism. In this study, rat hearts were suspended in Langendorff instrument and perfused with Krebs-Henseleit (KH) buffer containing DSS for 60 minutes, followed by 30 minutes of global ischemia. Parameters including heart rate, left ventricular developed pressure, and the rate of left ventricle diastolic pressure change were recorded to assess their cardiac function. All these indexes were improved in DSS group. The rate of cardiomyocytes apoptosis and MPTP opening were both inhibited in DSS group. In addition, DSS administration leads to downregulation of c-subunit of ATP synthase in both mRNA and protein levels. Consistently, when c-subunit of ATP synthase was overexpressed in H9C2 cells through pcDNA3/5G1 plasmid transfection, MPTP opening was enhanced when the cardioprotective effect of DSS also tapers. In conclusion, DSS could alleviate cardiac IR injury via inhibiting c-subunit of ATP synthase expression. PMID:29250127

Flavone inhibits nitric oxide synthase (NOS) activity, nitric oxide production and protein S-nitrosylation in breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Wenzhen; Yang, Bingwu; Fu, Huiling

As the core structure of flavonoids, flavone has been proved to possess anticancer effects. Flavone's growth inhibitory functions are related to NO. NO is synthesized by nitric oxide synthase (NOS), and generally increased in a variety of cancer cells. NO regulates multiple cellular responses by S-nitrosylation. In this study, we explored flavone-induced regulations on nitric oxide (NO)-related cellular processes in breast cancer cells. Our results showed that, flavone suppresses breast cancer cell proliferation and induces apoptosis. Flavone restrains NO synthesis by does-dependent inhibiting NOS enzymatic activity. The decrease of NO generation was detected by fluorescence microscopy and flow cytometry. Flavone-inducedmore » inhibitory effect on NOS activity is dependent on intact cell structure. For the NO-induced protein modification, flavone treatment significantly down-regulated protein S-nitrosylation, which was detected by “Biotin-switch” method. The present study provides a novel, NO-related mechanism for the anticancer function of flavone. - Highlights: • Flavone inhibits proliferation and induces apoptosis in MCF-7 cells. • Flavone decreases nitric oxide production by inhibiting NOS enzymatic activity in breast cancer cells. • Flavone down-regulates protein S-nitrosylation.« less

Glycogen Synthase Kinase 3 Inactivation Drives T-bet-Mediated Downregulation of Co-receptor PD-1 to Enhance CD8+ Cytolytic T Cell Responses

PubMed Central

Taylor, Alison; Harker, James A.; Chanthong, Kittiphat; Stevenson, Philip G.; Zuniga, Elina I.; Rudd, Christopher E.

2016-01-01

Summary Despite the importance of the co-receptor PD-1 in T cell immunity, the upstream signaling pathway that regulates PD-1 expression has not been defined. Glycogen synthase kinase 3 (GSK-3, isoforms α and β) is a serine-threonine kinase implicated in cellular processes. Here, we identified GSK-3 as a key upstream kinase that regulated PD-1 expression in CD8+ T cells. GSK-3 siRNA downregulation, or inhibition by small molecules, blocked PD-1 expression, resulting in increased CD8+ cytotoxic T lymphocyte (CTL) function. Mechanistically, GSK-3 inactivation increased Tbx21 transcription, promoting enhanced T-bet expression and subsequent suppression of Pdcd1 (encodes PD-1) transcription in CD8+ CTLs. Injection of GSK-3 inhibitors in mice increased in vivo CD8+ OT-I CTL function and the clearance of murine gamma-herpesvirus 68 and lymphocytic choriomeningitis clone 13 and reversed T cell exhaustion. Our findings identify GSK-3 as a regulator of PD-1 expression and demonstrate the applicability of GSK-3 inhibitors in the modulation of PD-1 in immunotherapy. PMID:26885856

Differential gene expression at different stages of mesocarp development in high- and low-yielding oil palm.

PubMed

Wong, Yick Ching; Teh, Huey Fang; Mebus, Katharina; Ooi, Tony Eng Keong; Kwong, Qi Bin; Koo, Ka Loo; Ong, Chuang Kee; Mayes, Sean; Chew, Fook Tim; Appleton, David R; Kulaveerasingam, Harikrishna

2017-06-21

The oil yield trait of oil palm is expected to involve multiple genes, environmental influences and interactions. Many of the underlying mechanisms that contribute to oil yield are still poorly understood. In this study, we used a microarray approach to study the gene expression profiles of mesocarp tissue at different developmental stages, comparing genetically related high- and low- oil yielding palms to identify genes that contributed to the higher oil-yielding palm and might contribute to the wider genetic improvement of oil palm breeding populations. A total of 3412 (2001 annotated) gene candidates were found to be significantly differentially expressed between high- and low-yielding palms at at least one of the different stages of mesocarp development evaluated. Gene Ontologies (GO) enrichment analysis identified 28 significantly enriched GO terms, including regulation of transcription, fatty acid biosynthesis and metabolic processes. These differentially expressed genes comprise several transcription factors, such as, bHLH, Dof zinc finger proteins and MADS box proteins. Several genes involved in glycolysis, TCA, and fatty acid biosynthesis pathways were also found up-regulated in high-yielding oil palm, among them; pyruvate dehydrogenase E1 component Subunit Beta (PDH), ATP-citrate lyase, β- ketoacyl-ACP synthases I (KAS I), β- ketoacyl-ACP synthases III (KAS III) and ketoacyl-ACP reductase (KAR). Sucrose metabolism-related genes such as Invertase, Sucrose Synthase 2 and Sucrose Phosphatase 2 were found to be down-regulated in high-yielding oil palms, compared to the lower yield palms. Our findings indicate that a higher carbon flux (channeled through down-regulation of the Sucrose Synthase 2 pathway) was being utilized by up-regulated genes involved in glycolysis, TCA and fatty acid biosynthesis leading to enhanced oil production in the high-yielding oil palm. These findings are an important stepping stone to understand the processes that lead to production of high-yielding oil palms and have implications for breeding to maximize oil production.

Gaucher's disease with myocardial involvement in pregnancy.

PubMed

Torloni, Maria Regina; Franco, Kátia; Sass, Nelson

2002-05-02

Described originally in 1882, Gaucher's disease is the most prevalent of storage disorders. This autosomal recessive disease is caused by a defective gene responsible for coding the beta-glucosidase enzyme, essential in the hydrolysis of glucosylceramide in glucose and ceramide. The accumulation of glucosylceramide in the lysosomes of the reticuloendothelial system produces a heterogeneous clinical picture with neurological involvement, liver and spleen enlargement, hematological disorders and bone lesions. Two pregnancies of a patient with Gaucher's disease are presented. The patient, who had been asymptomatic following earlier splenectomy, developed congestive heart failure due to myocardial involvement at the beginning of her first pregnancy, and responded to conservative treatment. In spite of this complication and also chronic anemia, hepatomegaly and ascites due to portal hypertension, the patient had two successful pregnancies with good perinatal results. No hemorrhagic complications were observed.

Glycogen synthase kinase-3 as drug target: from wallflower to center of attention.

PubMed

Van Wauwe, Jean; Haefner, Burkhard

2003-11-01

Some 20 years ago, glycogen synthase kinase-3 (GSK-3) was categorized as one of several protein kinases that could phosphorylate glycogen synthase and regulate the glucose metabolism pathway. Today, GSK-3 is being identified as a ubiquitous serine/threonine protein kinase that participates in a multitude of cellular processes, ranging from cell membrane-to-nucleus signaling, gene transcription, translation, cytoskeletal organization to cell cycle progression and survival. Two functional aspects make GSK-3 a peculiar kinase: its activity is constitutive and downregulated after cell activation by phosphorylation or interaction with inhibitory proteins, and the enzyme prefers substrates that are specifically prepared, that is prephosphorylated, by other kinases. Its pleiotropic but unique activities have made GSK-3 a much sought-after target for the treatment of prevalent human diseases such as type 2 diabetes and Alzheimer's disease. Recent drug discovery efforts have identified small-molecule, orally active inhibitors of GSK-3. This accomplishment may represent the first step toward the development of novel therapeutic agents.

Astaxanthin enhances pemetrexed-induced cytotoxicity by downregulation of thymidylate synthase expression in human lung cancer cells.

PubMed

Liao, Kai-Sheng; Wei, Chia-Li; Chen, Jyh-Cheng; Zheng, Hao-Yu; Chen, Wen-Ching; Wu, Chia-Hung; Wang, Tai-Jing; Peng, Yi-Shuan; Chang, Po-Yuan; Lin, Yun-Wei

2016-11-01

Pemetrexed, a multitargeted antifolate agent, has demonstrated clinical activity in non-small cell lung cancer (NSCLC) cells. Increased expression of thymidylate synthase (TS) is thought to be associated with resistance to pemetrexed. Astaxanthin exhibits a wide range of beneficial effects including anti-cancer and anti-inflammatory properties. In this study, we showed that down-regulating of TS expression in two NSCLC cell lines, human lung adenocarcinoma H1650 and squamous cell carcinoma H1703 cells, with astaxanthin were associated with decreased MKK1/2-ERK1/2 activity. Enforced expression of constitutively active MKK1 (MKK1-CA) vector significantly rescued the decreased TS mRNA and protein levels in astaxanthin-treated NSCLC cells. Combined treatment with a MKK1/2 inhibitor (U0126 or PD98059) further decreased the TS expression in astaxanthin-exposed NSCLC cells. Knockdown of TS using small interfering RNA (siRNA) or inhibiting ERK1/2 activity enhanced the cytotoxicity and cell growth inhibition of astaxanthin. Combination of pemetrexed and astaxanthin resulted in synergistic enhancing cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced activation of phospho-MKK1/2, phopho-ERK1/2, and TS expression. Overexpression of MKK1/2-CA reversed the astaxanthin and pemetrexed-induced synergistic cytotoxicity. Our findings suggested that the down-regulation of MKK1/2-ERK1/2-mediated TS expression by astaxanthin is an important regulator of enhancing the pemetrexed-induced cytotoxicity in NSCLC cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Chloroplast ATP Synthase Modulation of the Thylakoid Proton Motive Force: Implications for Photosystem I and Photosystem II Photoprotection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kanazawa, Atsuko; Ostendorf, Elisabeth; Kohzuma, Kaori

In wild type plants, decreasing CO 2 lowers the activity of the chloroplast ATP synthase, slowing proton efflux from the thylakoid lumen resulting in buildup of thylakoid proton motive force (pmf). The resulting acidification of the lumen regulates both light harvesting, via the qE mechanism, and photosynthetic electron transfer through the cytochrome b 6f complex. Here in this paper, we show that the cfq mutant of Arabidopsis, harboring single point mutation in its γ-subunit of the chloroplast ATP synthase, increases the specific activity of the ATP synthase and disables its down-regulation under low CO 2. The increased thylakoid proton conductivitymore » (g H +) in cfq results in decreased pmf and lumen acidification, preventing full activation of qE and more rapid electron transfer through the b6f complex, particularly under low CO 2 and fluctuating light. These conditions favor the accumulation of electrons on the acceptor side of PSI, and result in severe loss of PSI activity. Comparing the current results with previous work on the pgr5 mutant suggests a general mechanism where increased PSI photodamage in both mutants is caused by loss of pmf, rather than inhibition of CEF per se. Overall, our results support a critical role for ATP synthase regulation in maintaining photosynthetic control of electron transfer to prevent photodamage.« less

Chloroplast ATP Synthase Modulation of the Thylakoid Proton Motive Force: Implications for Photosystem I and Photosystem II Photoprotection

DOE PAGES

Kanazawa, Atsuko; Ostendorf, Elisabeth; Kohzuma, Kaori; ...

2017-05-03

In wild type plants, decreasing CO 2 lowers the activity of the chloroplast ATP synthase, slowing proton efflux from the thylakoid lumen resulting in buildup of thylakoid proton motive force (pmf). The resulting acidification of the lumen regulates both light harvesting, via the qE mechanism, and photosynthetic electron transfer through the cytochrome b 6f complex. Here in this paper, we show that the cfq mutant of Arabidopsis, harboring single point mutation in its γ-subunit of the chloroplast ATP synthase, increases the specific activity of the ATP synthase and disables its down-regulation under low CO 2. The increased thylakoid proton conductivitymore » (g H +) in cfq results in decreased pmf and lumen acidification, preventing full activation of qE and more rapid electron transfer through the b6f complex, particularly under low CO 2 and fluctuating light. These conditions favor the accumulation of electrons on the acceptor side of PSI, and result in severe loss of PSI activity. Comparing the current results with previous work on the pgr5 mutant suggests a general mechanism where increased PSI photodamage in both mutants is caused by loss of pmf, rather than inhibition of CEF per se. Overall, our results support a critical role for ATP synthase regulation in maintaining photosynthetic control of electron transfer to prevent photodamage.« less

Elk-3 is a transcriptional repressor of nitric-oxide synthase 2.

PubMed

Chen, Yen-Hsu; Layne, Matthew D; Chung, Su Wol; Ejima, Kuniaki; Baron, Rebecca M; Yet, Shaw-Fang; Perrella, Mark A

2003-10-10

The inducible isoform of nitric-oxide synthase (NOS2), a key enzyme catalyzing the dramatic increase in nitric oxide by lipopolysaccharide (LPS), plays an important role in the pathophysiology of endotoxemia and sepsis. Recent evidence suggests that Ets transcription factors may contribute to NOS2 induction by inflammatory stimuli. In this study, we investigated the role of Ets transcription factors in the regulation of NOS2 by LPS and transforming growth factor (TGF)-beta 1. Transient transfection assays in macrophages showed that Ets-2 produced an increase in NOS2 promoter activity, whereas the induction by Ets-1 was modest and NERF2 had no effect. Elk-3 (Net/Erp/Sap-2a) markedly repressed NOS2 promoter activity in a dose-dependent fashion, and overexpression of Elk-3 blunted the induction of endogenous NOS2 message. Mutation of the Net inhibitory domain of Elk-3, but not the C-terminal-binding protein interaction domain, partially alleviated this repressive effect. We also found that deletion of the Ets domain of Elk-3 completely abolished its repressive effect on the NOS2 promoter. LPS administration to macrophages led to a dose-dependent decrease in endogenous Elk-3 mRNA levels, and this decrease in Elk-3 preceded the induction of NOS2 mRNA. In a mouse model of endotoxemia, the expression of Elk-3 in kidney, lung, and heart was significantly down-regulated after systemic administration of LPS, and this down-regulation also preceded NOS2 induction. Moreover, TGF-beta 1 significantly increased endogenous Elk-3 mRNA levels that had been down-regulated by LPS in macrophages. This increase in Elk-3 correlated with a TGF-beta 1-induced down-regulation of NOS2. Taken together, our data suggest that Elk-3 is a strong repressor of NOS2 promoter activity and mRNA levels and that endogenous expression of Elk-3 inversely correlates with NOS2. Thus, Elk-3 may serve as an important mediator of NOS2 gene expression.

Down-regulation of the CSLF6 gene results in decreased (1,3;1,4)-beta-D-glucan in endosperm of wheat.

PubMed

Nemeth, Csilla; Freeman, Jackie; Jones, Huw D; Sparks, Caroline; Pellny, Till K; Wilkinson, Mark D; Dunwell, Jim; Andersson, Annica A M; Aman, Per; Guillon, Fabienne; Saulnier, Luc; Mitchell, Rowan A C; Shewry, Peter R

2010-03-01

(1,3;1,4)-beta-d-Glucan (beta-glucan) accounts for 20% of the total cell walls in the starchy endosperm of wheat (Triticum aestivum) and is an important source of dietary fiber for human nutrition with potential health benefits. Bioinformatic and array analyses of gene expression profiles in developing caryopses identified the CELLULOSE SYNTHASE-LIKE F6 (CSLF6) gene as encoding a putative beta-glucan synthase. RNA interference constructs were therefore designed to down-regulate CSLF6 gene expression and expressed in transgenic wheat under the control of a starchy endosperm-specific HMW subunit gene promoter. Analysis of wholemeal flours using an enzyme-based kit and by high-performance anion-exchange chromatography after digestion with lichenase showed decreases in total beta-glucan of between 30% and 52% and between 36% and 53%, respectively, in five transgenic lines compared to three control lines. The content of water-extractable beta-glucan was also reduced by about 50% in the transgenic lines, and the M(r) distribution of the fraction was decreased from an average of 79 to 85 x 10(4) g/mol in the controls and 36 to 57 x 10(4) g/mol in the transgenics. Immunolocalization of beta-glucan in semithin sections of mature and developing grains confirmed that the impact of the transgene was confined to the starchy endosperm with little or no effect on the aleurone or outer layers of the grain. The results confirm that the CSLF6 gene of wheat encodes a beta-glucan synthase and indicate that transgenic manipulation can be used to enhance the health benefits of wheat products.

Nitric-oxide synthase trafficking inducer is a pleiotropic regulator of endothelial cell function and signaling

PubMed Central

2017-01-01

Endothelial nitric-oxide synthase (eNOS) and its bioactive product, nitric oxide (NO), mediate many endothelial cell functions, including angiogenesis and vascular permeability. For example, vascular endothelial growth factor (VEGF)-mediated angiogenesis is inhibited upon reduction of NO bioactivity both in vitro and in vivo. Moreover, genetic disruption or pharmacological inhibition of eNOS attenuates angiogenesis during tissue repair, resulting in delayed wound closure. These observations emphasize that eNOS-derived NO can promote angiogenesis. Intriguingly, eNOS activity is regulated by nitric-oxide synthase trafficking inducer (NOSTRIN), which sequesters eNOS, thereby attenuating NO production. This has prompted significant interest in NOSTRIN's function in endothelial cells. We show here that NOSTRIN affects the functional transcriptome of endothelial cells by down-regulating several genes important for invasion and angiogenesis. Interestingly, the effects of NOSTRIN on endothelial gene expression were independent of eNOS activity. NOSTRIN also affected the expression of secreted cytokines involved in inflammatory responses, and ectopic NOSTRIN overexpression functionally restricted endothelial cell proliferation, invasion, adhesion, and VEGF-induced capillary tube formation. Furthermore, NOSTRIN interacted directly with TNF receptor-associated factor 6 (TRAF6), leading to the suppression of NFκB activity and inhibition of AKT activation via phosphorylation. Interestingly, TNF-α-induced NFκB pathway activation was reversed by NOSTRIN. We found that the SH3 domain of NOSTRIN is involved in the NOSTRIN-TRAF6 interaction and is required for NOSTRIN-induced down-regulation of endothelial cell proteins. These results have broad biological implications, as aberrant NOSTRIN expression leading to deactivation of the NFκB pathway, in turn triggering an anti-angiogenic cascade, might inhibit tumorigenesis and cancer progression. PMID:28235804

Nitric oxide donors or nitrite counteract copper-[dithiocarbamate](2)-mediated tumor cell death and inducible nitric oxide synthase down-regulation: possible role of a nitrosyl-copper [dithiocarbamate](2) complex.

PubMed

Rhenals, Maricela Viola; Strasberg-Rieber, Mary; Rieber, Manuel

2010-02-25

In contrast to other metal-dithiocarbamate [DEDTC] complexes, the copper-DEDTC complex is highly cytotoxic, inducing oxidative stress, preferentially in tumor cells. Because nitric oxide (NO) forms adducts with Cu[DEDTC](2), we investigated whether NO donors like S-nitroso-N-acetyl penicillamine (SNAP) or sodium nitroprusside (SNP), and nitrite, a NO decomposition product, modulate Cu[DEDTC](2) cytotoxicity against human tumor cells. We show that apoptosis-associated PARP cleavage and inducible nitric oxide synthase (iNOS) down-regulation induced by nanomolar Cu[DEDTC](2), are counteracted by 50 muM SNAP, SNP, or CoCl(2), an inducer of hypoxia and NO signaling. Nitrite was stochiometrically effective in antagonizing Cu[DEDTC](2) cytotoxicity and inducing shifts in the absorption spectrum of the binary complex in the 280 and 450 nm regions. Subtoxic concentrations of Cu[DEDTC](2) became lethal when tumor cells were pretreated with c-PTIO, a membrane-impermeable scavenger for extracellular NO. Our results suggest that: (a) reactive oxygen species induced by Cu[DEDTC](2) are scavenged by nitrite released from NO, (b) the extent of lethality of Cu[DEDTC](2) is dependent on the reciprocal formation of an inactive ternary Cu[DEDTC](2)NO copper-nitrosyl complex.

Phenotypic changes associated with RNA interference silencing of chalcone synthase in apple (Malus × domestica).

PubMed

Dare, Andrew P; Tomes, Sumathi; Jones, Midori; McGhie, Tony K; Stevenson, David E; Johnson, Ross A; Greenwood, David R; Hellens, Roger P

2013-05-01

We have identified in apple (Malus × domestica) three chalcone synthase (CHS) genes. In order to understand the functional redundancy of this gene family RNA interference knockout lines were generated where all three of these genes were down-regulated. These lines had no detectable anthocyanins and radically reduced concentrations of dihydrochalcones and flavonoids. Surprisingly, down-regulation of CHS also led to major changes in plant development, resulting in plants with shortened internode lengths, smaller leaves and a greatly reduced growth rate. Microscopic analysis revealed that these phenotypic changes extended down to the cellular level, with CHS-silenced lines showing aberrant cellular organisation in the leaves. Fruit collected from one CHS-silenced line was smaller than the 'Royal Gala' controls, lacked flavonoids in the skin and flesh and also had changes in cell morphology. Auxin transport experiments showed increased rates of auxin transport in a CHS-silenced line compared with the 'Royal Gala' control. As flavonoids are well known to be key modulators of auxin transport, we hypothesise that the removal of almost all flavonoids from the plant by CHS silencing creates a vastly altered environment for auxin transport to occur and results in the observed changes in growth and development. © 2013 The Authors The Plant Journal © 2013 Blackwell Publishing Ltd.

Transcriptional coupling of synaptic transmission and energy metabolism: role of nuclear respiratory factor 1 in co-regulating neuronal nitric oxide synthase and cytochrome c oxidase genes in neurons.

PubMed

Dhar, Shilpa S; Liang, Huan Ling; Wong-Riley, Margaret T T

2009-10-01

Neuronal activity is highly dependent on energy metabolism; yet, the two processes have traditionally been regarded as independently regulated at the transcriptional level. Recently, we found that the same transcription factor, nuclear respiratory factor 1 (NRF-1) co-regulates an important energy-generating enzyme, cytochrome c oxidase, as well as critical subunits of glutamatergic receptors. The present study tests our hypothesis that the co-regulation extends to the next level of glutamatergic synapses, namely, neuronal nitric oxide synthase, which generates nitric oxide as a downstream signaling molecule. Using in silico analysis, electrophoretic mobility shift assay, chromatin immunoprecipitation, promoter mutations, and NRF-1 silencing, we documented that NRF-1 functionally bound to Nos1, but not Nos2 (inducible) and Nos3 (endothelial) gene promoters. Both COX and Nos1 transcripts were up-regulated by depolarizing KCl treatment and down-regulated by TTX-mediated impulse blockade in neurons. However, NRF-1 silencing blocked the up-regulation of both Nos1 and COX induced by KCl depolarization, and over-expression of NRF-1 rescued both Nos1 and COX transcripts down-regulated by TTX. These findings are consistent with our hypothesis that synaptic neuronal transmission and energy metabolism are tightly coupled at the molecular level.

Ultraviolet A irradiation of the eye activates a nitric oxide-dependent hypothalamo-pituitary pro-opiomelanocortin pathway and modulates the functions of Langerhans cells.

PubMed

Hiramoto, Keiichi

2009-06-01

Ultraviolet A (UV-A) radiation decreases Langerhans cells (LC) in the skin specifically at the site of exposure. Unexpectedly, UV-A irradiation of the eye has been found systemically downregulating epidermal LC in mice. Male C57BL/6j mice and an inducible type of nitric oxide synthase knockout mice (iNOS(-/-)) were used in this study. The eye or ear was locally exposed to UV-A after covering the remaining body surface with aluminum foil at a dose of 110 kJ/m(2) using a sunlamp. Localized UV-A irradiation of the eye downregulated epidermal LC. The hypophysectomy strongly inhibited the UV-A-induced downregulation of LC. To elucidate the pathway by UV-A irradiation of the eye, the effect of a bilateral ciliary ganglionectomy and denervation of the optic nerves was examined. Optic nerve denervation strongly inhibited LC downregulation in response to localized irradiation of the eye. Furthermore, no LC downregulation in response to localized UV-A irradiation of the eye was observed in iNOS(-/-) mice. These results clearly indicate that a signal evoked by UV-A irradiation of the eye is transmitted in a nitric oxide-dependent manner through the optic nerves to the hypothalamo-pituitary pro-opiomelanocortin system.

Glucosylceramide transferase in Giardia preferentially catalyzes the synthesis of galactosylceramide during encystation.

PubMed

Robles-Martinez, Leobarda; Mendez, Tavis L; Apodaca, Jennifer; Das, Siddhartha

2017-01-01

The stage differentiation from trophozoite to cyst (i.e., encystation) is an essential step for Giardia to survive outside its human host and spread the infection via the fecal-oral route. We have previously shown that Giardia expresses glucosylceramide transferase 1 (GlcT1) enzyme, the activity of which is elevated during encystation. We have also reported that blocking the activity of gGlcT1 interferes with the biogenesis of encystation-specific vesicles (ESVs) and cyst viability in Giardia. To further understand the role of this enzyme and how it regulates encystation, we overexpressed, knocked down, and rescued the giardial GlcT1 (gGlcT1) gene and measured its enzymatic activity in live parasites as well as in isolated membrane fractions using NBD-ceramide and UDP-glucose or UDP-galactose. We observed that gGlcT1 is able to catalyze the synthesis of both glucosylceramide (GlcCer) and galactosylceramide (GalCer), however the synthesis of GalCer is 2-3 fold higher than of GlcCer. Although both activities follow Michaelis-Menten kinetics, the bindings of UDP-glucose and UDP-galactose with the enzyme appear to be non-competitive and independent of each other. The modulation of gGlcT1 synthesis concomitantly influenced the expression cyst-wall protein (CWP) and overall encystation. We propose that gGlcT1 is a unique enzyme and that Giardia uses this enzyme to synthesize both GlcCer and GalCer to facilitate the process of encystation/cyst production. Copyright © 2016 Elsevier B.V. All rights reserved.

Destabilization of fructose 1,6-bisphosphatase-Z-line interactions is a mechanism of glyconeogenesis down-regulation in vivo.

PubMed

Gizak, Agnieszka; Mazurek, Jakub; Wozniak, Marta; Maciaszczyk-Dziubinska, Ewa; Rakus, Dariusz

2013-03-01

Although it is well known that insulin controls the synthesis of glycogen from non-carbohydrates by down-regulating expression of several glyconeogenic enzymes, a mechanism of short-term inhibition of glyconeogenesis remains unknown. In recent years, we have shown that in skeletal muscle, fructose 1,6-bisphosphatase (FBPase) is a part of the hypothetical glyconeogenic complex located on sarcomeric Z-line. Here, we show that inhibition of glycogen synthase kinase-3 causes disruption of the FBPase-Z-line interactions and reduction of muscle glycogen content in vivo. The normal, striated pattern of muscle FBPase localization is also disturbed by insulin treatment but preserved when insulin is applied together with Akt inhibitor. We suggest that destabilization of FBPase-Z-line interaction is a universal cellular mechanism of glyconeogenesis down-regulation, allowing for preferential utilization of glucose for insulin-stimulated muscle glycogen synthesis. Copyright © 2012 Elsevier B.V. All rights reserved.

Neurofibromatosis-like phenotype in Drosophila caused by lack of glucosylceramide extension

PubMed Central

Dahlgaard, Katja; Jung, Anita; Qvortrup, Klaus; Clausen, Henrik; Kjaerulff, Ole; Wandall, Hans H.

2012-01-01

Glycosphingolipids (GSLs) are of fundamental importance in the nervous system. However, the molecular details associated with GSL function are largely unknown, in part because of the complexity of GSL biosynthesis in vertebrates. In Drosophila, only one major GSL biosynthetic pathway exists, controlled by the glycosyltransferase Egghead (Egh). Here we discovered that loss of Egh causes overgrowth of peripheral nerves and attraction of immune cells to the nerves. This phenotype is reminiscent of the human disorder neurofibromatosis type 1, which is characterized by disfiguring nerve sheath tumors with mast cell infiltration, increased cancer risk, and learning deficits. Neurofibromatosis type 1 is due to a reduction of the tumor suppressor neurofibromin, a negative regulator of the small GTPase Ras. Enhanced Ras signaling promotes glial growth through activation of phosphatidylinositol 3-kinase (PI3K) and its downstream kinase Akt. We find that overgrowth of peripheral nerves in egh mutants is suppressed by down-regulation of the PI3K signaling pathway by expression of either dominant-negative PI3K, the tumor suppressor PTEN, or the transcription factor FOXO in the subperineurial glia. These results show that loss of the glycosyltransferase Egh affects membrane signaling and activation of PI3K signaling in glia of the peripheral nervous system, and suggest that glycosyltransferases may suppress proliferation. PMID:22493273

Exogenous sucrose supply changes sugar metabolism and reduces photosynthesis of sugarcane through the down-regulation of Rubisco abundance and activity.

PubMed

Lobo, Ana Karla Moreira; de Oliveira Martins, Marcio; Lima Neto, Milton Costa; Machado, Eduardo Caruso; Ribeiro, Rafael Vasconcelos; Silveira, Joaquim Albenisio Gomes

2015-05-01

Photosynthetic modulation by sugars has been known for many years, but the biochemical and molecular comprehension of this process is lacking. We studied how the exogenous sucrose supplied to leaves could affect sugar metabolism in leaf, sheath and stalk and inhibit photosynthesis in four-month old sugarcane plants. Exogenous sucrose 50mM sprayed on attached leaves strongly impaired the net CO2 assimilation (PN) and decreased the instantaneous carboxylation efficiency (PN/Ci), suggesting that the impairment in photosynthesis was caused by biochemical restrictions. The photosystem II activity was also affected by excess sucrose as indicated by the reduction in the apparent electron transport rate, effective quantum yield and increase in non-photochemical quenching. In leaf segments, sucrose accumulation was related to increases in the activities of soluble acid and neutral invertases, sucrose synthase and sucrose phosphate synthase, whereas the contents of fructose increased and glucose slightly decreased. Changes in the activities of sucrose hydrolyzing and synthesizing enzymes in leaf, sheath and stalk and sugar profile in intact plants were not enough to identify which sugar(s) or enzyme(s) were directly involved in photosynthesis modulation. However, exogenous sucrose was able to trigger down-regulation in the Rubisco abundance, activation state and enzymatic activity. Despite the fact that PN/Ci had been notably decreased by sucrose, in vitro activity and abundance of PEPCase did not change, suggesting an in vivo modulation of this enzyme. The data reveal that sucrose and/or other derivative sugars in leaves inhibited sugarcane photosynthesis by down-regulation of Rubisco synthesis and activity. Our data also suggest that sugar modulation was not exerted by a feedback mechanism induced by the accumulation of sugars in immature sugarcane stalk. Copyright © 2015. Published by Elsevier GmbH.

Heat Shock Protein-70 Inducers and iNOS Inhibitors as Therapeutics to Ameliorate Hemorrhagic Shock

DTIC Science & Technology

2004-09-01

downregulation of iNOS can limit tissue injury caused by ischemia / reperfusion or hemorrhage/resuscitation. In our laboratory, geldanamycin, a member of... ischemia / reperfusion [Charier 1999]. Mice deficient in inducible NO synthase (iNOS) also demonstrate limited hemorrhage/resuscitation-induced injury ...tissues and leukotriene B4 (LTB4) generation increases. In a hemorrhage/resuscitation-induced injury model, iNOS, cyclooxygenase- 2 , and CD14 are all

Characterization of Scedosporium apiospermum Glucosylceramides and Their Involvement in Fungal Development and Macrophage Functions

PubMed Central

Rollin-Pinheiro, Rodrigo; Liporagi-Lopes, Livia Cristina; de Meirelles, Jardel Vieira; de Souza, Lauro M.; Barreto-Bergter, Eliana

2014-01-01

Scedosporium apiospermum is an emerging fungal pathogen that causes both localized and disseminated infections in immunocompromised patients. Glucosylceramides (CMH, GlcCer) are the main neutral glycosphingolipids expressed in fungal cells. In this study, glucosylceramides (GlcCer) were extracted and purified in several chromatographic steps. Using high-performance thin layer chromatography (HPTLC) and electrospray ionization mass spectrometry (ESI-MS), N-2′-hydroxyhexadecanoyl-1-β-D-glucopyranosyl-9-methyl-4,8-sphingadienine was identified as the main GlcCer in S. apiospermum. A monoclonal antibody (Mab) against this molecule was used for indirect immunofluorescence experiments, which revealed that this CMH is present on the surface of the mycelial and conidial forms of S. apiospermum. Treatment of S. apiospermum conidia with the Mab significantly reduced fungal growth. In addition, the Mab also enhanced the phagocytosis and killing of S. apiospermum by murine cells. In vitro assays were performed to evaluate the CMHs for their cytotoxic activities against the mammalian cell lines L.929 and RAW, and an inhibitory effect on cell proliferation was observed. Synergistic in vitro interactions were observed between the Mab against GlcCer and both amphotericin B (AmB) and itraconazole. Because Scedosporium species develop drug resistance, the number of available antifungal drugs is limited; our data indicate that combining immunotherapy with the available drugs might be a viable treatment option. These results suggest that in S. apiospermum, GlcCer are most likely cell wall components that are targeted by antifungal antibodies, which directly inhibit fungal development and enhance macrophage function; furthermore, these results suggest the combined use of monoclonal antibodies against GlcCer and antifungal drugs for antifungal immunotherapy. PMID:24878570

Characterization of Scedosporium apiospermum glucosylceramides and their involvement in fungal development and macrophage functions.

PubMed

Rollin-Pinheiro, Rodrigo; Liporagi-Lopes, Livia Cristina; de Meirelles, Jardel Vieira; Souza, Lauro M de; Barreto-Bergter, Eliana

2014-01-01

Scedosporium apiospermum is an emerging fungal pathogen that causes both localized and disseminated infections in immunocompromised patients. Glucosylceramides (CMH, GlcCer) are the main neutral glycosphingolipids expressed in fungal cells. In this study, glucosylceramides (GlcCer) were extracted and purified in several chromatographic steps. Using high-performance thin layer chromatography (HPTLC) and electrospray ionization mass spectrometry (ESI-MS), N-2'-hydroxyhexadecanoyl-1-β-D-glucopyranosyl-9-methyl-4,8-sphingadienine was identified as the main GlcCer in S. apiospermum. A monoclonal antibody (Mab) against this molecule was used for indirect immunofluorescence experiments, which revealed that this CMH is present on the surface of the mycelial and conidial forms of S. apiospermum. Treatment of S. apiospermum conidia with the Mab significantly reduced fungal growth. In addition, the Mab also enhanced the phagocytosis and killing of S. apiospermum by murine cells. In vitro assays were performed to evaluate the CMHs for their cytotoxic activities against the mammalian cell lines L.929 and RAW, and an inhibitory effect on cell proliferation was observed. Synergistic in vitro interactions were observed between the Mab against GlcCer and both amphotericin B (AmB) and itraconazole. Because Scedosporium species develop drug resistance, the number of available antifungal drugs is limited; our data indicate that combining immunotherapy with the available drugs might be a viable treatment option. These results suggest that in S. apiospermum, GlcCer are most likely cell wall components that are targeted by antifungal antibodies, which directly inhibit fungal development and enhance macrophage function; furthermore, these results suggest the combined use of monoclonal antibodies against GlcCer and antifungal drugs for antifungal immunotherapy.

A new glucocerebrosidase-deficient neuronal cell model provides a tool to probe pathophysiology and therapeutics for Gaucher disease

PubMed Central

Westbroek, Wendy; Nguyen, Matthew; Siebert, Marina; Lindstrom, Taylor; Burnett, Robert A.; Aflaki, Elma; Jung, Olive; Tamargo, Rafael; Rodriguez-Gil, Jorge L.; Acosta, Walter; Hendrix, An; Behre, Bahafta; Tayebi, Nahid; Fujiwara, Hideji; Sidhu, Rohini; Renvoise, Benoit; Ginns, Edward I.; Dutra, Amalia; Pak, Evgenia; Cramer, Carole; Ory, Daniel S.; Pavan, William J.

2016-01-01

ABSTRACT Glucocerebrosidase is a lysosomal hydrolase involved in the breakdown of glucosylceramide. Gaucher disease, a recessive lysosomal storage disorder, is caused by mutations in the gene GBA1. Dysfunctional glucocerebrosidase leads to accumulation of glucosylceramide and glycosylsphingosine in various cell types and organs. Mutations in GBA1 are also a common genetic risk factor for Parkinson disease and related synucleinopathies. In recent years, research on the pathophysiology of Gaucher disease, the molecular link between Gaucher and Parkinson disease, and novel therapeutics, have accelerated the need for relevant cell models with GBA1 mutations. Although induced pluripotent stem cells, primary rodent neurons, and transfected neuroblastoma cell lines have been used to study the effect of glucocerebrosidase deficiency on neuronal function, these models have limitations because of challenges in culturing and propagating the cells, low yield, and the introduction of exogenous mutant GBA1. To address some of these difficulties, we established a high yield, easy-to-culture mouse neuronal cell model with nearly complete glucocerebrosidase deficiency representative of Gaucher disease. We successfully immortalized cortical neurons from embryonic null allele gba−/− mice and the control littermate (gba+/+) by infecting differentiated primary cortical neurons in culture with an EF1α-SV40T lentivirus. Immortalized gba−/− neurons lack glucocerebrosidase protein and enzyme activity, and exhibit a dramatic increase in glucosylceramide and glucosylsphingosine accumulation, enlarged lysosomes, and an impaired ATP-dependent calcium-influx response; these phenotypical characteristics were absent in gba+/+ neurons. This null allele gba−/− mouse neuronal model provides a much-needed tool to study the pathophysiology of Gaucher disease and to evaluate new therapies. PMID:27482815

Highly efficient preparation of sphingoid bases from glucosylceramides by chemoenzymatic method[S

PubMed Central

Gowda, Siddabasave Gowda B.; Usuki, Seigo; Hammam, Mostafa A. S.; Murai, Yuta; Igarashi, Yasuyuki; Monde, Kenji

2016-01-01

Sphingoid base derivatives have attracted increasing attention as promising chemotherapeutic candidates against lifestyle diseases such as diabetes and cancer. Natural sphingoid bases can be a potential resource instead of those derived by time-consuming total organic synthesis. In particular, glucosylceramides (GlcCers) in food plants are enriched sources of sphingoid bases, differing from those of animals. Several chemical methodologies to transform GlcCers to sphingoid bases have already investigated; however, these conventional methods using acid or alkaline hydrolysis are not efficient due to poor reaction yield, producing complex by-products and resulting in separation problems. In this study, an extremely efficient and practical chemoenzymatic transformation method has been developed using microwave-enhanced butanolysis of GlcCers and a large amount of readily available almond β-glucosidase for its deglycosylation reaction of lysoGlcCers. The method is superior to conventional acid/base hydrolysis methods in its rapidity and its reaction cleanness (no isomerization, no rearrangement) with excellent overall yield. PMID:26667669

13C Metabolic Flux Analysis for Systematic Metabolic Engineering of S. cerevisiae for Overproduction of Fatty Acids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ghosh, Amit; Ando, David; Gin, Jennifer

Efficient redirection of microbial metabolism into the abundant production of desired bioproducts remains non-trivial. Here, we used flux-based modeling approaches to improve yields of fatty acids in Saccharomyces cerevisiae. We combined 13C labeling data with comprehensive genome-scale models to shed light onto microbial metabolism and improve metabolic engineering efforts. We concentrated on studying the balance of acetyl-CoA, a precursor metabolite for the biosynthesis of fatty acids. A genome-wide acetyl-CoA balance study showed ATP citrate lyase from Yarrowia lipolytica as a robust source of cytoplasmic acetyl-CoA and malate synthase as a desirable target for downregulation in terms of acetyl-CoA consumption. Thesemore » genetic modifications were applied to S. cerevisiae WRY2, a strain that is capable of producing 460 mg/L of free fatty acids. With the addition of ATP citrate lyase and downregulation of malate synthase, the engineered strain produced 26% more free fatty acids. Further increases in free fatty acid production of 33% were obtained by knocking out the cytoplasmic glycerol-3-phosphate dehydrogenase, which flux analysis had shown was competing for carbon flux upstream with the carbon flux through the acetyl-CoA production pathway in the cytoplasm. In total, the genetic interventions applied in this work increased fatty acid production by ~70%.« less

Near infrared radiation protects against oxygen-glucose deprivation-induced neurotoxicity by down-regulating neuronal nitric oxide synthase (nNOS) activity in vitro.

PubMed

Yu, Zhanyang; Li, Zhaoyu; Liu, Ning; Jizhang, Yunneng; McCarthy, Thomas J; Tedford, Clark E; Lo, Eng H; Wang, Xiaoying

2015-06-01

Near infrared radiation (NIR) has been shown to be neuroprotective against neurological diseases including stroke and brain trauma, but the underlying mechanisms remain poorly understood. In the current study we aimed to investigate the hypothesis that NIR may protect neurons by attenuating oxygen-glucose deprivation (OGD)-induced nitric oxide (NO) production and modulating cell survival/death signaling. Primary mouse cortical neurons were subjected to 4 h OGD and NIR was applied at 2 h reoxygenation. OGD significantly increased NO level in primary neurons compared to normal control, which was significantly ameliorated by NIR at 5 and 30 min post-NIR. Neither OGD nor NIR significantly changed neuronal nitric oxide synthase (nNOS) mRNA or total protein levels compared to control groups. However, OGD significantly increased nNOS activity compared to normal control, and this effect was significantly diminished by NIR. Moreover, NIR significantly ameliorated the neuronal death induced by S-Nitroso-N-acetyl-DL-penicillamine (SNAP), a NO donor. Finally, NIR significantly rescued OGD-induced suppression of p-Akt and Bcl-2 expression, and attenuated OGD-induced upregulation of Bax, BAD and caspase-3 activation. These results suggest NIR may protect against OGD at least partially through reducing NO production by down-regulating nNOS activity, and modulating cell survival/death signaling.

13C Metabolic Flux Analysis for Systematic Metabolic Engineering of S. cerevisiae for Overproduction of Fatty Acids

DOE PAGES

Ghosh, Amit; Ando, David; Gin, Jennifer; ...

2016-10-05

Efficient redirection of microbial metabolism into the abundant production of desired bioproducts remains non-trivial. Here, we used flux-based modeling approaches to improve yields of fatty acids in Saccharomyces cerevisiae. We combined 13C labeling data with comprehensive genome-scale models to shed light onto microbial metabolism and improve metabolic engineering efforts. We concentrated on studying the balance of acetyl-CoA, a precursor metabolite for the biosynthesis of fatty acids. A genome-wide acetyl-CoA balance study showed ATP citrate lyase from Yarrowia lipolytica as a robust source of cytoplasmic acetyl-CoA and malate synthase as a desirable target for downregulation in terms of acetyl-CoA consumption. Thesemore » genetic modifications were applied to S. cerevisiae WRY2, a strain that is capable of producing 460 mg/L of free fatty acids. With the addition of ATP citrate lyase and downregulation of malate synthase, the engineered strain produced 26% more free fatty acids. Further increases in free fatty acid production of 33% were obtained by knocking out the cytoplasmic glycerol-3-phosphate dehydrogenase, which flux analysis had shown was competing for carbon flux upstream with the carbon flux through the acetyl-CoA production pathway in the cytoplasm. In total, the genetic interventions applied in this work increased fatty acid production by ~70%.« less

Extra virgin olive oil polyphenolic extracts downregulate inflammatory responses in LPS-activated murine peritoneal macrophages suppressing NFκB and MAPK signalling pathways.

PubMed

Cárdeno, A; Sánchez-Hidalgo, M; Aparicio-Soto, M; Sánchez-Fidalgo, S; Alarcón-de-la-Lastra, C

2014-06-01

Extra virgin olive oil (EVOO) is obtained from the fruit of the olive tree Olea europaea L. Phenolic compounds present in EVOO have recognized anti-oxidant and anti-inflammatory properties. However, the activity of the total phenolic fraction extracted from EVOO and the action mechanisms involved are not well defined. The present study was designed to evaluate the potential anti-inflammatory mechanisms of the polyphenolic extract (PE) from EVOO on LPS-stimulated peritoneal murine macrophages. Nitric oxide (NO) production was analyzed by the Griess method and intracellular reactive oxygen species (ROS) by fluorescence analysis. Moreover, changes in the protein expression of the pro-inflammatory enzymes, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2 and microsomal prostaglandin E synthase-1 (mPGES-1), as well as the role of nuclear transcription factor kappa B (NFκB) and mitogen-activated protein kinase (MAPK) signalling pathways, were analyzed by Western blot. PE from EVOO reduced LPS-induced oxidative stress and inflammatory responses through decreasing NO and ROS generation. In addition, PE induced a significant down-regulation of iNOS, COX-2 and mPGES-1 protein expressions, reduced MAPK phosphorylation and prevented the nuclear NFκB translocation. This study establishes that PE from EVOO possesses anti-inflammatory activities on LPS-stimulated murine macrophages.

Transgene silencing of sucrose synthase in alfalfa (Medicago sativa L.) stem vascular tissue suggests a role for invertase in cell wall cellulose synthesis.

PubMed

Samac, Deborah A; Bucciarelli, Bruna; Miller, Susan S; Yang, S Samuel; O'Rourke, Jamie A; Shin, Sanghyun; Vance, Carroll P

2015-12-01

Alfalfa (Medicago sativa L.) is a widely adapted perennial forage crop that has high biomass production potential. Enhanced cellulose content in alfalfa stems would increase the value of the crop as a bioenergy feedstock. We examined if increased expression of sucrose synthase (SUS; EC 2.4.1.13) would increase cellulose in stem cell walls. Alfalfa plants were transformed with a truncated alfalfa phosphoenolpyruvate carboxylase gene promoter (PEPC7-P4) fused to an alfalfa nodule-enhanced SUS cDNA (MsSUS1) or the β-glucuronidase (GUS) gene. Strong GUS expression was detected in xylem and phloem indicating that the PEPC7-P4 promoter was active in stem vascular tissue. In contrast to expectations, MsSUS1 transcript accumulation was reduced 75-90 % in alfalfa plants containing the PEPC7-P4::MsSUS1 transgene compared to controls. Enzyme assays indicated that SUS activity in stems of selected down-regulated transformants was reduced by greater than 95 % compared to the controls. Although SUS activity was detected in xylem and phloem of control plants by in situ enzyme assays, plants with the PEPC7-P4::MsSUS1 transgene lacked detectable SUS activity in post-elongation stem (PES) internodes and had very low SUS activity in elongating stem (ES) internodes. Loss of SUS protein in PES internodes of down-regulated lines was confirmed by immunoblots. Down-regulation of SUS expression and activity in stem tissue resulted in no obvious phenotype or significant change in cell wall sugar composition. However, alkaline/neutral (A/N) invertase activity increased in SUS down-regulated lines and high levels of acid invertase activity were observed. In situ enzyme assays of stem tissue showed localization of neutral invertase in vascular tissues of ES and PES internodes. These results suggest that invertases play a primary role in providing glucose for cellulose biosynthesis or compensate for the loss of SUS1 activity in stem vascular tissue.

Internalization and sorting of a fluorescent analogue of glucosylceramide to the Golgi apparatus of human skin fibroblasts: utilization of endocytic and nonendocytic transport mechanisms

PubMed Central

1994-01-01

We examined the uptake and intracellular transport of the fluorescent glucosylceramide analogue N-[5-(5,7-dimethyl BODIPYTM)-1-pentanoyl]- glucosyl sphingosine (C5-DMB-GlcCer) in human skin fibroblasts, and we compared its behavior to that of the corresponding fluorescent analogues of sphingomyelin, galactosylceramide, and lactosylceramide. All four fluorescent analogues were readily transferred from defatted BSA to the plasma membrane during incubation at 4 degrees C. When cells treated with C5-DMB-GlcCer were washed, warmed to 37 degrees C, and subsequently incubated with defatted BSA to remove fluorescent lipid at the cell surface, strong fluorescence was observed at the Golgi apparatus, as well as weaker labeling at the nuclear envelope and other intracellular membranes. Similar results were obtained with C5-DMB- galactosylceramide, except that labeling of the Golgi apparatus was weaker than with C5-DMB-GlcCer. Internalization of C5-DMB-GlcCer was not inhibited by various treatments, including ATP depletion or warming to 19 degrees C, and biochemical analysis demonstrated that the lipid was not metabolized during its internalization. However, accumulation of C5-DMB-GlcCer at the Golgi apparatus was reduced when cells were treated with a nonfluorescent analogue of glucosylceramide, suggesting that accumulation of C5-DMB-GlcCer at the Golgi apparatus was a saturable process. In contrast, cells treated with C5-DMB-analogues of sphingomyelin or lactosylceramide internalized the fluorescent lipid into a punctate pattern of fluorescence during warming at 37 degrees C, and this process was temperature and energy dependent. These results with C5-DMB-sphingomyelin and C5-DMB-lactosylceramide were analogous to those obtained with another fluorescent analogue of sphingomyelin in which labeling of endocytic vesicles and plasma membrane lipid recycling were documented (Koval, M., and R. E. Pagano. 1990. J. Cell Biol. 111:429-442). Incubation of perforated cells with C5-DMB- sphingomyelin resulted in prominent labeling of the nuclear envelope and other intracellular membranes, similar to the pattern observed with C5-DMB-GlcCer in intact cells. These observations are consistent with the transbilayer movement of fluorescent analogues of glucosylceramide and galactosylceramide at the plasma membrane and early endosomes of human skin fibroblasts, and suggest that both endocytic and nonendocytic pathways are used in the internalization of these lipids from the plasma membrane. PMID:8188745

An ethanol extract of Piper betle Linn. mediates its anti-inflammatory activity via down-regulation of nitric oxide.

PubMed

Ganguly, Sudipto; Mula, Soumyaditya; Chattopadhyay, Subrata; Chatterjee, Mitali

2007-05-01

The leaves of Piper betle (locally known as Paan) have long been in use in the Indian indigenous system of medicine for the relief of pain; however, the underlying molecular mechanisms of this effect have not been elucidated. The anti-inflammatory and immunomodulatory effects of an ethanolic extract of the leaves of P. betle (100 mg kg(-1); PB) were demonstrated in a complete Freund's adjuvant-induced model of arthritis in rats with dexamethasone (0.1 mg kg(-1)) as the positive control. At non-toxic concentrations of PB (5-25 microg mL(-1)), a dose-dependent decrease in extracellular production of nitric oxide in murine peritoneal macrophages was measured by the Griess assay and corroborated by flow cytometry using the nitric oxide specific probe, 4,5-diaminofluorescein-2 diacetate. This decreased generation of reactive nitrogen species was mediated by PB progressively down-regulating transcription of inducible nitric oxide synthase in macrophages, and concomitantly causing a dose-dependent decrease in the expression of interleukin-12 p40, indicating the ability of PB to down-regulate T-helper 1 pro-inflammatory responses. Taken together, the anti-inflammatory and anti-arthrotic activity of PB is attributable to its ability to down-regulate the generation of reactive nitrogen species, thus meriting further pharmacological investigation.

Shikonin Isolated from Lithospermum erythrorhizon Downregulates Proinflammatory Mediators in Lipopolysaccharide-Stimulated BV2 Microglial Cells by Suppressing Crosstalk between Reactive Oxygen Species and NF-κB.

PubMed

Prasad, Rajapaksha Gedara; Choi, Yung Hyun; Kim, Gi-Young

2015-03-01

According to the expansion of lifespan, neuronal disorder based on inflammation has been social problem. Therefore, we isolated shikonin from Lithospermum erythrorhizon and evaluated anti-inflammatory effects of shikonin in lipopolysaccharide (LSP)-stimulated BV2 microglial cells. Shikonin dose-dependently inhibits the expression of the proinflammatory mediators, nitric oxide (NO), prostaglandin E2 (PGE2), and tumor necrosis factor-α (TNF-α) as well as their main regulatory genes and products such as inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and TNF-α in LPS-stimulated BV2 microglial cells. Additionally, shikonin suppressed the LPS-induced DNA-binding activity of nuclear factor-κB (NF-κB) to regulate the key regulatory genes of the proinflammatory mediators, such as iNOS, COX-2, and TNF-α, accompanied with downregulation of reactive oxygen species (ROS) generation. The results indicate that shikonin may downregulate the expression of proinflammatory genes involved in the synthesis of NO, PGE2, and TNF-α in LPS-treated BV2 microglial cells by suppressing ROS and NF-κB. Taken together, our results revealed that shikonin exerts downregulation of proinflammatory mediators by interference the ROS and NF-κB signaling pathway.

Shikonin Isolated from Lithospermum erythrorhizon Downregulates Proinflammatory Mediators in Lipopolysaccharide-Stimulated BV2 Microglial Cells by Suppressing Crosstalk between Reactive Oxygen Species and NF-κB

PubMed Central

Prasad, Rajapaksha Gedara; Choi, Yung Hyun; Kim, Gi-Young

2015-01-01

According to the expansion of lifespan, neuronal disorder based on inflammation has been social problem. Therefore, we isolated shikonin from Lithospermum erythrorhizon and evaluated anti-inflammatory effects of shikonin in lipopolysaccharide (LSP)-stimulated BV2 microglial cells. Shikonin dose-dependently inhibits the expression of the proinflammatory mediators, nitric oxide (NO), prostaglandin E2 (PGE2), and tumor necrosis factor-α (TNF-α) as well as their main regulatory genes and products such as inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and TNF-α in LPS-stimulated BV2 microglial cells. Additionally, shikonin suppressed the LPS-induced DNA-binding activity of nuclear factor-κB (NF-κB) to regulate the key regulatory genes of the proinflammatory mediators, such as iNOS, COX-2, and TNF-α, accompanied with downregulation of reactive oxygen species (ROS) generation. The results indicate that shikonin may downregulate the expression of proinflammatory genes involved in the synthesis of NO, PGE2, and TNF-α in LPS-treated BV2 microglial cells by suppressing ROS and NF-κB. Taken together, our results revealed that shikonin exerts downregulation of proinflammatory mediators by interference the ROS and NF-κB signaling pathway. PMID:25767678

The Tribolium chitin synthase genes TcCHS1 and TcCHS2 are specialized for synthesis of epidermal cuticle and midgut peritrophic matrix.

PubMed

Arakane, Y; Muthukrishnan, S; Kramer, K J; Specht, C A; Tomoyasu, Y; Lorenzen, M D; Kanost, M; Beeman, R W

2005-10-01

Functional analysis of the two chitin synthase genes, TcCHS1 and TcCHS2, in the red flour beetle, Tribolium castaneum, revealed unique and complementary roles for each gene. TcCHS1-specific RNA interference (RNAi) disrupted all three types of moult (larval-larval, larval-pupal and pupal-adult) and greatly reduced whole-body chitin content. Exon-specific RNAi showed that splice variant 8a of TcCHS1 was required for both the larval-pupal and pupal-adult moults, whereas splice variant 8b was required only for the latter. TcCHS2-specific RNAi had no effect on metamorphosis or on total body chitin content. However, RNAi-mediated down-regulation of TcCHS2, but not TcCHS1, led to cessation of feeding, a dramatic shrinkage in larval size and reduced chitin content in the midgut.

Selective downregulation of mitochondrial electron transport chain activity and increased oxidative stress in human atrial fibrillation.

PubMed

Emelyanova, Larisa; Ashary, Zain; Cosic, Milanka; Negmadjanov, Ulugbek; Ross, Gracious; Rizvi, Farhan; Olet, Susan; Kress, David; Sra, Jasbir; Tajik, A Jamil; Holmuhamedov, Ekhson L; Shi, Yang; Jahangir, Arshad

2016-07-01

Mitochondria are critical for maintaining normal cardiac function, and a deficit in mitochondrial energetics can lead to the development of the substrate that promotes atrial fibrillation (AF) and its progression. However, the link between mitochondrial dysfunction and AF in humans is still not fully defined. The aim of this study was to elucidate differences in the functional activity of mitochondrial oxidative phosphorylation (OXPHOS) complexes and oxidative stress in right atrial tissue from patients without (non-AF) and with AF (AF) who were undergoing open-heart surgery and were not significantly different for age, sex, major comorbidities, and medications. The overall functional activity of the electron transport chain (ETC), NADH:O2 oxidoreductase activity, was reduced by 30% in atrial tissue from AF compared with non-AF patients. This was predominantly due to a selective reduction in complex I (0.06 ± 0.007 vs. 0.09 ± 0.006 nmol·min(-1)·citrate synthase activity(-1), P = 0.02) and II (0.11 ± 0.012 vs. 0.16 ± 0.012 nmol·min(-1)·citrate synthase activity(-1), P = 0.003) functional activity in AF patients. Conversely, complex V activity was significantly increased in AF patients (0.21 ± 0.027 vs. 0.12 ± 0.01 nmol·min(-1)·citrate synthase activity(-1), P = 0.005). In addition, AF patients exhibited a higher oxidative stress with increased production of mitochondrial superoxide (73 ± 17 vs. 11 ± 2 arbitrary units, P = 0.03) and 4-hydroxynonenal level (77.64 ± 30.2 vs. 9.83 ± 2.83 ng·mg(-1) protein, P = 0.048). Our findings suggest that AF is associated with selective downregulation of ETC activity and increased oxidative stress that can contribute to the progression of the substrate for AF. Copyright © 2016 the American Physiological Society.

Prevention of renal damage caused by Shiga toxin type 2: Action of Miglustat on human endothelial and epithelial cells.

PubMed

Girard, Magalí C; Sacerdoti, Flavia; Rivera, Fulton P; Repetto, Horacio A; Ibarra, Cristina; Amaral, María M

2015-10-01

Typical hemolytic uremic syndrome (HUS) is responsible for acute and chronic renal failure in children younger than 5 years old in Argentina. Renal damages have been associated with Shiga toxin type 1 and/or 2 (Stx1, Stx2) produced by Escherichia coli O157:H7, although strains expressing Stx2 are highly prevalent in Argentina. Human glomerular endothelial cells (HGEC) and proximal tubule epithelial cells are very Stx-sensitive since they express high levels of Stx receptor (Gb3). Nowadays, there is no available therapy to protect patients from acute toxin-mediated cellular injury. New strategies have been developed based on the Gb3 biosynthesis inhibition through blocking the enzyme glucosylceramide (GL1) synthase. We assayed the action of a GL1 inhibitor (Miglustat: MG), on the prevention of the renal damage induced by Stx2. HGEC primary cultures and HK-2 cell line were pre-treated with MG and then incubated with Stx2. HK- 2 and HGEC express Gb3 and MG was able to decrease the levels of this receptor. As a consequence, both types of cells were protected from Stx2 cytotoxicity and morphology damage. MG was able to avoid Stx2 effects in human renal cells and could be a feasible strategy to protect kidney tissues from the cytotoxic effects of Stx2 in vivo. Copyright © 2015 Elsevier Ltd. All rights reserved.

Miglustat Improves Purkinje Cell Survival and Alters Microglial Phenotype in Feline Niemann-Pick Disease Type C

PubMed Central

Stein, Veronika M.; Crooks, Alexandra; Ding, Wenge; Prociuk, Maria; O’Donnell, Patricia; Bryan, Caroline; Sikora, Tracey; Dingemanse, Jasper; Vanier, Marie T.; Walkley, Steven U.; Vite, Charles H.

2012-01-01

Niemann-Pick disease type C (NPC disease) is an incurable cellular lipid trafficking disorder characterized by neurodegeneration and intralysosomal accumulation of cholesterol and glycosphingolipids. Treatment with miglustat, a small imino sugar that reversibly inhibits glucosylceramide synthase, which is necessary for glycosphingolipid synthesis, has been shown to benefit patients with NPC disease. The mechanism(s) and extent of brain cellular changes underlying this benefit are not understood. To investigate the basis of the efficacy of miglustat, cats with disease homologous to the juvenile-onset form of human NPC disease received daily miglustat orally beginning at 3 weeks of age. The plasma half-life of miglustat was 6.6 ± 1.1 hours, with a tmax, Cmax, and area under the plasma concentration-time curve of 1.7 ± 0.6 hours, 20.3 ± 4.6 μg/ml, and 104.1 ± 16.6 μg hours/ml, respectively. Miglustat delayed the onset of neurological signs and increased the lifespan of treated cats, and was associated with decreased GM2 ganglioside accumulation in the cerebellum and improved Purkinje cell survival. Ex vivo examination of microglia from the brains of treated cats revealed normalization of CD1c and class II major histocompatibility complex expression, as well as generation of reactive oxygen species. Together, these results suggest that prolonged Purkinje cell survival, reduced glycosphingolipid accumulation, and/or the modulation of microglial immunophenotype and function contribute to miglustat-induced neurological improvement in treated cats. PMID:22487861

Improving monoterpene geraniol production through geranyl diphosphate synthesis regulation in Saccharomyces cerevisiae.

PubMed

Zhao, Jianzhi; Bao, Xiaoming; Li, Chen; Shen, Yu; Hou, Jin

2016-05-01

Monoterpenes have wide applications in the food, cosmetics, and medicine industries and have recently received increased attention as advanced biofuels. However, compared with sesquiterpenes, monoterpene production is still lagging in Saccharomyces cerevisiae. In this study, geraniol, a valuable acyclic monoterpene alcohol, was synthesized in S. cerevisiae. We evaluated three geraniol synthases in S. cerevisiae, and the geraniol synthase Valeriana officinalis (tVoGES), which lacked a plastid-targeting peptide, yielded the highest geraniol production. To improve geraniol production, synthesis of the precursor geranyl diphosphate (GPP) was regulated by comparing three specific GPP synthase genes derived from different plants and the endogenous farnesyl diphosphate synthase gene variants ERG20 (G) (ERG20 (K197G) ) and ERG20 (WW) (ERG20 (F96W-N127W) ), and controlling endogenous ERG20 expression, coupled with increasing the expression of the mevalonate pathway by co-overexpressing IDI1, tHMG1, and UPC2-1. The results showed that overexpressing ERG20 (WW) and strengthening the mevalonate pathway significantly improved geraniol production, while expressing heterologous GPP synthase genes or down-regulating endogenous ERG20 expression did not show positive effect. In addition, we constructed an Erg20p(F96W-N127W)-tVoGES fusion protein, and geraniol production reached 66.2 mg/L after optimizing the amino acid linker and the order of the proteins. The best strain yielded 293 mg/L geraniol in a fed-batch cultivation, a sevenfold improvement over the highest titer previously reported in an engineered S. cerevisiae strain. Finally, we showed that the toxicity of geraniol limited its production. The platform developed here can be readily used to synthesize other monoterpenes.

l-Arginine Attenuates Cardiac Dysfunction, But Further Down-Regulates α-Myosin Heavy Chain Expression in Isoproterenol-Induced Cardiomyopathy.

PubMed

Kralova, Eva; Doka, Gabriel; Pivackova, Lenka; Srankova, Jasna; Kuracinova, Kristina; Janega, Pavol; Babal, Pavel; Klimas, Jan; Krenek, Peter

2015-10-01

In view of previously reported increased capacity for nitric oxide production, we suggested that l-arginine (ARG), the nitric oxide synthase (NOS) substrate, supplementation would improve cardiac function in isoproterenol (ISO)-induced heart failure. Male Wistar rats were treated with ISO for 8 days (5 mg/kg/day, i.p.) or vehicle. ARG was given to control (ARG) and ISO-treated (ISO+ARG) rats in water (0.4 g/kg/day). ISO administration was associated with 40% mortality, ventricular hypertrophy, decreased heart rate, left ventricular dysfunction, fibrosis and ECG signs of ischaemia. RT-PCR showed increased mRNA levels of cardiac hypertrophy marker atrial natriuretic peptide, but not BNP, decreased expression of myosin heavy chain isoform MYH6 and unaltered expression of pathological MYH7. ISO increased the protein levels of endothelial nitric oxide synthase, but at the same time it markedly up-regulated mRNA and protein levels of gp91phox, a catalytical subunit of superoxide-producing NADPH oxidase. Fibrosis was markedly increased by ISO. ARG treatment moderately ameliorated left ventricular dysfunction, but was without effect on cardiac hypertrophy and fibrosis. Combination of ISO and ARG led to a decrease in cav-1 expression, a further increase in MYH7 expression and a down-regulation of MYH6 that inversely correlated with gp91phox mRNA levels. Although ARG, at least partially, improved ISO-impaired basal left ventricular systolic function, it failed to reduce cardiac hypertrophy, fibrosis, oxidative stress and mortality. The protection of contractile performance might be related to increased capacity for nitric oxide production and the up-regulation of MYH7 which may compensate for the marked down-regulation of the major MYH6 isoform. © 2015 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

Drug development targeting the glycogen synthase kinase-3beta (GSK-3beta)-mediated signal transduction pathway: the role of GSK-3beta in the maintenance of steady-state levels of insulin receptor signaling molecules and Na(v)1.7 sodium channel in adrenal chromaffin cells.

PubMed

Nemoto, Takayuki; Yanagita, Toshihiko; Kanai, Tasuku; Wada, Akihiko

2009-02-01

Glycogen synthase kinase-3 (GSK-3) is constitutively active in nonstimulated cells, where the majority of its substrates undergo inactivation/proteolysis by phosphorylation. Extracellular stimuli (e.g., insulin) catalyze inhibitory Ser(9)-phosphorylation of GSK-3beta, turning on signaling and causing other biological consequences otherwise constitutively suppressed by GSK-3beta. Regulated and dysregulated activities of GSK-3beta are pivotal to health, disease, and therapeutics (e.g., insulin resistance, neurodegeneration, tumorigenesis, inflammation); however, the underlying mechanisms of multifunctional GSK-3beta remain elusive. In cultured bovine adrenal chromaffin cells, 1) constitutive and negatively-regulated activities of GSK-3beta up- and down-regulated insulin receptor, insulin receptor substrate-1 (IRS-1), IRS-2, and Akt levels via controlling proteasomal degradation and protein synthesis; 2) nicotinic receptor/protein kinase C-alpha (PKC-alpha)/extracellular signal-regulated kinase (ERK) pathway up-regulated IRS-1 and IRS-2 levels, enhancing insulin-induced the phosphoinositide 3-kinase (PI3K)/Akt/GSK-3beta pathway; 3) inhibition of calcineurin by cyclosporin A or FK506 down-regulated IRS-2 level, attenuating insulin-like growth factor-I (IGF-I)-induced ERK and GSK-3beta pathways; and 4) insulin, IGF-I or therapeutics (e.g., lithium) up-regulated the voltage-dependent Na(v)1.7 sodium channel.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Mi-Bo; Song, Youngwoo; Kim, Changhee

Highlights: • Kirenol inhibits the adipogenic transcription factors and lipogenic enzymes. • Kirenol stimulates the Wnt/β-catenin signaling pathway components. • Kirenol inhibits adipogenesis through activation of the Wnt/β-catenin signaling pathway. - Abstract: Kirenol, a natural diterpenoid compound, has been reported to possess anti-oxidant, anti-inflammatory, anti-allergic, and anti-arthritic activities; however, its anti-adipogenic effect remains to be studied. The present study evaluated the effect of kirenol on anti-adipogenesis through the activation of the Wnt/β-catenin signaling pathway. Kirenol prevented intracellular lipid accumulation by down-regulating key adipogenesis transcription factors [peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding proteins α (C/EBPα), and sterol regulatory element bindingmore » protein-1c (SREBP-1c)] and lipid biosynthesis-related enzymes [fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC)], as well as adipocytokines (adiponectin and leptin). Kirenol effectively activated the Wnt/β-catenin signaling pathway, in which kirenol up-regulated the expression of low density lipoprotein receptor related protein 6 (LRP6), disheveled 2 (DVL2), β-catenin, and cyclin D1 (CCND1), while it inactivated glycogen synthase kinase 3β (GSK3β) by increasing its phosphorylation. Kirenol down-regulated the expression levels of PPARγ and C/EBPα, which were up-regulated by siRNA knockdown of β-catenin. Overall, kirenol is capable of inhibiting the differentiation and lipogenesis of 3T3-L1 adipocytes through the activation of the Wnt/β-catenin signaling pathway, suggesting its potential as natural anti-obesity agent.« less

Stabilization of mismatch repair gene PMS2 by glycogen synthase kinase 3β is implicated in the treatment of cervical carcinoma

PubMed Central

2010-01-01

Background PMS2 expression loss was reported in a variety of human. However, its importance has not been fully understood in cervical carcinoma. The aim of this study was to determine the expression of PMS2 in cervical carcinoma and evaluate the significance of mismatch repair gene PMS2 regulated by glycogen synthase kinase 3β (GSK-3β) in chemosensitivity. Methods We examined PMS2 and phosphorylated GSK-3β(s9) expression in cervical carcinoma tissues using immunohistochemical staining. Furthermore, we detected PMS2 expression in HeLa cells and evaluate the interaction with GSK-3β after transfection with GSK-3β by small interference RNA (siRNA), co-immunoprecipitation and immunoblotting. We also evaluated the effect of PMS2 transfection on HeLa cells' chemosensitivity to cisplatin treatment. Results We found significant downregulation of PMS2 in cervical carcinoma, which was negatively associated with phosphorylated GSK-3β (s9). Furthermore, we demonstrated GSK-3β transfection was able to interact with PMS2 and enhance PMS2 production in HeLa cells, and increased PMS2 production was responsible for enhanced chemosensitivity. Conclusions Our results provide the evidence that stabilization of PMS2 production by GSK-3β was important to improve chemosensitization, indicating the significance of GSK-3β-related PMS2 downregulation in the development of cervical carcinoma and in developing a potential strategy for chemotherapy. PMID:20178594

Stabilization of mismatch repair gene PMS2 by glycogen synthase kinase 3beta is implicated in the treatment of cervical carcinoma.

PubMed

Zhang, Yuan; Shu, Yi Min; Wang, Shu Fang; Da, Bang Hong; Wang, Ze Hua; Li, Hua Bin

2010-02-23

PMS2 expression loss was reported in a variety of human. However, its importance has not been fully understood in cervical carcinoma. The aim of this study was to determine the expression of PMS2 in cervical carcinoma and evaluate the significance of mismatch repair gene PMS2 regulated by glycogen synthase kinase 3beta (GSK-3beta) in chemosensitivity. We examined PMS2 and phosphorylated GSK-3beta(s9) expression in cervical carcinoma tissues using immunohistochemical staining. Furthermore, we detected PMS2 expression in HeLa cells and evaluate the interaction with GSK-3beta after transfection with GSK-3beta by small interference RNA (siRNA), co-immunoprecipitation and immunoblotting. We also evaluated the effect of PMS2 transfection on HeLa cells' chemosensitivity to cisplatin treatment. We found significant downregulation of PMS2 in cervical carcinoma, which was negatively associated with phosphorylated GSK-3beta (s9). Furthermore, we demonstrated GSK-3beta transfection was able to interact with PMS2 and enhance PMS2 production in HeLa cells, and increased PMS2 production was responsible for enhanced chemosensitivity. Our results provide the evidence that stabilization of PMS2 production by GSK-3beta was important to improve chemosensitization, indicating the significance of GSK-3beta-related PMS2 downregulation in the development of cervical carcinoma and in developing a potential strategy for chemotherapy.

The depletion of F1 subunit ε in yeast leads to an uncoupled respiratory phenotype that is rescued by mutations in the proton-translocating subunits of F0

PubMed Central

Tetaud, Emmanuel; Godard, François; Giraud, Marie-France; Ackerman, Sharon H.; di Rago, Jean-Paul

2014-01-01

The central stalk of the ATP synthase is an elongated hetero-oligomeric structure providing a physical connection between the catalytic sites in F1 and the proton translocation channel in F0 for energy transduction between the two subdomains. The shape of the central stalk and relevance to energy coupling are essentially the same in ATP synthases from all forms of life, yet the protein composition of this domain changed during evolution of the mitochondrial enzyme from a two- to a three-subunit structure (γ, δ, ε). Whereas the mitochondrial γ- and δ-subunits are homologues of the bacterial central stalk proteins, the deliberate addition of subunit ε is poorly understood. Here we report that down-regulation of the gene (ATP15) encoding the ε-subunit rapidly leads to lethal F0-mediated proton leaks through the membrane because of the loss of stability of the ATP synthase. The ε-subunit is thus essential for oxidative phosphorylation. Moreover, mutations in F0 subunits a and c, which slow the proton translocation rate, are identified that prevent ε-deficient ATP synthases from dissipating the electrochemical potential. Cumulatively our data lead us to propose that the ε-subunit evolved to permit operation of the central stalk under the torque imposed at the normal speed of proton movement through mitochondrial F0. PMID:24451261

Inhibition of melanogenesis by Xanthium strumarium L.

PubMed

Li, Hailan; Min, Young Sil; Park, Kyoung-Chan; Kim, Dong-Seok

2012-01-01

Xanthium strumarium L. (Asteraceae) is traditionally used in Korea to treat skin diseases. In this study, we investigated the effects of a X. strumarium stem extract on melanin synthesis. It inhibited melanin synthesis in a concentration-dependent manner, but it did not directly inhibit tyrosinase, the rate-limiting melanogenic enzyme, and instead downregulated microphthalmia-associated transcription factor (MITF) and tyrosinase expression. MITF, the master regulator of pigmentation, is a target of the Wnt signaling pathway, which includes glycogen synthase kinase 3β (GSK3β) and β-catenin. Hence, the influence of X. strumarium stem extract on GSK3β and β-catenin was further investigated. X. strumarium induced GSK3β phosphorylation (inactivation), but the level of β-catenin did not change. Moreover, a specific GSK3β inhibitor restored X. strumarium-induced melanin reduction. Hence, we suggest that X. strumarium inhibits melanin synthesis through downregulation of tyrosinase via GSK3β phosphorylation.

ER stress upregulated PGE2/IFNγ-induced IL-6 expression and down-regulated iNOS expression in glial cells

NASA Astrophysics Data System (ADS)

Hosoi, Toru; Honda, Miya; Oba, Tatsuya; Ozawa, Koichiro

2013-12-01

The disruption of endoplasmic reticulum (ER) function can lead to neurodegenerative disorders, in which inflammation has also been implicated. We investigated the possible correlation between ER stress and immune function using glial cells. We demonstrated that ER stress synergistically enhanced prostaglandin (PG) E2 + interferon (IFN) γ-induced interleukin (IL)-6 production. This effect was mediated through cAMP. Immune-activated glial cells produced inducible nitric oxide synthase (iNOS). Interestingly, ER stress inhibited PGE2 + IFNγ-induced iNOS expression. Similar results were obtained when cells were treated with dbcAMP + IFNγ. Thus, cAMP has a dual effect on immune reactions; cAMP up-regulated IL-6 expression, but down-regulated iNOS expression under ER stress. Therefore, our results suggest a link between ER stress and immune reactions in neurodegenerative diseases.

Let-7i attenuates human brain microvascular endothelial cell damage in oxygen glucose deprivation model by decreasing toll-like receptor 4 expression.

PubMed

Xiang, Wei; Tian, Canhui; Peng, Shunli; Zhou, Liang; Pan, Suyue; Deng, Zhen

2017-11-04

The let-7 family of microRNAs (miRNAs) plays an important role on endothelial cell function. However, there have been few studies on their role under ischemic conditions. In this study, we demonstrate that let-7i, belonging to the let-7 family, rescues human brain microvascular endothelial cells (HBMECs) in an oxygen-glucose deprivation (OGD) model. Our data show that the expression of let-7 family miRNAs was downregulated after OGD. Overexpression of let-7i significantly alleviated cell death and improved survival of OGD-treated HBMECs. Let-7i also protected permeability in an in vitro blood brain barrier (BBB) model. Further, let-7i downregulated the expression of toll-like receptor 4 (TLR4), an inflammation trigger. Moreover, overexpression of let-7i decreased matrix metallopeptidase 9 (MMP9) and inducible nitric oxide synthase (iNOS) expression under OGD. Upon silencing TLR4 expression in HBMECs, the anti-inflammatory effect of let-7i was abolished. Our research suggests that let-7i promotes OGD-induced inflammation via downregulating TLR4 expression. Copyright © 2017 Elsevier Inc. All rights reserved.

Transcriptional profiling reveals elevated CO2 and elevated O3 alter resistance of soybean (Glycine max) to Japanese beetles (Popillia japonica).

PubMed

Casteel, Clare L; O'Neill, Bridget F; Zavala, Jorge A; Bilgin, Damla D; Berenbaum, May R; Delucia, Evan H

2008-04-01

The accumulation of CO2 and O3 in the troposphere alters phytochemistry which in turn influences the interactions between plants and insects. Using microarray analysis of field-grown soybean (Glycine max), we found that the number of transcripts in the leaves affected by herbivory by Japanese beetles (Popillia japonica) was greater when plants were grown under elevated CO2, elevated O3 and the combination of elevated CO2 plus elevated O3 than when grown in ambient atmosphere. The effect of herbivory on transcription diminished strongly with time (<1% of genes were affected by herbivory after 3 weeks), and elevated CO2 interacted more strongly with herbivory than elevated O3. The majority of transcripts affected by elevated O3 were related to antioxidant metabolism. Constitutive levels and the induction by herbivory of key transcripts associated with defence and hormone signalling were down-regulated under elevated CO2; 1-aminocyclopropane-1-carboxylate (ACC) synthase, lipoxygenase (LOX), allene oxide synthase (AOS), allene oxide cyclase (AOC), chalcone synthase (CHS), polyphenol oxidase (PPO) and cysteine protease inhibitor (CystPI) were lower in abundance compared with levels under ambient conditions. By suppressing the ability to mount an effective defence, elevated CO2 may decrease resistance of soybean to herbivory.

Role of the Tomato Non-Ripening Mutation in Regulating Fruit Quality Elucidated Using iTRAQ Protein Profile Analysis

PubMed Central

Yuan, Xin-Yu; Wang, Rui-Heng; Zhao, Xiao-Dan; Luo, Yun-Bo; Fu, Da-Qi

2016-01-01

Natural mutants of the Non-ripening (Nor) gene repress the normal ripening of tomato fruit. The molecular mechanism of fruit ripening regulation by the Nor gene is unclear. To elucidate how the Nor gene can affect ripening and fruit quality at the protein level, we used the fruits of Nor mutants and wild-type Ailsa Craig (AC) to perform iTRAQ (isobaric tags for relative and absolute quantitation) analysis. The Nor mutation altered tomato fruit ripening and affected quality in various respects, including ethylene biosynthesis by down-regulating the abundance of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO), pigment biosynthesis by repressing phytoene synthase 1 (PSY1), ζ-carotene isomerase (Z-ISO), chalcone synthase 1 (CHS1) and other proteins, enhancing fruit firmness by increasing the abundance of cellulose synthase protein, while reducing those of polygalacturonase 2 (PG2) and pectate lyase (PL), altering biosynthesis of nutrients such as carbohydrates, amino acids, and anthocyanins. Conversely, Nor mutation also enhanced the fruit’s resistance to some pathogens by up-regulating the expression of several genes associated with stress and defense. Therefore, the Nor gene is involved in the regulation of fruit ripening and quality. It is useful in the future as a means to improve fruit quality in tomato. PMID:27732677

Age-dependent changes in the sphingolipid composition of CD4+ T cell membranes and immune synapses implicate glucosylceramides in age-related T cell dysfunction

USDA-ARS?s Scientific Manuscript database

Sphingolipid (SL4) composition can influence the biophysical properties of cell membranes. Additionally, specific SL modulate signaling pathways involved in proliferation, senescence, and apoptosis. We investigated age-dependent changes in the SL composition of CD4+ T cells, and the impact of these ...

Hypothermia translocates nitric oxide synthase from cytosol to membrane in snail neurons.

PubMed

Rószer, Tamás; Kiss-Tóth, Eva; Rózsa, Dávid; Józsa, Tamás; Szentmiklósi, A József; Bánfalvi, Gáspár

2010-11-01

Neuronal nitric oxide (NO) levels are modulated through the control of catalytic activity of NO synthase (NOS). Although signals limiting excess NO synthesis are being extensively studied in the vertebrate nervous system, our knowledge is rather limited on the control of NOS in neurons of invertebrates. We have previously reported a transient inactivation of NOS in hibernating snails. In the present study, we aimed to understand the mechanism leading to blocked NO production during hypothermic periods of Helix pomatia. We have found that hypothermic challenge translocated NOS from the cytosol to the perinuclear endoplasmic reticulum, and that this cytosol to membrane trafficking was essential for inhibition of NO synthesis. Cold stress also downregulated NOS mRNA levels in snail neurons, although the amount of NOS protein remained unaffected in response to hypothermia. Our studies with cultured neurons and glia cells revealed that glia-neuron signaling may inhibit membrane binding and inactivation of NOS. We provide evidence that hypothermia keeps NO synthesis "hibernated" through subcellular redistribution of NOS.

Effect of caffeine on the expression pattern of water-soluble proteins in rice (Oryza sativa) seedlings.

PubMed

Deng, Wei-Wei; Sasamoto, Hamako; Ashihara, Hiroshi

2015-05-01

It has been suggested that caffeine acts as an allelochemical which influences the germination and growth of plants. The effect of caffeine on the expression profiles of proteins was investigated in shoot-root axes of rice (Oryza sativa) seedlings. Two-dimensional difference gel electrophoresis combined with Matrix-Assisted Laser Desorption/Ionization Time of Flight/Time of Flight Mass Spectrometry was employed for the separation and identification of proteins. The results indicated that amounts of 51 protein spots were reduced and 14 were increased by treatment with 1 mM caffeine. Twelve rice seedling proteins were identified. Down-regulated proteins were β-tubulin, sucrose synthase, glyceraldehyde-3-phosphate dehydrogenase, reversibly glycosylated polypeptide/α-1,4-glucan protein synthase and cytoplasmic malate dehydrogenase. In contrast, up-regulated proteins were alanyl-aminopeptidase, acetyl-CoA carboxylase, adenine phosphoribosyltransferase, NAD-malate dehydrogenase, ornithine carbamoyltransferase, glucose-6-phosphate isomerase and nuclear RNA binding protein. Possible alternation of metabolism caused by caffeine is discussed with the protein expression data.

Differential requirement for nitric oxide in IGF-1-induced anti-apoptotic, anti-oxidant and anti-atherosclerotic effects

PubMed Central

Sukhanov, Sergiy; Higashi, Yusuke; Shai, Shaw-Yung; Blackstock, Christopher; Galvez, Sarah; Vaughn, Charlotte; Titterington, Jane; Delafontaine, Patrick

2011-01-01

We have shown previously that insulin like-growth factor I (IGF-1) suppressed atherosclerosis in Apoe−/− mice and activated endothelial nitric oxide (NO) synthase. To determine whether IGF-1-induced atheroprotection depends on NO, IGF-1- or saline-infused mice were treated with L-NAME, the pan-NO synthase inhibitor or with D-NAME (control). IGF-1 reduced atherosclerosis in both the D-NAME and L-NAME groups suggesting that IGF-1’s anti-atherogenic effect was NO-independent. IGF-1 increased plaque smooth muscle cells, suppressed cell apoptosis and downregulated lipoprotein lipase and these effects were also NO-independent. On the contrary, IGF-1 decreased oxidative stress and suppressed TNF-α levels and these effects were blocked by L-NAME. Thus IGF-1’s anti-oxidant effect is dependent on its ability to increase NO but is distinct from its anti-atherosclerotic effect which is NO-independent. PMID:21872589

Impact of d-limonene synthase up- or down-regulation on sweet orange fruit and juice odor perception.

PubMed

Rodríguez, Ana; Peris, Josep E; Redondo, Ana; Shimada, Takehiko; Costell, Elvira; Carbonell, Inmaculada; Rojas, Cristina; Peña, Leandro

2017-02-15

Citrus fruits are characterized by a complex mixture of volatiles making up their characteristic aromas, being the d-limonene the most abundant one. However, its role on citrus fruit and juice odor is controversial. Transgenic oranges engineered for alterations in the presence or concentration of few related chemical groups enable asking precise questions about their contribution to overall odor, either positive or negative, as perceived by the human nose. Here, either down- or up-regulation of a d-limonene synthase allowed us to infer that a decrease of as much as 51 times in d-limonene and an increase of as much as 3.2 times in linalool in juice were neutral for odor perception while an increase of only 3 times in ethyl esters stimulated the preference of 66% of the judges. The ability to address these questions presents exciting opportunities to understand the basic principles of selection of food. Copyright © 2016 Elsevier Ltd. All rights reserved.

Altered transition metal homeostasis in Niemann-Pick disease, Type C1

PubMed Central

Hung, Ya Hui; Faux, Noel G.; Killilea, David W.; Yanjanin, Nicole; Firnkes, Sally; Volitakis, Irene; Ganio, George; Walterfang, Mark; Hastings, Caroline; Porter, Forbes D.; Ory, Daniel S.; Bush, Ashley I.

2014-01-01

The loss of NPC1 protein function is the predominant cause of Niemann-Pick type C1 disease (NP-C1), a systemic and neurodegenerative disorder characterized by late-endosomal/lysosomal accumulation of cholesterol and other lipids. Limited evidence from post-mortem human tissues, an Npc1−/− mouse model, and cell culture studies also suggest failure of metal homeostasis in NP-C1. To investigate these findings, we performed a comprehensive transition metal analysis of cerebrospinal fluid (CSF), plasma and tissue samples from human NP-C1 patients and an Npc1−/− mouse model. NPC1 deficiency in the Npc1−/− mouse model resulted in a perturbation of transition metal homeostasis in the plasma and key organs (brain, liver, spleen, heart, lungs, and kidneys). Analysis of human patient CSF, plasma and post-mortem brain tissues also indicated disrupted metal homeostasis. There was a disparity in the direction of metal changes between the human and the Npc1−/− mouse samples, which may reflect species-specific metal metabolism. Nevertheless, common to both species is brain zinc accumulation. Furthermore, treatment with the glucosylceramide synthase inhibitor miglustat, the only drug shown in a controlled clinical trial to have some efficacy for NP-C1, did not correct the alterations in CSF and plasma transition metal and ceruloplasmin (CP) metabolism in NP-C1 patients. These findings highlight the importance of NPC1 function in metal homeostasis, and indicate that metal-targeting therapy may be of value as a treatment for NP-C. PMID:24343124

Infection Structure–Specific Expression of β-1,3-Glucan Synthase Is Essential for Pathogenicity of Colletotrichum graminicola and Evasion of β-Glucan–Triggered Immunity in Maize[W

PubMed Central

Oliveira-Garcia, Ely; Deising, Holger B.

2013-01-01

β-1,3-Glucan and chitin are the most prominent polysaccharides of the fungal cell wall. Covalently linked, these polymers form a scaffold that determines the form and properties of vegetative and pathogenic hyphae. While the role of chitin in plant infection is well understood, the role of β-1,3-glucan is unknown. We functionally characterized the β-1,3-glucan synthase gene GLS1 of the maize (Zea mays) pathogen Colletotrichum graminicola, employing RNA interference (RNAi), GLS1 overexpression, live-cell imaging, and aniline blue fluorochrome staining. This hemibiotroph sequentially differentiates a melanized appressorium on the cuticle and biotrophic and necrotrophic hyphae in its host. Massive β-1,3-glucan contents were detected in cell walls of appressoria and necrotrophic hyphae. Unexpectedly, GLS1 expression and β-1,3-glucan contents were drastically reduced during biotrophic development. In appressoria of RNAi strains, downregulation of β-1,3-glucan synthesis increased cell wall elasticity, and the appressoria exploded. While the shape of biotrophic hyphae was unaffected in RNAi strains, necrotrophic hyphae showed severe distortions. Constitutive expression of GLS1 led to exposure of β-1,3-glucan on biotrophic hyphae, massive induction of broad-spectrum defense responses, and significantly reduced disease symptom severity. Thus, while β-1,3-glucan synthesis is required for cell wall rigidity in appressoria and fast-growing necrotrophic hyphae, its rigorous downregulation during biotrophic development represents a strategy for evading β-glucan–triggered immunity. PMID:23898035

RNA Interference Silencing of Glycogen Synthase Kinase 3β Inhibites Tau Phosphorylation in Mice with Alzheimer Disease.

PubMed

Bian, Hong; Bian, Wei; Lin, Xiaoying; Ma, Zhaoyin; Chen, Wen; Pu, Ying

2016-09-01

To explore the effect of glycogen synthase kinase 3β (GSK-3β) silencing on Tau-5 phosphorylation in mice suffering Alzheimer disease (AD). GSK-3β was firstly silenced in human neuroblastoma SH-SY5Y cells using special lentivirus (LV) and the content of Tau (A-12), p-Tau (Ser396) and p-Tau (PHF-6) proteins. GSK-3β was also silenced in APP/PS1 mouse model of AD mice, which were divided into three groups (n = 10): AD, vehicle, and LV group. Ten C57 mice were used as control. The memory ability of mice was tested by square water maze, and the morphological changes of hippocampus and neuron death were analyzed by haematoxylin-eosin staining. Moreover, the levels of Tau and phosphorylated Tau (p-Tau) were detected by western blotting and immunohistochemistry, respectively. The lentivirus-mediated GSK-3β silencing system was successfully developed and silencing GSK-3β at the cellular level reduced Tau phosphorylation obviously. Moreover, GSK-3β silence significantly improved the memory ability of AD mice in LV group compared with AD group (P < 0.05) according to the latency periods and error numbers. As for the hippocampus morphology and neuron death, no significant change was observed between LV group and normal control. Immunohistochemical detection and western blotting revealed that the levels of Tau and p-Tau were significantly down-regulated after GSK-3β silence. Silencing GSK-3β may have a positive effect on inhibiting the pathologic progression of AD through down-regulating the level of p-Tau.

A Metronidazole-Resistant Isolate of Blastocystis spp. Is Susceptible to Nitric Oxide and Downregulates Intestinal Epithelial Inducible Nitric Oxide Synthase by a Novel Parasite Survival Mechanism ▿

PubMed Central

Mirza, Haris; Wu, Zhaona; Kidwai, Fahad; Tan, Kevin S. W.

2011-01-01

Blastocystis, one of the most common parasites colonizing the human intestine, is an extracellular, noninvasive, luminal protozoan with controversial pathogenesis. Blastocystis infections can be asymptomatic or cause intestinal symptoms of vomiting, diarrhea, and abdominal pain. Although chronic infections are frequently reported, Blastocystis infections have also been reported to be self-limiting in immunocompetent patients. Characterizing the host innate response to Blastocystis would lead to a better understanding of the parasite's pathogenesis. Intestinal epithelial cells produce nitric oxide (NO), primarily on the apical side, in order to target luminal pathogens. In this study, we show that NO production by intestinal cells may be a host defense mechanism against Blastocystis. Two clinically relevant isolates of Blastocystis, ST-7 (B) and ST-4 (WR-1), were found to be susceptible to a range of NO donors. ST-7 (B), a metronidazole-resistant isolate, was found to be more sensitive to nitrosative stress. Using the Caco-2 model of human intestinal epithelium, Blastocystis ST-7 (B) but not ST-4 (WR-1) exhibited dose-dependent inhibition of Caco-2 NO production, and this was associated with downregulation of inducible nitric oxide synthase (iNOS). Despite its higher susceptibility to NO, Blastocystis ST-7 (B) may have evolved unique strategies to evade this potential host defense by depressing host NO production. This is the first study to highlight a strain-to-strain variation in the ability of Blastocystis to evade the host antiparasitic NO response. PMID:21930763

Hyaluronan synthase 3 (HAS3) overexpression downregulates MV3 melanoma cell proliferation, migration and adhesion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takabe, Piia, E-mail: piia.takabe@uef.fi; Bart, Geneviève; Ropponen, Antti

2015-09-10

Malignant skin melanoma is one of the most deadly human cancers. Extracellular matrix (ECM) influences the growth of malignant tumors by modulating tumor cells adhesion and migration. Hyaluronan is an essential component of the ECM, and its amount is altered in many tumors, suggesting an important role for hyaluronan in tumorigenesis. Nonetheless its role in melanomagenesis is not understood. In this study we produced a MV3 melanoma cell line with inducible expression of the hyaluronan synthase 3 (HAS3) and studied its effect on the behavior of the melanoma cells. HAS3 overexpression expanded the cell surface hyaluronan coat and decreased melanomamore » cell adhesion, migration and proliferation by cell cycle arrest at G1/G0. Melanoma cell migration was restored by removal of cell surface hyaluronan by Streptomyces hyaluronidase and by receptor blocking with hyaluronan oligosaccharides, while the effect on cell proliferation was receptor independent. Overexpression of HAS3 decreased ERK1/2 phosphorylation suggesting that inhibition of MAP-kinase signaling was responsible for these suppressive effects on the malignant phenotype of MV3 melanoma cells. - Highlights: • Inducible HAS3-MV3 melanoma cell line was generated using Lentiviral transduction. • HAS3 overexpression inhibits MV3 cell migration via hyaluronan–receptor interaction. • HAS3 overexpression decreases MV3 melanoma cell proliferation and adhesion. • ERK1/2 phosphorylation is downregulated by 50% in HAS3 overexpressing cells. • The results suggest that hyaluronan has anti-cancer like effects in melanoma.« less

Inhibitory effects and molecular mechanisms of garlic organosulfur compounds on the production of inflammatory mediators.

PubMed

You, Sixiang; Nakanishi, Eri; Kuwata, Hiroko; Chen, Jihua; Nakasone, Yasushi; He, Xi; He, Jianhua; Liu, Xiangxin; Zhang, Shirui; Zhang, Bin; Hou, De-Xing

2013-11-01

Garlic is used for both culinary and medicinal purposes by many cultures. The garlic organosulfur compounds (GOSCs) are thought to be bioactive components. This study aims to clarify the antiinflammatory effects and molecular mechanisms of GOSCs in both cell and animal models. RAW264.7 cells were treated with six kinds of GOSCs to screen their influence on cyclooxygenase-2 and inducible nitric oxide synthase expression by Western blotting. Prostaglandin E2 and nitrite were measured by ELISA and Griess reaction, respectively. Cytokines in culture medium were assayed by the multiplex technology. Proteins were detected by Western blotting. Mouse paw edema was induced by LPS. The results revealed that diallyl trisulfide (DATS) was a strongest inhibitor for cyclooxygenase and inducible nitric oxide synthase among GOSCs, and reduced the levels of LPS-induced IL-6, IL-10, IL-12(p70), KC, MCP-1, and TNF-α. Cellular signaling analysis revealed that DATS downregulated AKT1/TGF-β-activated kinase-mediated mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways. Furthermore, DATS activated Nrf2-mediated expression of HO-1 and NQO1 and reduced LPS-induced intracellular reactive oxygen species, which may contribute to suppress inflammatory mediator production. Finally, in vivo data demonstrated that DATS attenuated LPS-induced mouse paw edema. DATS as a potential inhibitor revealed antiinflammatory effect in both cell and animal models by downregulating AKT1/TGF-β-activated kinase-mediated NFκB and MAPK signaling pathways. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sphingolipid Metabolism Is Dysregulated at Transcriptomic and Metabolic Levels in the Spinal Cord of an Animal Model of Amyotrophic Lateral Sclerosis

PubMed Central

Henriques, Alexandre; Croixmarie, Vincent; Bouscary, Alexandra; Mosbach, Althéa; Keime, Céline; Boursier-Neyret, Claire; Walter, Bernard; Spedding, Michael; Loeffler, Jean-Philippe

2018-01-01

Lipid metabolism is drastically dysregulated in amyotrophic lateral sclerosis and impacts prognosis of patients. Animal models recapitulate alterations in the energy metabolism, including hypermetabolism and severe loss of adipose tissue. To gain insight into the molecular mechanisms underlying disease progression in amyotrophic lateral sclerosis, we have performed RNA-sequencing and lipidomic profiling in spinal cord of symptomatic SOD1G86R mice. Spinal transcriptome of SOD1G86R mice was characterized by differential expression of genes related to immune system, extracellular exosome, and lysosome. Hypothesis-driven identification of metabolites showed that lipids, including sphingomyelin(d18:0/26:1), ceramide(d18:1/22:0), and phosphatidylcholine(o-22:1/20:4) showed profound altered levels. A correlation between disease severity and gene expression or metabolite levels was found for sphingosine, ceramide(d18:1/26:0), Sgpp2, Sphk1, and Ugt8a. Joint-analysis revealed a significant enrichment of glycosphingolipid metabolism in SOD1G86R mice, due to the down-regulation of ceramide, glucosylceramide, and lactosylceramide and the overexpression of genes involved in their recycling in the lysosome. A drug-gene interaction database was interrogated to identify potential drugs able to modulate the dysregulated genes from the signaling pathway. Our results suggest that complex lipids are pivotally changed during the first phase of motor symptoms in an animal model of amyotrophic lateral sclerosis. PMID:29354030

Sphingolipid Metabolism Is Dysregulated at Transcriptomic and Metabolic Levels in the Spinal Cord of an Animal Model of Amyotrophic Lateral Sclerosis.

PubMed

Henriques, Alexandre; Croixmarie, Vincent; Bouscary, Alexandra; Mosbach, Althéa; Keime, Céline; Boursier-Neyret, Claire; Walter, Bernard; Spedding, Michael; Loeffler, Jean-Philippe

2017-01-01

Lipid metabolism is drastically dysregulated in amyotrophic lateral sclerosis and impacts prognosis of patients. Animal models recapitulate alterations in the energy metabolism, including hypermetabolism and severe loss of adipose tissue. To gain insight into the molecular mechanisms underlying disease progression in amyotrophic lateral sclerosis, we have performed RNA-sequencing and lipidomic profiling in spinal cord of symptomatic SOD1 G86R mice. Spinal transcriptome of SOD1 G86R mice was characterized by differential expression of genes related to immune system, extracellular exosome, and lysosome. Hypothesis-driven identification of metabolites showed that lipids, including sphingomyelin(d18:0/26:1), ceramide(d18:1/22:0), and phosphatidylcholine(o-22:1/20:4) showed profound altered levels. A correlation between disease severity and gene expression or metabolite levels was found for sphingosine, ceramide(d18:1/26:0), Sgpp2, Sphk1 , and Ugt8a . Joint-analysis revealed a significant enrichment of glycosphingolipid metabolism in SOD1 G86R mice, due to the down-regulation of ceramide, glucosylceramide, and lactosylceramide and the overexpression of genes involved in their recycling in the lysosome. A drug-gene interaction database was interrogated to identify potential drugs able to modulate the dysregulated genes from the signaling pathway. Our results suggest that complex lipids are pivotally changed during the first phase of motor symptoms in an animal model of amyotrophic lateral sclerosis.

Role of ghrelin in small intestinal motility following pediatric intracerebral hemorrhage in mice.

PubMed

Zan, Jieyu; Song, Lei; Wang, Jiejie; Zou, Rong; Hong, Fei; Zhao, Jinhua; Cheng, Yijun; Xu, Ming

2017-11-01

Small intestinal motility (SIM) disorder is a common complication following pediatric intracerebral hemorrhage (ICH), leading to a poor prognosis in patients. Previous studies have shown that ghrelin is involved in SIM in various diseases; however, the role of ghrelin in pediatric ICH‑induced SIM disorder remains to be elucidated. The present study was designed to investigate the association between ghrelin and SIM post‑ICH, and to examine the effect of exogenous ghrelin administration on SIM in vivo. An ICH model was induced in mice by autologous blood infusion. Neurobehavioral deficits were evaluated using a Rotarod test, forelimb placing test, and corner turn test. Intestinal mucosal damage was examined using hematoxylin and eosin staining. SIM was measured using charcoal meal staining. An enzyme‑linked immunosorbent assay was used to evaluate serum levels of ghrelin and nitric oxide (NO). Reverse transcription‑quantitative polymerase chain reaction and western blot analyses were performed to determine the levels of inducible nitric oxide synthase (iNOS), neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) at the mRNA and protein levels. Nω‑nitro‑L‑arginine methyl ester hydrochloride (L‑NAME), L‑arginine, atropine, phentolamine and propranolol were used to manipulate the putative pathways induced by ghrelin. Neurological dysfunction was observed post‑ICH. ICH caused damage to the intestinal mucosa and delayed SIM. Serum levels of ghrelin increased between 3 h and 3 days, peaking at 12 h, and showed a significant negative correlation with SIM post‑ICH. Ghrelin administration dose‑dependently attenua-ted ICH‑induced SIM disorder. Ghrelin also decreased NO levels by downregulating the mRNA and protein expression levels of iNOS, but not those of nNOS or eNOS, post‑ICH. Consistently, the effect was enhanced by L‑NAME and weakened by L‑arginine, respectively. The protective effect of ghrelin was eradicated by atropine, but not phentolamine or propranolol. These findings suggested that ghrelin ameliorated SIM disorder by downregulating iNOS/NO via the cholinergic pathway. Therefore, ghrelin may serve as a potential biomarker and useful target in ICH‑induced SIM disorder.

Effects of bile salts on glucosylceramide containing membranes.

PubMed

Halin, Josefin; Mattjus, Peter

2011-12-01

The glycolipid transfer protein (GLTP) is capable of transporting glycolipids from a donor membrane, through the aqueous environment, to an acceptor membrane. The GLTP mediated glycolipid transfer from sphingomyelin membranes is very slow. In contrast, the transfer is fast from membranes composed of phosphatidylcholine. The lateral glycolipid membrane organization is known to be driven by their tendency to mix non-randomly with different membrane lipids. Consequently, the properties of the membrane lipids surrounding the glycolipids play an important role in the ability of GLTP to bind and transfer its substrates. Since GLTP transfer of glycolipids is almost nonexistent from sphingomyelin membranes, we have used this exceptionality to investigate if membrane intercalators can alter the membrane packing and induce glycolipid transfer. We found that the bile salts cholate, deoxycholate, taurocholate and taurodeoxycholate, cause glucosylceramide to become transferrable by GLTP. Other compounds, such as single chain lipids, ceramide and nonionic surfactants, that have membrane-perturbing effects, did not affect the transfer capability of GLTP. We speculate that the strong hydrogen bonding network formed in the interfacial region of glycosphingolipid-sphingomyelin membranes is disrupted by the membrane partition of the bile salts causing the glycosphingolipid to become transferrable. Copyright © 2011 Elsevier B.V. All rights reserved.

Virus-induced gene silencing of Withania somnifera squalene synthase negatively regulates sterol and defence-related genes resulting in reduced withanolides and biotic stress tolerance.

PubMed

Singh, Anup Kumar; Dwivedi, Varun; Rai, Avanish; Pal, Shaifali; Reddy, Sajjalavarahalli Gangireddy Eswara; Rao, Dodaghatta Krishnarao Venkata; Shasany, Ajit Kumar; Nagegowda, Dinesh A

2015-12-01

Withania somnifera (L.) Dunal is an important Indian medicinal plant that produces withanolides, which are triterpenoid steroidal lactones having diverse biological activities. To enable fast and efficient functional characterization of genes in this slow-growing and difficult-to-transform plant, a virus-induced gene silencing (VIGS) was established by silencing phytoene desaturase (PDS) and squalene synthase (SQS). VIGS of the gene encoding SQS, which provides precursors for triterpenoids, resulted in significant reduction of squalene and withanolides, demonstrating its application in studying withanolides biosynthesis in W. somnifera leaves. A comprehensive analysis of gene expression and sterol pathway intermediates in WsSQS-vigs plants revealed transcriptional modulation with positive feedback regulation of mevalonate pathway genes, and negative feed-forward regulation of downstream sterol pathway genes including DWF1 (delta-24-sterol reductase) and CYP710A1 (C-22-sterol desaturase), resulting in significant reduction of sitosterol, campesterol and stigmasterol. However, there was little effect of SQS silencing on cholesterol, indicating the contribution of sitosterol, campesterol and stigmasterol, but not of cholesterol, towards withanolides formation. Branch-point oxidosqualene synthases in WsSQS-vigs plants exhibited differential regulation with reduced CAS (cycloartenol synthase) and cycloartenol, and induced BAS (β-amyrin synthase) and β-amyrin. Moreover, SQS silencing also led to the down-regulation of brassinosteroid-6-oxidase-2 (BR6OX2), pathogenesis-related (PR) and nonexpressor of PR (NPR) genes, resulting in reduced tolerance to bacterial and fungal infection as well as to insect feeding. Taken together, SQS silencing negatively regulated sterol and defence-related genes leading to reduced phytosterols, withanolides and biotic stress tolerance, thus implicating the application of VIGS for functional analysis of genes related to withanolides formation in W. somnifera leaves. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Parthenolide accumulation and expression of genes related to parthenolide biosynthesis affected by exogenous application of methyl jasmonate and salicylic acid in Tanacetum parthenium.

PubMed

Majdi, Mohammad; Abdollahi, Mohammad Reza; Maroufi, Asad

2015-11-01

Up-regulation of germacrene A synthase and down-regulation of parthenolide hydroxylase genes play key role in parthenolide accumulation of feverfew plants treated with methyl jasmonate and salicylic acid. Parthenolide is an important sesquiterpene lactone due to its anti-migraine and anti-cancer properties. Parthenolide amount was quantified by high-performance liquid chromatography after foliar application of methyl jasmonate (100 µM) or salicylic acid (1.0 mM) on feverfew leaves in time course experiment (3-96 h). Results indicate that exogenous application of methyl jasmonate or salicylic acid activated parthenolide biosynthesis. Parthenolide content reached its highest amount at 24 h after methyl jasmonate or salicylic acid treatments, which were 3.1- and 1.96-fold higher than control plants, respectively. Parthenolide transiently increased due to methyl jasmonate or salicylic acid treatments until 24 h, but did not show significant difference compared with control plants at 48 and 96 h time points in both treatments. Also, the transcript levels of early pathway (upstream) genes of terpene biosynthesis including 3-hydroxy-3-methylglutaryl-coenzyme A reductase, 1-deoxy-D-xylulose-5-phosphate reductoisomerase and hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase and the biosynthetic genes of parthenolide including germacrene A synthase, germacrene A oxidase, costunolide synthase and parthenolide synthase were increased by methyl jasmonate and salicylic acid treatments, but with different intensity. The transcriptional levels of these genes were higher in methyl jasmonate-treated plants than salicylic acid-treated plants. Parthenolide content measurements along with expression pattern analysis of the aforementioned genes and parthenolide hydroxylase as side branch gene of parthenolide suggest that the expression patterns of early pathway genes were not directly consistent with parthenolide accumulation pattern; hence, parthenolide accumulation is probably further modulated by the expression of its biosynthetic genes, especially germacrene A synthase and also its side branch gene, parthenolide hydroxylase.

Korean Red Ginseng Saponin Fraction Downregulates Proinflammatory Mediators in LPS Stimulated RAW264.7 Cells and Protects Mice against Endotoxic Shock.

PubMed

Yayeh, Taddessee; Jung, Kun-Ho; Jeong, Hye Yoon; Park, Ji-Hoon; Song, Yong-Bum; Kwak, Yi-Seong; Kang, Heun-Soo; Cho, Jae Youl; Oh, Jae-Wook; Kim, Sang-Keun; Rhee, Man Hee

2012-07-01

Korean red ginseng has shown therapeutic effects for a number of disease conditions. However, little is known about the antiinflammatory effect of Korean red ginseng saponin fraction (RGSF) in vitro and in vivo. Therefore, in this study, we showed that RGSF containing 20(S)-protopanaxadiol type saponins inhibited nitric oxide production and attenuated the release of tumor necrotic factor (TNF)-α, interleukin (IL)-6, granulocyte monocyte colony stimulating factor (GMCSF), and macrophage chemo-attractant protein-1 in lipopolysaccharide (LPS) stimulated murine macrophage RAW264.7 cells. Moreover, RGSF down-regulated the mRNA expressions of inducible nitric oxide synthase, cyclooxyginase-2, IL-1β, TNF-α, GMCSF, and IL-6. Furthermore, RGSF reduced the level of TNF-α in the serum and protected mice against LPS mediated endotoxic shock. In conclusion, these results indicated that ginsenosides from RGSF and their metabolites could be potential sources of therapeutic agents against inflammation.

Elucidating the Mechanisms of the Tomato ovate Mutation in Regulating Fruit Quality Using Proteomics Analysis.

PubMed

Liu, Juhua; Zhang, Jing; Miao, Hongxia; Jia, Caihong; Wang, Jingyi; Xu, Biyu; Jin, Zhiqiang

2017-11-22

The ovate mutation has frequently been used to study changes in fruit shape but not fruit quality. A deterioration in fruit quality associated with the ovate mutation was discovered in this study. To elucidate how ovate influences the quality of fruit, we performed a proteomics analysis of the fruits of the ovate mutant (LA3543) and wild-type ("Ailsa Craig", LA2838A) using tandem mass tag analysis. The results indicated that the ovate mutation significantly influences fruit quality in a number of ways, including by reducing the expression of 1-aminocyclopropane-1-carboxylic acid oxidase 3 (ACO3) in ethylene biosynthesis, improving firmness by reducing the amount of pectinesterase and polygalacturonase, reducing sugar accumulation by downregulating the abundance of mannan endo-1,4-β-mannosidase 4, β-galactosidase, and β-amylase, and reducing the malic acid content by downregulating the accumulation of malic enzymes and malate synthase. These findings could inform future improvements in fruit quality.

Differential expression of genes associated with lipid metabolism in longissimus dorsi of Korean bulls and steers.

PubMed

Bong, Jin Jong; Jeong, Jin Young; Rajasekar, Panchamoorthy; Cho, Young Moo; Kwon, Eung Gi; Kim, Hyeong Cheol; Paek, Bong Hyun; Baik, Myunggi

2012-07-01

The objective of this study was to compare expression of genes associated with lipid deposition and removal between bulls and steers in the longissimus dorsi muscle (LM) tissue of Korean cattle. Castration increased the expression of lipid uptake lipoprotein lipase, fatty acid translocase, and fatty acid transport protein 1 in LM. Castration increased lipogenic gene expression of both acetyl-CoA carboxylase and fatty acid synthase. In contrast, castration downregulated lipolytic gene expression of both adipose triglyceride lipase (ATGL) and monoglyceride lipase. Steers showed higher expression levels of insulin signaling phospho-v-akt murine thymoma viral oncogene homolog 1 than bulls but lower protein levels of nuclear Forkhead box O 1 (FoxO1) than bulls, suggesting that increased insulin signaling following castration decreases nuclear FoxO1 levels, leading to downregulation of ATGL gene expression. These findings suggest that castration contributes to increases in lipid uptake and lipogenesis and a decrease in lipolysis, resulting in improved marbling. Copyright © 2012 Elsevier Ltd. All rights reserved.

Metabolic and molecular analyses of white mutant Vaccinium berries show down-regulation of MYBPA1-type R2R3 MYB regulatory factor.

PubMed

Primetta, Anja K; Karppinen, Katja; Riihinen, Kaisu R; Jaakola, Laura

2015-09-01

MYBPA1-type R2R3 MYB transcription factor shows down-regulation in white mutant berries of Vaccinium uliginosum deficient in anthocyanins but not proanthocyanidins suggesting a role in the regulation of anthocyanin biosynthesis. Berries of the genus Vaccinium are among the best natural sources of flavonoids. In this study, the expression of structural and regulatory flavonoid biosynthetic genes and the accumulation of flavonoids in white mutant and blue-colored wild-type bog bilberry (V. uliginosum) fruits were measured at different stages of berry development. In contrast to high contents of anthocyanins in ripe blue-colored berries, only traces were detected by HPLC-ESI-MS in ripe white mutant berries. However, similar profile and high levels of flavonol glycosides and proanthocyanidins were quantified in both ripe white and ripe wild-type berries. Analysis with qRT-PCR showed strong down-regulation of structural genes chalcone synthase (VuCHS), dihydroflavonol 4-reductase (VuDFR) and anthocyanidin synthase (VuANS) as well as MYBPA1-type transcription factor VuMYBPA1 in white berries during ripening compared to wild-type berries. The profiles of transcript accumulation of chalcone isomerase (VuCHI), anthocyanidin reductase (VuANR), leucoanthocyanidin reductase (VuLAR) and flavonoid 3'5' hydroxylase (VuF3'5'H) were more similar between the white and the wild-type berries during fruit development, while expression of UDP-glucose: flavonoid 3-O-glucosyltransferase (VuUFGT) showed similar trend but fourfold lower level in white mutant. VuMYBPA1, the R2R3 MYB family member, is a homologue of VmMYB2 of V. myrtillus and VcMYBPA1 of V. corymbosum and belongs to MYBPA1-type MYB family which members are shown in some species to be related with proanthocyanidin biosynthesis in fruits. Our results combined with earlier data of the role of VmMYB2 in white mutant berries of V. myrtillus suggest that the regulation of anthocyanin biosynthesis in Vaccinium species could differ from other species studied.

Cardiac and renal upregulation of Nox2 and NF-κB and repression of Nox4 and Nrf2 in season- and diabetes-mediated models of vascular oxidative stress in guinea-pig and rat.

PubMed

Gajos-Draus, Anna; Duda, Monika; Beręsewicz, Andrzej

2017-11-01

The superoxide-forming NADPH oxidase homologues, Nox1, Nox2, and Nox5, seem to mediate the pro-atherosclerotic vascular phenotype. The hydrogen peroxide-forming Nox4 afforded vascular protection, likely via NF-E2-related factor-2 (Nrf2) activation and/or Nox2 downregulation in transgenic mice. We hypothesized that oxidative stress in the intact vasculature involves, aside from the upregulation of the superoxide-forming Noxs, the downregulation of the Nox4/Nrf2 pathway. Guinea-pigs and rats were studied either in winter or in summer, and the streptozotocin diabetic rats in winter. Plasma nitrite, and superoxide production by isolated hearts were measured, while frozen tissues served in biochemical analyses. Summer in both species and diabetes in rats downregulated myocardial Nox4 while reciprocally upregulating Nox2 and Nox5 in guinea-pigs, and Nox2 in rats. Simultaneously, myocardial Nrf2 activity and the expression of the Nrf2-directed heme oxygenase-1 and endothelial NO synthase were reduced while activity of the nuclear factor κ B (NF- κ B) and the expression of NF- κ B-directed inducible NO synthase and the vascular cell adhesion molecule-1 were increased. Cardiac superoxide production was increased while plasma nitrite was decreased reciprocally. Analogous disregulation of Noxs, Nrf2, and NF- κ B, occurred in diabetic rat kidneys. Given the diversity of the experimental settings and the uniform pattern of the responses, we speculate that: (1) chronic vascular oxidative stress is a nonspecific (model-, species-, organ-independent) response involving the induction of Nox2 (and Nox5 in guinea-pigs) and the NF- κ B pathway, and the repression of Nox4 and the Nrf2 pathway; and (2) the systems Nox2-NF- κ B and Nox4-Nrf2 regulate each other negatively. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Light Remodels Lipid Biosynthesis in Nannochloropsis gaditana by Modulating Carbon Partitioning between Organelles1[OPEN

PubMed Central

Vitulo, Nicola; Diretto, Gianfranco; Block, Maryse; Jouhet, Juliette; Meneghesso, Andrea; Valle, Giorgio; Giuliano, Giovanni; Maréchal, Eric

2016-01-01

The seawater microalga Nannochloropsis gaditana is capable of accumulating a large fraction of reduced carbon as lipids. To clarify the molecular bases of this metabolic feature, we investigated light-driven lipid biosynthesis in Nannochloropsis gaditana cultures combining the analysis of photosynthetic functionality with transcriptomic, lipidomic and metabolomic approaches. Light-dependent alterations are observed in amino acid, isoprenoid, nucleic acid, and vitamin biosynthesis, suggesting a deep remodeling in the microalgal metabolism triggered by photoadaptation. In particular, high light intensity is shown to affect lipid biosynthesis, inducing the accumulation of diacylglyceryl-N,N,N-trimethylhomo-Ser and triacylglycerols, together with the up-regulation of genes involved in their biosynthesis. Chloroplast polar lipids are instead decreased. This situation correlates with the induction of genes coding for a putative cytosolic fatty acid synthase of type 1 (FAS1) and polyketide synthase (PKS) and the down-regulation of the chloroplast fatty acid synthase of type 2 (FAS2). Lipid accumulation is accompanied by the regulation of triose phosphate/inorganic phosphate transport across the chloroplast membranes, tuning the carbon metabolic allocation between cell compartments, favoring the cytoplasm, mitochondrion, and endoplasmic reticulum at the expense of the chloroplast. These results highlight the high flexibility of lipid biosynthesis in N. gaditana and lay the foundations for a hypothetical mechanism of regulation of primary carbon partitioning by controlling metabolite allocation at the subcellular level. PMID:27325666

Transgenic expression of an unedited mitochondrial orfB gene product from wild abortive (WA) cytoplasm of rice (Oryza sativa L.) generates male sterility in fertile rice lines.

PubMed

Chakraborty, Anirban; Mitra, Joy; Bhattacharyya, Jagannath; Pradhan, Subrata; Sikdar, Narattam; Das, Srirupa; Chakraborty, Saikat; Kumar, Sachin; Lakhanpaul, Suman; Sen, Soumitra K

2015-06-01

Over-expression of the unedited mitochondrial orfB gene product generates male sterility in fertile indica rice lines in a dose-dependent manner. Cytoplasmic male sterility (CMS) and nuclear-controlled fertility restoration are widespread developmental features in plant reproductive systems. In self-pollinated crop plants, these processes often provide useful tools to exploit hybrid vigour. The wild abortive CMS has been employed in the majority of the "three-line" hybrid rice production since 1970s. In the present study, we provide experimental evidence for a positive functional relationship between the 1.1-kb unedited orfB gene transcript, and its translated product in the mitochondria with male sterility. The generation of the 1.1-kb unedited orfB gene transcripts increased during flowering, resulting in low ATP synthase activity in sterile plants. Following insertion of the unedited orfB gene into the genome of male-fertile plants, the plants became male sterile in a dose-dependent manner with concomitant reduction of ATPase activity of F1F0-ATP synthase (complex V). Fertility of the transgenic lines and normal activity of ATP synthase were restored by down-regulation of the unedited orfB gene expression through RNAi-mediated silencing. The genetic elements deciphered in this study could further be tested for their use in hybrid rice development.

Regulatory role of glycogen synthase kinase 3 for transcriptional activity of ADD1/SREBP1c.

PubMed

Kim, Kang Ho; Song, Min Jeong; Yoo, Eung Jae; Choe, Sung Sik; Park, Sang Dai; Kim, Jae Bum

2004-12-10

Adipocyte determination- and differentiation-dependent factor 1 (ADD1) plays important roles in lipid metabolism and insulin-dependent gene expression. Because insulin stimulates carbohydrate and lipid synthesis, it would be important to decipher how the transcriptional activity of ADD1/SREBP1c is regulated in the insulin signaling pathway. In this study, we demonstrated that glycogen synthase kinase (GSK)-3 negatively regulates the transcriptional activity of ADD1/SREBP1c. GSK3 inhibitors enhanced a transcriptional activity of ADD1/SREBP1c and expression of ADD1/SREBP1c target genes including fatty acid synthase (FAS), acetyl-CoA carboxylase 1 (ACC1), and steroyl-CoA desaturase 1 (SCD1) in adipocytes and hepatocytes. In contrast, overexpression of GSK3beta down-regulated the transcriptional activity of ADD1/SREBP1c. GSK3 inhibitor-mediated ADD1/SREBP1c target gene activation did not require de novo protein synthesis, implying that GSK3 might affect transcriptional activity of ADD1/SREBP1c at the level of post-translational modification. Additionally, we demonstrated that GSK3 efficiently phosphorylated ADD1/SREBP1c in vitro and in vivo. Therefore, these data suggest that GSK3 inactivation is crucial to confer stimulated transcriptional activity of ADD1/SREBP1c for insulin-dependent gene expression, which would coordinate lipid and glucose metabolism.

Pu-erh Tea Reduces Nitric Oxide Levels in Rats by Inhibiting Inducible Nitric Oxide Synthase Expression through Toll-Like Receptor 4

PubMed Central

Xu, Yang; Wang, Guan; Li, Chunjie; Zhang, Min; Zhao, Hang; Sheng, Jun; Shi, Wei

2012-01-01

Pu-erh tea undergoes a unique fermentation process and contains theabrownins, polysaccharides and caffeine; although it is unclear about which component is associated with the down regulation of nitric oxide levels or how this process is mediated. To address this question we examined the effects of pu-erh tea on nitric oxide synthase (NOS) genes. Cohorts of rats were separately given four-week treatments of water as control, pu-erh tea, or the tea components: theabrownins, caffeine or polysaccharides. Five experimental groups were injected with lipopolysaccharides (LPS) to induce nitric oxide (NO) production, while the corresponding five control groups were injected with saline as a negative control. The serum and liver NO concentrations were examined and the NOS expression of both mRNA and protein was measured in liver. The results showed that the rats which were fed pu-erh tea or polysaccharides had lower levels of NO which corresponded with the down-regulation of inducible nitric oxide synthase (iNOS) expression. We further demonstrate that this effect is mediated through reduction of Toll-like receptor 4 (TLR4) signaling. Thus we find that the polysaccharide components in pu-erh tea reduce NO levels in an animal model by inhibiting the iNOS expression via signaling through TLR4. PMID:22837686

α-Phellandrene alters expression of genes associated with DNA damage, cell cycle, and apoptosis in murine leukemia WEHI-3 cells.

PubMed

Lin, Jen-Jyh; Yu, Chien-Chih; Lu, Kung-Wen; Chang, Shu-Jen; Yu, Fu-Shun; Liao, Ching-Lung; Lin, Jaung-Geng; Chung, Jing-Gung

2014-08-01

α-phellandrene (α-PA) is a cyclic monoterpene, present in natural plants such as Schinus molle L. α-PA promotes immune responses in mice in vivo. However, there is no available information on whether α-PA affects gene expression in leukemia cells. The present study determined effects of α-PA on expression levels of genes associated with DNA damage, cell cycle and apoptotic cell death in mouse leukemia WEHI-3 cells. WEHI-3 cells were treated with 10 μM α-PA for 24 h, cells were harvested and total RNA was extracted, and gene expression was analyzed by cDNA microarray. Results indicated that α-PA up-regulated 10 genes 4-fold, 13 by over 3-fold and 175 by over 2-fold; 21 genes were down-regulated by over 4-fold, 26 genes by over 3-fold and expression of 204 genes was altered by at leas 2-fold compared with the untreated control cells. DNA damage-associated genes such as DNA damage-inducer transcript 4 and DNA fragmentation factor were up-regulated by 4-fold and over 2-fold, respectively; cell-cycle check point genes such as cyclin G2 and cyclin-dependent kinases inhibitor 2D and IA (p21) were up-regulated by over 3-fold and over 2-fold, respectively; apoptosis-associated genes such as BCL2/adenovirus EIB interacting protein 3, XIAP-associated factor 1, BCL2 modifying factor, caspase-8 and FADD-like apoptosis regulator were over 2-fold up-regulated. Furthermore, DNA damage-associated gene TATA box binding protein was over 4-fold down-regulated, and D19Ertd652c (DNA segment) over 2-fold down-regulated; cell cycle-associated gene cyclin E2 was over 2-fold down-regulated; apoptosis associated gene growth arrest-specific 5 was over 9-fold down-regulated, Gm5426 (ATP synthase) was over 3-fold down-regulated, and death box polypeptide 33 was over 2-fold down-regulated. Based on these observations, α-PA altered gene expression in WEHI-3 cells in vitro. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Inhibition of BRCA2 and Thymidylate Synthase Creates Multidrug Sensitive Tumor Cells via the Induction of Combined "Complementary Lethality".

PubMed

Rytelewski, Mateusz; Ferguson, Peter J; Maleki Vareki, Saman; Figueredo, Rene; Vincent, Mark; Koropatnick, James

2013-03-12

A high mutation rate leading to tumor cell heterogeneity is a driver of malignancy in human cancers. Paradoxically, however, genomic instability can also render tumors vulnerable to therapeutic attack. Thus, targeting DNA repair may induce an intolerable level of DNA damage in tumor cells. BRCA2 mediates homologous recombination repair, and BRCA2 polymorphisms increase cancer risk. However, tumors with BRCA2 mutations respond better to chemotherapy and are associated with improved patient prognosis. Thymidylate synthase (TS) is also involved in DNA maintenance and generates cellular thymidylate. We determined that antisense downregulation of BRCA2 synergistically potentiated drugs with mechanisms of action related to BRCA2 function (cisplatin, melphalan), a phenomenon we named "complementary lethality." TS knockdown induced complementary lethality to TS-targeting drugs (5-FUdR and pemetrexed) but not DNA cross-linking agents. Combined targeting of BRCA2 and TS induced complementary lethality to both DNA-damaging and TS-targeting agents, thus creating multidrug sensitive tumors. In addition, we demonstrated for the first time that simultaneous downregulation of both targets induced combined complementary lethality to multiple mechanistically different drugs in the same cell population. In this study, we propose and define the concept of "complementary lethality" and show that actively targeting BRCA2 and TS is of potential therapeutic benefit in multidrug treatment of human tumors. This work has contributed to the development of a BRCA2-targeting antisense oligdeoxynucleotide (ASO) "BR-1" which we will test in vivo in combination with our TS-targeting ASO "SARI 83" and attempt early clinical trials in the future.Molecular Therapy - Nucleic Acids (2013) 2, e78; doi:10.1038/mtna.2013.7 published online 12 March 2013.

SGK1 (glucose transport), dishevelled2 (wnt signaling), LC3/p62 (autophagy) and p53 (apoptosis) proteins are unaltered in Lafora disease.

PubMed

Wang, Peixiang; Israelian, Lori; Xue, Yunlin; Song, Siyuan; Attisano, Liliana; Minassian, Berge A

2016-01-01

Glycogen forms through the concerted actions of glycogen synthase (GS) which elongates glycogen strands, and glycogen branching enzyme (GBE). Lafora disease (LD) is a fatal neurodegenerative epilepsy that results from neuronal accumulation of hyperphosphorylated glycogen with excessively long strands (called polyglucosans). There is no GBE deficiency in LD. Instead, the disease is caused by loss-of-function mutations in the EPM2A or EPM2B genes, encoding, respectively, a phosphatase, laforin, and an E3 ubiquiting ligase, malin. A number of experimentally derived hypotheses have been published to explain LD, including: The SGK1 hypothesis - Phosphorylated SGK1 (pSGK1) raises cellular glucose uptake and levels, which would activate GS. Based on observing increased pSGK1 in LD mice it was proposed that raised pSGK1 leads to polyglucosan generation through GS hyperactivation. The Dishevelled2 hypothesis - Downregulating malin in cell culture was reported to increase levels of dishevelled2, which through the wnt/glycogen synthase kinase-3 pathway would likewise overactivate GS. The Autophagic defect hypothesis - Polyglucosans may be natural byproducts of normal glycogen metabolism. LD mice were reported to be autophagy-defective. LD would arise from failed autophagy leading to failed polyglucosan clearance. Finally, the p53 hypothesis - laforin and malin were reported to downregulate p53, their absence leading to increased p53, which would activate apoptosis, leading to the neurodegeneration of LD. In the present work we repeat key experiments that underlie these four hypotheses. We are unable to confirm increased pSGK1, dishevelled2, or p53 in LD mice, nor the reported autophagic defects. Our work does not support the above hypotheses in understanding this unique and severe form of epilepsy.

Suppressing Farnesyl Diphosphate Synthase Alters Chloroplast Development and Triggers Sterol-Dependent Induction of Jasmonate- and Fe-Related Responses1[OPEN

PubMed Central

Andrade, Paola; Caudepón, Daniel; Arró, Montserrat

2016-01-01

Farnesyl diphosphate synthase (FPS) catalyzes the synthesis of farnesyl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. Arabidopsis (Arabidopsis thaliana) contains two genes (FPS1 and FPS2) encoding FPS. Single fps1 and fps2 knockout mutants are phenotypically indistinguishable from wild-type plants, while fps1/fps2 double mutants are embryo lethal. To assess the effect of FPS down-regulation at postembryonic developmental stages, we generated Arabidopsis conditional knockdown mutants expressing artificial microRNAs devised to simultaneously silence both FPS genes. Induction of silencing from germination rapidly caused chlorosis and a strong developmental phenotype that led to seedling lethality. However, silencing of FPS after seed germination resulted in a slight developmental delay only, although leaves and cotyledons continued to show chlorosis and altered chloroplasts. Metabolomic analyses also revealed drastic changes in the profile of sterols, ubiquinones, and plastidial isoprenoids. RNA sequencing and reverse transcription-quantitative polymerase chain reaction transcriptomic analysis showed that a reduction in FPS activity levels triggers the misregulation of genes involved in biotic and abiotic stress responses, the most prominent one being the rapid induction of a set of genes related to the jasmonic acid pathway. Down-regulation of FPS also triggered an iron-deficiency transcriptional response that is consistent with the iron-deficient phenotype observed in FPS-silenced plants. The specific inhibition of the sterol biosynthesis pathway by chemical and genetic blockage mimicked these transcriptional responses, indicating that sterol depletion is the primary cause of the observed alterations. Our results highlight the importance of sterol homeostasis for normal chloroplast development and function and reveal important clues about how isoprenoid and sterol metabolism is integrated within plant physiology and development. PMID:27382138

Suppressing Farnesyl Diphosphate Synthase Alters Chloroplast Development and Triggers Sterol-Dependent Induction of Jasmonate- and Fe-Related Responses.

PubMed

Manzano, David; Andrade, Paola; Caudepón, Daniel; Altabella, Teresa; Arró, Montserrat; Ferrer, Albert

2016-09-01

Farnesyl diphosphate synthase (FPS) catalyzes the synthesis of farnesyl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. Arabidopsis (Arabidopsis thaliana) contains two genes (FPS1 and FPS2) encoding FPS. Single fps1 and fps2 knockout mutants are phenotypically indistinguishable from wild-type plants, while fps1/fps2 double mutants are embryo lethal. To assess the effect of FPS down-regulation at postembryonic developmental stages, we generated Arabidopsis conditional knockdown mutants expressing artificial microRNAs devised to simultaneously silence both FPS genes. Induction of silencing from germination rapidly caused chlorosis and a strong developmental phenotype that led to seedling lethality. However, silencing of FPS after seed germination resulted in a slight developmental delay only, although leaves and cotyledons continued to show chlorosis and altered chloroplasts. Metabolomic analyses also revealed drastic changes in the profile of sterols, ubiquinones, and plastidial isoprenoids. RNA sequencing and reverse transcription-quantitative polymerase chain reaction transcriptomic analysis showed that a reduction in FPS activity levels triggers the misregulation of genes involved in biotic and abiotic stress responses, the most prominent one being the rapid induction of a set of genes related to the jasmonic acid pathway. Down-regulation of FPS also triggered an iron-deficiency transcriptional response that is consistent with the iron-deficient phenotype observed in FPS-silenced plants. The specific inhibition of the sterol biosynthesis pathway by chemical and genetic blockage mimicked these transcriptional responses, indicating that sterol depletion is the primary cause of the observed alterations. Our results highlight the importance of sterol homeostasis for normal chloroplast development and function and reveal important clues about how isoprenoid and sterol metabolism is integrated within plant physiology and development. © 2016 American Society of Plant Biologists. All rights reserved.

Inhibiting glycogen synthase kinase-3 and transforming growth factor-β signaling to promote epithelial transition of human adipose mesenchymal stem cells.

PubMed

Setiawan, Melina; Tan, Xiao-Wei; Goh, Tze-Wei; Hin-Fai Yam, Gary; Mehta, Jodhbir S

2017-09-02

This study was aimed to investigate the epithelial differentiation of human adipose-derived mesenchymal stem cells (ADSCs) by inhibiting glycogen synthase kinase-3 (GSK3) and transforming growth factor β (TGFβ) signaling. STEMPRO human ADSCs at passage 2 were treated with CHIR99021 (GSK3 inhibitor), E-616452 (TGFβ1 receptor kinase inhibitor), A-83-01 (TGFβ type 1 receptor inhibitor), valproic acid (histone deacetylase inhibitor), tranylcypromine (monoamine oxidase inhibitor) and all-trans retinoic acid for 72 h. The mesenchymal-epithelial transition was shown by down-regulation of mesenchymal genes (Slug, Zinc Finger E-box Binding Homeobox 1 ZEB1, integrin α5 ITGA5 and vimentin VIM) and up-regulation of epithelial genes (E-cadherin, Epithelial Cell Adhesion Molecule EpCAM, Zonula Occludens-1 ZO-1, occludin, deltaN p63 δNp63, Transcription Factor 4 TCF4 and Twist Family bHLH Transcription Factor TWIST), compared to untreated ADSCs. Cell morphology and stress fiber pattern were examined and the treated cells became less migratory in scratch wound closure assay. The formation of cell junction complexes was observed under transmission electron microscopy. Global gene expression using GeneChip ® Human Genome U133 Array (Affymetrix) showed that the treatment up-regulated 540 genes (containing genes for cell cycle, cytoskeleton reorganization, chemotaxis, epithelium development and regulation of cell migration) and down-regulated 483 genes. Human ADSCs were transited to epithelial lineage by inhibiting GSK3 and TGFβ signaling. It can be an adult stem cell source for epithelial cell-based therapy. Copyright © 2017 Elsevier Inc. All rights reserved.

Temozolomide downregulates P-glycoprotein expression in glioblastoma stem cells by interfering with the Wnt3a/glycogen synthase-3 kinase/β-catenin pathway

PubMed Central

Riganti, Chiara; Salaroglio, Iris Chiara; Caldera, Valentina; Campia, Ivana; Kopecka, Joanna; Mellai, Marta; Annovazzi, Laura; Bosia, Amalia; Ghigo, Dario; Schiffer, Davide

2013-01-01

Background Glioblastoma multiforme stem cells display a highly chemoresistant phenotype, whose molecular basis is poorly known. We aim to clarify this issue and to investigate the effects of temozolomide on chemoresistant stem cells. Methods A panel of human glioblastoma cultures, grown as stem cells (neurospheres) and adherent cells, was used. Results Neurospheres had a multidrug resistant phenotype compared with adherent cells. Such chemoresistance was overcome by apparently noncytotoxic doses of temozolomide, which chemosensitized glioblastoma cells to doxorubicin, vinblastine, and etoposide. This effect was selective for P-glycoprotein (Pgp) substrates and for stem cells, leading to an investigation of whether there was a correlation between the expression of Pgp and the activity of typical stemness pathways. We found that Wnt3a and ABCB1, which encodes for Pgp, were both highly expressed in glioblastoma stem cells and reduced by temozolomide. Temozolomide-treated cells had increased methylation of the cytosine–phosphate–guanine islands in the Wnt3a gene promoter, decreased expression of Wnt3a, disrupted glycogen synthase-3 kinase/β-catenin axis, reduced transcriptional activation of ABCB1, and a lower amount and activity of Pgp. Wnt3a overexpression was sufficient to transform adherent cells into neurospheres and to simultaneously increase proliferation and ABCB1 expression. On the contrary, glioblastoma stem cells silenced for Wnt3a lost the ability to form neurospheres and reduced at the same time the proliferation rate and ABCB1 levels. Conclusions Our work suggests that Wnt3a is an autocrine mediator of stemness, proliferation, and chemoresistance in human glioblastoma and that temozolomide may chemosensitize the stem cell population by downregulating Wnt3a signaling. PMID:23897632

Identification of Genes Encoding Granule-Bound Starch Synthase Involved in Amylose Metabolism in Banana Fruit

PubMed Central

Liu, Weixin; Xu, Biyu; Jin, Zhiqiang

2014-01-01

Granule-bound starch synthase (GBSS) is responsible for amylose synthesis, but the role of GBSS genes and their encoded proteins remains poorly understood in banana. In this study, amylose content and GBSS activity gradually increased during development of the banana fruit, and decreased during storage of the mature fruit. GBSS protein in banana starch granules was approximately 55.0 kDa. The protein was up-regulated expression during development while it was down-regulated expression during storage. Six genes, designated as MaGBSSI-1, MaGBSSI-2, MaGBSSI-3, MaGBSSI-4, MaGBSSII-1, and MaGBSSII-2, were cloned and characterized from banana fruit. Among the six genes, the expression pattern of MaGBSSI-3 was the most consistent with the changes in amylose content, GBSS enzyme activity, GBSS protein levels, and the quantity or size of starch granules in banana fruit. These results suggest that MaGBSSI-3 might regulate amylose metabolism by affecting the variation of GBSS levels and the quantity or size of starch granules in banana fruit during development or storage. PMID:24505384

Virus-Induced Gene Silencing of the Eggplant Chalcone Synthase Gene during Fruit Ripening Modifies Epidermal Cells and Gravitropism.

PubMed

Wang, Cuicui; Fu, Daqi

2018-03-21

Eggplant ( Solanum melongena L.) fruits accumulate flavonoids in their cuticle and epidermal cells during ripening. Although many mutants available in model plant species, such as Arabidopsis thaliana and Medicago truncatula, are enabling the intricacies of flavonoid-related physiology to be deduced, the mechanisms whereby flavonoids influence eggplant fruit physiology are unknown. Virus-induced gene silencing (VIGS) is a reliable tool for the study of flavonoid function in fruit, and in this study, we successfully applied this technique to downregulate S. melongena chalcone synthase gene ( SmCHS) expression during eggplant fruit ripening. In addition to the expected change in fruit color attributable to a lack of anthocyanins, several other modifications, including differences in epidermal cell size and shape, were observed in the different sectors. We also found that silencing of CHS gene expression was associated with a negative gravitropic response in eggplant fruits. These observations indicate that epidermal cell expansion during ripening is dependent upon CHS expression and that there may be a relationship between CHS expression and gravitropism during eggplant fruit ripening.

Human Isoprenoid Synthase Enzymes as Therapeutic Targets

NASA Astrophysics Data System (ADS)

Park, Jaeok; Matralis, Alexios; Berghuis, Albert; Tsantrizos, Youla

2014-07-01

The complex biochemical network known as the mevalonate pathway is responsible for the biosynthesis of all isoprenoids in the human body, which consists of a vast array of metabolites that are vital for proper cellular functions. Two key isoprenoids, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) are responsible for the post-translational prenylation of small GTP-binding proteins, and serve as the biosynthetic precursors to numerous other biomolecules. The down-stream metabolite of FPP and GGPP is squalene, the precursor to steroids, bile acids, lipoproteins and vitamin D. In the past, interest in prenyl synthase inhibitors focused mainly on the role of the FPP in lytic bone diseases. More recently, pre-clinical and clinical studies have strongly implicated high levels of protein prenylation in a plethora of human diseases, including non-skeletal cancers, the progression of neurodegenerative diseases and cardiovascular diseases. In this review, we focus mainly on the potential therapeutic value of down-regulating the biosynthesis of FPP, GGPP and squalene. We summarize the most recent drug discovery efforts and the structural data available that support the current on-going studies.

Expressed proteins of Herbaspirillum seropedicae in maize (DKB240) roots-bacteria interaction revealed using proteomics.

PubMed

Ferrari, Cibele Santos; Amaral, Fernanda Plucani; Bueno, Jessica Cavalheiro Ferreira; Scariot, Mirella Christine; Valentim-Neto, Pedro Alexandre; Arisi, Ana Carolina Maisonnave

2014-11-01

Several molecular tools have been used to clarify the basis of plant-bacteria interaction; however, the mechanism behind the association is still unclear. In this study, we used a proteomic approach to investigate the root proteome of Zea mays (cv. DKB240) inoculated with Herbaspirillum seropedicae strain SmR1 grown in vitro and harvested 7 days after inoculation. Eighteen differentially accumulated proteins were observed in root samples, ten of which were identified by MALDI-TOF mass spectrometry peptide mass fingerprint. Among the identified proteins, we observed three proteins present exclusively in inoculated root samples and six upregulated proteins and one downregulated protein relative to control. Differentially expressed maize proteins were identified as hypothetical protein ZEAMMB73_483204, hypothetical protein ZEAMMB73_269466, and tubulin beta-7 chain. The following were identified as H. seropedicae proteins: peroxiredoxin protein, EF-Tu elongation factor protein, cation transport ATPase, NADPH:quinone oxidoreductase, dinitrogenase reductase, and type III secretion ATP synthase. Our results presented the first evidence of type III secretion ATP synthase expression during H. seropedicae-maize root interaction.

Human isoprenoid synthase enzymes as therapeutic targets

PubMed Central

Park, Jaeok; Matralis, Alexios N.; Berghuis, Albert M.; Tsantrizos, Youla S.

2014-01-01

In the human body, the complex biochemical network known as the mevalonate pathway is responsible for the biosynthesis of all isoprenoids, which consists of a vast array of metabolites that are vital for proper cellular functions. Two key isoprenoids, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) are responsible for the post-translational prenylation of small GTP-binding proteins, and serve as the biosynthetic precursors to numerous other biomolecules. The down-stream metabolite of FPP and GGPP is squalene, the precursor to steroids, bile acids, lipoproteins, and vitamin D. In the past, interest in prenyl synthase inhibitors focused mainly on the role of the FPP in lytic bone diseases. More recently pre-clinical and clinical studies have strongly implicated high levels of protein prenylation in a plethora of human diseases, including non-skeletal cancers, the progression of neurodegenerative diseases and cardiovascular diseases. In this review, we focus mainly on the potential therapeutic value of down-regulating the biosynthesis of FPP, GGPP, and squalene. We summarize the most recent drug discovery efforts and the structural data available that support the current on-going studies. PMID:25101260

Temporal system-level organization of the switch from glycolytic to gluconeogenic operation in yeast

PubMed Central

Zampar, Guillermo G; Kümmel, Anne; Ewald, Jennifer; Jol, Stefan; Niebel, Bastian; Picotti, Paola; Aebersold, Ruedi; Sauer, Uwe; Zamboni, Nicola; Heinemann, Matthias

2013-01-01

The diauxic shift in Saccharomyces cerevisiae is an ideal model to study how eukaryotic cells readjust their metabolism from glycolytic to gluconeogenic operation. In this work, we generated time-resolved physiological data, quantitative metabolome (69 intracellular metabolites) and proteome (72 enzymes) profiles. We found that the diauxic shift is accomplished by three key events that are temporally organized: (i) a reduction in the glycolytic flux and the production of storage compounds before glucose depletion, mediated by downregulation of phosphofructokinase and pyruvate kinase reactions; (ii) upon glucose exhaustion, the reversion of carbon flow through glycolysis and onset of the glyoxylate cycle operation triggered by an increased expression of the enzymes that catalyze the malate synthase and cytosolic citrate synthase reactions; and (iii) in the later stages of the adaptation, the shutting down of the pentose phosphate pathway with a change in NADPH regeneration. Moreover, we identified the transcription factors associated with the observed changes in protein abundances. Taken together, our results represent an important contribution toward a systems-level understanding of how this adaptation is realized. PMID:23549479

Glucosylceramide accumulation is not confined to the lysosome in fibroblasts from patients with Gaucher disease.

PubMed

Fuller, Maria; Rozaklis, Tina; Lovejoy, Melanie; Zarrinkalam, Krystyna; Hopwood, John J; Meikle, Peter J

2008-04-01

Gaucher disease (GD) is an inborn error of glycosphingolipid metabolism resulting from a deficiency of the lysosomal enzyme beta-glucosidase leading to the accumulation of glucosylceramide (GC) in lysosomes of affected cells. In order to determine the effect of GC accumulation on intracellular lipid content in fibroblasts from patients with GD, we measured individual species of ceramide, di- and trihexosylceramide, sphingomyelin, phosphatidylcholine, phosphatidylinositol and phosphatidylglycerol using electrospray ionisation-tandem mass spectrometry. The different subspecies of each lipid class correlated with each other and were summed to give total lipid concentrations. In addition to GC, we also noted secondary elevations in other lipids, especially in type 2 GD. Sub-cellular fractionation showed that GC was not confined to the lysosome but increased throughout the cell. The sequelae of extra-lysosomal accumulation may have implications in the pathogenic mechanisms of GD by interaction with biochemical and metabolic pathways located outside the lysosome. The elevation of ceramide in confluent type 2 GD fibroblasts redistributed from its primary site of accumulation in the lysosome to the endosomal region at four-weeks post-confluence. The accumulation of lipids in the endosome and lysosome suggests both impaired trafficking of lipids and reduced capacity of the lysosome to degrade lipids.

Lentiviral Gene Therapy Using Cellular Promoters Cures Type 1 Gaucher Disease in Mice

PubMed Central

Dahl, Maria; Doyle, Alexander; Olsson, Karin; Månsson, Jan-Eric; Marques, André R A; Mirzaian, Mina; Aerts, Johannes M; Ehinger, Mats; Rothe, Michael; Modlich, Ute; Schambach, Axel; Karlsson, Stefan

2015-01-01

Gaucher disease is caused by an inherited deficiency of the enzyme glucosylceramidase. Due to the lack of a fully functional enzyme, there is progressive build-up of the lipid component glucosylceramide. Insufficient glucosylceramidase activity results in hepatosplenomegaly, cytopenias, and bone disease in patients. Gene therapy represents a future therapeutic option for patients unresponsive to enzyme replacement therapy and lacking a suitable bone marrow donor. By proof-of-principle experiments, we have previously demonstrated a reversal of symptoms in a murine disease model of type 1 Gaucher disease, using gammaretroviral vectors harboring strong viral promoters to drive glucosidase β-acid (GBA) gene expression. To investigate whether safer vectors can correct the enzyme deficiency, we utilized self-inactivating lentiviral vectors (SIN LVs) with the GBA gene under the control of human phosphoglycerate kinase (PGK) and CD68 promoter, respectively. Here, we report prevention of, as well as reversal of, manifest disease symptoms after lentiviral gene transfer. Glucosylceramidase activity above levels required for clearance of glucosylceramide from tissues resulted in reversal of splenomegaly, reduced Gaucher cell infiltration and a restoration of hematological parameters. These findings support the use of SIN-LVs with cellular promoters in future clinical gene therapy protocols for type 1 Gaucher disease. PMID:25655314

Activation of iNKT cells by a distinct constituent of the endogenous glucosylceramide fraction

PubMed Central

Brennan, Patrick J.; Tatituri, Raju V. V.; Heiss, Christian; Watts, Gerald F. M.; Hsu, Fong-Fu; Veerapen, Natacha; Cox, Liam R.; Azadi, Parastoo; Besra, Gurdyal S.; Brenner, Michael B.

2014-01-01

Invariant natural killer T (iNKT) cells are a specialized T-cell subset that recognizes lipids as antigens, contributing to immune responses in diverse disease processes. Experimental data suggests that iNKT cells can recognize both microbial and endogenous lipid antigens. Several candidate endogenous lipid antigens have been proposed, although the contextual role of specific antigens during immune responses remains largely unknown. We have previously reported that mammalian glucosylceramides (GlcCers) activate iNKT cells. GlcCers are found in most mammalian tissues, and exist in variable molecular forms that differ mainly in N-acyl fatty acid chain use. In this report, we purified, characterized, and tested the GlcCer fractions from multiple animal species. Although activity was broadly identified in these GlcCer fractions from mammalian sources, we also found activity properties that could not be reconciled by differences in fatty acid chain use. Enzymatic digestion of β-GlcCer and a chromatographic separation method demonstrated that the activity in the GlcCer fraction was limited to a rare component of this fraction, and was not contained within the bulk of β-GlcCer molecular species. Our data suggest that a minor lipid species that copurifies with β-GlcCer in mammals functions as a lipid self antigen for iNKT cells. PMID:25197085

Glioma Cell Death Induced by Irradiation or Alkylating Agent Chemotherapy Is Independent of the Intrinsic Ceramide Pathway

PubMed Central

Gramatzki, Dorothee; Herrmann, Caroline; Happold, Caroline; Becker, Katrin Anne; Gulbins, Erich; Weller, Michael; Tabatabai, Ghazaleh

2013-01-01

Background/Aims Resistance to genotoxic therapy is a characteristic feature of glioma cells. Acid sphingomyelinase (ASM) hydrolyzes sphingomyelin to ceramide and glucosylceramide synthase (GCS) catalyzes ceramide metabolism. Increased ceramide levels have been suggested to enhance chemotherapy-induced death of cancer cells. Methods Microarray and clinical data for ASM and GCS in astrocytomas WHO grade II–IV were acquired from the Rembrandt database. Moreover, the glioblastoma database of the Cancer Genome Atlas network (TCGA) was used for survival data of glioblastoma patients. For in vitro studies, increases in ceramide levels were achieved either by ASM overexpression or by the GCS inhibitor DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) in human glioma cell lines. Combinations of alkylating chemotherapy or irradiation and ASM overexpression, PPMP or exogenous ceramide were applied in parental cells. The anti-glioma effects were investigated by assessing proliferation, metabolic activity, viability and clonogenicity. Finally, viability and clonogenicity were assessed in temozolomide (TMZ)-resistant cells upon treatment with PPMP, exogenous ceramide, alkylating chemotherapy, irradiation or their combinations. Results Interrogations from the Rembrandt and TCGA database showed a better survival of glioblastoma patients with low expression of ASM or GCS. ASM overexpression or PPMP treatment alone led to ceramide accumulation but did not enhance the anti-glioma activity of alkylating chemotherapy or irradiation. PPMP or exogenous ceramide induced acute cytotoxicity in glioblastoma cells. Combined treatments with chemotherapy or irradiation led to additive, but not synergistic effects. Finally, no synergy was found when TMZ-resistant cells were treated with exogenous ceramide or PPMP alone or in combination with TMZ or irradiation. Conclusion Modulation of intrinsic glioma cell ceramide levels by ASM overexpression or GCS inhibition does not enhance the anti-glioma activity of alkylating chemotherapy or irradiation. PMID:23667632

Treatment of cataplexy in Niemann-Pick disease type C with the use of miglustat.

PubMed

Zarowski, Marcin; Steinborn, Barbara; Gurda, Barbara; Dvorakova, Lenka; Vlaskova, Hana; Kothare, Sanjeev V

2011-01-01

Cataplexy is the sudden muscle weakness brought on by strong emotions, particularly joking, laughter, or anger. Cataplexy may involve only certain group of muscles or the entire voluntary musculature. In rare cases, symptoms of cataplexy can be seen during the course of some inherited diseases (Niemann-Pick type C (NPC), Prader-Willi syndrome, myotonic dystrophy, Norrie disease). We report the successful use of miglustat, a reversible inhibitor of the enzyme glucosylceramide synthase, approved for use in Gaucher's disease, and which catalyses the first step in the biosynthesis of most glycosphingolipid, in a boy with NPC with cataplexy. A 9-year-old boy was admitted for assessments of frequent "drop attacks" while laughing. The filipin fluorescence tests of cultured skin fibroblasts revealed massive accumulation of unesterified cholesterol, confirming the diagnosis of NPC disease. Molecular studies confirmed the diagnosis of NPC too. After approval from the bioethics committee, miglustat was initiated on the child at 100mg three times a day. Cataplectic attacks disappeared completely after 6 months on treatment, and patient continues to be in remission from the cataplectic attacks at 16 months follow-up. There was no further progression of neurological signs or symptoms or splenomegaly, with some improvement in cognitive function as well as social, affective and attention problems, up-gaze, and gait. Miglustat was well tolerated with no side effects observed. In summary, this is the first report of miglustat treatment of cataplexy in NPC. Long-term follow-up for continuing efficacy and tolerability in a larger cohort with NPC is needed to substantiate our observation. © 2010 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.

Impairment of ceramide synthesis causes a novel progressive myoclonus epilepsy.

PubMed

Vanni, Nicola; Fruscione, Floriana; Ferlazzo, Edoardo; Striano, Pasquale; Robbiano, Angela; Traverso, Monica; Sander, Thomas; Falace, Antonio; Gazzerro, Elisabetta; Bramanti, Placido; Bielawski, Jacek; Fassio, Anna; Minetti, Carlo; Genton, Pierre; Zara, Federico

2014-08-01

Alterations of sphingolipid metabolism are implicated in the pathogenesis of many neurodegenerative disorders. We identified a homozygous nonsynonymous mutation in CERS1, the gene encoding ceramide synthase 1, in 4 siblings affected by a progressive disorder with myoclonic epilepsy and dementia. CerS1, a transmembrane protein of the endoplasmic reticulum (ER), catalyzes the biosynthesis of C18-ceramides. We demonstrated that the mutation decreases C18-ceramide levels. In addition, we showed that downregulation of CerS1 in a neuroblastoma cell line triggers ER stress response and induces proapoptotic pathways. This study demonstrates that impairment of ceramide biosynthesis underlies neurodegeneration in humans. © 2014 American Neurological Association.

Partial deletion of argininosuccinate synthase protects from pyrazole plus lipopolysaccharide-induced liver injury by decreasing nitrosative stress

PubMed Central

Lu, Yongke; Leung, Tung Ming; Ward, Stephen C.

2012-01-01

Argininosuccinate synthase (ASS) is the rate-limiting enzyme in the urea cycle. Along with nitric oxide synthase (NOS)-2, ASS endows cells with the l-citrulline/nitric oxide (NO·) salvage pathway to continually supply l-arginine from l-citrulline for sustained NO· generation. Because of the relevant role of NOS in liver injury, we hypothesized that downregulation of ASS could decrease the availability of intracellular substrate for NO· synthesis by NOS-2 and, hence, decrease liver damage. Previous work demonstrated that pyrazole plus LPS caused significant liver injury involving NO· generation and formation of 3-nitrotyrosine protein adducts; thus, wild-type (WT) and Ass+/− mice (Ass−/− mice are lethal) were treated with pyrazole plus LPS, and markers of nitrosative stress, as well as liver injury, were analyzed. Partial ablation of Ass protected from pyrazole plus LPS-induced liver injury by decreasing nitrosative stress and hepatic and circulating TNFα. Moreover, apoptosis was prevented, since pyrazole plus LPS-treated Ass+/− mice showed decreased phosphorylation of JNK; increased MAPK phosphatase-1, which is known to deactivate JNK signaling; and lower cleaved caspase-3 than treated WT mice, and this was accompanied by less TdT-mediated dUTP nick end labeling-positive staining. Lastly, hepatic neutrophil accumulation was almost absent in pyrazole plus LPS-treated Ass+/− compared with WT mice. Partial Ass ablation prevents pyrazole plus LPS-mediated liver injury by reducing nitrosative stress, TNFα, apoptosis, and neutrophil infiltration. PMID:22052013

The polyketide synthase gene pks4 is essential for sexual development and regulates fruiting body morphology in Sordaria macrospora.

PubMed

Schindler, Daniel; Nowrousian, Minou

2014-07-01

Filamentous ascomycetes have long been known as producers of a variety of secondary metabolites, many of which have toxic effects on other organisms. However, the role of these metabolites in the biology of the fungi that produce them remains in most cases enigmatic. A major group of fungal secondary metabolites are polyketides. They are chemically diverse, but have in common that their chemical scaffolds are synthesized by polyketide synthases (PKSs). In a previous study, we analyzed development-dependent expression of pks genes in the filamentous ascomycete Sordaria macrospora. Here, we show that a deletion mutant of the pks4 gene is sterile, producing only protoperithecia but no mature perithecia, whereas overexpression of pks4 leads to enlarged, malformed fruiting bodies. Thus, correct expression levels of pks4 are essential for wild type-like perithecia formation. The predicted PKS4 protein has a domain structure that is similar to homologs in other fungi, but conserved residues of a methyl transferase domain present in other fungi are mutated in PKS4. Expression of several developmental genes is misregulated in the pks4 mutant. Surprisingly, the development-associated app gene is not downregulated in the mutant, in contrast to all other previously studied mutants with a block at the protoperithecial stage. Our data show that the polyketide synthase gene pks4 is essential for sexual development and plays a role in regulating fruiting body morphology. Copyright © 2014 Elsevier Inc. All rights reserved.

Silencing of the ACC synthase gene ACACS2 causes delayed flowering in pineapple [Ananas comosus (L.) Merr.].

PubMed

Trusov, Yuri; Botella, José Ramón

2006-01-01

Flowering is a crucial developmental stage in the plant life cycle. A number of different factors, from environmental to chemical, can trigger flowering. In pineapple, and other bromeliads, it has been proposed that flowering is triggered by a small burst of ethylene production in the meristem in response to environmental cues. A 1-amino-cyclopropane-1-carboxylate synthase (ACC synthase) gene has been cloned from pineapple (ACACS2), which is induced in the meristem under the same environmental conditions that induce flowering. Two transgenic pineapple lines have been produced containing co-suppression constructs designed to down-regulate the expression of the ACACS2 gene. Northern analysis revealed that the ACACS2 gene was silenced in a number of transgenic plants in both lines. Southern hybridization revealed clear differences in the methylation status of silenced versus non-silenced plants by the inability of a methylation-sensitive enzyme to digest within the ACACS2 DNA extracted from silenced plants, indicating that methylation is the cause of the observed co-suppression of the ACACS2 gene. Flowering characteristics of the transgenic plants were studied under field conditions in South East Queensland, Australia. Flowering dynamics studies revealed significant differences in flowering behaviour, with transgenic plants exhibiting silencing showing a marked delay in flowering when compared with non-silenced transgenic plants and control non-transformed plants. It is argued that the ACACS2 gene is one of the key contributors towards triggering 'natural flowering' in mature pineapples under commercial field conditions.

Terpene Down-Regulation Triggers Defense Responses in Transgenic Orange Leading to Resistance against Fungal Pathogens1[W

PubMed Central

Rodríguez, Ana; Shimada, Takehiko; Cervera, Magdalena; Alquézar, Berta; Gadea, José; Gómez-Cadenas, Aurelio; De Ollas, Carlos José; Rodrigo, María Jesús; Zacarías, Lorenzo; Peña, Leandro

2014-01-01

Terpenoid volatiles are isoprene compounds that are emitted by plants to communicate with the environment. In addition to their function in repelling herbivores and attracting carnivorous predators in green tissues, the presumed primary function of terpenoid volatiles released from mature fruits is the attraction of seed-dispersing animals. Mature oranges (Citrus sinensis) primarily accumulate terpenes in peel oil glands, with d-limonene accounting for approximately 97% of the total volatile terpenes. In a previous report, we showed that down-regulation of a d-limonene synthase gene alters monoterpene levels in orange antisense (AS) fruits, leading to resistance against Penicillium digitatum infection. A global gene expression analysis of AS versus empty vector (EV) transgenic fruits revealed that the down-regulation of d-limonene up-regulated genes involved in the innate immune response. Basal levels of jasmonic acid were substantially higher in the EV compared with AS oranges. Upon fungal challenge, salicylic acid levels were triggered in EV samples, while jasmonic acid metabolism and signaling were drastically increased in AS orange peels. In nature, d-limonene levels increase in orange fruit once the seeds are fully viable. The inverse correlation between the increase in d-limonene content and the decrease in the defense response suggests that d-limonene promotes infection by microorganisms that are likely involved in facilitating access to the pulp for seed-dispersing frugivores. PMID:24192451

Terpene Down-Regulation in Orange Reveals the Role of Fruit Aromas in Mediating Interactions with Insect Herbivores and Pathogens1[C][W

PubMed Central

Rodríguez, Ana; San Andrés, Victoria; Cervera, Magdalena; Redondo, Ana; Alquézar, Berta; Shimada, Takehiko; Gadea, José; Rodrigo, María Jesús; Zacarías, Lorenzo; Palou, Lluís; López, María M.; Castañera, Pedro; Peña, Leandro

2011-01-01

Plants use volatile terpene compounds as odor cues for communicating with the environment. Fleshy fruits are particularly rich in volatiles that deter herbivores and attract seed dispersal agents. We have investigated how terpenes in citrus fruit peels affect the interaction between the plant, insects, and microorganisms. Because limonene represents up to 97% of the total volatiles in orange (Citrus sinensis) fruit peel, we chose to down-regulate the expression of a limonene synthase gene in orange plants by introducing an antisense construct of this gene. Transgenic fruits showed reduced accumulation of limonene in the peel. When these fruits were challenged with either the fungus Penicillium digitatum or with the bacterium Xanthomonas citri subsp. citri, they showed marked resistance against these pathogens that were unable to infect the peel tissues. Moreover, males of the citrus pest medfly (Ceratitis capitata) were less attracted to low limonene-expressing fruits than to control fruits. These results indicate that limonene accumulation in the peel of citrus fruit appears to be involved in the successful trophic interaction between fruits, insects, and microorganisms. Terpene down-regulation might be a strategy to generate broad-spectrum resistance against pests and pathogens in fleshy fruits from economically important crops. In addition, terpene engineering may be important for studying the basic ecological interactions between fruits, herbivores, and pathogens. PMID:21525333

Terpene down-regulation in orange reveals the role of fruit aromas in mediating interactions with insect herbivores and pathogens.

PubMed

Rodríguez, Ana; San Andrés, Victoria; Cervera, Magdalena; Redondo, Ana; Alquézar, Berta; Shimada, Takehiko; Gadea, José; Rodrigo, María Jesús; Zacarías, Lorenzo; Palou, Lluís; López, María M; Castañera, Pedro; Peña, Leandro

2011-06-01

Plants use volatile terpene compounds as odor cues for communicating with the environment. Fleshy fruits are particularly rich in volatiles that deter herbivores and attract seed dispersal agents. We have investigated how terpenes in citrus fruit peels affect the interaction between the plant, insects, and microorganisms. Because limonene represents up to 97% of the total volatiles in orange (Citrus sinensis) fruit peel, we chose to down-regulate the expression of a limonene synthase gene in orange plants by introducing an antisense construct of this gene. Transgenic fruits showed reduced accumulation of limonene in the peel. When these fruits were challenged with either the fungus Penicillium digitatum or with the bacterium Xanthomonas citri subsp. citri, they showed marked resistance against these pathogens that were unable to infect the peel tissues. Moreover, males of the citrus pest medfly (Ceratitis capitata) were less attracted to low limonene-expressing fruits than to control fruits. These results indicate that limonene accumulation in the peel of citrus fruit appears to be involved in the successful trophic interaction between fruits, insects, and microorganisms. Terpene down-regulation might be a strategy to generate broad-spectrum resistance against pests and pathogens in fleshy fruits from economically important crops. In addition, terpene engineering may be important for studying the basic ecological interactions between fruits, herbivores, and pathogens.

Effects of cannabidiol interactions with Wnt/β-catenin pathway and PPARγ on oxidative stress and neuroinflammation in Alzheimer's disease.

PubMed

Vallée, Alexandre; Lecarpentier, Yves; Guillevin, Rémy; Vallée, Jean-Noël

2017-10-01

Alzheimer's disease (AD) is a neurodegenerative disease, in which the primary etiology remains unknown. AD presents amyloid beta (Aβ) protein aggregation and neurofibrillary plaque deposits. AD shows oxidative stress and chronic inflammation. In AD, canonical Wingless-Int (Wnt)/β-catenin pathway is downregulated, whereas peroxisome proliferator-activated receptor γ (PPARγ) is increased. Downregulation of Wnt/β-catenin, through activation of glycogen synthase kinase-3β (GSK-3β) by Aβ, and inactivation of phosphatidylinositol 3-kinase/Akt signaling involve oxidative stress in AD. Cannabidiol (CBD) is a non-psychotomimetic phytocannabinoid from Cannabis sativa plant. In PC12 cells, Aβ-induced tau protein hyperphosphorylation is inhibited by CBD. This inhibition is associated with a downregulation of p-GSK-3β, an inhibitor of Wnt pathway. CBD may also increase Wnt/β-catenin by stimulation of PPARγ, inhibition of Aβ and ubiquitination of amyloid precursor protein. CBD attenuates oxidative stress and diminishes mitochondrial dysfunction and reactive oxygen species generation. CBD suppresses, through activation of PPARγ, pro-inflammatory signaling and may be a potential new candidate for AD therapy. © The Author 2017. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Identification of inflammation-related proteins in a murine colitis model by 2D fluorescence difference gel electrophoresis and mass spectrometry.

PubMed

Naito, Yuji; Takagi, Tomohisa; Okada, Hitomi; Omatsu, Tatsushi; Mizushima, Katsura; Handa, Osamu; Kokura, Satoshi; Ichikawa, Hiroshi; Fujiwake, Hideshi; Yoshikawa, Toshikazu

2010-05-01

The aim of this study was to identify new intestinal proteins potentially associated with acute inflammation using proteomic profiling of an in vivo mice model of ulcerative colitis. 2D fluorescence difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight spectrometer (MALDI-TOF) peptide mass fingerprinting were used to determine differentially expressed proteins between normal and inflamed intestinal mucosa. Acute colitis was induced by 8.0% dextran sodium sulfate (DSS) given p.o. for 7 days. Among a total of seven protein spots showing differential expression, we identified five different proteins, of which two were upregulated and three downregulated in colitis in comparison to normal mucosa, using the MASCOT search engine. 3-Hydroxy-3-methylglutaryl-coenzyme A synthase 2 and serpin b1a were upregulated proteins, and protein disulfide-isomerase A3, peroxiredoxin-6 and vimentin were identified as downregulated proteins. These identified proteins may be responsible for the development of the intestinal inflammation. 2D-DIGE and MALDI-TOF mass spectrometry are useful in the search for the differentially expressed proteins.

Celecoxib can suppress expression of genes associated with PGE2 pathway in chondrocytes under inflammatory conditions.

PubMed

Sun, Tian-Wen; Wu, Zhi-Hong; Weng, Xi-Sheng

2015-01-01

This study aimed to investigate the effect of a selective cyclooxygenase-2 (COX-2) inhibitor (celecoxib) on the expression of arachidonate-associated inflammatory genes in cultured human normal chondrocytes. Normal chondrocytes were obtained from the cartilage of three different amputated patients without osteoarthritis (OA). Affymetrix Human microarray was used to assess the alterations in gene expression in three groups of cells: untreated cells (negative control group), cells treated with interleukin-1β (IL-1β) (positive control group), and cells treated with IL-1β and celecoxib. The patterns of up-regulation and down-regulation of gene expression were further validated by real-time PCR. A total of 1091 up-regulated genes and 1252 down-regulated genes were identified in the positive control group compared with the negative control group. Among them, PTGS2, ADAMTS5, PTGER2, mPTGES and PTGER4 are known to be involved in chondrocyte inflammation, while VEGFA, BCL2, TRAF1, CYR61, BMP6, DAPK1, DUSP7, IL1RN, MMP13 and TNFSF10 were reported being associated with cytokine and chemokine signaling. 189 up-regulated genes and 177 down-regulated genes were identified in the positive control group compared with intervention group. PTGS1, PTGS2, ADAMTS5, PTGER2, mPTGES and PTGER4 were among the genes down-regulated upon the treatment with celecoxib. Our results demonstrated that the OA chondrocytes are the site of active eicosanoid production. IL-1β can activate inflammation in chondrocytes and trigger the production of various proteins involved in cyclooxygenase pathway. The expression of genes corresponding to these proteins can be down-regulated by celecoxib. The findings indicate that the therapy with prostaglandin E2 (PGE2)-blocking agents may decrease the PGE2 production not only by direct inhibition of COX-2 activity, but also by down-regulating the expression of genes encoding for COX-2, microsomal prostaglandin-endoperoxide synthase 1 (mPGES-1) and prostaglandin E receptors 4 (EP4) in the articular chondrocytes.

PCI-24781 down-regulates EZH2 expression and then promotes glioma apoptosis by suppressing the PIK3K/Akt/mTOR pathway.

PubMed

Zhang, Wei; Lv, Shengqing; Liu, Jun; Zang, Zhenle; Yin, Junyi; An, Ning; Yang, Hui; Song, Yechun

2014-10-01

PCI-24781 is a novel histone deacetylase inhibitor that inhibits tumor proliferation and promotes cell apoptosis. However, it is unclear whether PCI-24781 inhibits Enhancer of Zeste 2 (EZH2) expression in malignant gliomas. In this work, three glioma cell lines were incubated with various concentrations of PCI-24781 (0, 0.25, 0.5, 1, 2.5 and 5 μM) and analyzed for cell proliferation by the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay and colony formation, and cell cycle and apoptosis were assessed by flow cytometry. The expression of EZH2 and apoptosis-related proteins was assessed by western blotting. Malignant glioma cells were also transfected with EZH2 siRNA to examine how PCI-24781 suppresses tumor cells. EZH2 was highly expressed in the three glioma cell lines. Incubation with PCI-24781 reduced cell proliferation and increased cell apoptosis by down-regulating EZH2 in a concentration-dependent manner. These effects were simulated by EZH2 siRNA. In addition, PCI-24781 or EZH2 siRNA accelerated cell apoptosis by down-regulating the expression of AKT, mTOR, p70 ribosomal protein S6 kinase (p70s6k), glycogen synthase kinase 3A and B (GSK3a/b) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1). These data suggest that PCI-24781 may be a promising therapeutic agent for treating gliomas by down-regulating EZH2 which promotes cell apoptosis by suppressing the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of the rapamycin (mTOR) pathway.

The effects of whole ovarian perfusion and cryopreservation on endothelial cell-related gene expression in the ovarian medulla and pedicle.

PubMed

Onions, V J; Webb, R; Pincott-Allen, C; Picton, H M; Campbell, B K

2013-04-01

Fertility preservation by whole ovarian cryopreservation requires successful cryopreservation of both the ovary and its vascular supply. Previous work has indicated detrimental effects of both perfusion and cryopreservation on the ovarian vasculature. This study assessed the effects of blood perfusion, alone or in combination with cryopreservation, on functional effects in the follicle population and ovarian function in vivo following short-term autotransplantation of the tissue after vascular reanastomosis and measured acute changes in endothelial cell-related gene expression within the ovarian medulla and pedicle. Following autotransplantation for 7 days, primordial, transitional and primary follicle densities were significantly reduced (P < 0.05) and stromal Ki67 and caspase-3 expression significantly increased (P < 0.05) in cryopreserved but not fresh or perfused whole ovaries. There was evidence of clot formation and fluorescent microsphere (FMS) extravasation in the medulla of all cryopreserved ovaries, indicating vascular damage. Utilizing a customized RT-PCR array or conventional RT-PCR, we found that perfusion alone resulted in down-regulation in the expression of caspase 6 and thrombospondin 1 (THBS1) genes in the medulla. Following additional cryopreservation, endothelial nitric oxide synthase (eNOS), endothelin 1, endothelin receptor A and Bcl-2 expression were significantly (P < 0.05) down-regulated. In the pedicle, both perfusion and cryopreservation caused a (P < 0.05) down-regulation of eNOS and THBS1, and an up-regulation in Bax expression. Perfusion also caused a down-regulation of TNF and up-regulation of endothelin-2 expression (P < 0.05). In conclusion, this study has identified a number of endothelial cell-related genes expressed in the medulla which are acutely affected by both cryopreservation and perfusion, supporting the hypothesis that both interventions have deleterious effects on endothelial cell function.

Downregulation of lipin-1 induces insulin resistance by increasing intracellular ceramide accumulation in C2C12 myotubes

PubMed Central

Huang, Shujuan; Huang, Suling; Wang, Xi; Zhang, Qingli; Liu, Jia; Leng, Ying

2017-01-01

Dysregulation of lipid metabolism in skeletal muscle is involved in the development of insulin resistance. Mutations in lipin-1, a key lipid metabolism regulator leads to significant systemic insulin resistance in fld mice. However, the function of lipin-1 on lipid metabolism and insulin sensitivity in skeletal muscle is still unclear. Herein we demonstrated that downregulation of lipin-1 in C2C12 myotubes by siRNA transfection suppressed insulin action, characterized by reduced insulin stimulated Akt phosphorylation and glucose uptake. Correspondingly, decreased lipin-1 expression was observed in palmitate-induced insulin resistance in C2C12 myotubes, suggested that lipin-1 might play a role in the etiology of insulin resistance in skeletal muscle. The insulin resistance induced by lipin-1 downregulation was related to the disturbance of lipid homeostasis. Lipin-1 silencing reduced intracellular DAG and TAG levels, but elevated ceramide accumulation in C2C12 myotubes. Moreover, the impaired insulin stimulated Akt phosphorylation and glucose uptake caused by lipin-1 silencing could be blocked by the pretreatment with SPT inhibitor myriocin, ceramide synthase inhibitor FB1, or PP2A inhibitor okadaic acid, suggested that the increased ceramide accumulation might be responsible for the development of insulin resistance induced by lipin-1 silencing in C2C12 myotubes. Meanwhile, decreased lipin-1 expression also impaired mitochondrial function in C2C12 myotubes. Therefore, our study suggests that lipin-1 plays an important role in lipid metabolism and downregulation of lipin-1 induces insulin resistance by increasing intracellular ceramide accumulation in C2C12 myotubes. These results offer a molecular insight into the role of lipin-1 in the development of insulin resistance in skeletal muscle. PMID:28123341

Downregulation of lipin-1 induces insulin resistance by increasing intracellular ceramide accumulation in C2C12 myotubes.

PubMed

Huang, Shujuan; Huang, Suling; Wang, Xi; Zhang, Qingli; Liu, Jia; Leng, Ying

2017-01-01

Dysregulation of lipid metabolism in skeletal muscle is involved in the development of insulin resistance. Mutations in lipin-1, a key lipid metabolism regulator leads to significant systemic insulin resistance in fld mice. However, the function of lipin-1 on lipid metabolism and insulin sensitivity in skeletal muscle is still unclear. Herein we demonstrated that downregulation of lipin-1 in C2C12 myotubes by siRNA transfection suppressed insulin action, characterized by reduced insulin stimulated Akt phosphorylation and glucose uptake. Correspondingly, decreased lipin-1 expression was observed in palmitate-induced insulin resistance in C2C12 myotubes, suggested that lipin-1 might play a role in the etiology of insulin resistance in skeletal muscle. The insulin resistance induced by lipin-1 downregulation was related to the disturbance of lipid homeostasis. Lipin-1 silencing reduced intracellular DAG and TAG levels, but elevated ceramide accumulation in C2C12 myotubes. Moreover, the impaired insulin stimulated Akt phosphorylation and glucose uptake caused by lipin-1 silencing could be blocked by the pretreatment with SPT inhibitor myriocin, ceramide synthase inhibitor FB1, or PP2A inhibitor okadaic acid, suggested that the increased ceramide accumulation might be responsible for the development of insulin resistance induced by lipin-1 silencing in C2C12 myotubes. Meanwhile, decreased lipin-1 expression also impaired mitochondrial function in C2C12 myotubes. Therefore, our study suggests that lipin-1 plays an important role in lipid metabolism and downregulation of lipin-1 induces insulin resistance by increasing intracellular ceramide accumulation in C2C12 myotubes. These results offer a molecular insight into the role of lipin-1 in the development of insulin resistance in skeletal muscle.

Glucosylceramide Administration as a Vaccination Strategy in Mouse Models of Cryptococcosis.

PubMed

Mor, Visesato; Farnoud, Amir M; Singh, Ashutosh; Rella, Antonella; Tanno, Hiromasa; Ishii, Keiko; Kawakami, Kazuyoshi; Sato, Toshiya; Del Poeta, Maurizio

2016-01-01

Cryptococcus neoformans is an opportunistic fungal pathogen and the causative agent of the disease cryptococcosis. Cryptococcosis is initiated as a pulmonary infection and in conditions of immune deficiency disseminates to the blood stream and central nervous system, resulting in life-threatening meningoencephalitis. A number of studies have focused on the development of a vaccine against Cryptococcus, primarily utilizing protein-conjugated components of the Cryptococcus polysaccharide capsule as antigen. However, there is currently no vaccine against Cryptococcus in the clinic. Previous studies have shown that the glycosphingolipid, glucosylceramide (GlcCer), is a virulence factor in C. neoformans and antibodies against this lipid inhibit fungal growth and cell division. In the present study, we have investigated the possibility of using GlcCer as a therapeutic agent against C. neoformans infections in mouse models of cryptococcosis. GlcCer purified from a non-pathogenic fungus, Candida utilis, was administered intraperitoneally, prior to infecting mice with a lethal dose of C. neoformans. GlcCer administration prevented the dissemination of C. neoformans from the lungs to the brain and led to 60% mouse survival. GlcCer administration did not cause hepatic injury and elicited an anti-GlcCer antibody response, which was observed independent of the route of administration and the strains of mouse. Taken together, our results suggest that fungal GlcCer can protect mice against lethal doses of C. neoformans infection and can provide a viable vaccination strategy against Cryptococcus.

Glucosylceramide Contained in Koji Mold-Cultured Cereal Confers Membrane and Flavor Modification and Stress Tolerance to Saccharomyces cerevisiae during Coculture Fermentation.

PubMed

Sawada, Kazutaka; Sato, Tomoya; Hamajima, Hiroshi; Jayakody, Lahiru Niroshan; Hirata, Miyo; Yamashiro, Mikako; Tajima, Marie; Mitsutake, Susumu; Nagao, Koji; Tsuge, Keisuke; Abe, Fumiyoshi; Hanada, Kentaro; Kitagaki, Hiroshi

2015-06-01

In nature, different microorganisms create communities through their physiochemical and metabolic interactions. Many fermenting microbes, such as yeasts, lactic acid bacteria, and acetic acid bacteria, secrete acidic substances and grow faster at acidic pH values. However, on the surface of cereals, the pH is neutral to alkaline. Therefore, in order to grow on cereals, microbes must adapt to the alkaline environment at the initial stage of colonization; such adaptations are also crucial for industrial fermentation. Here, we show that the yeast Saccharomyces cerevisiae, which is incapable of synthesizing glucosylceramide (GlcCer), adapted to alkaline conditions after exposure to GlcCer from koji cereal cultured with Aspergillus kawachii. We also show that various species of GlcCer derived from different plants and fungi similarly conferred alkali tolerance to yeast. Although exogenous ceramide also enhanced the alkali tolerance of yeast, no discernible degradation of GlcCer to ceramide was observed in the yeast culture, suggesting that exogenous GlcCer itself exerted the activity. Exogenous GlcCer also increased ethanol tolerance and modified the flavor profile of the yeast cells by altering the membrane properties. These results indicate that GlcCer from A. kawachii modifies the physiology of the yeast S. cerevisiae and demonstrate a new mechanism for cooperation between microbes in food fermentation. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Glucosylceramide Contained in Koji Mold-Cultured Cereal Confers Membrane and Flavor Modification and Stress Tolerance to Saccharomyces cerevisiae during Coculture Fermentation

PubMed Central

Sawada, Kazutaka; Sato, Tomoya; Hamajima, Hiroshi; Jayakody, Lahiru Niroshan; Hirata, Miyo; Yamashiro, Mikako; Tajima, Marie; Mitsutake, Susumu; Nagao, Koji; Tsuge, Keisuke; Abe, Fumiyoshi; Hanada, Kentaro

2015-01-01

In nature, different microorganisms create communities through their physiochemical and metabolic interactions. Many fermenting microbes, such as yeasts, lactic acid bacteria, and acetic acid bacteria, secrete acidic substances and grow faster at acidic pH values. However, on the surface of cereals, the pH is neutral to alkaline. Therefore, in order to grow on cereals, microbes must adapt to the alkaline environment at the initial stage of colonization; such adaptations are also crucial for industrial fermentation. Here, we show that the yeast Saccharomyces cerevisiae, which is incapable of synthesizing glucosylceramide (GlcCer), adapted to alkaline conditions after exposure to GlcCer from koji cereal cultured with Aspergillus kawachii. We also show that various species of GlcCer derived from different plants and fungi similarly conferred alkali tolerance to yeast. Although exogenous ceramide also enhanced the alkali tolerance of yeast, no discernible degradation of GlcCer to ceramide was observed in the yeast culture, suggesting that exogenous GlcCer itself exerted the activity. Exogenous GlcCer also increased ethanol tolerance and modified the flavor profile of the yeast cells by altering the membrane properties. These results indicate that GlcCer from A. kawachii modifies the physiology of the yeast S. cerevisiae and demonstrate a new mechanism for cooperation between microbes in food fermentation. PMID:25795678

Traditional Chinese medical therapy for erectile dysfunction

PubMed Central

Li, Hao; Jiang, Hongyang

2017-01-01

Traditional Chinese medicine (TCM), including acupuncture and Chinese herbs, is used as an alternative therapy to increase the curative effect for erectile dysfunction (ED). A large number of studies have been conducted to investigate the effect and mechanism of TCM for treating ED. The therapeutic effect of acupuncture on ED is still controversial at present. However, some Chinese herbs exhibited satisfying outcomes and they might improve erectile function by activating nitric oxide synthase (NOS)-cyclic guanosine monophosphate (cGMP) pathway, increasing cyclic adenosine monophosphate (cAMP) expression, elevating testosterone level, reducing intracellular Ca2+ concentration, down-regulating transforming growth factor β1 (TGFβ1)/Smad2 signaling pathway, or ameliorating the oxidative stress. PMID:28540226

Glycogen synthase kinase 3 regulates PAX3-FKHR-mediated cell proliferation in human alveolar rhabdomyosarcoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zeng, Fu-Yue; Dong, Hanqing; Cui, Jimmy

2010-01-01

Patients with alveolar rhabdomyosarcoma (ARMS) have poorer response to conventional chemotherapy and lower survival rates than those with embryonal RMS (ERMS). To identify compounds that preferentially block the growth of ARMS, we conducted a small-scale screen of 160 kinase inhibitors against the ARMS cell line Rh30 and ERMS cell line RD and identified inhibitors of glycogen synthase kinase 3 (GSK3), including TWS119 as ARMS-selective inhibitors. GSK3 inhibitors inhibited cell proliferation and induced apoptosis more effectively in Rh30 than RD cells. Ectopic expression of fusion protein PAX3-FKHR in RD cells significantly increased their sensitivity to TWS119. Down-regulation of GSK3 by GSK3more » inhibitors or siRNA significantly reduced the transcriptional activity of PAX3-FKHR. These results suggest that GSK3 is directly involved in regulating the transcriptional activity of PAX3-FKHR. Also, GSK3 phosphorylated PAX3-FKHR in vitro, suggesting that GSK3 might regulate PAX3-FKHR activity via phosphorylation. These findings support a novel mechanism of PAX3-FKHR regulation by GSK3 and provide a novel strategy to develop GSK inhibitors as anti-ARMS therapies.« less

Glycogen synthase kinase-3 regulates inflammatory tolerance in astrocytes

PubMed Central

Beurel, Eléonore; Jope, Richard S.

2010-01-01

Inflammatory tolerance is the down-regulation of inflammation upon repeated stimuli, which is well-established to occur in peripheral immune cells. However, less is known about inflammatory tolerance in the brain although it may provide an important protective mechanism from detrimental consequences of prolonged inflammation, which appears to occur in many psychiatric and neurodegenerative conditions. Array analysis of 308 inflammatory molecules produced by mouse primary astrocytes after two sequential stimulations with lipopolysaccharide (LPS) distinguished three classes, tolerant, sensitized and unaltered groups. For many of these inflammatory molecules, inhibition of glycogen synthase kinase-3 (GSK3) increased tolerance and reduced sensitization. Focusing on LPS-tolerance in interleukin-6 (IL-6) production, we found that microglia exhibited a strong tolerance response that matched that of macrophages, whereas astrocytes exhibited only partial tolerance. The astrocyte semi-tolerance was found to be regulated by GSK3. GSK3 inhibitors or knocking down GSK3 levels promoted LPS-tolerance and astrocytes expressing constitutively active GSK3 did not develop LPS-tolerance. These findings identify the critical role of GSK3 in counteracting IL-6 inflammatory tolerance in cells of the CNS, supporting the therapeutic potential of GSK3 inhibitors to reduce neuroinflammation by promoting tolerance. PMID:20553816

Androstenediol inhibits the trauma-hemorrhage-induced increase in caspase-3 by downregulating the inducible nitric oxide synthase pathway.

PubMed

Kiang, Juliann G; Peckham, Russell M; Duke, Leah E; Shimizu, Tomoharu; Chaudry, Irshad H; Tsokos, George C

2007-03-01

Soft tissue trauma and hemorrhage (T-H) diminishes various aspects of liver function, while it increases hepatic nitrate/nitrite, inducible nitric oxide synthase (iNOS), and endothelin-1 levels. Treatment with androstenediol (AED) inhibits the T-H-induced alterations of the above parameters. We sought to identify the molecular events underlying the beneficial effect of AED. Exposure of rats to T-H significantly increased the caspase-3 activity and protein, whereas treatment with AED significantly limited these increases. AED treatment also suppressed the T-H-induced increase in iNOS by effectively altering the levels of key transcription factors involved in the regulation of iNOS expression. Immunoprecipitation and immunoblotting analyses indicate that T-H increased apoptosome formation, and AED treatment significantly decreased it. Modulating the iNOS protein by transfecting cells with iNOS gene or small interfering RNA further confirmed the correlation between iNOS and caspase-3. Our data indicate that AED limits caspase-3 expression by suppressing the expression of transcription factors involved in the production of iNOS, resulting in decreased apoptosome. AED can potentially be a useful adjuvant for limiting liver apoptosis following T-H shock.

Chalcone Synthase (CHS) Gene Suppression in Flax Leads to Changes in Wall Synthesis and Sensing Genes, Cell Wall Chemistry and Stem Morphology Parameters

PubMed Central

Zuk, Magdalena; Działo, Magdalena; Richter, Dorota; Dymińska, Lucyna; Matuła, Jan; Kotecki, Andrzej; Hanuza, Jerzy; Szopa, Jan

2016-01-01

The chalcone synthase (CHS) gene controls the first step in the flavonoid biosynthesis. In flax, CHS down-regulation resulted in tannin accumulation and reduction in lignin synthesis, but plant growth was not affected. This suggests that lignin content and thus cell wall characteristics might be modulated through CHS activity. This study investigated the possibility that CHS affects cell wall sensing as well as polymer content and arrangement. CHS-suppressed and thus lignin-reduced plants showed significant changes in expression of genes involved in both synthesis of components and cell wall sensing. This was accompanied by increased levels of cellulose and hemicellulose. CHS-reduced flax also showed significant changes in morphology and arrangement of the cell wall. The stem tissue layers were enlarged averagely twofold compared to the control, and the number of fiber cells more than doubled. The stem morphology changes were accompanied by reduction of the crystallinity index of the cell wall. CHS silencing induces a signal transduction cascade that leads to modification of plant metabolism in a wide range and thus cell wall structure. PMID:27446124

Two Atypical l-Cysteine-regulated NADPH-dependent Oxidoreductases Involved in Redox Maintenance, l-Cystine and Iron Reduction, and Metronidazole Activation in the Enteric Protozoan Entamoeba histolytica*

PubMed Central

Jeelani, Ghulam; Husain, Afzal; Sato, Dan; Ali, Vahab; Suematsu, Makoto; Soga, Tomoyoshi; Nozaki, Tomoyoshi

2010-01-01

We discovered novel catalytic activities of two atypical NADPH-dependent oxidoreductases (EhNO1/2) from the enteric protozoan parasite Entamoeba histolytica. EhNO1/2 were previously annotated as the small subunit of glutamate synthase (glutamine:2-oxoglutarate amidotransferase) based on similarity to authentic bacterial homologs. As E. histolytica lacks the large subunit of glutamate synthase, EhNO1/2 were presumed to play an unknown role other than glutamine/glutamate conversion. Transcriptomic and quantitative reverse PCR analyses revealed that supplementation or deprivation of extracellular l-cysteine caused dramatic up- or down-regulation, respectively, of EhNO2, but not EhNO1 expression. Biochemical analysis showed that these FAD- and 2[4Fe-4S]-containing enzymes do not act as glutamate synthases, a conclusion which was supported by phylogenetic analyses. Rather, they catalyze the NADPH-dependent reduction of oxygen to hydrogen peroxide and l-cystine to l-cysteine and also function as ferric and ferredoxin-NADP+ reductases. EhNO1/2 showed notable differences in substrate specificity and catalytic efficiency; EhNO1 had lower Km and higher kcat/Km values for ferric ion and ferredoxin than EhNO2, whereas EhNO2 preferred l-cystine as a substrate. In accordance with these properties, only EhNO1 was observed to physically interact with intrinsic ferredoxin. Interestingly, EhNO1/2 also reduced metronidazole, and E. histolytica transformants overexpressing either of these proteins were more sensitive to metronidazole, suggesting that EhNO1/2 are targets of this anti-amebic drug. To date, this is the first report to demonstrate that small subunit-like proteins of glutamate synthase could play an important role in redox maintenance, l-cysteine/l-cystine homeostasis, iron reduction, and the activation of metronidazole. PMID:20592025

Seasonal superoxide overproduction and endothelial activation in guinea-pig heart; seasonal oxidative stress in rats and humans.

PubMed

Konior, Anna; Klemenska, Emilia; Brudek, Magdalena; Podolecka, Ewa; Czarnowska, Elżbieta; Beręsewicz, Andrzej

2011-04-01

Seasonality in endothelial dysfunction and oxidative stress was noted in humans and rats, suggesting it is a common phenomenon of a potential clinical relevance. We aimed at studying (i) seasonal variations in cardiac superoxide (O(2)(-)) production in rodents and in 8-isoprostane urinary excretion in humans, (ii) the mechanism of cardiac O(2)(-) overproduction occurring in late spring/summer months in rodents, (iii) whether this seasonal O(2)(-)-overproduction is associated with a pro-inflammatory endothelial activation, and (iv) how the summer-associated changes compare to those caused by diabetes, a classical cardiovascular risk factor. Langendorff-perfused guinea-pig and rat hearts generated ~100% more O(2)(-), and human subjects excreted 65% more 8-isoprostane in the summer vs. other seasons. Inhibitors of NADPH oxidase, xanthine oxidase, and NO synthase inhibited the seasonal O(2)(-)-overproduction. In the summer vs. other seasons, cardiac NADPH oxidase and xanthine oxidase activity, and protein expression were increased, the endothelial NO synthase and superoxide dismutases were downregulated, and, in guinea-pig hearts, adhesion molecules upregulation and the endothelial glycocalyx destruction associated these changes. In guinea-pig hearts, the summer and a streptozotocin-induced diabetes mediated similar changes, yet, more severe endothelial activation associated the diabetes. These findings suggest that the seasonal oxidative stress is a common phenomenon, associated, at least in guinea-pigs, with the endothelial activation. Nonetheless, its biological meaning (regulatory vs. deleterious) remains unclear. Upregulated NADPH oxidase and xanthine oxidase and uncoupled NO synthase are the sources of the seasonal O(2)(-)-overproduction. Copyright © 2010 Elsevier Ltd. All rights reserved.

Alteration of S-adenosylhomocysteine levels affects lignin biosynthesis in switchgrass.

PubMed

Bai, Zetao; Qi, Tianxiong; Liu, Yuchen; Wu, Zhenying; Ma, Lichao; Liu, Wenwen; Cao, Yingping; Bao, Yan; Fu, Chunxiang

2018-04-28

Methionine (Met) synthesized from aspartate is a fundamental amino acid needed to produce S-adenosylmethionine (SAM) that is an important cofactor for the methylation of monolignols. As a competitive inhibitor of SAM-dependent methylation, the effect of S-adenosylhomocysteine (SAH) on lignin biosynthesis, however, is still largely unknown in plants. Expression levels of Cystathionine γ-synthase (PvCGS) and S-adenosylhomocysteine hydrolase1 (PvSAHH1) were downregulated by RNAi technology, respectively, in switchgrass, a dual-purpose forage and biofuel crop. The transgenic switchgrass lines were subjected to studying the impact of SAH on lignin biosynthesis. Our results showed that downregulation of PvCGS in switchgrass altered the accumulation of aspartate-derived and aromatic amino acids, reduced the content of SAH, enhanced lignin biosynthesis, and stunted plant growth. In contrast, downregulation of PvSAHH1 raised SAH levels in switchgrass, impaired the biosynthesis of both guaiacyl and syringyl lignins, and therefore significantly increased saccharification efficiency of cell walls. This work indicates that SAH plays a crucial role in monolignol methylation in switchgrass. Genetic regulation of either PvCGS or PvSAHH1 expression in switchgrass can change intracellular SAH contents and SAM to SAH ratios and therefore affect lignin biosynthesis. Thus, our study suggests that genes involved in Met metabolism are of interest as new valuable targets for cell wall bioengineering in future. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Inhibition of Adipogenesis and Induction of Apoptosis and Lipolysis by Stem Bromelain in 3T3-L1 Adipocytes

PubMed Central

Dave, Sandeep; Kaur, Naval Jit; Nanduri, Ravikanth; Dkhar, H. Kitdorlang; Kumar, Ashwani; Gupta, Pawan

2012-01-01

The phytotherapeutic protein stem bromelain (SBM) is used as an anti-obesity alternative medicine. We show at the cellular level that SBM irreversibly inhibits 3T3-L1 adipocyte differentiation by reducing adipogenic gene expression and induces apoptosis and lipolysis in mature adipocytes. At the molecular level, SBM suppressed adipogenesis by downregulating C/EBPα and PPARγ independent of C/EBPβ gene expression. Moreover, mRNA levels of adipocyte fatty acid-binding protein (ap2), fatty acid synthase (FAS), lipoprotein lipase (LPL), CD36, and acetyl-CoA carboxylase (ACC) were also downregulated by SBM. Additionally, SBM reduced adiponectin expression and secretion. SBM's ability to repress PPARγ expression seems to stem from its ability to inhibit Akt and augment the TNFα pathway. The Akt–TSC2–mTORC1 pathway has recently been described for PPARγ expression in adipocytes. In our experiments, TNFα upregulation compromised cell viability of mature adipocytes (via apoptosis) and induced lipolysis. Lipolytic response was evident by downregulation of anti-lipolytic genes perilipin, phosphodiestersae-3B (PDE3B), and GTP binding protein Giα1, as well as sustained expression of hormone sensitive lipase (HSL). These data indicate that SBM, together with all-trans retinoic-acid (atRA), may be a potent modulator of obesity by repressing the PPARγ-regulated adipogenesis pathway at all stages and by augmenting TNFα-induced lipolysis and apoptosis in mature adipocytes. PMID:22292054

Protein-protein interactions among enzymes of starch biosynthesis in high-amylose barley genotypes reveal differential roles of heteromeric enzyme complexes in the synthesis of A and B granules.

PubMed

Ahmed, Zaheer; Tetlow, Ian J; Ahmed, Regina; Morell, Matthew K; Emes, Michael J

2015-04-01

The present study investigated the role of protein phosphorylation, and protein complex formation between key enzymes of amylopectin synthesis, in barley genotypes exhibiting "high amylose" phenotypes. Starch branching enzyme (SBE) down-regulated lines (ΔSBEIIa and ΔSBEIIb), starch synthase (SS)IIa (ssiia(-), sex6) and SSIII (ssiii(-), amo1) mutants were compared to a reference genotype, OAC Baxter. Down-regulation of either SBEIIa or IIb caused pleiotropic effects on SSI and starch phosphorylase (SP) and resulted in formation of novel protein complexes in which the missing SBEII isoform was substituted by SBEI and SP. In the ΔSBEIIb down-regulated line, soluble SP activity was undetectable. Nonetheless, SP was incorporated into a heteromeric protein complex with SBEI and SBEIIa and was readily detected in starch granules. In amo1, unlike other mutants, the data suggest that both SBEIIa and SBEIIb are in a protein complex with SSI and SSIIa. In the sex6 mutant no protein complexes involving SBEIIa or SBEIIb were detected in amyloplasts. Studies with Pro-Q Diamond revealed that GBSS, SSI, SSIIa, SBEIIb and SP are phosphorylated in their granule bound state. Alteration in the granule proteome in ΔSBEIIa and ΔSBEIIb lines, suggests that different protein complexes are involved in the synthesis of A and B granules. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Expression of a nitric oxide degrading enzyme induces a senescence programme in Arabidopsis.

PubMed

Mishina, Tatiana E; Lamb, Chris; Zeier, Jürgen

2007-01-01

Nitric oxide (NO) has been proposed to act as a factor delaying leaf senescence and fruit maturation in plants. Here we show that expression of a NO degrading dioxygenase (NOD) in Arabidopsis thaliana initiates a senescence-like phenotype, an effect that proved to be more pronounced in older than in younger leaves. This senescence phenotype was preceded by a massive switch in gene expression in which photosynthetic genes were down-regulated, whereas many senescence-associated genes (SAGs) and the 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene ACS6 involved in ethylene synthesis were up-regulated. External fumigation of NOD plants with NO as well as environmental conditions known to stimulate endogenous NO production attenuated the induced senescence programme. For instance, both high light conditions and nitrate feeding reduced the senescence phenotype and attenuated the down-regulation of photosynthetic genes as well as the up-regulation of SAGs. Treatment of plants with the cytokinin 6-benzylaminopurin (BAP) reduced the down-regulation of photosynthesis, although it had no consistent effect on SAG expression. Metabolic changes during NOD-induced senescence comprehended increases in salicylic acid (SA) levels, accumulation of the phytoalexin camalexin and elevation of leaf gamma-tocopherol contents, all of which occurred during natural senescence in Arabidopsis leaves as well. Moreover, NO fumigation delayed the senescence process induced by darkening individual Arabidopsis Columbia-0 (Col-0) leaves. Our data thus support the notion that NO acts as a negative regulator of leaf senescence.

Low concentrations of methylmercury inhibit neural progenitor cell proliferation associated with up-regulation of glycogen synthase kinase 3β and subsequent degradation of cyclin E in rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fujimura, Masatake, E-mail: fujimura@nimd.go.jp; Usuki, Fusako

2015-10-01

Methylmercury (MeHg) is an environmental neurotoxicant. The developing nervous system is susceptible to low concentrations of MeHg; however, the effect of MeHg on neural progenitor cell (NPC) proliferation, a key stage of neurogenesis during development, remains to be clarified. In this study, we investigated the effect of low concentrations of MeHg on NPCs by using a primary culture system developed using the embryonic rat cerebral cortex. NPC proliferation was suppressed 48 h after exposure to 10 nM MeHg, but cell death was not observed. Western blot analyses for cyclins A, B, D1, and E demonstrated that MeHg down-regulated cyclin E,more » a promoter of the G1/S cell cycle transition. Cyclin E has been shown to be degraded following the phosphorylation by glycogen synthase kinase 3β (GSK-3β). The time course study showed that GSK-3β was up-regulated 3 h after exposure to 10 nM MeHg, and cyclin E degradation 48 h after MeHg exposure. We further demonstrated that GSK-3β inhibitors, lithium and SB-415286, suppressed MeHg-induced inhibition of NPC proliferation by preventing cyclin E degradation. These results suggest that the inhibition of NPC proliferation induced by low concentration of MeHg was associated with up-regulation of GSK-3β at the early stage and subsequent degeneration of cyclin E. - Highlights: • NPC proliferation was suppressed by 10 nM MeHg, but cell death was not observed. • MeHg induced down-regulation of cyclin E, a promoter of cell cycle progression. • GSK-3β was up-regulated by 10 nM MeHg, leading to cyclin E degradation. • GSK-3β inhibitors suppressed MeHg-induced degradation of cyclin E.« less

Inhibiting glycogen synthase kinase-3 reduces endotoxaemic acute renal failure by down-regulating inflammation and renal cell apoptosis

PubMed Central

Wang, Y; Huang, WC; Wang, CY; Tsai, CC; Chen, CL; Chang, YT; Kai, JI; Lin, CF

2009-01-01

Background and purpose: Excessive inflammation and apoptosis are pathological features of endotoxaemic acute renal failure. Activation of glycogen synthase kinase-3 (GSK-3) is involved in inflammation and apoptosis. We investigated the effects of inhibiting GSK-3 on lipopolysaccharide (LPS)-induced acute renal failure, nuclear factor-κB (NF-κB), inflammation and apoptosis. Experimental approach: The effects of inhibiting GSK-3 with inhibitors, including lithium chloride (LiCl) and 6-bromo-indirubin-3′-oxime (BIO), on LPS-treated (15 mg·kg−1) C3H/HeN mice (LiCl, 40 mg·kg−1 and BIO, 2 mg·kg−1) and LPS-treated (1 µg·mL−1) renal epithelial cells (LiCl, 20 mM and BIO, 5 µM) were studied. Mouse survival was monitored and renal function was analysed by histological and serological examination. Cytokine and chemokine production, and cell apoptosis were measured by enzyme-linked immunosorbent assay and terminal deoxynucleotidyl transferase-mediated dUTP–biotin nick-end labelling staining, respectively. Activation of NF-κB and GSK-3 was determined by immunostaining and Western blotting, respectively. Key results: Mice treated with GSK-3 inhibitors showed decreased mortality, renal tubular dilatation, vacuolization and sloughing, blood urea nitrogen, creatinine and renal cell apoptosis in response to endotoxaemia. Inhibiting GSK-3 reduced LPS-induced tumour necrosis factor-α (TNF-α) and CCL5/RANTES (released upon activation of normal T-cells) in vivo in mice and in vitro in murine kidney cortical collecting duct epithelial M1 cells. Inhibiting GSK-3 did not block TNF-α-induced cytotoxicity in rat kidney proximal tubular epithelial NRK52E or in M1 cells. Conclusions and implications: These results suggest that GSK-3 inhibition protects against endotoxaemic acute renal failure mainly by down-regulating pro-inflammatory TNF-α and RANTES. PMID:19508392

Regulation of Hyaluronan (HA) Metabolism Mediated by HYBID (Hyaluronan-binding Protein Involved in HA Depolymerization, KIAA1199) and HA Synthases in Growth Factor-stimulated Fibroblasts.

PubMed

Nagaoka, Aya; Yoshida, Hiroyuki; Nakamura, Sachiko; Morikawa, Tomohiko; Kawabata, Keigo; Kobayashi, Masaki; Sakai, Shingo; Takahashi, Yoshito; Okada, Yasunori; Inoue, Shintaro

2015-12-25

Regulation of hyaluronan (HA) synthesis and degradation is essential to maintenance of extracellular matrix homeostasis. We recently reported that HYBID (HYaluronan-Binding protein Involved in hyaluronan Depolymerization), also called KIAA1199, plays a key role in HA depolymerization in skin and arthritic synovial fibroblasts. However, regulation of HA metabolism mediated by HYBID and HA synthases (HASs) under stimulation with growth factors remains obscure. Here we report that TGF-β1, basic FGF, EGF, and PDGF-BB commonly enhance total amount of HA in skin fibroblasts through up-regulation of HAS expression, but molecular size of newly produced HA is dependent on HYBID expression levels. Stimulation of HAS1/2 expression and suppression of HYBID expression by TGF-β1 were abrogated by blockade of the MAPK and/or Smad signaling and the PI3K-Akt signaling, respectively. In normal human skin, expression of the TGF-β1 receptors correlated positively with HAS2 expression and inversely with HYBID expression. On the other hand, TGF-β1 up-regulated HAS1/2 expression but exerted only a slight suppressive effect on HYBID expression in synovial fibroblasts from the patients with osteoarthritis or rheumatoid arthritis, resulting in the production of lower molecular weight HA compared with normal skin and synovial fibroblasts. These data demonstrate that although TGF-β1, basic FGF, EGF, and PDGF-BB enhance HA production in skin fibroblasts, TGF-β1 most efficiently contributes to production of high molecular weight HA by HAS up-regulation and HYBID down-regulation and suggests that inefficient down-regulation of HYBID by TGF-β1 in arthritic synovial fibroblasts may be linked to accumulation of depolymerized HA in synovial fluids in arthritis patients. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Dianthus superbus fructus suppresses airway inflammation by downregulating of inducible nitric oxide synthase in an ovalbumin-induced murine model of asthma

PubMed Central

2012-01-01

Background Dianthus superbus has long been used as a herbal medicine in Asia and as an anti-inflammatory agent. In this study, we evaluated the anti-inflammatory effects of Dianthus superbus fructus ethanolic extract (DSE) on Th2-type cytokines, eosinophil infiltration, and other factors in an ovalbumin (OVA)-induced murine asthma model. To study the possible mechanism of the anti-inflammatory effect of DSE, we also evaluated the expression of inducible nitric oxide synthase (iNOS) in the respiratory tract. Methods Mice were sensitized on days 0 and 14 by intraperitoneal injection of OVA. On days 21, 22 and 23 after initial sensitization, mice received an airway challenge with OVA for 1 h using an ultrasonic nebulizer. DSE was applied 1 h prior to OVA challenge. Mice were administered DSE orally at doses of 100 mg/kg or 200 mg/kg once daily from day 18 to 23. Bronchoalveolar lavage fluid (BALF) was collected 48 h after the final OVA challenge. Levels of interleukin (IL)-4, IL-13 and eotaxin in BALF were measured using enzyme-linked immunosorbent assays (ELISAs). Lung tissue sections were stained with hematoxylin and eosin for assessment of cell infiltration and mucus production with periodic acid shift staining, in conjunction with ELISA and western blot analyses for iNOS expression. Results DSE significantly reduced the levels of IL-4, IL-13, eotaxin, and immunoglobulin (Ig) E, number of inflammatory cells in BALF, and inflammatory cell infiltration and mucus production in the respiratory tract. DSE also attenuated the overexpression of iNOS protein induced by OVA challenge. Conclusion Our results suggest that DSE effectively protects against allergic airway inflammation by downregulating of iNOS expression and that DSE has potential as a therapeutic agent for allergic asthma. PMID:23110404

Effects of an Antimutagenic 1,4-Dihydropyridine AV-153 on Expression of Nitric Oxide Synthases and DNA Repair-related Enzymes and Genes in Kidneys of Rats with a Streptozotocin Model of Diabetes Mellitus.

PubMed

Ošiņa, Kristīne; Rostoka, Evita; Isajevs, Sergejs; Sokolovska, Jelizaveta; Sjakste, Tatjana; Sjakste, Nikolajs

2016-11-01

Development of complications of diabetes mellitus (DM), including diabetic nephropathy, is a complex multi-stage process, dependent on many factors including the modification of nitric oxide (NO) production and an impaired DNA repair. The goal of this work was to study in vivo effects of 1,4-dihydropyridine AV-153, known as antimutagen and DNA binder, on the expression of several genes and proteins involved in NO metabolism and DNA repair in the kidneys of rats with a streptozotocin (STZ)-induced model of DM. Transcription intensity was monitored by means of real-time RT-PCR and the expression of proteins by immunohistochemistry. Development of DM significantly induced PARP1 protein expression, while AV-153 (0.5 mg/kg) administration decreased it. AV-153 increased the expression of Parp1 gene in the kidneys of both intact and diabetic animals. Expression of H2afx mRNA and γH2AX histone protein, a marker of DNA breakage, was not changed in diabetic animals, but AV-153 up-regulated the expression of the gene without any impact on the protein expression. Development of DM was followed by a significant increase in iNOS enzyme expression, while AV-153 down-regulated the enzyme expression up to normal levels. iNos gene expression was also found to be increased in diabetic animals, but unlike the protein, the expression of mRNA was found to be enhanced by AV-153 administration. Expression of both eNOS protein and eNos gene in the kidneys was down-regulated, and the administration of AV-153 normalized the expression level. The effects of the compound in the kidneys of diabetic animals appear to be beneficial, as a trend for the normalization of expression of NO synthases is observed. © 2016 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

Korean red ginseng extract induces apoptosis and decreases telomerase activity in human leukemia cells.

PubMed

Park, Sang Eun; Park, Cheol; Kim, Sun Hee; Hossain, Mohammad Akbar; Kim, Min Young; Chung, Hae Young; Son, Woo Sung; Kim, Gi-Young; Choi, Yung Hyun; Kim, Nam Deuk

2009-01-21

Korean red ginseng (KRG, Panax ginseng C.A. Meyer Radix rubra) has been used to treat various diseases including cancer. However, the molecular mechanisms responsible for KRG extract induced apoptosis and telomerase inhibition remain unclear. The hot water extract from KRG was used to evaluate the mechanism of induction of apoptosis in U937 human leukemia cells and its effects on cyclooxgenase-2 (COX-2) and telomerase activity. KRG extract treatment to U937 cells resulted in growth inhibition and induction of apoptosis in a concentration-dependent manner as measured by hemacytometer counts, MTT assay, fluorescence microscopy, agarose gel electrophoresis and flow cytometry analysis. The increase in apoptosis was associated with the down-regulation of antiapoptotic Bcl-2, Bcl-X(L), and IAPs family members, and the activation of caspase-3. KRG extract treatment also decreased the expression levels of COX-2 and inducible nitric oxide synthase. Furthermore, KRG extract treatment progressively down-regulated the expression of human telomerase reverse transcriptase, a main determinant of the telomerase enzymatic activity, with inhibiting the expression of c-Myc in a concentration-dependent manner. These results provide important new insights into the possible molecular mechanisms of the anticancer activity of KRG extract.

Glucosylceramide Administration as a Vaccination Strategy in Mouse Models of Cryptococcosis

PubMed Central

Mor, Visesato; Farnoud, Amir M.; Singh, Ashutosh; Rella, Antonella; Tanno, Hiromasa; Ishii, Keiko; Kawakami, Kazuyoshi; Sato, Toshiya; Del Poeta, Maurizio

2016-01-01

Cryptococcus neoformans is an opportunistic fungal pathogen and the causative agent of the disease cryptococcosis. Cryptococcosis is initiated as a pulmonary infection and in conditions of immune deficiency disseminates to the blood stream and central nervous system, resulting in life-threatening meningoencephalitis. A number of studies have focused on the development of a vaccine against Cryptococcus, primarily utilizing protein-conjugated components of the Cryptococcus polysaccharide capsule as antigen. However, there is currently no vaccine against Cryptococcus in the clinic. Previous studies have shown that the glycosphingolipid, glucosylceramide (GlcCer), is a virulence factor in C. neoformans and antibodies against this lipid inhibit fungal growth and cell division. In the present study, we have investigated the possibility of using GlcCer as a therapeutic agent against C. neoformans infections in mouse models of cryptococcosis. GlcCer purified from a non-pathogenic fungus, Candida utilis, was administered intraperitoneally, prior to infecting mice with a lethal dose of C. neoformans. GlcCer administration prevented the dissemination of C. neoformans from the lungs to the brain and led to 60% mouse survival. GlcCer administration did not cause hepatic injury and elicited an anti-GlcCer antibody response, which was observed independent of the route of administration and the strains of mouse. Taken together, our results suggest that fungal GlcCer can protect mice against lethal doses of C. neoformans infection and can provide a viable vaccination strategy against Cryptococcus. PMID:27082428

The reconstituted P-glycoprotein multidrug transporter is a flippase for glucosylceramide and other simple glycosphingolipids.

PubMed

Eckford, Paul D W; Sharom, Frances J

2005-07-15

The Pgp (P-glycoprotein) multidrug transporter, which is linked to multidrug resistance in human cancers, functions as an efflux pump for non-polar drugs, powered by the hydrolysis of ATP at its nucleotide binding domains. The drug binding sites of Pgp appear to be located within the cytoplasmic leaflet of the membrane bilayer, suggesting that Pgp may function as a 'flippase' for hydrophobic compounds. Pgp has been shown to translocate fluorescent phospholipids, and it has been suggested that it may also interact with GlcCer (glucosylceramide). Here we use a dithionite fluorescence quenching technique to show that reconstituted Pgp can flip several NBD (nitrobenzo-2-oxa-1,3-diazole)-labelled simple glycosphingolipids, including NBD-GlcCer, from one leaflet of the bilayer to the other in an ATP-dependent, vanadate-sensitive fashion. The rate of NBD-GlcCer flipping was similar to that observed for NBD-labelled PC (phosphatidylcholine). NBD-GlcCer flipping was inhibited in a concentration-dependent, saturable fashion by various Pgp substrates and modulators, and inhibition correlated well with the Kd for binding to the protein. The addition of a second sugar to the headgroup of the glycolipid to form NBD-lactosylceramide drastically reduced the rate of flipping compared with NBD-PC, probably because of the increased size and polarity contributed by the additional sugar residue. We conclude that Pgp functions as a broad-specificity outwardly-directed flippase for simple glycosphingolipids and membrane phospholipids.

A Review of Gaucher Disease Pathophysiology, Clinical Presentation and Treatments

PubMed Central

Stirnemann, Jérôme; Belmatoug, Nadia; Camou, Fabrice; Serratrice, Christine; Froissart, Roseline; Caillaud, Catherine; Levade, Thierry; Astudillo, Leonardo; Serratrice, Jacques; Brassier, Anaïs; Rose, Christian; Billette de Villemeur, Thierry; Berger, Marc G.

2017-01-01

Gaucher disease (GD, ORPHA355) is a rare, autosomal recessive genetic disorder. It is caused by a deficiency of the lysosomal enzyme, glucocerebrosidase, which leads to an accumulation of its substrate, glucosylceramide, in macrophages. In the general population, its incidence is approximately 1/40,000 to 1/60,000 births, rising to 1/800 in Ashkenazi Jews. The main cause of the cytopenia, splenomegaly, hepatomegaly, and bone lesions associated with the disease is considered to be the infiltration of the bone marrow, spleen, and liver by Gaucher cells. Type-1 Gaucher disease, which affects the majority of patients (90% in Europe and USA, but less in other regions), is characterized by effects on the viscera, whereas types 2 and 3 are also associated with neurological impairment, either severe in type 2 or variable in type 3. A diagnosis of GD can be confirmed by demonstrating the deficiency of acid glucocerebrosidase activity in leukocytes. Mutations in the GBA1 gene should be identified as they may be of prognostic value in some cases. Patients with type-1 GD—but also carriers of GBA1 mutation—have been found to be predisposed to developing Parkinson’s disease, and the risk of neoplasia associated with the disease is still subject to discussion. Disease-specific treatment consists of intravenous enzyme replacement therapy (ERT) using one of the currently available molecules (imiglucerase, velaglucerase, or taliglucerase). Orally administered inhibitors of glucosylceramide biosynthesis can also be used (miglustat or eliglustat). PMID:28218669

Glucerabacter canisensis gen. nov., sp. nov., isolated from dog feces and its effect on the hydrolysis of plant glucosylceramide in the intestine of dogs.

PubMed

Kawata, Misho; Tsukamoto, Ami; Isozaki, Ryohei; Nobukawa, Shohei; Kawahara, Natsuki; Akutsu, Shoko; Suzuki, Masato; Asanuma, Narito

2018-04-01

A Gram-positive, obligately anaerobic, oval-rod shaped, non-spore-forming, and non-pigmented bacterium, designated strain NATH-2371 T (= JCM31739 T  = DSM105698 T ), was isolated from dog feces. Comparative 16S rRNA gene sequence analysis revealed that strain NATH-2371 T belongs to Clostridium cluster XIVa, and the closest strains were Coprococcus comes ATCC 27758 T (94.8% 16S rRNA gene sequence similarity) and Clostridium nexile DSM 1787 T (94.0%). Strain NATH-2371 T produced acetate, formate, and ethanol from glucose. Predominant cellular fatty acids are C 16:0 and C 16:0 DMA. On the basis of the phenotypic and genotypic differences, strain NATH-2371 T represents a novel species in a new genus of the family Lachnospiraceae, for which the name Glucerabacter canisensis gen. nov., sp. nov., is proposed. This strain was found to efficiently hydrolyze plant glucosylceramide (GluCer). The abundance of strain NATH-2371 T in dog feces was higher in young dogs than in old dogs. Incubation of dog fecal bacteria showed that GluCer-hydrolyzing activity decreased with the age of dogs. The cell number of strain NATH-2371 T in dog feces appeared to be correlated with GluCer hydrolysis. Thus, this bacterium is likely to play a major role in GluCer hydrolysis in the dog intestine.

Virus-induced gene silencing of the two squalene synthase isoforms of apple tree (Malus × domestica L.) negatively impacts phytosterol biosynthesis, plastid pigmentation and leaf growth.

PubMed

Navarro Gallón, Sandra M; Elejalde-Palmett, Carolina; Daudu, Dimitri; Liesecke, Franziska; Jullien, Frédéric; Papon, Nicolas; Dugé de Bernonville, Thomas; Courdavault, Vincent; Lanoue, Arnaud; Oudin, Audrey; Glévarec, Gaëlle; Pichon, Olivier; Clastre, Marc; St-Pierre, Benoit; Atehortùa, Lucia; Yoshikawa, Nobuyuki; Giglioli-Guivarc'h, Nathalie; Besseau, Sébastien

2017-07-01

The use of a VIGS approach to silence the newly characterized apple tree SQS isoforms points out the biological function of phytosterols in plastid pigmentation and leaf development. Triterpenoids are beneficial health compounds highly accumulated in apple; however, their metabolic regulation is poorly understood. Squalene synthase (SQS) is a key branch point enzyme involved in both phytosterol and triterpene biosynthesis. In this study, two SQS isoforms were identified in apple tree genome. Both isoforms are located at the endoplasmic reticulum surface and were demonstrated to be functional SQS enzymes using an in vitro activity assay. MdSQS1 and MdSQS2 display specificities in their expression profiles with respect to plant organs and environmental constraints. This indicates a possible preferential involvement of each isoform in phytosterol and/or triterpene metabolic pathways as further argued using RNAseq meta-transcriptomic analyses. Finally, a virus-induced gene silencing (VIGS) approach was used to silence MdSQS1 and MdSQS2. The concomitant down-regulation of both MdSQS isoforms strongly affected phytosterol synthesis without alteration in triterpene accumulation, since triterpene-specific oxidosqualene synthases were found to be up-regulated to compensate metabolic flux reduction. Phytosterol deficiencies in silenced plants clearly disturbed chloroplast pigmentation and led to abnormal development impacting leaf division rather than elongation or differentiation. In conclusion, beyond the characterization of two SQS isoforms in apple tree, this work brings clues for a specific involvement of each isoform in phytosterol and triterpene pathways and emphasizes the biological function of phytosterols in development and chloroplast integrity. Our report also opens the door to metabolism studies in Malus domestica using the apple latent spherical virus-based VIGS method.

Evaluation of deoxyhypusine synthase inhibitors targeting BCR-ABL positive leukemias.

PubMed

Ziegler, Patrick; Chahoud, Tuhama; Wilhelm, Thomas; Pällman, Nora; Braig, Melanie; Wiehle, Valeska; Ziegler, Susanne; Schröder, Marcus; Meier, Chris; Kolodzik, Adrian; Rarey, Matthias; Panse, Jens; Hauber, Joachim; Balabanov, Stefan; Brümmendorf, Tim H

2012-12-01

Effective inhibition of BCR-ABL tyrosine kinase activity with Imatinib represents a breakthrough in the treatment of patients with chronic myeloid leukemia (CML). However, more than 30 % of patients with CML in chronic phase do not respond adequately to Imatinib and the drug seems not to affect the quiescent pool of BCR-ABL positive leukemic stem and progenitor cells. Therefore, despite encouraging clinical results, Imatinib can still not be considered a curative treatment option in CML. We recently reported downregulation of eukaryotic initiation factor 5A (eIF5A) in Imatinib treated K562 cells. Furthermore, the inhibition of eIF5A by siRNA in combination with Imatinib has been shown to exert synergistic cytotoxic effects on BCR-ABL positive cell lines. Based on the structure of known deoxyhypusine synthase (DHS) inhibitors such as CNI-1493, a drug design approach was applied to develop potential compounds targeting DHS. Here we report the biological evaluation of selected novel (DHSI-15) as compared to established (CNI-1493, deoxyspergualin) DHS inhibitors. We show that upon the compounds tested, DHSI-15 and deoxyspergualin exert strongest antiproliferative effects on BCR-ABL cells including Imatinib resistant mutants. However, this effect did not seem to be restricted to BCR-ABL positive cell lines or primary cells. Both compounds are able to induce apoptosis/necrosis during long term incubation of BCR-ABL positive BA/F3 derivates. Pharmacological synergism can be observed for deoxyspergualin and Imatinib, but not for DHSI-15 and Imatinib. Finally we show that deoxyspergualin is able to inhibit proliferation of CD34+ progenitor cells from CML patients. We conclude that inhibition of deoxyhypusine synthase (DHS) can be supportive for the anti-proliferative treatment of leukemia and merits further investigation including other cancers.

Anti-obesity effects of 3-hydroxychromone derivative, a novel small-molecule inhibitor of glycogen synthase kinase-3.

PubMed

Lee, Sooho; Yang, Woo Kyeom; Song, Ji Ho; Ra, Young Min; Jeong, Jin-Hyun; Choe, Wonchae; Kang, Insug; Kim, Sung-Soo; Ha, Joohun

2013-04-01

Glycogen synthase kinase 3 (GSK-3) plays a central role in cellular energy metabolism, and dysregulation of GSK-3 activity is implicated in a variety of metabolic disorders, including obesity, type 2 diabetes, and cancer. Hence, GSK-3 has emerged as an attractive target molecule for the treatment of metabolic disorders. Therefore, this research focused on identification and characterization of a novel small-molecule GSK-3 inhibitor. Compound 1a, a structure based on 3-hydroxychromone bearing isothiazolidine-1,1-dione, was identified from chemical library as a highly potent GSK-3 inhibitor. An in vitro kinase assay utilizing a panel of kinases demonstrated that compound 1a strongly inhibits GSK-3β. The potential effects of compound 1a on the inactivation of GSK-3 were confirmed in human liver HepG2 and human embryonic kidney HEK293 cells. Stabilization of glycogen synthase and β-catenin, which are direct targets of GSK-3, by compound 1a was assessed in comparison with two other GSK-3 inhibitors: LiCl and SB-415286. In mouse 3T3-L1 preadipocytes, compound 1a markedly blocked adipocyte differentiation. Consistently, intraperitoneal administration of compound 1a to diet-induced obese mice significantly ameliorated their key symptoms such as body weight gain, increased adiposity, dyslipidemia, and hepatic steatosis due to the marked reduction of whole-body lipid level. In vitro and in vivo effects were accompanied by upregulation of β-catenin stability and downregulation of the expression of several critical genes related to lipid metabolism. From these results, it can be concluded that compound 1a, a novel small-molecule inhibitor of GSK-3, has potential as a new class of therapeutic agent for obesity treatment. Copyright © 2013 Elsevier Inc. All rights reserved.

IFN-gamma synergizes with LPS to induce nitric oxide biosynthesis through glycogen synthase kinase-3-inhibited IL-10.

PubMed

Lin, Chiou-Feng; Tsai, Cheng-Chieh; Huang, Wei-Ching; Wang, Chi-Yun; Tseng, Hsiang-Chi; Wang, Yi; Kai, Jui-In; Wang, Szu-Wen; Cheng, Yi-Lin

2008-10-15

Interferon-gamma (IFN-gamma) plays a crucial role in innate immunity and inflammation. It causes the synergistic effect on endotoxin lipopolysaccharide (LPS)-stimulated inducible nitric oxide synthase (iNOS)/NO biosynthesis; however, the mechanism remains unclear. In the present study, we investigated the effects of glycogen synthase kinase-3 (GSK-3)-mediated inhibition of anti-inflammatory interleukin-10 (IL-10). We found, in LPS-stimulated macrophages, that IFN-gamma increased iNOS expression and NO production in a time-dependent manner. In addition, ELISA analysis showed the upregulation of tumor necrosis factor-alpha and regulated on activation, normal T expressed and secreted, and the downregulation of IL-10. RT-PCR further showed changes in the IL-10 mRNA level as well. Treating cells with recombinant IL-10 showed a decrease in IFN-gamma/LPS-induced iNOS/NO biosynthesis, whereas anti-IL-10 neutralizing antibodies enhanced this effect, suggesting that IL-10 acts in an anti-inflammatory role. GSK-3-inhibitor treatment blocked IFN-gamma/LPS-induced iNOS/NO biosynthesis but upregulated IL-10 production. Inhibiting GSK-3 using short-interference RNA showed similar results. Additionally, treating cells with anti-IL-10 neutralizing antibodies blocked these effects. We further showed that inhibiting GSK-3 increased phosphorylation of transcription factor cyclic AMP response element binding protein. Inhibiting protein tyrosine kinase Pyk2, an upstream regulator of GSK-3beta, caused inhibition on IFN-gamma/LPS-induced GSK-3beta phosphorylation at tyrosine 216 and iNOS/NO biosynthesis. Taken together, these findings reveal the involvement of GSK-3-inhibited IL-10 on the induction of iNOS/NO biosynthesis by IFN-gamma synergized with LPS. (c) 2008 Wiley-Liss, Inc.

RNAi and Homologous Over-Expression Based Functional Approaches Reveal Triterpenoid Synthase Gene-Cycloartenol Synthase Is Involved in Downstream Withanolide Biosynthesis in Withania somnifera

PubMed Central

Mishra, Bhawana; Sangwan, Rajender Singh; Asha; Jadaun, Jyoti Singh; Sangwan, Neelam S.

2016-01-01

Withania somnifera Dunal, is one of the most commonly used medicinal plant in Ayurvedic and indigenous medicine traditionally owing to its therapeutic potential, because of major chemical constituents, withanolides. Withanolide biosynthesis requires the activities of several enzymes in vivo. Cycloartenol synthase (CAS) is an important enzyme in the withanolide biosynthetic pathway, catalyzing cyclization of 2, 3 oxidosqualene into cycloartenol. In the present study, we have cloned full-length WsCAS from Withania somnifera by homology-based PCR method. For gene function investigation, we constructed three RNAi gene-silencing constructs in backbone of RNAi vector pGSA and a full-length over-expression construct. These constructs were transformed in Agrobacterium strain GV3101 for plant transformation in W. somnifera. Molecular and metabolite analysis was performed in putative Withania transformants. The PCR and Southern blot results showed the genomic integration of these RNAi and overexpression construct(s) in Withania genome. The qRT-PCR analysis showed that the expression of WsCAS gene was considerably downregulated in stable transgenic silenced Withania lines compared with the non-transformed control and HPLC analysis showed that withanolide content was greatly reduced in silenced lines. Transgenic plants over expressing CAS gene displayed enhanced level of CAS transcript and withanolide content compared to non-transformed controls. This work is the first full proof report of functional validation of any metabolic pathway gene in W. somnifera at whole plant level as per our knowledge and it will be further useful to understand the regulatory role of different genes involved in the biosynthesis of withanolides. PMID:26919744

ARGININOSUCCINATE SYNTHASE CONDITIONS THE RESPONSE TO ACUTE AND CHRONIC ETHANOL-INDUCED LIVER INJURY IN MICE

PubMed Central

Yan, Wei; Morón-Concepción, Jose A.; Ward, Stephen C.; Ge, Xiaodong; de la Rosa, Laura Conde; Nieto, Natalia

2012-01-01

Background and Aim Argininosuccinate synthase (ASS) is the rate-limiting enzyme in both the urea and the l-citrulline/nitric oxide (NO·) cycles regulating protein catabolism, ammonia levels and NO· generation (1-2). Since a proteomics analysis identified ASS and nitric oxide synthase-2 (NOS2) as co-induced in rat hepatocytes by chronic ethanol consumption, which also occurred in alcoholic liver disease (ALD) and in cirrhotic patients, we hypothesized that ASS could play a role in ethanol binge and chronic ethanol-induced liver damage. Methods To investigate the contribution of ASS to the pathophysiology of ALD, wild-type (WT) and Ass+/− mice (Ass−/− are lethal due to hyperammonemia) were exposed to an ethanol binge or to chronic ethanol drinking. Results Compared with WT, Ass+/− mice given an ethanol binge exhibited decreased steatosis, lower NOS2 induction and less 3-nitrotyrosine (3-NT) protein residues, indicating that reducing nitrosative stress via the l-citrulline/NO· pathway plays a significant role in preventing liver damage. However, chronic ethanol treated Ass+/− mice displayed enhanced liver injury compared with WT mice. This was due to hyperammonemia, lower phosphorylated AMP-activated protein kinase (pAMPKα) to total AMPKα ratio, decreased sirtuin (Sirt-1) and peroxisomal proliferator-activated receptor coactivator-1α (Pgc1α) mRNAs, lower fatty acid β-oxidation due to down-regulation of carnitine palmitoyl transferase-II (CPT-II), decreased antioxidant defense and elevated lipid peroxidation end-products in spite of comparable nitrosative stress but likely reduced NOS3. Conclusion Partial Ass ablation protects only in acute ethanol-induced liver injury by decreasing nitrosative stress but not in a more chronic scenario where oxidative stress and impaired fatty acid β-oxidation are key events. PMID:22213272

A Phase I, Pharmacokinetic, and Pharmacodynamic Study of Vorinostat in Combination with 5-Fluorouracil, Leucovorin, and Oxaliplatin in Patients with Refractory Colorectal Cancer

PubMed Central

Fakih, Marwan G.; Pendyala, Lakshmi; Fetterly, Gerald; Toth, Karoli; Zwiebel, James A; Espinoza-Delgado, Igor; Litwin, Alan; Rustum, Youcef M.; Ross, Mary Ellen; Holleran, Julianne L.; Egorin, Merrill J.

2014-01-01

Purpose We conducted a phase I study to determine the maximum tolerated dose (MTD) of vorinostat in combination with fixed doses of 5-Fluorouracil (5-FU), leucovorin, and oxaliplatin (FOLFOX). Experimental Design Vorinostat was given PO BID for 1 week every 2 weeks. FOLFOX was given on days 4 and 5 of vorinostat. The vorinostat starting dose was 100 mg BID. Escalation occurred in cohorts of 3–6 patients. Pharmacokinetics of vorinostat, 5-FU, and oxaliplatin were studied. Results Twenty-one patients were enrolled. Thrombocytopenia, neutropenia, gastrointestinal toxicities, and fatigue increased in frequency and severity at higher dose-levels (DL) of vorinostat. Two of 4 evaluable patients at DL 4 (vorinostat 400 mg PO BID) developed dose-limiting fatigue. One of 10 evaluable patients at DL3 (vorinostat 300 mg PO BID) had dose-limiting fatigue, anorexia, and dehydration. There were significant relationships between vorinostat dose and AUC on days 1 and 5 (Pearson, < 0.001). Vorinostat AUC increased (p = 0.005) and clearance decreased (p = 0.003) on day 5 compared to day 1. The median Cmax of 5-FU at each DL increased significantly with increasing doses of vorinostat, suggesting a pharmacokinetic interaction between 5-FU and vorinostat. Vorinostat-induced thymidylate synthase modulation was not consistent; only two of six patients had a decrease in intra-tumoral thymidylate synthase expression by RT-PCR. Conclusions The MTD of vorinostat in combination with FOLFOX is 300 mg PO BID x 1 week every two weeks. Alternative vorinostat dosing schedules may be needed for optimal down-regulation of thymidylate synthase expression. PMID:19383814

Argininosuccinate synthase conditions the response to acute and chronic ethanol-induced liver injury in mice.

PubMed

Leung, Tung Ming; Lu, Yongke; Yan, Wei; Morón-Concepción, Jose A; Ward, Stephen C; Ge, Xiaodong; Conde de la Rosa, Laura; Nieto, Natalia

2012-05-01

Argininosuccinate synthase (ASS) is the rate-limiting enzyme in both the urea and the L-citrulline/nitric oxide (NO·) cycles regulating protein catabolism, ammonia levels, and NO· generation. Because a proteomics analysis identified ASS and nitric oxide synthase-2 (NOS2) as coinduced in rat hepatocytes by chronic ethanol consumption, which also occurred in alcoholic liver disease (ALD) and in cirrhosis patients, we hypothesized that ASS could play a role in ethanol binge and chronic ethanol-induced liver damage. To investigate the contribution of ASS to the pathophysiology of ALD, wildtype (WT) and Ass(+/-) mice (Ass(-/-) are lethal due to hyperammonemia) were exposed to an ethanol binge or to chronic ethanol drinking. Compared with WT, Ass(+/-) mice given an ethanol binge exhibited decreased steatosis, lower NOS2 induction, and less 3-nitrotyrosine (3-NT) protein residues, indicating that reducing nitrosative stress by way of the L-citrulline/NO· pathway plays a significant role in preventing liver damage. However, chronic ethanol-treated Ass(+/-) mice displayed enhanced liver injury compared with WT mice. This was due to hyperammonemia, lower phosphorylated AMP-activated protein kinase alpha (pAMPKα) to total AMPKα ratio, decreased sirtuin-1 (Sirt-1) and peroxisomal proliferator-activated receptor coactivator-1α (Pgc1α) messenger RNAs (mRNAs), lower fatty acid β-oxidation due to down-regulation of carnitine palmitoyl transferase-II (CPT-II), decreased antioxidant defense, and elevated lipid peroxidation end-products in spite of comparable nitrosative stress but likely reduced NOS3. Partial Ass ablation protects only in acute ethanol-induced liver injury by decreasing nitrosative stress but not in a more chronic scenario where oxidative stress and impaired fatty acid β-oxidation are key events. Copyright © 2011 American Association for the Study of Liver Diseases.

Transient Silencing of CHALCONE SYNTHASE during Fruit Ripening Modifies Tomato Epidermal Cells and Cuticle Properties1[C][W

PubMed Central

España, Laura; Heredia-Guerrero, José A.; Reina-Pinto, José J.; Fernández-Muñoz, Rafael; Heredia, Antonio; Domínguez, Eva

2014-01-01

Tomato (Solanum lycopersicum) fruit ripening is accompanied by an increase in CHALCONE SYNTHASE (CHS) activity and flavonoid biosynthesis. Flavonoids accumulate in the cuticle, giving its characteristic orange color that contributes to the eventual red color of the ripe fruit. Using virus-induced gene silencing in fruits, we have down-regulated the expression of SlCHS during ripening and compared the cuticles derived from silenced and nonsilenced regions. Silenced regions showed a pink color due to the lack of flavonoids incorporated to the cuticle. This change in color was accompanied by several other changes in the cuticle and epidermis. The epidermal cells displayed a decreased tangential cell width; a decrease in the amount of cuticle and its main components, cutin and polysaccharides, was also observed. Flavonoids dramatically altered the cuticle biomechanical properties by stiffening the elastic and viscoelastic phase and by reducing the ability of the cuticle to deform. There seemed to be a negative relation between SlCHS expression and wax accumulation during ripening that could be related to the decreased cuticle permeability to water observed in the regions silencing SlCHS. A reduction in the overall number of ester linkages present in the cutin matrix was also dependent on the presence of flavonoids. PMID:25277718

Differential expression proteomics of human colorectal cancer based on a syngeneic cellular model for the progression of adenoma to carcinoma.

PubMed

Roth, Udo; Razawi, Hanieh; Hommer, Julia; Engelmann, Katja; Schwientek, Tilo; Müller, Stefan; Baldus, Stephan E; Patsos, Georgios; Corfield, Anthony P; Paraskeva, Christos; Hanisch, Franz-Georg

2010-01-01

This is the first differential expression proteomics study on a human syngeneic cellular in vitro progression model of the colorectal adenoma-to-carcinoma sequence, the anchorage-dependent non-tumorigenic adenoma derived cell line AA/C1 and the derived anchorage-independent and tumorigenic carcinoma cell line AA/C1/SB10C. The study is based on quantitative 2-DE and is complemented by Western blot validation. Excluding redundancies due to proteolysis and post-translational modified isoforms of over 2000 protein spots, 13 proteins were revealed as regulated with statistical variance being within the 95th confidence level and were identified by peptide mass fingerprinting in MALDI MS. Progression-associated proteins belong to the functional complexes of anaerobic glycolysis/gluconeogenesis, steroid biosynthesis, prostaglandin biosynthesis, the regulation and maintenance of the cytoskeleton, protein biosynthesis and degradation, the regulation of apoptosis or other functions. Partial but significant overlap was revealed with previous proteomics and transcriptomics studies in colorectal carcinoma. Among upregulated proteins we identified 3-HMG-CoA synthase, protein phosphatase 1, prostaglandin E synthase 2, villin 1, annexin A1, triosephosphate isomerase, phosphoserine aminotransferase 1, fumarylacetoacetate hydrolase and pyrroline-5-carboxylate reductase 1 (PYCR1), while glucose-regulated protein 78, cathepsin D, lamin A/C and quinolate phosphoribosyltransferase were downregulated.

Caveolin-1 down-regulates inducible nitric oxide synthase via the proteasome pathway in human colon carcinoma cells

PubMed Central

Felley-Bosco, Emanuela; Bender, Florent C.; Courjault-Gautier, Françoise; Bron, Claude; Quest, Andrew F. G.

2000-01-01

To investigate whether caveolin-1 (cav-1) may modulate inducible nitric oxide synthase (iNOS) function in intact cells, the human intestinal carcinoma cell lines HT29 and DLD1 that have low endogenous cav-1 levels were transfected with cav-1 cDNA. In nontransfected cells, iNOS mRNA and protein levels were increased by the addition of a mix of cytokines. Ectopic expression of cav-1 in both cell lines correlated with significantly decreased iNOS activity and protein levels. This effect was linked to a posttranscriptional mechanism involving enhanced iNOS protein degradation by the proteasome pathway, because (i) induction of iNOS mRNA by cytokines was not affected and (ii) iNOS protein levels increased in the presence of the proteasome inhibitors N-acetyl-Leu-Leu-Norleucinal and lactacystin. In addition, a small amount of iNOS was found to cofractionate with cav-1 in Triton X-100-insoluble membrane fractions where also iNOS degradation was apparent. As has been described for endothelial and neuronal NOS isoenzymes, direct binding between cav-1 and human iNOS was detected in vitro. Taken together, these results suggest that cav-1 promotes iNOS presence in detergent-insoluble membrane fractions and degradation there via the proteasome pathway. PMID:11114180

Increased expression of fatty acid synthase provides a survival advantage to colorectal cancer cells via upregulation of cellular respiration

PubMed Central

Zaytseva, Yekaterina Y.; Harris, Jennifer W.; Mitov, Mihail I.; Kim, Ji Tae; Butterfield, D. Allan; Lee, Eun Y.; Weiss, Heidi L.; Gao, Tianyan; Evers, B. Mark

2015-01-01

Fatty acid synthase (FASN), a lipogenic enzyme, is upregulated in colorectal cancer (CRC). Increased de novo lipid synthesis is thought to be a metabolic adaptation of cancer cells that promotes survival and metastasis; however, the mechanisms for this phenomenon are not fully understood. We show that FASN plays a role in regulation of energy homeostasis by enhancing cellular respiration in CRC. We demonstrate that endogenously synthesized lipids fuel fatty acid oxidation, particularly during metabolic stress, and maintain energy homeostasis. Increased FASN expression is associated with a decrease in activation of energy-sensing pathways and accumulation of lipid droplets in CRC cells and orthotopic CRCs. Immunohistochemical evaluation demonstrated increased expression of FASN and p62, a marker of autophagy inhibition, in primary CRCs and liver metastases compared to matched normal colonic mucosa. Our findings indicate that overexpression of FASN plays a crucial role in maintaining energy homeostasis in CRC via increased oxidation of endogenously synthesized lipids. Importantly, activation of fatty acid oxidation and consequent downregulation of stress-response signaling pathways may be key adaptation mechanisms that mediate the effects of FASN on cancer cell survival and metastasis, providing a strong rationale for targeting this pathway in advanced CRC. PMID:25970773

Increased expression of fatty acid synthase provides a survival advantage to colorectal cancer cells via upregulation of cellular respiration.

PubMed

Zaytseva, Yekaterina Y; Harris, Jennifer W; Mitov, Mihail I; Kim, Ji Tae; Butterfield, D Allan; Lee, Eun Y; Weiss, Heidi L; Gao, Tianyan; Evers, B Mark

2015-08-07

Fatty acid synthase (FASN), a lipogenic enzyme, is upregulated in colorectal cancer (CRC). Increased de novo lipid synthesis is thought to be a metabolic adaptation of cancer cells that promotes survival and metastasis; however, the mechanisms for this phenomenon are not fully understood. We show that FASN plays a role in regulation of energy homeostasis by enhancing cellular respiration in CRC. We demonstrate that endogenously synthesized lipids fuel fatty acid oxidation, particularly during metabolic stress, and maintain energy homeostasis. Increased FASN expression is associated with a decrease in activation of energy-sensing pathways and accumulation of lipid droplets in CRC cells and orthotopic CRCs. Immunohistochemical evaluation demonstrated increased expression of FASN and p62, a marker of autophagy inhibition, in primary CRCs and liver metastases compared to matched normal colonic mucosa. Our findings indicate that overexpression of FASN plays a crucial role in maintaining energy homeostasis in CRC via increased oxidation of endogenously synthesized lipids. Importantly, activation of fatty acid oxidation and consequent downregulation of stress-response signaling pathways may be key adaptation mechanisms that mediate the effects of FASN on cancer cell survival and metastasis, providing a strong rationale for targeting this pathway in advanced CRC.

Autophagy regulates the therapeutic potential of mesenchymal stem cells in experimental autoimmune encephalomyelitis

PubMed Central

Dang, Shipeng; Xu, Huanbai; Xu, Congfeng; Cai, Wei; Li, Qian; Cheng, Yiji; Jin, Min; Wang, Ru-Xing; Peng, Yongde; Zhang, Yi; Wu, Changping; He, Xiaozhou; Wan, Bing; Zhang, Yanyun

2014-01-01

Mesenchymal stem cell (MSC)-based therapy is a promising approach to treat various inflammatory disorders including multiple sclerosis. However, the fate of MSCs in the inflammatory microenvironment is largely unknown. Experimental autoimmune encephalomyelitis (EAE) is a well-studied animal model of multiple sclerosis. We demonstrated that autophagy occurred in MSCs during their application for EAE treatment. Inflammatory cytokines, e.g., interferon gamma and tumor necrosis factor, induced autophagy in MSCs synergistically by inducing expression of BECN1/Beclin 1. Inhibition of autophagy by knockdown of Becn1 significantly improved the therapeutic effects of MSCs on EAE, which was mainly attributable to enhanced suppression upon activation and expansion of CD4+ T cells. Mechanistically, inhibition of autophagy increased reactive oxygen species generation and mitogen-activated protein kinase 1/3 activation in MSCs, which were essential for PTGS2 (prostaglandin-endoperoxide synthase 2 [prostaglandin G/H synthase and cyclooxygenase]) and downstream prostaglandin E2 expression to exert immunoregulatory function. Furthermore, pharmacological treatment of MSCs to inhibit autophagy increased their immunosuppressive effects on T cell-mediated EAE. Our findings indicate that inflammatory microenvironment-induced autophagy downregulates the immunosuppressive function of MSCs. Therefore, modulation of autophagy in MSCs would provide a novel strategy to improve MSC-based immunotherapy. PMID:24905997

Autophagy regulates the therapeutic potential of mesenchymal stem cells in experimental autoimmune encephalomyelitis.

PubMed

Dang, Shipeng; Xu, Huanbai; Xu, Congfeng; Cai, Wei; Li, Qian; Cheng, Yiji; Jin, Min; Wang, Ru-Xing; Peng, Yongde; Zhang, Yi; Wu, Changping; He, Xiaozhou; Wan, Bing; Zhang, Yanyun

2014-07-01

Mesenchymal stem cell (MSC)-based therapy is a promising approach to treat various inflammatory disorders including multiple sclerosis. However, the fate of MSCs in the inflammatory microenvironment is largely unknown. Experimental autoimmune encephalomyelitis (EAE) is a well-studied animal model of multiple sclerosis. We demonstrated that autophagy occurred in MSCs during their application for EAE treatment. Inflammatory cytokines, e.g., interferon gamma and tumor necrosis factor, induced autophagy in MSCs synergistically by inducing expression of BECN1/Beclin 1. Inhibition of autophagy by knockdown of Becn1 significantly improved the therapeutic effects of MSCs on EAE, which was mainly attributable to enhanced suppression upon activation and expansion of CD4(+) T cells. Mechanistically, inhibition of autophagy increased reactive oxygen species generation and mitogen-activated protein kinase 1/3 activation in MSCs, which were essential for PTGS2 (prostaglandin-endoperoxide synthase 2 [prostaglandin G/H synthase and cyclooxygenase]) and downstream prostaglandin E2 expression to exert immunoregulatory function. Furthermore, pharmacological treatment of MSCs to inhibit autophagy increased their immunosuppressive effects on T cell-mediated EAE. Our findings indicate that inflammatory microenvironment-induced autophagy downregulates the immunosuppressive function of MSCs. Therefore, modulation of autophagy in MSCs would provide a novel strategy to improve MSC-based immunotherapy.

2C-Methyl- D- erythritol 2,4-cyclodiphosphate synthase from Stevia rebaudiana Bertoni is a functional gene.

PubMed

Kumar, Hitesh; Singh, Kashmir; Kumar, Sanjay

2012-12-01

Stevia [Stevia rebaudiana (Bertoni)] is a perennial herb which accumulates sweet diterpenoid steviol glycosides (SGs) in its leaf tissue. SGs are synthesized by 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. Of the various enzymes of the MEP pathway, 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MDS) (encoded by MDS) catalyzes the cyclization of 4-(cytidine 5' diphospho)-2C-methyl-D-erythritol 2-phosphate into 2C-methyl-D-erythritol 2,4-cyclodiphosphate. Complementation of the MDS knockout mutant strain of Escherichia coli, EB370 with putative MDS of stevia (SrMDS) rescued the lethal mutant, suggesting SrMDS to be a functional gene. Experiments conducted in plant growth chamber and in the field suggested SrMDS to be a light regulated gene. Indole 3-acetic acid (IAA; 50, 100 μM) down-regulated the expression of SrMDS at 4 h of the treatment, whereas, abscisic acid did not modulate its expression. A high expression of SrMDS was observed during the light hours of the day as compared to the dark hours. The present work established functionality of SrMDS and showed the role of light and IAA in regulating expression of SrMDS.

Adiponectin inhibits insulin function in primary trophoblasts by PPARα-mediated ceramide synthesis.

PubMed

Aye, Irving L M H; Gao, Xiaoli; Weintraub, Susan T; Jansson, Thomas; Powell, Theresa L

2014-04-01

Maternal adiponectin (ADN) levels are inversely correlated with birth weight, and ADN infusion in pregnant mice down-regulates placental nutrient transporters and decreases fetal growth. In contrast to the insulin-sensitizing effects in adipose tissue and muscle, ADN inhibits insulin signaling in the placenta. However, the molecular mechanisms involved are unknown. We hypothesized that ADN inhibits insulin signaling and insulin-stimulated amino acid transport in primary human trophoblasts by peroxisome proliferator-activated receptor-α (PPARα)-mediated ceramide synthesis. Primary human term trophoblast cells were treated with ADN and/or insulin. ADN increased the phosphorylation of p38 MAPK and PPARα. ADN inhibited insulin signaling and insulin-stimulated amino acid transport. This effect was dependent on PPARα, because activation of PPARα with an agonist (GW7647) inhibited insulin signaling and function, whereas PPARα-small interfering RNA reversed the effects of ADN on the insulin response. ADN increased ceramide synthase expression and stimulated ceramide production. C2-ceramide inhibited insulin signaling and function, whereas inhibition of ceramide synthase (with Fumonisin B1) reversed the effects of ADN on insulin signaling and amino acid transport. These findings are consistent with the model that maternal ADN limits fetal growth mediated by activation of placental PPARα and ceramide synthesis, which inhibits placental insulin signaling and amino acid transport, resulting in reduced fetal nutrient availability.

Transcriptional Profiling of Canker-Resistant Transgenic Sweet Orange (Citrus sinensis Osbeck) Constitutively Overexpressing a Spermidine Synthase Gene

PubMed Central

Fu, Xing-Zheng; Liu, Ji-Hong

2013-01-01

Citrus canker disease caused by Xanthomonas citri subsp. citri (Xcc) is one of the most devastating diseases affecting the citrus industry worldwide. In our previous study, the canker-resistant transgenic sweet orange (Citrus sinensis Osbeck) plants were produced via constitutively overexpressing a spermidine synthase. To unravel the molecular mechanisms underlying Xcc resistance of the transgenic plants, in the present study global transcriptional profiling was compared between untransformed line (WT) and the transgenic line (TG9) by hybridizing with Affymetrix Citrus GeneChip. In total, 666 differentially expressed genes (DEGs) were identified, 448 upregulated, and 218 downregulated. The DEGs were classified into 33 categories after Gene ontology (GO) annotation, in which 68 genes are in response to stimulus and involved in immune system process, 12 genes are related to cell wall, and 13 genes belong to transcription factors. These genes and those related to starch and sucrose metabolism, glutathione metabolism, biosynthesis of phenylpropanoids, and plant hormones were hypothesized to play major roles in the canker resistance of TG9. Semiquantitative RT-PCR analysis showed that the transcript levels of several candidate genes in TG9 were significantly higher than in WT both before and after Xcc inoculation, indicating their potential association with canker disease. PMID:23509803

The distribution of renal hyaluronan and the expression of hyaluronan synthases during water deprivation in the Spinifex hopping mouse, Notomys alexis.

PubMed

Bartolo, Ray C; Donald, John A

2007-12-01

Hyaluronan (HA) is a glycosaminoglycan that is synthesized by a family of enzymes called hyaluronan synthases (HASs), of which there are three isoforms (HAS1, 2 and 3) in mammals. The HASs have different tissue expression patterns and function, indicating that synthesis of HA and formation of the HA matrix may be regulated by various factors. The HA matrix has an important role in renal water handling and the production of a concentrated urine. We investigated the distribution of HA and the expression of HAS1, HAS2 and HAS3 mRNAs in the kidney of the Spinifex hopping mouse, Notomys alexis, a native Australian desert rodent that is reported to produce the most concentrated urine of any mammal. After periods of three, seven and fourteen days of water deprivation, the distribution of renal HA changed considerably, and there was a general down-regulation of HAS mRNA expression. It is proposed that the regulation of HA synthesis by the different HAS isoforms during water deprivation in N. alexis, could be influenced by the molecular mass of the HA chains produced by each isoform, followed by the rate at which the individual HAS produces HA.

Isoeugenin, a Novel Nitric Oxide Synthase Inhibitor Isolated from the Rhizomes of Imperata cylindrica.

PubMed

An, Hyo-Jin; Nugroho, Agung; Song, Byong-Min; Park, Hee-Juhn

2015-12-01

Phytochemical studies on the constituents of the rhizomes of Imperata cylindrica (Gramineae) were performed using high-performance liquid chromatography (HPLC). We also aimed to search for any biologically active substance capable of inhibiting nitric oxide (NO) formation in lipopolysaccharide (LPS)-activated macrophage 264.7 cells, by testing four compounds isolated from this plant. Four compounds, including a new chromone, isoeugenin, along with ferulic acid, p-coumaric acid, and caffeic acid were isolated and identified by NMR spectroscopy. The structure of isoeugenin was determined as 7-hydroxy-5-methoxy-2-methylchromone by the 2D-NMR technique. Among the four compounds, isoeugenin has the lowest IC50 value on the inhibition of NO production in LPS-activated macrophage RAW264.7 cells (IC50, 9.33 μg/mL). In addition, isoeugenin significantly suppressed the LPS-induced expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and proinflammatory cytokines mRNA levels. Taken together, these results suggest that the anti-inflammatory activity of isoeugenin is associated with the down-regulation of iNOS, COX-2, and pro-inflammatory cytokines in RAW264.7 cells. Accordingly, our results suggest that the new chromone isoegenin should be considered a potential treatment for inflammatory disease.

Baicalin Protects the Cardiomyocytes from ER Stress-Induced Apoptosis: Inhibition of CHOP through Induction of Endothelial Nitric Oxide Synthase

PubMed Central

Wang, Bo; Guo, Xiaowang; Zeng, Chao; Xu, Yong; Shen, Liangliang; Cheng, Ke; Xia, Yuesheng; Li, Xiumin; Wang, Haichang; Fan, Li; Wang, Xiaoming

2014-01-01

Baicalin, the main active ingredient of the Scutellaria root, exerts anti-oxidant and anti-apoptotic effects in cardiovascular diseases. However, the therapeutic mechanism of baicalin remains unknown. Cultured neonatal rat cardiomyocytes were pre-treated with baicalin (0–50 µM) for 24 h, and subsequently treated with tunicamycin (100 ng/ml). Cell viability was detected by MTT assay, and cell damage was determined by LDH release and TUNEL assay. The expression of CHOP, JNK, caspase-3, eNOS was analyzed by western blot. NO was measured by DAF-FM staining. As a result, treatment with baicalin significantly reduced apoptosis induced by ER stress inducer tunicamycin in cardiomyocytes. Molecularly, baicalin ameliorated tunicamycin-induced ER stress by downregulation of CHOP. In addition, baicalin inverted tunicamycin-induced decreases of eNOS mRNA and protein levels, phospho eNOS and NO production through CHOP pathway. However, the protective effects of baicalin were significantly decreased in cardiomyocytes treated with L-NAME, which suppressed activation of nitric oxide synthase. In conclusion, our results implicate that baicalin could protect cardiomyocytes from ER stress-induced apoptosis via CHOP/eNOS/NO pathway, and suggest the therapeutic values of baicalin against ER stress-associated cardiomyocyte apoptosis. PMID:24520378

Phytotoxic effects of Sicyos deppei (Cucurbitaceae) in germinating tomato seeds.

PubMed

Lara-Núñez, Aurora; Sánchez-Nieto, Sobeida; Luisa Anaya, Ana; Cruz-Ortega, Rocio

2009-06-01

The phytotoxic effect of allelochemicals is referred to as allelochemical stress and it is considered a biotic stress. Sicyos deppei G. Don (Cucurbitaceae) is an allelopathic weed that causes phytotoxicity in Lycopersicon esculentum, delaying seed germination and severely inhibiting radicle growth. This paper reports in in vitro conditions, the effects of the aqueous leachate of S. deppei-throughout tomato germination times-on (1) the dynamics of starch and sugars metabolism, (2) activity and expression of the cell wall enzymes involved in endosperm weakening that allows the protrusion of the radicle, and (3) whether abscisic acid (ABA) is involved in this altered metabolic processes. Results showed that S. deppei leachate on tomato seed germination mainly caused: (1) delay in starch degradation as well as in sucrose hydrolysis; (2) lower activity of sucrose phosphate synthase, cell wall invertase, and alpha-amylase; being sucrose phosphate synthase (SPS) gene expression down-regulated, and the last two up regulated; (3) also, lower activity of endo beta-mannanase, beta-1,3 glucanase, alpha-galactosidase, and exo-polygalacturonase with altered gene expression; and (4) higher content of ABA during all times of germination. The phytotoxic effect of S. deppei aqueous leachate is because of the sum of many metabolic processes affected during tomato seed germination that finally is evidenced by a strong inhibition of radicle growth.

The glycogen metabolism via Akt signaling is important for the secretion of enamel matrix in tooth development.

PubMed

Ida-Yonemochi, Hiroko; Otsu, Keishi; Ohshima, Hayato; Harada, Hidemitsu

2016-02-01

Cells alter their energy metabolism depending on the stage of differentiation or various environments. In the ameloblast differentiation of continuous growing mouse incisors, we found temporary glycogen storage in preameloblasts before the start of enamel matrix secretion and investigated the relationship between enamel matrix secretion and glycogen metabolism. Immunohistochemistry showed that in the transitional stage from preameloblasts to secretory ameloblasts, the glycogen synthase changed from the inactive form to the active form, the expression of glycogen phosphorylase increased, and further, the levels of IGF-1, IGF-1 receptor and activated Akt increased. These results suggested that the activation of Akt signaling via IGF is linked to the onset of both glycogen metabolism and enamel matrix deposition. In the experiments using organ culture and ameloblast cell line, the activation of Akt signaling by IGF-1 stimulated glycogen metabolism through the up-regulation of Glut-1,-4 and Gsk-3β and the dephosphorylation of glycogen synthase. Subsequently, they resulted in increased enamel matrix secretion. In contrast, some inhibitors of Akt signals and glycogen synthesis/degradation down-regulated enamel matrix secretion. Taking these findings together, glycogen metabolism via Akt signaling is an essential system for the secretion of enamel matrix in ameloblast differentiation. Copyright © 2016 Elsevier B.V. All rights reserved.

Application of cranberry concentrate (Vaccinium macrocarpon) to control Escherichia coli O157:H7 in ground beef and its antimicrobial mechanism related to the downregulated slp, hdeA and cfa.

PubMed

Wu, Vivian C H; Qiu, Xujian; de los Reyes, Benildo G; Lin, Chih-Sheng; Pan, Yingjie

2009-02-01

The possible use of cranberry concentrate (CC) as a natural food preservative was studied by examining its antimicrobial effect on the growth of Escherichia coli O157:H7 inoculated in ground beef, its organoleptical effect on beef patties, and its antimicrobial mechanism on the gene regulation level. Inoculated ground beef was added with CC and stored at 4 degrees C for 5 days. Bacteria were detected on day 0, 1, 3, and 5. Cranberry concentrate (2.5%, 5%, and 7.5% w/w) reduced total aerobic bacteria 1.5 log, 2.1 log, and 2.7 log CFU/g and E. coli O157:H7 0.4 log, 0.7 log, and 2.4 log CFU/g, respectively, when compared to the control on day 5. Fifty panelists evaluated the burgers supplemented with CC. No differences in appearance, flavor, and taste were found among burgers with 0%, 2.5%, and 5% CC. The expression of E. coli O157:H7 cyclopropane fatty acyl phospholipid synthase (cfa), hypothetical protein (hdeA), outer membrane porin protein C (ompC), hyperosmotically inducible periplasmic protein (osmY), and outer membrane protein induced after carbon starvation (slp) genes with or without CC (2.5% v/v) treatment was investigated by quantitative real-time PCR. Compared to the control, slp, hdeA, and cfa were markedly downregulated, ompC was slightly downregulated, while osmY was slightly affected.

Aronia melanocarpa Extract Ameliorates Hepatic Lipid Metabolism through PPARγ2 Downregulation

PubMed Central

Kim, Jung-Hee; Lee, Eun Byul; Hur, Wonhee; Kwon, Oh-Joo; Park, Hyoung-Jin; Yoon, Seung Kew

2017-01-01

Nonalcoholic fatty liver disease (NAFLD) is a hepatic manifestation of metabolic syndrome. Studies have demonstrated that anthocyanin-rich foods may improve hyperlipidemia and ameliorate hepatic steatosis. Here, effects of Aronia melanocarpa (AM), known to be rich of anthocyanins, on hepatic lipid metabolism and adipogenic genes were determined. AM was treated to C57BL/6N mice fed with high fat diet (HFD) or to FL83B cells treated with free fatty acid (FFA). Changes in levels of lipids, enzymes and hormones were observed, and expressions of adipogenic genes involved in hepatic lipid metabolism were detected by PCR, Western blotting and luciferase assay. In mice, AM significantly reduced the body and liver weight, lipid accumulation in the liver, and levels of biochemical markers such as fatty acid synthase, hepatic triglyceride and leptin. Serum transaminases, indicators for hepatocyte injury, were also suppressed, while superoxide dismutase activity and liver antioxidant capacity were significantly increased. In FL83B cells, AM significantly reduced FFA-induced lipid droplet accumulation. Protein synthesis of an adipogenic transcription factor, peroxisome proliferator-activated receptor γ2 (PPARγ2) was inhibited in vivo. Furthermore, transcriptional activity of PPARγ2 was down-regulated in vitro, and mRNA expression of PPARγ2 and its downstream target genes, adipocyte protein 2 and lipoprotein lipase were down-regulated by AM both in vitro and in vivo. These results show beneficial effects of AM against hepatic lipid accumulation through the inhibition of PPARγ2 expression along with improvements in body weight, liver functions, lipid profiles and antioxidant capacity suggesting the potential therapeutic efficacy of AM on NAFLD. PMID:28081181

Aronia melanocarpa Extract Ameliorates Hepatic Lipid Metabolism through PPARγ2 Downregulation.

PubMed

Park, Chung-Hwa; Kim, Jung-Hee; Lee, Eun Byul; Hur, Wonhee; Kwon, Oh-Joo; Park, Hyoung-Jin; Yoon, Seung Kew

2017-01-01

Nonalcoholic fatty liver disease (NAFLD) is a hepatic manifestation of metabolic syndrome. Studies have demonstrated that anthocyanin-rich foods may improve hyperlipidemia and ameliorate hepatic steatosis. Here, effects of Aronia melanocarpa (AM), known to be rich of anthocyanins, on hepatic lipid metabolism and adipogenic genes were determined. AM was treated to C57BL/6N mice fed with high fat diet (HFD) or to FL83B cells treated with free fatty acid (FFA). Changes in levels of lipids, enzymes and hormones were observed, and expressions of adipogenic genes involved in hepatic lipid metabolism were detected by PCR, Western blotting and luciferase assay. In mice, AM significantly reduced the body and liver weight, lipid accumulation in the liver, and levels of biochemical markers such as fatty acid synthase, hepatic triglyceride and leptin. Serum transaminases, indicators for hepatocyte injury, were also suppressed, while superoxide dismutase activity and liver antioxidant capacity were significantly increased. In FL83B cells, AM significantly reduced FFA-induced lipid droplet accumulation. Protein synthesis of an adipogenic transcription factor, peroxisome proliferator-activated receptor γ2 (PPARγ2) was inhibited in vivo. Furthermore, transcriptional activity of PPARγ2 was down-regulated in vitro, and mRNA expression of PPARγ2 and its downstream target genes, adipocyte protein 2 and lipoprotein lipase were down-regulated by AM both in vitro and in vivo. These results show beneficial effects of AM against hepatic lipid accumulation through the inhibition of PPARγ2 expression along with improvements in body weight, liver functions, lipid profiles and antioxidant capacity suggesting the potential therapeutic efficacy of AM on NAFLD.

Small interfering RNA-mediated down-regulation of caveolin-1 differentially modulates signaling pathways in endothelial cells.

PubMed

Gonzalez, Eva; Nagiel, Aaron; Lin, Alison J; Golan, David E; Michel, Thomas

2004-09-24

Caveolin-1 is a scaffolding/regulatory protein that interacts with diverse signaling molecules in endothelial cells. To explore the role of this protein in receptor-modulated signaling pathways, we transfected bovine aortic endothelial cells (BAEC) with small interfering RNA (siRNA) duplexes to down-regulate caveolin-1 expression. Transfection of BAEC with duplex siRNA targeted against caveolin-1 mRNA selectively "knocked-down" the expression of caveolin-1 by approximately 90%, as demonstrated by immunoblot analyses of BAEC lysates. We used discontinuous sucrose gradients to purify caveolin-containing lipid rafts from siRNA-treated endothelial cells. Despite the near-total down-regulation of caveolin-1 expression, the lipid raft targeting of diverse signaling proteins (including the endothelial isoform of nitric-oxide synthase, Src-family tyrosine kinases, Galphaq and the insulin receptor) was unchanged. We explored the consequences of caveolin-1 knockdown on kinase pathways modulated by the agonists sphingosine-1 phosphate (S1P) and vascular endothelial growth factor (VEGF). siRNA-mediated caveolin-1 knockdown enhanced basal as well as S1P- and VEGF-induced phosphorylation of the protein kinase Akt and did not modify the basal or agonist-induced phosphorylation of extracellular signal-regulated kinases 1/2. Caveolin-1 knock-down also significantly enhanced the basal and agonist-induced activity of the small GTPase Rac. We used siRNA to down-regulate Rac expression in BAEC, and we observed that Rac knockdown significantly reduced basal, S1P-, and VEGF-induced Akt phosphorylation, suggesting a role for Rac activation in the caveolin siRNA-mediated increase in Akt phosphorylation. By using siRNA to knockdown caveolin-1 and Rac expression in cultured endothelial cells, we have found that caveolin-1 does not seem to be required for the targeting of signaling molecules to caveolae/lipid rafts and that caveolin-1 differentially modulates specific kinase pathways in endothelial cells. Copyright 2004 American Society for Biochemistry and Molecular Biology, Inc.

Pre-clinical assessment of A-674563 as an anti-melanoma agent

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zou, Ying; Fan, Guobiao; Wang, Xuemin, E-mail: wangxuemeidr@yeah.net

The present study aims to investigate the anti-melanoma activity by an Akt1 specific inhibitor A-674563. We showed that A-674563 was anti-proliferative and cytotoxic when added to human melanoma cells (A375, WM-115 and SK-Mel-2 lines). A-674563 induced caspase-dependent apoptotic death of human melanoma cells, and its cytotoxicity was inhibited with pre-treatment of caspase inhibitors. Further, A-674563 treatment blocked Akt and its downstream S6 Kinase 1 (S6K1) activation in A375 melanoma cells. Significantly, restoring Akt-S6K1 activation via introduction of constitutively-active Akt1 (ca-Akt1) only partially attenuated A-674563's cytotoxicity against A375 cells. Further, A-674563 induced pro-apoptotic ceramide production in A375 cells. Significantly, sphingosine-1-phosphate (S1P) inhibited A-674563-inducedmore » ceramide production and subsequent A375 cell apoptosis. On the other hand, co-treatment with the glucosylceramide synthase (GCS) inhibitor PDMP or the cell permeable short-chain ceramide (C6) potentiated A-674563's cytotoxicity against A375 cells. In vivo, A-674563 oral gavage inhibited A375 xenograft growth in severe combined immunodeficiency (scid) mice. Akt inactivation, caspase-3 activation and ceramide production were also observed in A-674563-treated A375 xenografts. Together, these results suggest that A-674563 exerts potent anti-melanoma activity, involving Akt-dependent and Akt-independent mechanisms. - Highlights: • A-674563 inhibits human melanoma cell survival and proliferation. • A-674563 induces melanoma cell apoptotic death, inhibited by caspase inhibitors. • A-674563 inhibits melanoma cells via Akt-dependent and -independent mechanisms. • A-674563 induces ceramide production in melanoma cells, independent of Akt inhibition. • A-674563 oral administration potently inhibits A375 xenograft growth in mice.« less

Epidermal Hydration Is Improved by Enhanced Ceramide Metabolism in Aged C57BL/6J Mice After Dietary Supplementation of Royal Jelly.

PubMed

Jeon, Sanghun; Cho, Yunhi

2015-09-01

Epidermal hydration is maintained by the epidermal lipid barrier, of which ceramide (Cer) is the major constituent. We examined the dietary effect of royal jelly (RJ) on epidermal hydration in aged mice. Altered Cer metabolism was further determined by measuring epidermal levels of individual Cer, glucosylceramide (GC), and sphingomyelin (SM) species, and of Cer-metabolizing enzymes. Aged C57BL/6J mice were fed a control diet (group AGED) or diets with 1% RJ harvested from two different areas (groups AGED+RJ1:AGED + RJ2) for 16 weeks. Aged C57BL/6J mice with no dietary intervention (the control group: group C) represented the onset of aging. In group AGED, epidermal levels of hydration, Cer1/2/5/6/7, GC-A/B/C/D, SM1/2/3, and β-glucocerebrosidase (GCase) protein, an enzyme of GC hydrolysis for Cer generation, were lower than in group C; these levels, as well as those of Cer3/4 and acidic sphingomyelinase (aSMase) protein, an enzyme of SM hydrolysis for Cer generation, were higher in group AGED + RJ1 than in group AGED. Despite increases in GC-B, SM1/2/3, and serine palmitoyltransferase2 protein, an enzyme of de novo Cer synthesis, in group AGED + RJ2 to levels higher than in group AGED, epidermal levels of hydration, Cer1-7, GC-A/C/D, GCase, and aSMase proteins were similar in these two groups. Expression of GCase and aSMase mRNAs, and of Cer synthase3 and ceramidase proteins, enzymes of de novo Cer synthesis and degradation, did not differ among groups. Dietary RJ1 improved epidermal hydration by enhancing Cer metabolism with increased levels of all Cer, GC, and SM species, and of GCase and aSMase proteins.

Pathological levels of glucosylceramide change the biophysical properties of artificial and cell membranes.

PubMed

Varela, Ana R P; Ventura, Ana E; Carreira, Ana C; Fedorov, Aleksander; Futerman, Anthony H; Prieto, Manuel; Silva, Liana C

2016-12-21

Glucosylceramide (GlcCer) plays an active role in the regulation of various cellular events. Moreover, GlcCer is also a key modulator of membrane biophysical properties, which might be linked to the mechanism of its biological action. In order to understand the biophysical implications of GlcCer on membranes of living cells, we first studied the effect of GlcCer on artificial membranes containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), sphingomyelin (SM) and cholesterol (Chol). Using an array of biophysical methods, we demonstrate that at lower GlcCer/Chol ratios, GlcCer stabilizes SM/Chol-enriched liquid-ordered domains. However, upon decreasing the Chol content, GlcCer significantly increased membrane order through the formation of gel domains. Changes in pH disturbed the packing properties of GlcCer-containing membranes, leading to an increase in membrane fluidity and reduced membrane electronegativity. To address the biophysical impact of GlcCer in biological membranes, studies were performed in wild type and in fibroblasts treated with conduritol-B-epoxide (CBE), which causes intracellular GlcCer accumulation, and in fibroblasts from patients with type I Gaucher disease (GD). Decreased membrane fluidity was observed in cells containing higher levels of GlcCer, such as in CBE-treated and GD cells. Together, we demonstrate that elevated GlcCer levels change the biophysical properties of cellular membranes, which might compromise membrane-associated cellular events and be of relevance for understanding the pathology of diseases, such as GD, in which GlcCer accumulates at high levels.

Gromwell (Lithospermum erythrorhizon) supplementation enhances epidermal levels of ceramides, glucosylceramides, β-glucocerebrosidase, and acidic sphingomyelinase in NC/Nga mice.

PubMed

Kim, Jungmin; Cho, Yunhi

2013-10-01

We have previously reported that dietary gromwell (Lithospermum erythrorhizon; LE) prevents the development of atopic dermatitis (AD) with increased epidermal levels of total ceramide (Cer), the major lipid maintaining epidermal barrier. In this study, we investigated whether the increased level of total Cer induced by dietary LE would be related to the altered metabolism of glucosylceramide (GlcCer) and sphingomyelin (SM), two major precursor lipids in Cer generation. NC/Nga mice, an animal model of AD, were fed a control diet (group CA: atopic control) or a diet with 70% ethanol LE extracts (1% in diet; group LE) for 10 weeks. Individual species of Cer, GlcCer, and SM were analyzed by high-performance thin layer chromatography. In the epidermis of group CA, total Cer (including Cer2 and Cer5-7) and total GlcCer (including GlcCer-B/C/D) were significantly reduced; these levels in group LE were increased to levels similar to the normal control group of BALB/c mice (group C). In addition, protein expressions and activities of β-glucocerebrosidase (β-GlcCer'ase) and acidic sphingomyelinase (aSMase), enzymes for GlcCer or SM hydrolysis, respectively, were increased in group LE. However, alterations of Cer1, Cer3/4, GlcCer-A, and all SM species (including SM1-3) were not significant among groups C, CA, and LE. Dietary gromwell increases GlcCer-B/C/D, and further enhances the generation of Cer2 and Cer5-7 with high protein expressions and activities of β-GlcCer'ase and aSMase.

Fusion or Fission: The Destiny of Mitochondria In Traumatic Brain Injury of Different Severities.

PubMed

Di Pietro, Valentina; Lazzarino, Giacomo; Amorini, Angela Maria; Signoretti, Stefano; Hill, Lisa J; Porto, Edoardo; Tavazzi, Barbara; Lazzarino, Giuseppe; Belli, Antonio

2017-08-23

Mitochondrial dynamics are regulated by a complex system of proteins representing the mitochondrial quality control (MQC). MQC balances antagonistic forces of fusion and fission determining mitochondrial and cell fates. In several neurological disorders, dysfunctional mitochondria show significant changes in gene and protein expression of the MQC and contribute to the pathophysiological mechanisms of cell damage. In this study, we evaluated the main gene and protein expression involved in the MQC in rats receiving traumatic brain injury (TBI) of different severities. At 6, 24, 48 and 120 hours after mild TBI (mTBI) or severe TBI (sTBI), gene and protein expressions of fusion and fission were measured in brain tissue homogenates. Compared to intact brain controls, results showed that genes and proteins inducing fusion or fission were upregulated and downregulated, respectively, in mTBI, but downregulated and upregulated, respectively, in sTBI. In particular, OPA1, regulating inner membrane dynamics, cristae remodelling, oxidative phosphorylation, was post-translationally cleaved generating differential amounts of long and short OPA1 in mTBI and sTBI. Corroborated by data referring to citrate synthase, these results confirm the transitory (mTBI) or permanent (sTBI) mitochondrial dysfunction, enhancing MQC importance to maintain cell functions and indicating in OPA1 an attractive potential therapeutic target for TBI.

Thymoquinone Defeats Diabetes-Induced Testicular Damage in Rats Targeting Antioxidant, Inflammatory and Aromatase Expression

PubMed Central

Atta, Mustafa S.; Almadaly, Essam A.; El-Far, Ali H.; Saleh, Rasha M.; Assar, Doaa H.; Al Jaouni, Soad K.; Mousa, Shaker A.

2017-01-01

Antioxidants have valuable effects on the process of spermatogenesis, particularly with diabetes mellitus (DM). Therefore, the present study investigated the impact and the intracellular mechanisms by which thymoquinone (TQ) works against diabetes-induced testicular deteriorations in rats. Wistar male rats (n = 60) were randomly allocated into four groups; Control, Diabetic (streptozotocin (STZ)-treated rats where diabetes was induced by intraperitoneal injection of STZ, 65 mg/kg), Diabetic + TQ (diabetic rats treated with TQ (50 mg/kg) orally once daily), and TQ (non-diabetic rats treated with TQ) for 12 weeks. Results revealed that TQ significantly improved the sperm parameters with a reduction in nitric oxide (NO) and malondialdehyde (MDA) levels in testicular tissue. Also, it increased testicular reduced glutathione (GSH) levels and superoxide dismutase (SOD) activity. Interestingly, TQ induced downregulation of testicular inducible nitric oxide synthase (iNOS) and nuclear factor kappa-B (NF-κB) and significantly upregulated the aromatase protein expression levels in testicles in comparison with the diabetic rats. In conclusion, TQ treatment exerted a protective effect against reproductive dysfunction induced by diabetes not only through its powerful antioxidant and hypoglycemic effects but also through its downregulation of testicular iNOS and NF-κB along with upregulation of aromatase expression levels in diabetic rats. PMID:28448463

Rice ethylene-response AP2/ERF factor OsEATB restricts internode elongation by down-regulating a gibberellin biosynthetic gene.

PubMed

Qi, Weiwei; Sun, Fan; Wang, Qianjie; Chen, Mingluan; Huang, Yunqing; Feng, Yu-Qi; Luo, Xiaojin; Yang, Jinshui

2011-09-01

Plant height is a decisive factor in plant architecture. Rice (Oryza sativa) plants have the potential for rapid internodal elongation, which determines plant height. A large body of physiological research has shown that ethylene and gibberellin are involved in this process. The APETALA2 (AP2)/Ethylene-Responsive Element Binding Factor (ERF) family of transcriptional factors is only present in the plant kingdom. This family has various developmental and physiological functions. A rice AP2/ERF gene, OsEATB (for ERF protein associated with tillering and panicle branching) was cloned from indica rice variety 9311. Bioinformatic analysis suggested that this ERF has a potential new function. Ectopic expression of OsEATB showed that the cross talk between ethylene and gibberellin, which is mediated by OsEATB, might underlie differences in rice internode elongation. Analyses of gene expression demonstrated that OsEATB restricts ethylene-induced enhancement of gibberellin responsiveness during the internode elongation process by down-regulating the gibberellin biosynthetic gene, ent-kaurene synthase A. Plant height is negatively correlated with tiller number, and higher yields are typically obtained from dwarf crops. OsEATB reduces rice plant height and panicle length at maturity, promoting the branching potential of both tillers and spikelets. These are useful traits for breeding high-yielding crops.

Anthocyanins Downregulate Lipopolysaccharide-Induced Inflammatory Responses in BV2 Microglial Cells by Suppressing the NF-κB and Akt/MAPKs Signaling Pathways

PubMed Central

Jeong, Jin-Woo; Lee, Won Sup; Shin, Sung Chul; Kim, Gi-Young; Choi, Byung Tae; Choi, Yung Hyun

2013-01-01

Anthocyanins are naturally occurring polyphenols that impart bright color to fruits, vegetables and plants and have a variety of protective properties, which have generally been attributed to their antioxidant capacity. However, little is known about the molecular mechanisms underlying anti-inflammatory effects of anthocyanins related to neurodegenerative diseases. Therefore, we determined whether anthocyanins isolated from black soybean seed coats would inhibit pro-inflammatory mediators and cytokines in lipopolysaccharide (LPS)-stimulated murine BV2 microglial cells. Our results showed that anthocyanins significantly inhibited LPS-induced pro-inflammatory mediators, such as nitric oxide (NO) and prostaglandin E2, and pro-inflammatory cytokines including tumor necrosis factor (TNF)-α and interleukin (IL)-1β, without significant cytotoxicity. Anthocyanins also downregulated excessive expression of inducible NO synthase, cyclooxygenase-2, TNF-α, and IL-1β in LPS-stimulated BV2 cells. Moreover, anthocyanins inhibited nuclear translocation of nuclear factor-kappa B (NF-κB) by reducing inhibitor of NF-κB alpha degradation as well as phosphorylating extracellular signal-regulated kinase, c-Jun N-terminal kinase, p38 mitogen-activated protein kinase, and Akt. These findings suggest that anthocyanins may offer substantial therapeutic potential for treating inflammatory and neurodegenerative diseases accompanied by microglial activation. PMID:23344054

Mechanism of arctigenin-mediated specific cytotoxicity against human lung adenocarcinoma cell lines.

PubMed

Susanti, Siti; Iwasaki, Hironori; Inafuku, Masashi; Taira, Naoyuki; Oku, Hirosuke

2013-12-15

The lignan arctigenin (ARG) from the herb Arctium lappa L. possesses anti-cancer activity, however the mechanism of action of ARG has been found to vary among tissues and types of cancer cells. The current study aims to gain insight into the ARG mediated mechanism of action involved in inhibiting proliferation and inducing apoptosis in lung adenocarcinoma cells. This study also delineates the cancer cell specificity of ARG by comparison with its effects on various normal cell lines. ARG selectively arrested the proliferation of cancer cells at the G0/G1 phase through the down-regulation of NPAT protein expression. This down-regulation occurred via the suppression of either cyclin E/CDK2 or cyclin H/CDK7, while apoptosis was induced through the modulation of the Akt-1-related signaling pathway. Furthermore, a GSH synthase inhibitor specifically enhanced the cytotoxicity of ARG against cancer cells, suggesting that the intracellular GSH content was another factor influencing the susceptibility of cancer cells to ARG. These findings suggest that specific cytotoxicity of ARG against lung cancer cells was explained by its selective modulation of the expression of NPAT, which is involved in histone biosynthesis. The cytotoxicity of ARG appeared to be dependent on the intracellular GSH level. Copyright © 2013 Elsevier GmbH. All rights reserved.

Sulfur amino acid metabolism in doxorubicin-resistant breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ryu, Chang Seon; Kwak, Hui Chan; Lee, Kye Sook

2011-08-15

Although methionine dependency is a phenotypic characteristic of tumor cells, it remains to be determined whether changes in sulfur amino acid metabolism occur in cancer cells resistant to chemotherapeutic medications. We compared expression/activity of sulfur amino acid metabolizing enzymes and cellular levels of sulfur amino acids and their metabolites between normal MCF-7 cells and doxorubicin-resistant MCF-7 (MCF-7/Adr) cells. The S-adenosylmethionine/S-adenosylhomocysteine ratio, an index of transmethylation potential, in MCF-7/Adr cells decreased to {approx} 10% relative to that in MCF-7 cells, which may have resulted from down-regulation of S-adenosylhomocysteine hydrolase. Expression of homocysteine-clearing enzymes, such as cystathionine beta-synthase, methionine synthase/methylene tetrahydrofolate reductase,more » and betaine homocysteine methyltransferase, was up-regulated in MCF-7/Adr cells, suggesting that acquiring doxorubicin resistance attenuated methionine-dependence and activated transsulfuration from methionine to cysteine. Homocysteine was similar, which is associated with a balance between the increased expressions of homocysteine-clearing enzymes and decreased extracellular homocysteine. Despite an elevation in cysteine, cellular GSH decreased in MCF-7/Adr cells, which was attributed to over-efflux of GSH into the medium and down-regulation of the GSH synthesis enzyme. Consequently, MCF-7/Adr cells were more sensitive to the oxidative stress induced by bleomycin and menadione than MCF-7 cells. In conclusion, our results suggest that regulating sulfur amino acid metabolism may be a possible therapeutic target for chemoresistant cancer cells. These results warrant further investigations to determine the role of sulfur amino acid metabolism in acquiring anticancer drug resistance in cancer cells using chemical and biological regulators involved in sulfur amino acid metabolism. - Research Highlights: > MCF-7/Adr cells showed decreases in cellular GSH, which were attributed to increase efflux of GSH. > MCF-7/Adr was more sensitive to oxidative stress induced by bleomycin and menadione. > Hcy-clearing enzymes involved in were up-regulated in MCF-7/Adr cells. > Doxorubicin-resistance attenuated Met-dependence and activated transsulfuration. > Regulating sulfur amino acid metabolism may be a possible therapeutic target.« less

Transcriptional analysis of late ripening stages of grapevine berry

PubMed Central

2011-01-01

Background The composition of grapevine berry at harvest is a major determinant of wine quality. Optimal oenological maturity of berries is characterized by a high sugar/acidity ratio, high anthocyanin content in the skin, and low astringency. However, harvest time is still mostly determined empirically, based on crude biochemical composition and berry tasting. In this context, it is interesting to identify genes that are expressed/repressed specifically at the late stages of ripening and which may be used as indicators of maturity. Results Whole bunches and berries sorted by density were collected in vineyard on Chardonnay (white cultivar) grapevines for two consecutive years at three stages of ripening (7-days before harvest (TH-7), harvest (TH), and 10-days after harvest (TH+10)). Microvinification and sensory analysis indicate that the quality of the wines made from the whole bunches collected at TH-7, TH and TH+10 differed, TH providing the highest quality wines. In parallel, gene expression was studied with Qiagen/Operon microarrays using two types of samples, i.e. whole bunches and berries sorted by density. Only 12 genes were consistently up- or down-regulated in whole bunches and density sorted berries for the two years studied in Chardonnay. 52 genes were differentially expressed between the TH-7 and TH samples. In order to determine whether these genes followed a similar pattern of expression during the late stages of berry ripening in a red cultivar, nine genes were selected for RT-PCR analysis with Cabernet Sauvignon grown under two different temperature regimes affecting the precocity of ripening. The expression profiles and their relationship to ripening were confirmed in Cabernet Sauvignon for seven genes, encoding a carotenoid cleavage dioxygenase, a galactinol synthase, a late embryogenesis abundant protein, a dirigent-like protein, a histidine kinase receptor, a valencene synthase and a putative S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase. Conclusions This set of up- and down-regulated genes characterize the late stages of berry ripening in the two cultivars studied, and are indirectly linked to wine quality. They might be used directly or indirectly to design immunological, biochemical or molecular tools aimed at the determination of optimal ripening in these cultivars. PMID:22098939

Transient Receptor Potential Melastatin 7 Cation Channel Kinase: New Player in Angiotensin II-Induced Hypertension.

PubMed

Antunes, Tayze T; Callera, Glaucia E; He, Ying; Yogi, Alvaro; Ryazanov, Alexey G; Ryazanova, Lillia V; Zhai, Alexander; Stewart, Duncan J; Shrier, Alvin; Touyz, Rhian M

2016-04-01

Transient receptor potential melastatin 7 (TRPM7) is a bifunctional protein comprising a magnesium (Mg(2+))/cation channel and a kinase domain. We previously demonstrated that vasoactive agents regulate vascular TRPM7. Whether TRPM7 plays a role in the pathophysiology of hypertension and associated cardiovascular dysfunction is unknown. We studied TRPM7 kinase-deficient mice (TRPM7Δkinase; heterozygous for TRPM7 kinase) and wild-type (WT) mice infused with angiotensin II (Ang II; 400 ng/kg per minute, 4 weeks). TRPM7 kinase expression was lower in heart and aorta from TRPM7Δkinase versus WT mice, effects that were further reduced by Ang II infusion. Plasma Mg(2+) was lower in TRPM7Δkinase versus WT mice in basal and stimulated conditions. Ang II increased blood pressure in both strains with exaggerated responses in TRPM7Δkinase versus WT groups (P<0.05). Acetylcholine-induced vasorelaxation was reduced in Ang II-infused TRPM7Δkinase mice, an effect associated with Akt and endothelial nitric oxide synthase downregulation. Vascular cell adhesion molecule-1 expression was increased in Ang II-infused TRPM7 kinase-deficient mice. TRPM7 kinase targets, calpain, and annexin-1, were activated by Ang II in WT but not in TRPM7Δkinase mice. Echocardiographic and histopathologic analysis demonstrated cardiac hypertrophy and left ventricular dysfunction in Ang II-treated groups. In TRPM7 kinase-deficient mice, Ang II-induced cardiac functional and structural effects were amplified compared with WT counterparts. Our data demonstrate that in TRPM7Δkinase mice, Ang II-induced hypertension is exaggerated, cardiac remodeling and left ventricular dysfunction are amplified, and endothelial function is impaired. These processes are associated with hypomagnesemia, blunted TRPM7 kinase expression/signaling, endothelial nitric oxide synthase downregulation, and proinflammatory vascular responses. Our findings identify TRPM7 kinase as a novel player in Ang II-induced hypertension and associated vascular and target organ damage. © 2016 American Heart Association, Inc.

Antidiabetic activity of Ganoderma lucidum polysaccharides F31 down-regulated hepatic glucose regulatory enzymes in diabetic mice.

PubMed

Xiao, Chun; Wu, Qingping; Zhang, Jumei; Xie, Yizhen; Cai, Wen; Tan, Jianbin

2017-01-20

Ganoderma lucidum (Lin Zhi) has been used to treat diabetes in Chinese folk for centuries. Our laboratory previously demonstrated that Ganoderma lucidum polysaccharides (GLPs) had hypoglycemic effects in diabetic mice. Our aim was to identify the main bioactives in GLPs and corresponding mechanism of action. Four polysaccharide-enriched fraction were isolated from GLPs and the antidiabetic activities were evaluated by type 2 diabetic mice. Fasting serum glucose (FSG), fasting serum insulin (FSI) and epididymal fat/BW ratio were measured at the end of the experiment. In liver, the mRNA levels of hepatic glucose regulatory enzymes were determined by quantitative polymerase chain reaction (qPCR) and the protein levels of phospho-AMP-activated protein kinase (p-AMPK)/AMPK were determined by western blotting test. In epididymal fat tissue, the mRNA and protein levels GLUT4, resistin, fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC1) were determined by qPCR and immuno-histochemistry. The structure of polysaccharide F31 was obtained from GPC, FTIR NMR and GC-MS spectroscopy, RESULTS: F31 significantly decreased FSG (P<0.05), FSI and epididymal fat/BW ratio (P<0.01). In liver, F31 decreased the mRNA levels of hepatic glucose regulatory enzymes, and up-regulated the ratio of phospho-AMP-activated protein kinase (p-AMPK)/AMPK. In epididymal fat tissue, F31 increased the mRNA levels of GLUT4 but decreased fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC1) and resistin. Immuno-histochemistry results revealed F31 increased the protein levels of GLUT4 and decreased resistin. Data suggested that the main bioactives in GLPs was F31, which was determined to be a β-heteropolysaccharide with the weight-average molecular weight of 15.9kDa. The possible action mechanism of F31 may be associated with down-regulation of the hepatic glucose regulated enzyme mRNA levels via AMPK activation, improvement of insulin resistance and decrease of epididymal fat/BW ratio. These results strongly suggest that F31 has antidiabetic potential. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Statin-induced inhibition of breast cancer proliferation and invasion involves attenuation of iron transport: intermediacy of nitric oxide and antioxidant defence mechanisms.

PubMed

Kanugula, Anantha Koteswararao; Gollavilli, Paradesi Naidu; Vasamsetti, Sathish Babu; Karnewar, Santosh; Gopoju, Raja; Ummanni, Ramesh; Kotamraju, Srigiridhar

2014-08-01

Accumulating evidence from in vitro, in vivo, clinical and epidemiological studies shows promising results for the use of statins against many cancers including breast carcinoma. However, the molecular mechanisms responsible for the anti-proliferative and anti-invasive properties of statins still remain elusive. In this study, we investigated the involvement of nitric oxide, iron homeostasis and antioxidant defence mechanisms in mediating the anti-proliferative and anti-invasive properties of hydrophobic statins in MDA-MB-231, MDA-MB-453 and BT-549 metastatic triple negative breast cancer cells. Fluvastatin and simvastatin significantly increased cytotoxicity which was reversed with mevalonate. Interestingly, fluvastatin downregulated transferrin receptor (TfR1), with a concomitant depletion of intracellular iron levels in these cells. Statin-induced effects were mimicked by geranylgeranyl transferase inhibitor (GGTI-298) but not farnesyl transferase inhibitor (FTI-277). Further, it was observed that TfR1 downregulation is mediated by increased nitric oxide levels via inducible nitric oxide synthase (iNOS) expression. NOS inhibitors (asymmetric dimethylarginine and 1400W) counteracted and sepiapterin, a precursor of tetrahydrobiopterin, exacerbated statin-induced depletion of intracellular iron levels. Notably, fluvastatin increased manganese superoxide dismutase (by repressing the transcription factor DNA damage-binding protein 2), catalase and glutathione which, in turn, diminished H2 O2 levels. Fluvastatin-induced downregulation of TfR1, matrix metalloproteinase-2, -9 and inhibition of invasion were reversed in the presence of aminotriazole, a specific inhibitor of catalase. Finally, we conclude that fluvastatin, by altering iron homeostasis, nitric oxide generation and antioxidant defence mechanisms, induces triple negative breast cancer cell death. © 2014 FEBS.

WAFs lead molting retardation of naupliar stages with down-regulated expression profiles of chitin metabolic pathway and related genes in the copepod Tigriopus japonicus.

PubMed

Hwang, Dae-Sik; Lee, Min-Chul; Kyung, Do-Hyun; Kim, Hui-Su; Han, Jeonghoon; Kim, Il-Chan; Puthumana, Jayesh; Lee, Jae-Seong

2017-03-01

Oil pollution is considered being disastrous to marine organisms and ecosystems. As molting is critical in the developmental process of arthropods in general and copepods, in particular, the impact will be adverse if the target of spilled oil is on molting. Thus, we investigated the harmful effects of water accommodated fractions (WAFs) of crude oil with an emphasis on inhibition of chitin metabolic pathways related genes and developmental retardation in the copepod Tigriopus japonicus. Also, we analysed the ontology and domain of chitin metabolic pathway genes and mRNA expression patterns of developmental stage-specific genes. Further, the developmental retardation followed by transcriptional modulations in nuclear receptor genes (NR) and chitin metabolic pathway-related genes were observed in the WAFs-exposed T. japonicus. As a result, the developmental time was found significantly (P<0.05) delayed in response to 40% WAFs in comparison with that of control. Moreover, the NR gene, HR3 and chitinases (CHT9 and CHT10) were up-regulated in N4-5 stages, while chitin synthase genes (CHS-1, CHS-2-1, and CHS-2-2) down-regulated in response to WAFs. In brief, a high concentration of WAFs repressed nuclear receptor genes but elicited activation of some of the transcription factors at low concentration of WAFs, resulting in suppression of chitin synthesis. Thus, we suggest that WAF can lead molting retardation of naupliar stages in T. japonicus through down-regulations of chitin metabolism. These findings will provide a better understanding of the mode of action of chitin biosynthesis associated with molting mechanism in WAF-exposed T. japonicus. Copyright © 2016 Elsevier Inc. All rights reserved.

A transcriptomic approach to search for novel phenotypic regulators in McArdle disease.

PubMed

Nogales-Gadea, Gisela; Consuegra-García, Inés; Rubio, Juan C; Arenas, Joaquin; Cuadros, Marc; Camara, Yolanda; Torres-Torronteras, Javier; Fiuza-Luces, Carmen; Lucia, Alejandro; Martín, Miguel A; García-Arumí, Elena; Andreu, Antoni L

2012-01-01

McArdle disease is caused by lack of glycogen phosphorylase (GP) activity in skeletal muscle. Patients experience exercise intolerance, presenting as early fatigue and contractures. In this study, we investigated the effects produced by a lack of GP on several genes and proteins of skeletal muscle in McArdle patients. Muscle tissue of 35 patients and 7 healthy controls were used to identify abnormalities in the patients' transcriptomic profile using low-density arrays. Gene expression was analyzed for the influence of variables such as sex and clinical severity. Differences in protein expression were studied by immunoblotting and 2D electrophoresis analysis, and protein complexes were examined by two-dimensional, blue native gel electrophoresis (BN-PAGE). A number of genes including those encoding acetyl-coA carboxylase beta, m-cadherin, calpain III, creatine kinase, glycogen synthase (GS), and sarcoplasmic reticulum calcium ATPase 1 (SERCA1), were found to be downregulated in patients. Specifically, compared to controls, GS and SERCA1 proteins were reduced by 50% and 75% respectively; also, unphosphorylated GS and SERCA1 were highly downregulated. On BN-PAGE analysis, GP was present with GS in two muscle protein complexes. Our findings revealed some issues that could be important in understanding the physiological consequences of McArdle disease: (i) SERCA1 downregulation in patients could result in impaired calcium transport in type II (fast-twitch) muscle fibers, leading to early fatigability during exercise tasks involving type II fibers (which mostly use glycolytic metabolism), i.e. isometric exercise, lifting weights or intense dynamic exercise (stair climbing, bicycling, walking at a very brisk pace), (ii) GP and GS were found together in two protein complexes, which suggests a new regulatory mechanism in the activity of these glycogen enzymes.

Monocrotaline-Induced Pulmonary Hypertension Involves Downregulation of Antiaging Protein Klotho and eNOS Activity.

PubMed

Varshney, Rohan; Ali, Quaisar; Wu, Chengxiang; Sun, Zhongjie

2016-11-01

The objective of this study is to investigate whether stem cell delivery of secreted Klotho (SKL), an aging-suppressor protein, attenuates monocrotaline-induced pulmonary vascular dysfunction and remodeling. Overexpression of SKL in mesenchymal stem cells (MSCs) was achieved by transfecting MSCs with lentiviral vectors expressing SKL-green fluorescent protein (GFP). Four groups of rats were treated with monocrotaline, whereas an additional group was given saline (control). Three days later, 4 monocrotaline-treated groups received intravenous delivery of nontransfected MSCs, MSC-GFP, MSC-SKL-GFP, and PBS, respectively. Ex vivo vascular relaxing responses to acetylcholine were diminished in small pulmonary arteries (PAs) in monocrotaline-treated rats, indicating pulmonary vascular endothelial dysfunction. Interestingly, delivery of MSCs overexpressing SKL (MSC-SKL-GFP) abolished monocrotaline-induced pulmonary vascular endothelial dysfunction and PA remodeling. Monocrotaline significantly increased right ventricular systolic blood pressure, which was attenuated significantly by MSC-SKL-GFP, indicating improved PA hypertension. MSC-SKL-GFP also attenuated right ventricular hypertrophy. Nontransfected MSCs slightly, but not significantly, improved PA hypertension and pulmonary vascular endothelial dysfunction. MSC-SKL-GFP attenuated monocrotaline-induced inflammation, as evidenced by decreased macrophage infiltration around PAs. MSC-SKL-GFP increased SKL levels, which rescued the downregulation of SIRT1 (Sirtuin 1) expression and endothelial NO synthase (eNOS) phosphorylation in the lungs of monocrotaline-treated rats. In cultured endothelial cells, SKL abolished monocrotaline-induced downregulation of eNOS activity and NO levels and enhanced cell viability. Therefore, stem cell delivery of SKL is an effective therapeutic strategy for pulmonary vascular endothelial dysfunction and PA remodeling. SKL attenuates monocrotaline-induced PA remodeling and PA smooth muscle cell proliferation, likely by reducing inflammation and restoring SIRT1 levels and eNOS activity. © 2016 American Heart Association, Inc.

Aging-related renal injury and inflammation are associated with downregulation of Klotho and induction of RIG-I/NF-κB signaling pathway in senescence-accelerated mice.

PubMed

Zeng, Yi; Wang, Ping-Han; Zhang, Mao; Du, Jun-Rong

2016-02-01

The predominant distribution of the antiaging Klotho protein in both the kidneys and brain may point to its essential role in protecting against dysfunction of the kidney-brain axis during the aging process. Our previous study showed that the downregulation of Klotho was involved in aging-related cognitive impairment in aged senescence-accelerated mouse prone-8 (SAMP8) mice. The present study investigated the potential role of Klotho in aging-associated inflammation and renal injury. Age- and gender-matched groups of SAMP8 mice and their corresponding normal control senescence-accelerated mouse resistant-1 (SAMR1) were used to investigate the potential role of Klotho in aging-associated inflammation and renal injury. Compared with aged SAMR1 controls, early-stage chronic kidney disease (CKD), which is associated with an increase in the urinary albumin-to-creatinine ratio, inflammatory cell infiltration, glomerulosclerosis, and tubulointerstitial fibrosis, was observed in aged SAMP8 mice. Furthermore, the aging-related loss of Klotho-induced activation of the retinoic acid-inducible gene 1/nuclear factor-κB (RIG-I/NF-κB) signaling pathway and subsequent production of the proinflammatory mediators tumor necrosis factor α, interleukin-6, and inducible nitric oxide synthase in the kidneys of aged SAMP8 mice compared with SAMR1 controls. The present results suggest that aging-related inflammation and the development of early-stage CKD are likely associated with the downregulation of Klotho and induction of the RIG-I/NF-κB signaling pathway in 12-month-old SAMP8 mice. Moreover, aged SAMP8 mice with cognitive deficits and renal damage may be a potential mouse model for investigating the kidney-brain axis in the aging process.

AMPK inhibits MTDH expression via GSK3β and SIRT1 activation: potential role in triple negative breast cancer cell proliferation.

PubMed

Gollavilli, Paradesi Naidu; Kanugula, Anantha Koteswararao; Koyyada, Rajeswari; Karnewar, Santosh; Neeli, Praveen Kumar; Kotamraju, Srigiridhar

2015-10-01

Recent studies have highlighted the involvement of metadherin (MTDH), an oncogenic protein, in promoting cancer progression, metastasis and chemoresistance in many cancers including mammary carcinomas. However, the molecular regulation of MTDH is still not completely understood. In this study we document that AMP activated protein kinase (AMPK) activation-induced anti-proliferative effects are, in part, mediated by inhibiting MTDH expression in MDA-MB-231 and BT-549 triple negative breast cancer (TNBC) cells. 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMPK activator, caused growth arrest, inhibition of migration and invasion of TNBC cells. Intriguingly, AICAR or metformin treatment resulted in significant downregulation of MTDH expression via inhibiting c-Myc expression. In contrast, treatment of cells with compound C, an inhibitor of AMPK, increased both c-Myc and MTDH expressions in TNBC cells. Also, AMPK activation caused increased glycogen synthase kinase 3β (GSK3β) activity by inhibiting the inactive phosphorylation at Ser9, on the one hand, and activation of sirtuin1 (SIRT1) by inhibiting Ser47 phosphorylation, as evidenced by deacetylation of p53, on the other hand. Moreover, AMPK-induced GSK3β and SIRT1 activities were found to be responsible for inhibiting c-Myc-mediated upregulation of MTDH, as LiCl (an inhibitor of GSK3β) and EX-527 (an inhibitor of SIRT1) reversed AICAR-mediated downregulation of c-Myc and MTDH expressions. Similar results were observed with siSIRT1 treatment. Furthermore, AICAR and EX-527 treatments caused increased cell death under MTDH-depleted conditions. Finally, we uncovered a novel regulation of MTDH expression and showed that AMPK activation by inducing GSK3β and SIRT1 downregulates MTDH expression via inhibiting c-Myc in TNBC cells. © 2015 FEBS.

Nanotized PPARα Overexpression Targeted to Hypertrophied Myocardium Improves Cardiac Function by Attenuating the p53-GSK3β-Mediated Mitochondrial Death Pathway.

PubMed

Rana, Santanu; Datta, Ritwik; Chaudhuri, Ratul Datta; Chatterjee, Emeli; Chawla-Sarkar, Mamta; Sarkar, Sagartirtha

2018-05-09

Metabolic remodeling of cardiac muscles during pathological hypertrophy is characterized by downregulation of fatty acid oxidation (FAO) regulator, peroxisome proliferator-activated receptor alpha (PPARα). Thereby, we hypothesized that a cardiac-specific induction of PPARα might restore the FAO-related protein expression and resultant energy deficit. In the present study, consequences of PPARα augmentation were evaluated for amelioration of chronic oxidative stress, myocyte apoptosis, and cardiac function during pathological cardiac hypertrophy. Nanotized PPARα overexpression targeted to myocardium was done by a stearic acid-modified carboxymethyl-chitosan (CMC) conjugated to a 20-mer myocyte-targeted peptide (CMCP). Overexpression of PPARα ameliorated pathological hypertrophy and improved cardiac function. Augmented PPARα in hypertrophied myocytes revealed downregulated p53 acetylation (lys 382), leading to reduced apoptosis. Such cells showed increased binding of PPARα with p53 that in turn reduced interaction of p53 with glycogen synthase kinase-3β (GSK3β), which upregulated inactive phospho-GSK3β (serine [Ser]9) expression within mitochondrial protein fraction. Altogether, the altered molecular milieu in PPARα-overexpressed hypertrophy groups restored mitochondrial structure and function both in vitro and in vivo. Cardiomyocyte-targeted overexpression of a protein of interest (PPARα) by nanotized plasmid has been described for the first time in this study. Our data provide a novel insight towards regression of pathological hypertrophy by ameliorating mitochondrial oxidative stress in targeted PPARα-overexpressed myocardium. PPARα-overexpression during pathological hypertrophy showed substantial betterment of mitochondrial structure and function, along with downregulated apoptosis. Myocardium-targeted overexpression of PPARα during pathological cardiac hypertrophy led to an overall improvement of cardiac energy deficit and subsequent cardiac function, thereby, opening up a potential avenue for cardiac tissue engineering during hypertrophic cardiac pathophysiology.

The reciprocal relationship between heme oxygenase and nitric oxide synthase in the organs of lipopolysaccharide-treated rodents.

PubMed

Furuichi, Masayuki; Yokozuka, Motoi; Takemori, Ken; Yamanashi, Yoshitaka; Sakamoto, Atsuhiro

2009-08-01

The production of nitric oxide (NO) by inducible NO synthase (NOS) and carbon monoxide (CO) by inducible heme oxygenase (HO) contributes greatly to endotoxemia. Reciprocal relationships have been proposed between the NO/NOS and CO/HO systems. However, the interaction between these systems during endotoxemia is unclear, and it is unknown whether the interactive behavior differs among organs. Using endotoxic rats, we studied the effects of the inducible NOS (iNOS) inhibitor L-canavanine (CAN), and the HO inhibitor zinc protoporphyrin (ZPP) on gene expression and protein levels of iNOS, endothelial NOS (eNOS), inducible HO (HO-1), and constitutive HO (HO-2) in the brain, lung, heart, liver and kidney tissue. Intravenous injection of LPS significantly increased iNOS and HO-1 gene expression in all organs. The effects of LPS on eNOS gene expression differed among organs, with increased expression in the liver and kidney, and no change in the lung, brain and heart. ZPP administration down-regulated the LPS-induced increase in HO-1 expression and produced a further increase in iNOS expression in all organs. These data suggest that the CO/HO system modifies the NO/NOS system in endotoxic organs, and that there were only minor organ-specific behaviors in terms of the relationship between these systems in the organs examined.

CHP1002, a novel andrographolide derivative, inhibits pro-inflammatory inducible nitric oxide synthase and cyclooxygenase-2 expressions in RAW264.7 macrophages via up-regulation of heme oxygenase-1 expression.

PubMed

Zhang, Bo; Yan, Lingdi; Zhou, Peilan; Dong, Zhaoqi; Feng, Siliang; Liu, Keliang; Gong, Zehui

2013-02-01

Andrographolides, a type of diterpene lactone, are widely known to have anti-inflammatory and anti-oxidative properties. CHP1002, a synthetic derivative of andrographolide, has similar anti-inflammatory action in mouse ear swelling test and rat paw edema test. In the present study, the mechanism of anti-inflammatory effects of CHP1002 was investigated in RAW264.7 macrophages. CHP1002 potently suppressed inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expressions in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. CHP1002 reduced the production of iNOS-derived nitric oxide (NO) and COX-2-derived prostaglandin E2 (PGE2). CHP1002 induced heme oxygenase-1 (HO-1) expression via activation of extracellular signal-regulated kinase (ERK) and NF-E2 related factor 2 transcription factor (Nrf2). Down-regulation of LPS-induced iNOS and COX-2 expressions was partially reversed by the HO-1 inhibitor zinc protoporphyrin (ZnPP). In addition, CHP1002 significantly attenuated LPS-induced TNF-α, IL-1β and IL-6 production. CHP1002 effectively induced HO-1 and was capable of inhibiting some macrophage-derived pro-inflammatory mediators, which may be closely correlated with its anti-inflammatory action. Copyright © 2012 Elsevier B.V. All rights reserved.

Silencing of long noncoding RNA AK139328 attenuates ischemia/reperfusion injury in mouse livers.

PubMed

Chen, Zhenzhen; Jia, Shi; Li, Danhua; Cai, Junyan; Tu, Jian; Geng, Bin; Guan, Youfei; Cui, Qinghua; Yang, Jichun

2013-01-01

Recently, increasing evidences had suggested that long noncoding RNAs (LncRNAs) are involved in a wide range of physiological and pathophysiological processes. Here we determined the LncRNA expression profile using microarray technology in mouse livers after ischemia/reperfusion treatment. Seventy one LncRNAs were upregulated, and 27 LncRNAs were downregulated in ischemia/reperfusion-treated mouse livers. Eleven of the most significantly deregulated LncRNAs were further validated by quantitative PCR assays. Among the upregulated LncRNAs confirmed by quantitative PCR assays, AK139328 exhibited the highest expression level in normal mouse livers. siRNA-mediated knockdown of hepatic AK139328 decreased plasma aminotransferase activities, and reduced necrosis area in the livers with a decrease in caspase-3 activation after ischemia/reperfusion treatment. In ischemia/reperfusion liver, knockdown of AK139328 increased survival signaling proteins including phosphorylated Akt (pAkt), glycogen synthase kinase 3 (pGSK3) and endothelial nitric oxide synthase (peNOS). Furthermore, knockdown of AK139328 also reduced macrophage infitration and inhibited NF-κB activity and inflammatory cytokines expression. In conclusion, these findings revealed that deregulated LncRNAs are involved in liver ischemia/reperfusion injury. Silencing of AK139328 ameliorated ischemia/reperfusion injury in the liver with the activation of Akt signaling pathway and inhibition of NF-κB activity. LncRNA AK139328 might be a novel target for diagnosis and treatment of liver surgery or transplantation.

Baicalein Ameliorates Pulmonary Arterial Hypertension Caused by Monocrotaline through Downregulation of ET-1 and ETAR in Pneumonectomized Rats.

PubMed

Hsu, Wen-Lin; Lin, Yu-Chieh; Jeng, Jing-Ren; Chang, Heng-Yuan; Chou, Tz-Chong

2018-05-08

Baicalein (BE) extracted from Scutellaria baicalensis Georgi is able to alleviate various cardiovascular and inflammatory diseases. However, the effects of BE on pulmonary arterial hypertension (PAH) remain unknown. Therefore, the present study aimed to examine whether BE ameliorates pneumonectomy and monocrotaline-induced PAH in rats and further investigate the underlying molecular mechanisms. Administration of BE greatly attenuated the development of PAH as evidenced by an improvement of its characteristic features, including elevation of right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary vascular remodeling. Moreover, the increased protein expression of endothelin-1 (ET-1) and ET A receptor (ET A R), superoxide overproduction, and activation of Akt/ERK1/2/GSK3[Formula: see text]/[Formula: see text]-catenin pathway that occurred in the lungs of PAH rats were markedly reversed by BE treatment. Compared with the untreated PAH rats, higher expression of endothelial nitric oxide synthase (eNOS), but lower levels of inducible nitric oxide synthase and vWF were observed in BE-treated PAH rats. Collectively, treatment with BE remarkably attenuates the pathogenesis of PAH, and the protection of BE may be associated with suppressing Akt/Erk1/2/GSK3[Formula: see text]/[Formula: see text]-catenin/ET-1/ET A R signaling and preventing endothelial dysfunction. These results suggest that BE is a potential agent for treatment of PAH.

Inhibition of Cycloartenol Synthase (CAS) Function in Tobacco BY-2 Cells.

PubMed

Gas-Pascual, Elisabet; Simonovik, Biljana; Schaller, Hubert; Bach, Thomas J

2015-08-01

Tobacco BY-2 cell suspensions are our preferred model for studying isoprenoid biosynthesis pathways, due to their easy genetic transformation and the efficient absorption of metabolic precursors, intermediates, and/or inhibitors. Using this model system, we have analyzed the effects of chemical and genetic blockage of cycloartenol synthase (CAS, EC 5.4.99.8), an oxidosqualene cyclase that catalyzes the first committed step in the sterol pathway of plants. BY-2 cells were treated with RO 48-8071, a potent inhibitor of oxidosqualene cyclization. Short-term treatments (24 h) resulted in accumulation of oxidosqualene with no changes in the final sterol products. Interestingly, long-term treatments (6 days) induced down-regulation in gene expression not only of CAS but also of the SMT2 gene coding sterol methyltransferase 2 (EC 2.1.1.41). This explains some of the increase in 24-methyl sterols at the expense of the 24-ethyl sterols stigmasterol and sitosterol. In our alternative strategy, CAS gene expression was partially blocked by using an inducible artificial microRNA. The limited effectiveness of this approach might be explained by some dependence of the machinery for RNAi formation on an operating MVA/sterol pathway. For comparison we checked the effect of RO 48-8071 on a green cell suspension of Arabidopsis and on seedlings, containing a small spectrum of triterpenes besides phytosterols. Triterpenes remained essentially unaffected, but phytosterol accumulation was clearly diminished.

Cuminum cyminum, a dietary spice, attenuates hypertension via endothelial nitric oxide synthase and NO pathway in renovascular hypertensive rats.

PubMed

Kalaivani, Periyathambi; Saranya, Ramesh Babu; Ramakrishnan, Ganapathy; Ranju, Vijayan; Sathiya, Sekar; Gayathri, Veeraraghavan; Thiyagarajan, Lakshmi Kantham; Venkhatesh, Jayakothanda Ramaswamy; Babu, Chidambaram Saravana; Thanikachalam, Sadagopan

2013-01-01

Cuminum cyminum (CC) is a commonly used spice in South Indian foods. It has been traditionally used for the treatment and management of sleep disorders, indigestion, and hypertension. The present study was carried out to scientifically evaluate the anti-hypertensive potential of standardized aqueous extract of CC seeds and its role in arterial endothelial nitric oxide synthase expression, inflammation, and oxidative stress in renal hypertensive rats. Renal hypertension was induced by the two-kidney one-clip (2K/1C) method in rats. Systolic blood pressure (SBP), plasma nitrate/nitrite, carotid-eNOS, renal-TNF-α, IL-6, Bax, Bcl-2, thioredoxin 1 (TRX1), and thioredoxin reductase 1 (TRXR1) mRNA expressions were studied to demonstrate the anti-hypertensive action of CC. Cuminum cyminum was administered orally (200 mg/kg b.wt) for a period of 9 weeks; it improved plasma nitric oxide and decreased the systolic blood pressure in hypertensive rats. It also up-regulated the gene expression of eNOS, Bcl-2, TRX1, and TRXR1; and down-regulated Bax, TNF-α, and IL-6. These data reveal that CC seeds augment endothelial functions and ameliorate inflammatory and oxidative stress in hypertensive rats. The present report is the first of its kind to demonstrate the mechanism of anti-hypertensive action of CC seeds in an animal model of renovascular hypertension.

Adiponectin Inhibits Insulin Function in Primary Trophoblasts by PPARα-Mediated Ceramide Synthesis

PubMed Central

Gao, Xiaoli; Weintraub, Susan T.; Jansson, Thomas; Powell, Theresa L.

2014-01-01

Maternal adiponectin (ADN) levels are inversely correlated with birth weight, and ADN infusion in pregnant mice down-regulates placental nutrient transporters and decreases fetal growth. In contrast to the insulin-sensitizing effects in adipose tissue and muscle, ADN inhibits insulin signaling in the placenta. However, the molecular mechanisms involved are unknown. We hypothesized that ADN inhibits insulin signaling and insulin-stimulated amino acid transport in primary human trophoblasts by peroxisome proliferator-activated receptor-α (PPARα)-mediated ceramide synthesis. Primary human term trophoblast cells were treated with ADN and/or insulin. ADN increased the phosphorylation of p38 MAPK and PPARα. ADN inhibited insulin signaling and insulin-stimulated amino acid transport. This effect was dependent on PPARα, because activation of PPARα with an agonist (GW7647) inhibited insulin signaling and function, whereas PPARα-small interfering RNA reversed the effects of ADN on the insulin response. ADN increased ceramide synthase expression and stimulated ceramide production. C2-ceramide inhibited insulin signaling and function, whereas inhibition of ceramide synthase (with Fumonisin B1) reversed the effects of ADN on insulin signaling and amino acid transport. These findings are consistent with the model that maternal ADN limits fetal growth mediated by activation of placental PPARα and ceramide synthesis, which inhibits placental insulin signaling and amino acid transport, resulting in reduced fetal nutrient availability. PMID:24606127

Involvement of ethylene and polyamines biosynthesis and abdominal phloem tissues characters of wheat caryopsis during grain filling under stress conditions

PubMed Central

Yang, Weibing; Li, Yanxia; Yin, Yanping; Qin, Zhilie; Zheng, Mengjing; Chen, Jin; Luo, Yongli; Pang, Dangwei; Jiang, Wenwen; Li, Yong; Wang, Zhenlin

2017-01-01

Severe water deficit (SD) severely limited the photo-assimilate supply during the grain-filling stages. Although the ethylene and polyamines (PAs) have been identified as important signaling molecules involved in stress tolerance, it is yet unclear how 1-Aminocylopropane-1-carboxylic acid (ACC) and PA biosynthesis involving wheat abdominal phloem characters mitigate SD-induced filling inhibition. The results obtained indicated that the SD down-regulated the TaSUT1 expression and decreased the activities of sucrose synthase (SuSase, EC2.4.1.13), ADP glucose pyrophosphorylase (AGPase, EC2.7.7.27), soluble starch synthase (SSSase, EC2.4.1.21), then substantially limited grain filling. As a result, increased ACC and putrescine (Put) concentrations and their biosynthesis-related gene expression reduced spermidine (Spd) biosynthesis under SD condition. And, the ACC and PA biosynthesis in inferior grains was more sensitive to SD than that in superior grains. Intermediary cells (ICs) of caryopsis emerged prematurely under SD to compensate for the weakened photo-assimilate transport functions of sieve elements (SEs). Finally, plasmolysis and nuclear chromatin condensation of phloem parenchyma cells (PPC) and membrane degradation of SEs, as well as the decreased ATPase activity on plasma membranes of ICs and PPC at the later filling stage under SD were responsible for the considerably decreased weight of inferior grains. PMID:28383077

Solanesyl Diphosphate Synthase, an Enzyme of the Ubiquinone Synthetic Pathway, Is Required throughout the Life Cycle of Trypanosoma brucei

PubMed Central

Lai, De-Hua; Poropat, Estefanía; Pravia, Carlos; Landoni, Malena; Couto, Alicia S.; Pérez Rojo, Fernando G.; Fuchs, Alicia G.; Dubin, Marta; Elingold, Igal; Rodríguez, Juan B.; Ferella, Marcela; Esteva, Mónica I.

2014-01-01

Ubiquinone 9 (UQ9), the expected product of the long-chain solanesyl diphosphate synthase of Trypanosoma brucei (TbSPPS), has a central role in reoxidation of reducing equivalents in the mitochondrion of T. brucei. The ablation of TbSPPS gene expression by RNA interference increased the generation of reactive oxygen species and reduced cell growth and oxygen consumption. The addition of glycerol to the culture medium exacerbated the phenotype by blocking its endogenous generation and excretion. The participation of TbSPPS in UQ synthesis was further confirmed by growth rescue using UQ with 10 isoprenyl subunits (UQ10). Furthermore, the survival of infected mice was prolonged upon the downregulation of TbSPPS and/or the addition of glycerol to drinking water. TbSPPS is inhibited by 1-[(n-oct-1-ylamino)ethyl] 1,1-bisphosphonic acid, and treatment with this compound was lethal for the cells. The findings that both UQ9 and ATP pools were severely depleted by the drug and that exogenous UQ10 was able to fully rescue growth of the inhibited parasites strongly suggest that TbSPPS and UQ synthesis are the main targets of the drug. These two strategies highlight the importance of TbSPPS for T. brucei, justifying further efforts to validate it as a new drug target. PMID:24376001

Ruscogenin inhibits lipopolysaccharide-induced acute lung injury in mice: involvement of tissue factor, inducible NO synthase and nuclear factor (NF)-κB.

PubMed

Sun, Qi; Chen, Ling; Gao, Mengyu; Jiang, Wenwen; Shao, Fangxian; Li, Jingjing; Wang, Jun; Kou, Junping; Yu, Boyang

2012-01-01

Acute lung injury is still a significant clinical problem with a high mortality rate and there are few effective therapies in clinic. Here, we studied the inhibitory effect of ruscogenin, an anti-inflammatory and anti-thrombotic natural product, on lipopolysaccharide (LPS)-induced acute lung injury in mice basing on our previous studies. The results showed that a single oral administration of ruscogenin significantly decreased lung wet to dry weight (W/D) ratio at doses of 0.3, 1.0 and 3.0 mg/kg 1 h prior to LPS challenge (30 mg/kg, intravenous injection). Histopathological changes such as pulmonary edema, coagulation and infiltration of inflammatory cells were also attenuated by ruscogenin. In addition, ruscogenin markedly decreased LPS-induced myeloperoxidase (MPO) activity and nitrate/nitrite content, and also downregulated expression of tissue factor (TF), inducible NO synthase (iNOS) and nuclear factor (NF)-κB p-p65 (Ser 536) in the lung tissue at three doses. Furthermore, ruscogenin reduced plasma TF procoagulant activity and nitrate/nitrite content in LPS-induced ALI mice. These findings confirmed that ruscogenin significantly attenuate LPS-induced acute lung injury via inhibiting expressions of TF and iNOS and NF-κB p65 activation, indicating it as a potential therapeutic agent for ALI or sepsis. Copyright © 2011 Elsevier B.V. All rights reserved.

15-Deoxy-{delta}{sup 12,14}-prostaglandin J{sub 2}-induced down-regulation of endothelial nitric oxide synthase in association with HSP70 induction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hwang, Jinah; Lee, Hyun-Il; Chang, Young-Sun

2007-05-25

A natural ligand of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), 15-deoxy-{delta}{sup 12,14}-prostaglandin J{sub 2} (15d-PGJ{sub 2}), decreases endothelial nitric oxide synthase (eNOS) expression by an unknown mechanism. Here we found that 15d-PGJ{sub 2}-induced eNOS reduction is inversely associated with heat shock protein 70 (HSP70) induction in endothelial cells. Treatment of cells with 15d-PGJ{sub 2} decreased eNOS protein expression in a concentration- and time-dependent manner, but independently of PPAR{gamma} with no effect on mRNA levels. Although 15d-PGJ{sub 2} elicited endothelial apoptosis, inhibition of both pan-caspases and cathepsins failed to reverse reduction of eNOS protein. Interestingly, we observed that 15d-PGJ{sub 2} induced HSP70more » in a dose-dependent manner. Immunoprecipitation and heat shock treatment demonstrated that eNOS reduction was strongly related to HSP70 induction. Cellular fractionation revealed that treatment with 15d-PGJ{sub 2} increased eNOS distribution 2.5-fold from soluble to insoluble fractions. These findings provide new insights into mechanisms whereby eNOS regulation by 15d-PGJ{sub 2} is related to HSP70 induction.« less

Remote Control by Inter-Enzyme Allostery: A Novel Paradigm for Regulation of the Shikimate Pathway.

PubMed

Munack, Steffi; Roderer, Kathrin; Ökvist, Mats; Kamarauskaite, Jurate; Sasso, Severin; van Eerde, André; Kast, Peter; Krengel, Ute

2016-03-27

DAHP synthase and chorismate mutase catalyze key steps in the shikimate biosynthetic pathway en route to aromatic amino acids. In Mycobacterium tuberculosis, chorismate mutase (MtCM; Rv0948c), located at the branch point toward phenylalanine and tyrosine, has poor activity on its own. However, it is efficiently activated by the first enzyme of the pathway, DAHP synthase (MtDS; Rv2178c), through formation of a non-covalent MtCM-MtDS complex. Here, we show how MtDS serves as an allosteric platform for feedback regulation of both enzymes, using X-ray crystallography, small-angle X-ray scattering, size-exclusion chromatography, and multi-angle light scattering. Crystal structures of the fully inhibited MtDS and the allosterically down-regulated MtCM-MtDS complex, solved at 2.8 and 2.7Å, respectively, reveal how effector binding at the internal MtDS subunit interfaces regulates the activity of MtDS and MtCM. While binding of all three metabolic end products to MtDS shuts down the entire pathway, the binding of phenylalanine jointly with tyrosine releases MtCM from the MtCM-MtDS complex, hence suppressing MtCM activation by 'inter-enzyme allostery'. This elegant regulatory principle, invoking a transient allosteric enzyme interaction, seems to be driven by dynamics and is likely a general strategy used by nature. Copyright © 2016 Elsevier Ltd. All rights reserved.

Environmental metabolomics reveal geographic variation in aerobic metabolism and metabolic substrates in Mongolian gerbils (Meriones unguiculatus).

PubMed

Shi, Yao-Long; Chi, Qing-Sheng; Liu, Wei; Fu, He-Ping; Wang, De-Hua

2015-06-01

Mongolian gerbils (Meriones unguiculatus) have a large-scale distribution in northern China. Geographic physiological variations which related to energy and water metabolism are critical to animals' local adaptation and distribution. However, the underlying biochemical mechanism of such variation and its role in adaptation remains largely unknown. We used GC-MS metabolomics approach to investigate the biochemical adaptation of Mongolian gerbils from xeric (desert), transition (desert steppe) and mesic (typical steppe) environments. Gerbils in desert population had lower resting metabolic rate (RMR) and total evaporative water loss (TEWL) than mesic population. Serum metabolomics revealed that concentrations of five tricarboxylic acid cycle intermediates (citrate, cis-aconitate, α-ketoglutarate, fumarate and malate) were lower in desert population than mesic population. Gastrocnemius metabolomics and citrate synthase activity analysis showed a lower concentration of citrate and lower citrate synthase activity in desert population. These findings suggest that desert dwelling gerbils decrease RMR and TEWL via down-regulation of aerobic respiration. Gastrocnemius metabolomics also revealed that there were higher concentrations of glucose and glycolytic intermediates, but lower concentrations of lipids, amino acids and urea in desert population than mesic population. This geographic variation in metabolic substrates may enhance metabolic water production per oxygen molecule for desert population while constraining aerobic respiration to reduce RMR and TEWL. Copyright © 2015 Elsevier Inc. All rights reserved.

Autosomal recessive progressive myoclonus epilepsy due to impaired ceramide synthesis.

PubMed

Ferlazzo, Edoardo; Striano, Pasquale; Italiano, Domenico; Calarese, Tiziana; Gasparini, Sara; Vanni, Nicola; Fruscione, Floriana; Genton, Pierre; Zara, Federico

2016-09-01

Autosomal recessive progressive myoclonus epilepsy due to impaired ceramide synthesis is an extremely rare condition, so far reported in a single family of Algerian origin presenting an unusual, severe form of progressive myoclonus epilepsy characterized by myoclonus, generalized tonic-clonic seizures and moderate to severe cognitive impairment, with probable autosomal recessive inheritance. Disease onset was between 6 and 16 years of age. Genetic study allowed to identify a homozygous nonsynonymous mutation in CERS1, the gene encoding ceramide synthase 1, a transmembrane protein of the endoplasmic reticulum (ER), catalyzes the biosynthesis of C18-ceramides. The mutation decreased C18-ceramide levels. In addition, downregulation of CerS1 in neuroblastoma cell line showed activation of ER stress response and induction of proapoptotic pathways. This observation demonstrates that impairment of ceramide biosynthesis underlies neurodegeneration in humans.

Identification of a New Class of Antifungals Targeting the Synthesis of Fungal Sphingolipids.

PubMed

Mor, Visesato; Rella, Antonella; Farnoud, Amir M; Singh, Ashutosh; Munshi, Mansa; Bryan, Arielle; Naseem, Shamoon; Konopka, James B; Ojima, Iwao; Bullesbach, Erika; Ashbaugh, Alan; Linke, Michael J; Cushion, Melanie; Collins, Margaret; Ananthula, Hari Krishna; Sallans, Larry; Desai, Pankaj B; Wiederhold, Nathan P; Fothergill, Annette W; Kirkpatrick, William R; Patterson, Thomas; Wong, Lai Hong; Sinha, Sunita; Giaever, Guri; Nislow, Corey; Flaherty, Patrick; Pan, Xuewen; Cesar, Gabriele Vargas; de Melo Tavares, Patricia; Frases, Susana; Miranda, Kildare; Rodrigues, Marcio L; Luberto, Chiara; Nimrichter, Leonardo; Del Poeta, Maurizio

2015-06-23

Recent estimates suggest that >300 million people are afflicted by serious fungal infections worldwide. Current antifungal drugs are static and toxic and/or have a narrow spectrum of activity. Thus, there is an urgent need for the development of new antifungal drugs. The fungal sphingolipid glucosylceramide (GlcCer) is critical in promoting virulence of a variety of human-pathogenic fungi. In this study, we screened a synthetic drug library for compounds that target the synthesis of fungal, but not mammalian, GlcCer and found two compounds [N'-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) and its derivative, 3-bromo-N'-(3-bromo-4-hydroxybenzylidene) benzohydrazide (D0)] that were highly effective in vitro and in vivo against several pathogenic fungi. BHBM and D0 were well tolerated in animals and are highly synergistic or additive to current antifungals. BHBM and D0 significantly affected fungal cell morphology and resulted in the accumulation of intracellular vesicles. Deep-sequencing analysis of drug-resistant mutants revealed that four protein products, encoded by genes APL5, COS111, MKK1, and STE2, which are involved in vesicular transport and cell cycle progression, are targeted by BHBM. Fungal infections are a significant cause of morbidity and mortality worldwide. Current antifungal drugs suffer from various drawbacks, including toxicity, drug resistance, and narrow spectrum of activity. In this study, we have demonstrated that pharmaceutical inhibition of fungal glucosylceramide presents a new opportunity to treat cryptococcosis and various other fungal infections. In addition to being effective against pathogenic fungi, the compounds discovered in this study were well tolerated by animals and additive to current antifungals. These findings suggest that these drugs might pave the way for the development of a new class of antifungals. Copyright © 2015 Mor et al.

Gromwell (Lithospermum erythrorhizon) Supplementation Enhances Epidermal Levels of Ceramides, Glucosylceramides, β-Glucocerebrosidase, and Acidic Sphingomyelinase in NC/Nga Mice

PubMed Central

Kim, Jungmin; Cho, Yunhi

2013-01-01

Abstract We have previously reported that dietary gromwell (Lithospermum erythrorhizon; LE) prevents the development of atopic dermatitis (AD) with increased epidermal levels of total ceramide (Cer), the major lipid maintaining epidermal barrier. In this study, we investigated whether the increased level of total Cer induced by dietary LE would be related to the altered metabolism of glucosylceramide (GlcCer) and sphingomyelin (SM), two major precursor lipids in Cer generation. NC/Nga mice, an animal model of AD, were fed a control diet (group CA: atopic control) or a diet with 70% ethanol LE extracts (1% in diet; group LE) for 10 weeks. Individual species of Cer, GlcCer, and SM were analyzed by high-performance thin layer chromatography. In the epidermis of group CA, total Cer (including Cer2 and Cer5–7) and total GlcCer (including GlcCer-B/C/D) were significantly reduced; these levels in group LE were increased to levels similar to the normal control group of BALB/c mice (group C). In addition, protein expressions and activities of β-glucocerebrosidase (β-GlcCer'ase) and acidic sphingomyelinase (aSMase), enzymes for GlcCer or SM hydrolysis, respectively, were increased in group LE. However, alterations of Cer1, Cer3/4, GlcCer-A, and all SM species (including SM1–3) were not significant among groups C, CA, and LE. Dietary gromwell increases GlcCer-B/C/D, and further enhances the generation of Cer2 and Cer5–7 with high protein expressions and activities of β-GlcCer'ase and aSMase. PMID:24074295

Oral administration of glucosylceramide ameliorates inflammatory dry-skin condition in chronic oxazolone-induced irritant contact dermatitis in the mouse ear.

PubMed

Yeom, Mijung; Kim, Sung-Hun; Lee, Bombi; Han, Jeong-Jun; Chung, Guk Hoon; Choi, Hee-Don; Lee, Hyejung; Hahm, Dae-Hyun

2012-08-01

Irritant contact dermatitis (ICD) is an inflammatory skin disease triggered by exposure to a chemical that is toxic or irritating to the skin. A major characteristic of chronic ICD is an inflammatory dry-skin condition with associated itching. Although glucosylceramide (GlcCer) is known to improve the skin barrier function, its mechanism of action is unknown. Using a mouse model of oxazolone-induced chronic ICD, this study investigated the effects of oral administration of GlcCer on inflammatory dry skin. Chronic ICD was induced by repeated application of oxazolone in mice. GlcCer was orally administered once daily throughout the elicitation phase. The beneficial efficacy of GlcCer on cutaneous inflammation was evaluated by assessing ear thickness, lymph node weight, histological findings, and mRNA expression of pro-inflammatory cytokines such as IL-1β and IL-6. Additionally, parameters of the itch-associated response, including scratching behavior, water content of the skin, and aquaporin-3 levels in the lesional ear, were measured. Oral GlcCer administration significantly suppressed mRNA expression of the pro-inflammatory cytokines IL-1β and IL-6. GlcCer also suppressed ear swelling, lymph node weight gains, and infiltration of leukocytes and mast cells in ICD mice. In oxazolone-induced ICD mice, GlcCer significantly inhibited irritant-related scratching behavior and dehydration of the stratum corneum, and decreased aquaporin-3 expression. Our results indicate that GlcCer suppressed inflammation not only by inhibiting cytokine production but also by repairing the skin barrier function, suggesting a potential beneficial role for GlcCer in the improvement of chronic ICD. Copyright © 2012 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Selective chaperone effect of aminocyclitol derivatives on G202R and other mutant glucocerebrosidases causing Gaucher disease.

PubMed

Serra-Vinardell, Jenny; Díaz, Lucía; Gutiérrez-de Terán, Hugo; Sánchez-Ollé, Gessamí; Bujons, Jordi; Michelakakis, Helen; Mavridou, Irene; Aerts, Johannes M F G; Delgado, Antonio; Grinberg, Daniel; Vilageliu, Lluïsa; Casas, Josefina

2014-09-01

Gaucher disease is an autosomal recessive lysosomal disorder characterized by the accumulation of glucosylceramide as a result of a deficiency of the enzyme glucocerebrosidase. Several competitive glucocerebrosidase inhibitors are able to act as pharmacological chaperones for an efficient rescue of the mutated, misfolded forms of the enzyme. Along this line, we report in this work on the ability of several aminocyclitols to increase the residual glucocerebrosidase activity in patient fibroblasts with different genotypes. Some of the compounds were slightly active on fibroblasts bearing some mutations, including the highly prevalent N370S mutation. All compounds were highly active as enzyme activity enhancers on fibroblasts from Gaucher disease patients containing the G202R mutation. Moreover, using the novel tagged sphingolipid ω-azidosphingosine, a reduction in the tagged glucosylceramide accumulation was also observed for selected aminocyclitols. Attempts to explain the activity impairment observed in glucocerebrosidase bearing the G202R mutation by comparative molecular dynamic studies on wild type and the G202R mutated proteins (free and isofagomine-bound, in both cases) were unsuccessful. Under the simulation conditions used, no clear effect of the G202R mutation neither over the global structure of the protein nor on the loops that constitute the glucocerebrosidase active site was observed. Since the G202R residue is located on the protein surface, altered protein-membrane or protein-protein interactions could account for the observed differences. In conclusion, we have tested novel compounds that have shown some chaperone effect on particular glucocerebrosidase mutant enzymes, supporting the enhancement therapy as an alternative approach for Gaucher disease. Copyright © 2014 Elsevier Ltd. All rights reserved.

Glucosylceramide and Lysophosphatidylcholines as Potential Blood Biomarkers for Drug-Induced Hepatic Phospholipidosis

PubMed Central

Saito, Kosuke; Maekawa, Keiko; Ishikawa, Masaki; Senoo, Yuya; Urata, Masayo; Murayama, Mayumi; Nakatsu, Noriyuki; Yamada, Hiroshi; Saito, Yoshiro

2014-01-01

Drug-induced phospholipidosis is one of the major concerns in drug development and clinical treatment. The present study involved the use of a nontargeting lipidomic analysis with liquid chromatography-mass spectrometry to explore noninvasive blood biomarkers for hepatic phospholipidosis from rat plasma. We used three tricyclic antidepressants (clomipramine [CPM], imipramine [IMI], and amitriptyline [AMT]) for the model of phospholipidosis in hepatocytes and ketoconazole (KC) for the model of phospholipidosis in cholangiocytes and administered treatment for 3 and 28 days each. Total plasma lipids were extracted and measured. Lipid molecules contributing to the separation of control and drug-treated rat plasma in a multivariate orthogonal partial least squares discriminant analysis were identified. Four lysophosphatidylcholines (LPCs) (16:1, 18:1, 18:2, and 20:4) and 42:1 hexosylceramide (HexCer) were identified as molecules separating control and drug-treated rats in all models of phospholipidosis in hepatocytes. In addition, 16:1, 18:2, and 20:4 LPCs and 42:1 HexCer were identified in a model of hepatic phospholipidosis in cholangiocytes, although LPCs were identified only in the case of 3-day treatment with KC. The levels of LPCs were decreased by drug-induced phospholipidosis, whereas those of 42:1 HexCer were increased. The increase in 42:1 HexCer was much higher in the case of IMI and AMT than in the case of CPM; moreover, the increase induced by IMI was dose-dependent. Structural characterization determining long-chain base and hexose delineated that 42:1 HexCer was d18:1/24:0 glucosylceramide (GluCer). In summary, our study demonstrated that d18:1/24:0 GluCer and LPCs are potential novel biomarkers for drug-induced hepatic phospholipidosis. PMID:24980264

Ambient pH Controls Glycogen Levels by Regulating Glycogen Synthase Gene Expression in Neurospora crassa. New Insights into the pH Signaling Pathway

PubMed Central

Cupertino, Fernanda Barbosa; Freitas, Fernanda Zanolli; de Paula, Renato Magalhães; Bertolini, Maria Célia

2012-01-01

Glycogen is a polysaccharide widely distributed in microorganisms and animal cells and its metabolism is under intricate regulation. Its accumulation in a specific situation results from the balance between glycogen synthase and glycogen phosphorylase activities that control synthesis and degradation, respectively. These enzymes are highly regulated at transcriptional and post-translational levels. The existence of a DNA motif for the Aspergillus nidulans pH responsive transcription factor PacC in the promoter of the gene encoding glycogen synthase (gsn) in Neurospora crassa prompted us to investigate whether this transcription factor regulates glycogen accumulation. Transcription factors such as PacC in A. nidulans and Rim101p in Saccharomyces cerevisiae play a role in the signaling pathway that mediates adaptation to ambient pH by inducing the expression of alkaline genes and repressing acidic genes. We showed here that at pH 7.8 pacC was over-expressed and gsn was down-regulated in wild-type N. crassa coinciding with low glycogen accumulation. In the pacCKO strain the glycogen levels and gsn expression at alkaline pH were, respectively, similar to and higher than the wild-type strain at normal pH (5.8). These results characterize gsn as an acidic gene and suggest a regulatory role for PACC in gsn expression. The truncated recombinant protein, containing the DNA-binding domain specifically bound to a gsn DNA fragment containing the PacC motif. DNA-protein complexes were observed with extracts from cells grown at normal and alkaline pH and confirmed by ChIP-PCR analysis. The PACC present in these extracts showed equal molecular mass, indicating that the protein is already processed at normal pH, in contrast to A. nidulans. Together, these results show that the pH signaling pathway controls glycogen accumulation by regulating gsn expression and suggest the existence of a different mechanism for PACC activation in N. crassa. PMID:22952943

Stable expression of lipocalin-type prostaglandin D synthase in cultured preadipocytes impairs adipogenesis program independently of endogenous prostanoids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hossain, Mohammad Salim; Chowdhury, Abu Asad; Rahman, Mohammad Sharifur

Lipocalin-type prostaglandin D synthase (L-PGDS) expressed preferentially in adipocytes is responsible for the synthesis of PGD{sub 2} and its non-enzymatic dehydration products, PGJ{sub 2} series, serving as pro-adipogenic factors. However, the role of L-PGDS in the regulation of adipogenesis is complex because of the occurrence of several derivatives from PGD{sub 2} and their distinct receptor subtypes as well as other functions such as a transporter of lipophilic molecules. To manipulate the expression levels of L-PGDS in cultured adipocytes, cultured preadipogenic 3T3-L1 cells were transfected stably with a mammalian expression vector having cDNA encoding murine L-PGDS oriented in the sense direction.more » The isolated cloned stable transfectants with L-PGDS expressed higher levels of the transcript and protein levels of L-PGDS, and synthesized PGD{sub 2} from exogenous arachidonic acid at significantly higher levels. By contrast, the synthesis of PGE{sub 2} remained unchanged, indicating no influence on the reactions of cyclooxygenase (COX) and PGE synthase. Furthermore, the ability of those transfectants to synthesize {Delta}{sup 12}-PGJ{sub 2} increased more greatly during the maturation phase. The sustained expression of L-PGDS in cultured stable transfectants hampered the storage of fats during the maturation phase of adipocytes, which was accompanied by the reduced gene expression of adipocyte-specific markers reflecting the down-regulation of the adipogenesis program. The suppressed adipogenesis was not rescued by either exogenous aspirin or peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonists including troglitazone and {Delta}{sup 12}-PGJ{sub 2}. Taken together, the results indicate the negative regulation of the adipogenesis program by the enhanced expression of L-PGDS through a cellular mechanism involving the interference of the PPAR{gamma} signaling pathway without the contribution of endogenous pro-adipogenic prostanoids. -- Highlights: Black-Right-Pointing-Pointer Cultured preadipocytes were transfected with sense lipocalin-type PGD synthase. Black-Right-Pointing-Pointer The cloned transfectants had a higher ability to synthesize PGD{sub 2} and PGJ{sub 2} series. Black-Right-Pointing-Pointer The sustained expression of sense L-PGDS hampered the storage of fats. Black-Right-Pointing-Pointer Adipogenesis program was suppressed independently of endogenous prostanoids.« less

Impact of down-regulation of starch branching enzyme IIb in rice by artificial microRNA- and hairpin RNA-mediated RNA silencing

PubMed Central

Butardo, Vito M.; Fitzgerald, Melissa A.; Bird, Anthony R.; Gidley, Michael J.; Flanagan, Bernadine M.; Larroque, Oscar; Resurreccion, Adoracion P.; Laidlaw, Hunter K. C.; Jobling, Stephen A.; Morell, Matthew K.; Rahman, Sadequr

2011-01-01

The inactivation of starch branching IIb (SBEIIb) in rice is traditionally associated with elevated apparent amylose content, increased peak gelatinization temperature, and a decreased proportion of short amylopectin branches. To elucidate further the structural and functional role of this enzyme, the phenotypic effects of down-regulating SBEIIb expression in rice endosperm were characterized by artificial microRNA (amiRNA) and hairpin RNA (hp-RNA) gene silencing. The results showed that RNA silencing of SBEIIb expression in rice grains did not affect the expression of other major isoforms of starch branching enzymes or starch synthases. Structural analyses of debranched starch showed that the doubling of apparent amylose content was not due to an increase in the relative proportion of amylose chains but instead was due to significantly elevated levels of long amylopectin and intermediate chains. Rices altered by the amiRNA technique produced a more extreme starch phenotype than those modified using the hp-RNA technique, with a greater increase in the proportion of long amylopectin and intermediate chains. The more pronounced starch structural modifications produced in the amiRNA lines led to more severe alterations in starch granule morphology and crystallinity as well as digestibility of freshly cooked grains. The potential role of attenuating SBEIIb expression in generating starch with elevated levels of resistant starch and lower glycaemic index is discussed. PMID:21791436

miR-143 suppresses epithelial-mesenchymal transition and inhibits tumor growth of breast cancer through down-regulation of ERK5.

PubMed

Zhai, Limin; Ma, Chuanxiang; Li, Wentong; Yang, Shuo; Liu, Zhijun

2016-12-01

Epithelial-mesenchymal transition (EMT) plays a pivotal role in the development of cancer invasion and metastasis. Many studies have significantly enhanced the knowledge on EMT through the characterization of microRNAs (miRNAs) influencing the signaling pathways and downstream events that define EMT on a molecular level. In this study, we found that miR-143 suppressed EMT. Up-regulating miR-143 enhanced E-cadherin-mediated cell-cell adhesion ability, reduced mesenchymal markers, and decreased cell proliferation, migration, and invasion in vitro. In vivo, the xenograft mouse model also unveiled the suppressive effects of miR-143 on tumor growth. Additionally, we demonstrated that up-regulating extracellular signal regulated kinase 5 (ERK5) was associated with poor prognosis of breast cancer patients. Moreover, we observed an inverse correlation between miR-143 and ERK5 in breast cancer tissues. miR-143 directly targeted seed sequences in the 3'-untranslated regions of ERK5. Furthermore, we revealed that the downstream molecules of glycogen synthase kinase 3 beta (GSK-3β)/Snail signaling were involved in EMT and modulated by ERK5. In summary, our findings demonstrated that miR-143 down-regulated its target ERK5, leading to the suppression of EMT induced by GSK-3β/Snail signaling of breast cancer. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

PORPHOBILINOGEN DEAMINASE Deficiency Alters Vegetative and Reproductive Development and Causes Lesions in Arabidopsis

PubMed Central

Quesada, Víctor; Hricová, Andrea; Ponce, María Rosa; Micol, José Luis

2013-01-01

The Arabidopsis rugosa1 (rug1) mutant has irregularly shaped leaves and reduced growth. In the absence of pathogens, leaves of rug1 plants have spontaneous lesions reminiscent of those seen in lesion-mimic mutants; rug1 plants also express cytological and molecular markers associated with defence against pathogens. These rug1 phenotypes are made stronger by dark/light transitions. The rug1 mutant also has delayed flowering time, upregulation of the floral repressor FLOWERING LOCUS C (FLC) and downregulation of the flowering promoters FT and SOC1/AGL20. Vernalization suppresses the late flowering phenotype of rug1 by repressing FLC. Microarray analysis revealed that 280 nuclear genes are differentially expressed between rug1 and wild type; almost a quarter of these genes are involved in plant defence. In rug1, the auxin response is also affected and several auxin-responsive genes are downregulated. We identified the RUG1 gene by map-based cloning and found that it encodes porphobilinogen deaminase (PBGD), also known as hydroxymethylbilane synthase, an enzyme of the tetrapyrrole biosynthesis pathway, which produces chlorophyll, heme, siroheme and phytochromobilin in plants. PBGD activity is reduced in rug1 plants, which accumulate porphobilinogen. Our results indicate that Arabidopsis PBGD deficiency impairs the porphyrin pathway and triggers constitutive activation of plant defence mechanisms leading to leaf lesions and affecting vegetative and reproductive development. PMID:23308205

In vivo preventive effects of insect tea on buccal mucosa cancer in ICR mice.

PubMed

Zhao, Xin; Wang, Rui; Qian, Yu; Li, Guijie; Zhou, Yalin; Sun, Peng

2014-01-01

Insect tea is a particular drink or health product in China and it is also used as Chinese medicine now. Its functional effects need to be proved. The ICR mice buccal mucosa cancer model was established by injecting the mice with U14 cells and mice was treated with insect tea. Tumor volumes and lymph node metastasis rates were determined. And the buccal mucosa tissues and cancer cervical lymph node were also checked by histology test, real-time polymerase chain reaction, and western blot assays. The tumor volumes for the group treated with insect tea mice was smaller than those from the control mice. The sections of buccal mucosa cancer tissue showed that the canceration of insect tea mice was weaker than control mice. Insect tea significantly induced apoptosis in buccal mucosa tissues by upregulating Bax, caspases, and downregulating Bcl-2. Nuclear factor-κB, inducible nitric oxide synthase, and COX-2 gene, was significantly downregulated by insect tea, demonstrating its anti-inflammatory properties. Insect tea also exerted a great anti-metastasis effect on tissues as demonstrated by decreased expression of Matrix metalloproteinases genes and increased expression of tissue inhibitors of matrix metalloproteinases. The highest concentration of 1600 mg/kg oral gavage and 400 mg/mL smear insect tea showed the best anticancer effects. Based on the results, insect tea showed the strong in vivo buccal mucosa cancer preventive effects.

[Screening cold-acclimation differential expression candidate genes in the brain of common carp (Cyprinus carpio)].

PubMed

Xu, Li-Hua; Chang, Yu-Mei; Liu, Chun-Lei; Liang, Li-Qun; Liu, Jin-Liang; Chi, Bing-Jie

2011-03-01

In this study, 26 candidate genes were quantified and normalized in the brain cDNA of common carp (Cyprinus carpio) at 23°C and 6°C using double-standard curve method of real-time quantitative PCR. The results showed that five candidates up-regulated in the samples at 6°C (P<0.01) and quantified 2.11, 13.9, 2.52, 7.38, and 1.83 times more than in the samples at 23°C, respectively. Gene function searching indicated that the protein products of these five candidates were elongation of very long chain fatty acids protein, Acyl-CoA desaturase, Transcription initiation factor IIB, Myo-inositol- 1-phosphate synthase, and Blood-brain barrier HT7 antigen individually. Moreover, seven down-regulated candidates were also identified in the same samples at 6°C (P>0.05), and their expression levels were decreased by 21.8%, 25.9%, 16.6%, 23.7%, 15.8%, 16.3%, and 42.5%, respectively, in comparison with the samples at 23°C. These seven down-regulated candidates mainly participated in the inhibition of glycolysis, improvement of cell apoptosis, and intervention of synapse remodeling based on the results of function searching. The five cold-induced genes identified in this study will be used as important elements for fish with cold sensitive through transgenic technology in future.

Antimicrobial activity of apple cider vinegar against Escherichia coli, Staphylococcus aureus and Candida albicans; downregulating cytokine and microbial protein expression.

PubMed

Yagnik, Darshna; Serafin, Vlad; J Shah, Ajit

2018-01-29

The global escalation in antibiotic resistance cases means alternative antimicrobials are essential. The aim of this study was to investigate the antimicrobial capacity of apple cider vinegar (ACV) against E. coli, S. aureus and C. albicans. The minimum dilution of ACV required for growth inhibition varied for each microbial species. For C. albicans, a 1/2 ACV had the strongest effect, S. aureus, a 1/25 dilution ACV was required, whereas for E-coli cultures, a 1/50 ACV dilution was required (p < 0.05). Monocyte co-culture with microbes alongside ACV resulted in dose dependent downregulation of inflammatory cytokines (TNFα, IL-6). Results are expressed as percentage decreases in cytokine secretion comparing ACV treated with non-ACV treated monocytes cultured with E-coli (TNFα, 99.2%; IL-6, 98%), S. aureus (TNFα, 90%; IL-6, 83%) and C. albicans (TNFα, 83.3%; IL-6, 90.1%) respectively. Proteomic analyses of microbes demonstrated that ACV impaired cell integrity, organelles and protein expression. ACV treatment resulted in an absence in expression of DNA starvation protein, citrate synthase, isocitrate and malate dehydrogenases in E-coli; chaperone protein DNak and ftsz in S. aureus and pyruvate kinase, 6-phosphogluconate dehydrogenase, fructose bisphosphate were among the enzymes absent in C.albican cultures. The results demonstrate ACV has multiple antimicrobial potential with clinical therapeutic implications.

Phloem loading in Verbascum phoeniceum L. depends on the synthesis of raffinose-family oligosaccharides

PubMed Central

McCaskill, Ashlee; Turgeon, Robert

2007-01-01

Phloem loading is the initial step in photoassimilate export and the one that creates the driving force for mass flow. It has been proposed that loading occurs symplastically in species that translocate carbohydrate primarily as raffinose family oligosaccharides (RFOs). In these plants, dense fields of plasmodesmata connect bundle sheath cells to specialized companion cells (intermediary cells) in the minor veins. According to the polymer trap model, advanced as a mechanism of symplastic loading, sucrose from the mesophyll diffuses into intermediary cells and is converted there to RFOs. This process keeps the sucrose concentration low and, because of the larger size of the RFOs, prevents back diffusion. To test this model, the RFO pathway was down-regulated in Verbascum phoeniceum L. by suppressing the synthesis of galactinol synthase (GAS), which catalyzes the first committed step in RFO production. Two GAS genes (VpGAS1 and VpGAS2) were cloned and shown to be expressed in intermediary cells. Simultaneous RNAi suppression of both genes resulted in pronounced inhibition of RFO synthesis. Phloem transport was negatively affected, as evidenced by the accumulation of carbohydrate in the lamina and the reduced capacity of leaves to export sugars during a prolonged dark period. In plants with severe down-regulation, additional symptoms of reduced export were obvious, including impaired growth, leaf chlorosis, and necrosis and curling of leaf margins. PMID:18048337

Rice Ethylene-Response AP2/ERF Factor OsEATB Restricts Internode Elongation by Down-Regulating a Gibberellin Biosynthetic Gene1[W][OA

PubMed Central

Qi, Weiwei; Sun, Fan; Wang, Qianjie; Chen, Mingluan; Huang, Yunqing; Feng, Yu-Qi; Luo, Xiaojin; Yang, Jinshui

2011-01-01

Plant height is a decisive factor in plant architecture. Rice (Oryza sativa) plants have the potential for rapid internodal elongation, which determines plant height. A large body of physiological research has shown that ethylene and gibberellin are involved in this process. The APETALA2 (AP2)/Ethylene-Responsive Element Binding Factor (ERF) family of transcriptional factors is only present in the plant kingdom. This family has various developmental and physiological functions. A rice AP2/ERF gene, OsEATB (for ERF protein associated with tillering and panicle branching) was cloned from indica rice variety 9311. Bioinformatic analysis suggested that this ERF has a potential new function. Ectopic expression of OsEATB showed that the cross talk between ethylene and gibberellin, which is mediated by OsEATB, might underlie differences in rice internode elongation. Analyses of gene expression demonstrated that OsEATB restricts ethylene-induced enhancement of gibberellin responsiveness during the internode elongation process by down-regulating the gibberellin biosynthetic gene, ent-kaurene synthase A. Plant height is negatively correlated with tiller number, and higher yields are typically obtained from dwarf crops. OsEATB reduces rice plant height and panicle length at maturity, promoting the branching potential of both tillers and spikelets. These are useful traits for breeding high-yielding crops. PMID:21753115

Propolis prevents diet-induced hyperlipidemia and mitigates weight gain in diet-induced obesity in mice.

PubMed

Koya-Miyata, Satomi; Arai, Norie; Mizote, Akiko; Taniguchi, Yoshifumi; Ushio, Shimpei; Iwaki, Kanso; Fukuda, Shigeharu

2009-12-01

We examined the hypolipidemic effect of propolis in a mouse obesity model induced by a high fat-diet. C57BL/6N mice were fed a high-fat diet ad libitum and given propolis extract intragastrically at 0 mg/kg (control), 5 mg/kg or 50 mg/kg twice daily for 10 d. Compared with mice in the control group, mice in the propolis extract-administrated groups displayed a reduction in all of the following parameters: body weight gain, weight of visceral adipose tissue, liver and serum triglycerides, cholesterol, and non-esterified fatty acids. Real-time polymerase chain reaction analysis of the liver showed down-regulation of mRNA expression associated with fatty acid biosynthesis, including fatty acid synthase, acetyl-CoA carboxylase alpha, and sterol regulatory element binding protein in the propolis-administrated mice. Subsequently, obese C57BL/6N mice that had been administered a high-fat diet were given propolis extract at 0 mg/kg (control), 2.5 mg/kg or 25 mg/kg for 4 weeks. The propolis extract treated mice showed a decrease in weight gain, a reduction of serum non-esterified fatty acids, and lipid accumulation in the liver. These results suggest that propolis extract prevented and mitigated high-fat diet-induced hyperlipidemia by down-regulating the expression of genes associated with lipid metabolism.

Local gene silencing in plants via synthetic dsRNA and carrier peptide.

PubMed

Numata, Keiji; Ohtani, Misato; Yoshizumi, Takeshi; Demura, Taku; Kodama, Yutaka

2014-10-01

Quick and facile transient RNA interference (RNAi) is one of the most valuable plant biotechnologies for analysing plant gene functions. To establish a novel double-strand RNA (dsRNA) delivery system for plants, we developed an ionic complex of synthetic dsRNA with a carrier peptide in which a cell-penetrating peptide is fused with a polycation sequence as a gene carrier. The dsRNA-peptide complex is 100-300 nm in diameter and positively charged. Infiltration of the complex into intact leaf cells of Arabidopsis thaliana successfully induced rapid and efficient down-regulation of exogenous and endogenous genes such as yellow fluorescent protein and chalcone synthase. The present method realizes quick and local gene silencing in specific tissues and/or organs in plants. © 2014 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Glycogen Synthase Kinase 3 (GSK-3) influences epithelial barrier function by regulating Occludin, Claudin-1 and E-cadherin expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Severson, Eric A.; Kwon, Mike; Hilgarth, Roland S.

2010-07-02

The Apical Junctional Complex (AJC) encompassing the tight junction (TJ) and adherens junction (AJ) plays a pivotal role in regulating epithelial barrier function and epithelial cell proliferative processes through signaling events that remain poorly characterized. A potential regulator of AJC protein expression is Glycogen Synthase Kinase-3 (GSK-3). GSK-3 is a constitutively active kinase that is repressed during epithelial-mesenchymal transition (EMT). In the present study, we report that GSK-3 activity regulates the structure and function of the AJC in polarized model intestinal (SK-CO15) and kidney (Madin-Darby Canine Kidney (MDCK)) epithelial cells. Reduction of GSK-3 activity, either by small molecule inhibitors ormore » siRNA targeting GSK-3 alpha and beta mRNA, resulted in increased permeability to both ions and bulk solutes. Immunofluorescence labeling and immunoblot analyses revealed that the barrier defects correlated with decreased protein expression of AJC transmembrane proteins Occludin, Claudin-1 and E-cadherin without influencing other TJ proteins, Zonula Occludens-1 (ZO-1) and Junctional Adhesion Molecule A (JAM-A). The decrease in Occludin and E-cadherin protein expression correlated with downregulation of the corresponding mRNA levels for these respective proteins following GSK-3 inhibition. These observations implicate an important role of GSK-3 in the regulation of the structure and function of the AJC that is mediated by differential modulation of mRNA transcription of key AJC proteins, Occludin, Claudin-1 and E-cadherin.« less

Proteomic analysis of enterotoxigenic Escherichia coli (ETEC) in neutral and alkaline conditions.

PubMed

Gonzales-Siles, Lucia; Karlsson, Roger; Kenny, Diarmuid; Karlsson, Anders; Sjöling, Åsa

2017-01-07

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in children and travelers to endemic areas. Secretion of the heat labile AB 5 toxin (LT) is induced by alkaline conditions. In this study, we determined the surface proteome of ETEC exposed to alkaline conditions (pH 9) as compared to neutral conditions (pH 7) using a LPI Hexalane FlowCell combined with quantitative proteomics. Relative quantitation with isobaric labeling (TMT) was used to compare peptide abundance and their corresponding proteins in multiple samples at MS/MS level. For protein identification and quantification samples were analyzed using either a 1D-LCMS or a 2D-LCMS approach. Strong up-regulation of the ATP synthase operon encoding F1Fo ATP synthase and down-regulation of proton pumping proteins NuoF, NuoG, Ndh and WrbA were detected among proteins involved in regulating the proton and electron transport under alkaline conditions. Reduced expression of proteins involved in osmotic stress was found at alkaline conditions while the Sec-dependent transport over the inner membrane and outer membrane protein proteins such as OmpA and the β-Barrel Assembly Machinery (BAM) complex were up-regulated. ETEC exposed to alkaline environments express a specific proteome profile characterized by up-regulation of membrane proteins and secretion of LT toxin. Alkaline microenvironments have been reported close to the intestinal epithelium and the alkaline proteome may hence represent a better view of ETEC during infection.

Proteomic analysis of common bean seed with storage protein deficiency reveals up-regulation of sulfur-rich proteins and starch and raffinose metabolic enzymes, and down-regulation of the secretory pathway.

PubMed

Marsolais, Frédéric; Pajak, Agnieszka; Yin, Fuqiang; Taylor, Meghan; Gabriel, Michelle; Merino, Diana M; Ma, Vanessa; Kameka, Alexander; Vijayan, Perumal; Pham, Hai; Huang, Shangzhi; Rivoal, Jean; Bett, Kirstin; Hernández-Sebastià, Cinta; Liu, Qiang; Bertrand, Annick; Chapman, Ralph

2010-06-16

A deficiency in major seed storage proteins is associated with a nearly two-fold increase in sulfur amino acid content in genetically related lines of common bean (Phaseolus vulgaris). Their mature seed proteome was compared by an approach combining label-free quantification by spectral counting, 2-DE, and analysis of selective extracts. Lack of phaseolin, phytohemagglutinin and arcelin was mainly compensated by increases in legumin, alpha-amylase inhibitors and mannose lectin FRIL. Along with legumin, albumin-2, defensin and albumin-1 were major contributors to the elevated sulfur amino acid content. Coordinate induction of granule-bound starch synthase I, starch synthase II-2 and starch branching enzyme were associated with minor alteration of starch composition, whereas increased levels of UDP-glucose 4-epimerase were correlated with a 30% increase in raffinose content. Induction of cell division cycle protein 48 and ubiquitin suggested enhanced ER-associated degradation. This was not associated with a classical unfolded protein response as the levels of ER HSC70-cognate binding protein were actually reduced in the mutant. Repression of rab1 GTPase was consistent with decreased traffic through the secretory pathway. Collectively, these results have implications for the nutritional quality of common bean, and provide information on the pleiotropic phenotype associated with storage protein deficiency in a dicotyledonous seed. Crown Copyright 2010. Published by Elsevier B.V. All rights reserved.

Promoting PGC-1α-driven mitochondrial biogenesis is detrimental in pressure-overloaded mouse hearts

PubMed Central

Karamanlidis, Georgios; Garcia-Menendez, Lorena; Kolwicz, Stephen C.; Lee, Chi Fung

2014-01-01

Mitochondrial dysfunction in animal models of heart failure is associated with downregulation of the peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α pathway. To test whether PGC-1α is an appropriate therapeutic target for increasing mitochondrial biogenesis and improving function in heart failure, we used a transgenic (TG) mouse model of moderate overexpression of PGC-1α (∼3-fold) in the heart. TG mice had small increases in citrate synthase activity and mitochondria size in the heart without alterations in myocardial energetics or cardiac function at baseline. In vivo dobutamine stress increased fractional shortening in wild-type mice, but this increase was attenuated in TG mice, whereas ex vivo isolated perfused TG hearts demonstrated normal functional and energetic response to high workload challenge. When subjected to pressure overload by transverse aortic constriction (TAC), TG mice displayed a significantly greater acute mortality for both male and female mice; however, long-term survival up to 8 wk was similar between the two groups. TG mice also showed a greater decrease in fractional shortening and a greater increase in left ventricular chamber dimension in response to TAC. Mitochondrial gene expression and citrate synthase activity were mildly increased in TG mice compared with wild-type mice, and this difference was also maintained after TAC. Our data suggest that a moderate level of PGC-1α overexpression in the heart compromises acute survival and does not improve cardiac function during chronic pressure overload in mice. PMID:25172896

Crocin Suppresses LPS-Stimulated Expression of Inducible Nitric Oxide Synthase by Upregulation of Heme Oxygenase-1 via Calcium/Calmodulin-Dependent Protein Kinase 4

PubMed Central

Kim, Ji-Hee; Park, Ga-Young; Bang, Soo Young; Park, Sun Young; Bae, Soo-Kyung; Kim, YoungHee

2014-01-01

Crocin is a water-soluble carotenoid pigment that is primarily used in various cuisines as a seasoning and coloring agent, as well as in traditional medicines for the treatment of edema, fever, and hepatic disorder. In this study, we demonstrated that crocin markedly induces the expression of heme oxygenase-1 (HO-1) which leads to an anti-inflammatory response. Crocin inhibited inducible nitric oxide synthase (iNOS) expression and nitric oxide production via downregulation of nuclear factor kappa B activity in lipopolysaccharide- (LPS-) stimulated RAW 264.7 macrophages. These effects were abrogated by blocking of HO-1 expression or activity. Crocin also induced Ca2+ mobilization from intracellular pools and phosphorylation of Ca2+/calmodulin-dependent protein kinase 4 (CAMK4). CAMK4 knockdown and kinase-dead mutant inhibited crocin-mediated HO-1 expression, Nrf2 activation, and phosphorylation of Akt, indicating that HO-1 expression is mediated by CAMK4 and that Akt is a downstream mediator of CAMK4 in crocin signaling. Moreover, crocin-mediated suppression of iNOS expression was blocked by CAMK4 inhibition. Overall, these results suggest that crocin suppresses LPS-stimulated expression of iNOS by inducing HO-1 expression via Ca2+/calmodulin-CAMK4-PI3K/Akt-Nrf2 signaling cascades. Our findings provide a novel molecular mechanism for the inhibitory effects of crocin against endotoxin-mediated inflammation. PMID:24839356

Comprehensive transcriptome-based characterization of differentially expressed genes involved in microsporogenesis of radish CMS line and its maintainer.

PubMed

Xie, Yang; Zhang, Wei; Wang, Yan; Xu, Liang; Zhu, Xianwen; Muleke, Everlyne M; Liu, Liwang

2016-09-01

Microsporogenesis is an indispensable period for investigating microspore development and cytoplasmic male sterility (CMS) occurrence. Radish CMS line plays a critical role in elite F1 hybrid seed production and heterosis utilization. However, the molecular mechanisms of microspore development and CMS occurrence have not been thoroughly uncovered in radish. In this study, a comparative analysis of radish floral buds from a CMS line (NAU-WA) and its maintainer (NAU-WB) was conducted using next generation sequencing (NGS) technology. Digital gene expression (DGE) profiling revealed that 3504 genes were significantly differentially expressed between NAU-WA and NAU-WB library, among which 1910 were upregulated and 1594 were downregulated. Gene ontology (GO) analysis showed that these differentially expressed genes (DEGs) were mainly enriched in extracellular region, catalytic activity, and response to stimulus. KEGG enrichment analysis revealed that the DEGs were predominantly associated with flavonoid biosynthesis, glycolysis, and biosynthesis of secondary metabolites. Real-time quantitative PCR analysis showed that the expression profiles of 13 randomly selected DEGs were in high agreement with results from Illumina sequencing. Several candidate genes encoding ATP synthase, auxin response factor (ARF), transcription factors (TFs), chalcone synthase (CHS), and male sterility (MS) were responsible for microsporogenesis. Furthermore, a schematic diagram for functional interaction of DEGs from NAU-WA vs. NAU-WB library in radish plants was proposed. These results could provide new information on the dissection of the molecular mechanisms underlying microspore development and CMS occurrence in radish.

Andrographolide Enhances Nuclear Factor-κB Subunit p65 Ser536 Dephosphorylation through Activation of Protein Phosphatase 2A in Vascular Smooth Muscle Cells*

PubMed Central

Hsieh, Cheng Y.; Hsu, Ming J.; Hsiao, George; Wang, Yi H.; Huang, Chi W.; Chen, Shiuan W.; Jayakumar, Thanasekaran; Chiu, Pei T.; Chiu, Yi H.; Sheu, Joen R.

2011-01-01

Recent studies have demonstrated that transcription factor nuclear factor (NF)-κB inhibition may contribute to the protective anti-inflammatory actions of andrographolide, an abundant component of plants of the genus Andrographis. However, the precise mechanism by which andrographolide inhibits NF-κB signaling remains unclear. We thus investigated the mechanism involved in andrographolide suppression of NF-κB signaling in rat vascular smooth muscle cells (VSMCs) exposed to proinflammatory stimuli, LPS, and IFN-γ. Andrographolide was shown to suppress LPS/IFN-γ-induced inducible nitric-oxide synthase and matrix metalloprotease 9 expression in rat VSMCs. Andrographolide also inhibited LPS/IFN-γ-induced p65 nuclear translocation, DNA binding activity, p65 Ser536 phosphorylation, and NF-κB reporter activity. However, IKK phosphorylation and downstream inhibitory κBα phosphorylation and degradation were not altered by the presence of andrographolide in LPS/IFN-γ-stimulated VSMCs. These andrographolide inhibitory actions could be prevented by selective inhibition of neutral sphingomyelinase and protein phosphatase 2A (PP2A). Furthermore, andrographolide was demonstrated to increase ceramide formation and PP2A activity in VSMCs and to inhibit neointimal formation in rat carotid injury models. These results suggest that andrographolide caused neutral sphingomyelinase-mediated ceramide formation and PP2A activation to dephosphorylate p65 Ser536, leading to NF-κB inactivation and subsequent inducible nitric-oxide synthase down-regulation in rat VSMCs stimulated by LPS and IFN-γ. PMID:21169355

HLA-B35 Upregulates Endothelin-1 and Downregulates Endothelial Nitric Oxide Synthase via Endoplasmic Reticulum Stress Response in Endothelial Cells

PubMed Central

Lenna, Stefania; Townsend, Danyelle M.; Tan, Filemon K.; Kapanadze, Bagrat; Markiewicz, Malgorzata; Trojanowska, Maria; Scorza, Raffaella

2013-01-01

The presence of the HLA-B35 allele has emerged as an important risk factor for the development of isolated pulmonary hypertension in patients with scleroderma, however the mechanisms underlying this association have not been fully elucidated. The goal of our study was to determine the molecular mechanisms that mediate the biological effects of HLA-B35 in endothelial cells (ECs). Our data demonstrate that HLA-B35 expression at physiological levels via adenoviral vector resulted in significantly increased endothelin-1 (ET-1) and a significantly decreased endothelial NO synthase (eNOS), mRNA, and protein levels. Furthermore, HLA-B35 greatly upregulated expression of chaperones, including heat shock proteins (HSPs) HSP70 (HSPA1A and HSPA1B) and HSP40 (DNAJB1 and DNAJB9), suggesting that HLA-B35 induces the endoplasmic reticulum (ER) stress and unfolded protein response in ECs. Examination of selected mediators of the unfolded protein response, including H chain binding protein (BiP; GRP78), C/Ebp homologous protein (CHOP; GADD153), endoplasmic reticulum oxidase, and protein disulfide isomerase has revealed a consistent increase of BiP expression levels. Accordingly, thapsigargin, a known ER stress inducer, stimulated ET-1mRNAand protein levels in ECs. This study suggests that HLA-B35 could contribute to EC dysfunction via ER stress-mediated induction of ET-1 in patients with pulmonary hypertension. PMID:20335527

HLA-B35 upregulates endothelin-1 and downregulates endothelial nitric oxide synthase via endoplasmic reticulum stress response in endothelial cells.

PubMed

Lenna, Stefania; Townsend, Danyelle M; Tan, Filemon K; Kapanadze, Bagrat; Markiewicz, Malgorzata; Trojanowska, Maria; Scorza, Raffaella

2010-05-01

The presence of the HLA-B35 allele has emerged as an important risk factor for the development of isolated pulmonary hypertension in patients with scleroderma, however the mechanisms underlying this association have not been fully elucidated. The goal of our study was to determine the molecular mechanisms that mediate the biological effects of HLA-B35 in endothelial cells (ECs). Our data demonstrate that HLA-B35 expression at physiological levels via adenoviral vector resulted in significantly increased endothelin-1 (ET-1) and a significantly decreased endothelial NO synthase (eNOS), mRNA, and protein levels. Furthermore, HLA-B35 greatly upregulated expression of chaperones, including heat shock proteins (HSPs) HSP70 (HSPA1A and HSPA1B) and HSP40 (DNAJB1 and DNAJB9), suggesting that HLA-B35 induces the endoplasmic reticulum (ER) stress and unfolded protein response in ECs. Examination of selected mediators of the unfolded protein response, including H chain binding protein (BiP; GRP78), C/Ebp homologous protein (CHOP; GADD153), endoplasmic reticulum oxidase, and protein disulfide isomerase has revealed a consistent increase of BiP expression levels. Accordingly, thapsigargin, a known ER stress inducer, stimulated ET-1 mRNA and protein levels in ECs. This study suggests that HLA-B35 could contribute to EC dysfunction via ER stress-mediated induction of ET-1 in patients with pulmonary hypertension.

Monoterpene synthases from common sage (Salvia officinalis)

DOEpatents

Croteau, Rodney Bruce; Wise, Mitchell Lynn; Katahira, Eva Joy; Savage, Thomas Jonathan

1999-01-01

cDNAs encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase from common sage (Salvia officinalis) have been isolated and sequenced, and the corresponding amino acid sequences has been determined. Accordingly, isolated DNA sequences (SEQ ID No:1; SEQ ID No:3 and SEQ ID No:5) are provided which code for the expression of (+)-bornyl diphosphate synthase (SEQ ID No:2), 1,8-cineole synthase (SEQ ID No:4) and (+)-sabinene synthase SEQ ID No:6), respectively, from sage (Salvia officinalis). In other aspects, replicable recombinant cloning vehicles are provided which code for (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase, or for a base sequence sufficiently complementary to at least a portion of (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant monoterpene synthases that may be used to facilitate their production, isolation and purification in significant amounts. Recombinant (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase may be used to obtain expression or enhanced expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase in plants in order to enhance the production of monoterpenoids, or may be otherwise employed for the regulation or expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase, or the production of their products.

Regulation of CDP-diacylglycerol synthesis and utilization by inositol and choline in Schizosaccharomyces pombe.

PubMed Central

Gaynor, P M; Greenberg, M L

1992-01-01

CDP-diacylglycerol (CDP-DG) is an important branchpoint intermediate in eucaryotic phospholipid biosynthesis and could be a key regulatory site in phospholipid metabolism. Therefore, we examined the effects of growth phase, phospholipid precursors, and the disruption of phosphatidylcholine (PC) synthesis on the membrane-associated phospholipid biosynthetic enzymes CDP-DG synthase, phosphatidylglycerolphosphate (PGP) synthase, phosphatidylinositol (PI) synthase, and phosphatidylserine (PS) synthase in cell extracts of the fission yeast Schizosaccharomyces pombe. In complete synthetic medium containing inositol, maximal expression of CDP-DG synthase, PGP synthase, PI synthase, and PS synthase in wild-type cells occurred in the exponential phase of growth and decreased two- to fourfold in the stationary phase of growth. In cells starved for inositol, this decrease in PGP synthase, PI synthase, and PS synthase expression was not observed. Starvation for inositol resulted in a twofold derepression of PGP synthase and PS synthase expression, while PI synthase expression decreased initially and then remained constant. Upon the addition of inositol to inositol-starved cells, there was a rapid and continued increase in PI synthase expression. We examined expression of these enzymes in cho2 and cho1 mutants, which are blocked in the methylation pathway for synthesis of PC. Choline starvation resulted in a decrease in PS synthase and CDP-DG synthase expression in cho1 but not cho2 cells. Expression of PGP synthase and PI synthase was not affected by choline starvation. Inositol starvation resulted in a 1.7-fold derepression of PGP synthase expression in cho2 but not cho1 cells when PC was synthesized. PS synthase expression was not depressed, while CDP-DG synthase and PI synthase expression decreased in cho2 and cho1 cells in the absence of inositol. These results demonstrate that (i) CDP-DG synthase, PGP synthase, PI synthase, and PS synthase are similarly regulated by growth phase; (ii) inositol affects the expression of PGP synthase, PI synthase, and PS synthase; (iii) disruption of the methylation pathway results in aberrant patterns of regulation of growth phase and phospholipid precursors. Important differences between S. pombe and Saccharomyces cerevisiae with regard to regulation of these enzymes are discussed. PMID:1324908

Aluminium induced oxidative stress results in decreased mitochondrial biogenesis via modulation of PGC-1α expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sharma, Deep Raj; Sunkaria, Aditya; Wani, Willayat Yousuf

The present investigation was carried out to elucidate a possible molecular mechanism related to the effects of aluminium-induced oxidative stress on various mitochondrial respiratory complex subunits with special emphasis on the role of Peroxisome proliferator activated receptor gamma co-activator 1α (PGC-1α) and its downstream targets i.e. Nuclear respiratory factor-1(NRF-1), Nuclear respiratory factor-2(NRF-2) and Mitochondrial transcription factor A (Tfam) in mitochondrial biogenesis. Aluminium lactate (10 mg/kg b.wt./day) was administered intragastrically to rats for 12 weeks. After 12 weeks of exposure, we found an increase in ROS levels, mitochondrial DNA oxidation and decrease in citrate synthase activity in the Hippocampus (HC) andmore » Corpus striatum (CS) regions of rat brain. On the other hand, there was a decrease in the mRNA levels of the mitochondrial encoded subunits–NADH dehydrogenase (ND) subunits i.e. ND1, ND2, ND3, Cytochrome b (Cytb), Cytochrome oxidase (COX) subunits i.e. COX1, COX3, ATP synthase (ATPase) subunit 6 along with reduced expression of nuclear encoded subunits COX4, COX5A, COX5B of Electron transport chain (ETC). Besides, a decrease in mitochondrial DNA copy number and mitochondrial content in both regions of rat brain was observed. The PGC-1α was down-regulated in aluminium treated rats along with NRF-1, NRF-2 and Tfam, which act downstream from PGC-1α in aluminium treated rats. Electron microscopy results revealed a significant increase in the mitochondrial swelling, loss of cristae, chromatin condensation and decreases in mitochondrial number in case of aluminium treated rats as compared to control. So, PGC-1α seems to be a potent target for aluminium neurotoxicity, which makes it an almost ideal target to control or limit the damage that has been associated with the defective mitochondrial function seen in neurodegenerative diseases. - Highlights: • Aluminium decreases the mRNA levels of mitochondrial and nuclear encoded subunits. • It decreases the mtDNA copy number and mitochondrial content in rat brain. • It down-regulates the mRNA and protein levels of PGC-1α, NRF-1, NRF-2 and Tfam. • It also disturbs the mitochondrial or nuclear architecture of neurons. • Finally it also decreases mitochondrial number in HC and CS regions of rat brain.« less

Regulation of basal gastric acid secretion by the glycogen synthase kinase GSK3.

PubMed

Rotte, Anand; Pasham, Venkanna; Eichenmüller, Melanie; Yang, Wenting; Qadri, Syed M; Bhandaru, Madhuri; Lang, Florian

2010-10-01

According to previous observations, basal gastric acid secretion is downregulated by phosphoinositol-3-(PI3)-kinase, phosphoinositide-dependent kinase (PDK1), and protein kinase B (PKBβ/Akt2) signaling. PKB/Akt phosphorylates glycogen synthase kinase GSK3. The present study explored whether PKB/Akt-dependent GSK3-phosphorylation modifies gastric acid secretion. Utilizing 2',7'-bis-(carboxyethyl)-5(6')-carboxyfluorescein (BCECF)-fluorescence, basal gastric acid secretion was determined from Na(+)-independent pH recovery (∆pH/min) following an ammonium pulse, which reflects H(+)/K(+)-ATPase activity. Experiments were performed in gastric glands from gene-targeted mice (gsk3 ( KI )) with PKB/serum and glucocorticoid-inducible kinase (SGK)-insensitive GSKα,β, in which the serines within the PKB/SGK phosphorylation site were replaced by alanine (GSK3α(21A/21A), GSK3β(9A/9A)). The cytosolic pH in isolated gastric glands was similar in gsk3 ( KI ) and their wild-type littermates (gsk3 ( WT )). However, ∆pH/min was significantly larger in gsk3 ( KI ) than in gsk3 ( WT ) mice and ∆pH/min was virtually abolished by the H(+)/K(+)-ATPase inhibitor omeprazole (100 μM) in gastric glands from both gsk3 ( KI ) and gsk3 ( WT ). Plasma gastrin levels were lower in gsk3 ( KI ) than in gsk3 ( WT ). Both, an increase of extracellular K(+) concentration to 35 mM [replacing Na(+)/N-methyl-D: -glucamine (NMDG)] and treatment with forskolin (5 μM), significantly increased ∆pH/min to virtually the same value in both genotypes. The protein kinase A (PKA) inhibitor H89 (150 nM) and the H(2)-receptor antagonist ranitidine (100 μM) decreased ∆pH/min in gsk3 ( KI ) but not gsk3 ( WT ) and again abrogated the differences between the genotypes. The protein abundance of phosphorylated but not of total PKA was significantly larger in gsk3 ( KI ) than in gsk3 ( WT ). Basal gastric acid secretion is enhanced by the disruption of PKB/SGK-dependent phosphorylation and the inhibition of GSK3. Thus, the inhibition of GSK3 participates in the signaling of PI3-kinase-dependent downregulation of basal gastric acid secretion.

Crosstalk Between Peroxisome Proliferator-Activated Receptor Gamma and the Canonical WNT/β-Catenin Pathway in Chronic Inflammation and Oxidative Stress During Carcinogenesis

PubMed Central

Vallée, Alexandre; Lecarpentier, Yves

2018-01-01

Inflammation and oxidative stress are common and co-substantial pathological processes accompanying, promoting, and even initiating numerous cancers. The canonical WNT/β-catenin pathway and peroxisome proliferator-activated receptor gamma (PPARγ) generally work in opposition. If one of them is upregulated, the other one is downregulated and vice versa. WNT/β-catenin signaling is upregulated in inflammatory processes and oxidative stress and in many cancers, although there are some exceptions for cancers. The opposite is observed with PPARγ, which is generally downregulated during inflammation and oxidative stress and in many cancers. This helps to explain in part the opposite and unidirectional profile of the canonical WNT/β-catenin signaling and PPARγ in these three frequent and morbid processes that potentiate each other and create a vicious circle. Many intracellular pathways commonly involved downstream will help maintain and amplify inflammation, oxidative stress, and cancer. Thus, many WNT/β-catenin target genes such as c-Myc, cyclin D1, and HIF-1α are involved in the development of cancers. Nuclear factor-kappaB (NFκB) can activate many inflammatory factors such as TNF-α, TGF-β, interleukin-6 (IL-6), IL-8, MMP, vascular endothelial growth factor, COX2, Bcl2, and inducible nitric oxide synthase. These factors are often associated with cancerous processes and may even promote them. Reactive oxygen species (ROS), generated by cellular alterations, stimulate the production of inflammatory factors such as NFκB, signal transducer and activator transcription, activator protein-1, and HIF-α. NFκB inhibits glycogen synthase kinase-3β (GSK-3β) and therefore activates the canonical WNT pathway. ROS activates the phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) signaling in many cancers. PI3K/Akt also inhibits GSK-3β. Many gene mutations of the canonical WNT/β-catenin pathway giving rise to cancers have been reported (CTNNB1, AXIN, APC). Conversely, a significant reduction in the expression of PPARγ has been observed in many cancers. Moreover, PPARγ agonists promote cell cycle arrest, cell differentiation, and apoptosis and reduce inflammation, angiogenesis, oxidative stress, cell proliferation, invasion, and cell migration. All these complex and opposing interactions between the canonical WNT/β-catenin pathway and PPARγ appear to be fairly common in inflammation, oxidative stress, and cancers. PMID:29706964

Functional genomics of fuzzless-lintless mutant of Gossypium hirsutum L. cv. MCU5 reveal key genes and pathways involved in cotton fibre initiation and elongation

PubMed Central

2012-01-01

Background Fuzzless-lintless cotton mutants are considered to be the ideal material to understand the molecular mechanisms involved in fibre cell development. Although there are few reports on transcriptome and proteome analyses in cotton at fibre initiation and elongation stages, there is no comprehensive comparative transcriptome analysis of fibre-bearing and fuzzless-lintless cotton ovules covering fibre initiation to secondary cell wall (SCW) synthesis stages. In the present study, a comparative transcriptome analysis was carried out using G. hirsutum L. cv. MCU5 wild-type (WT) and it’s near isogenic fuzzless-lintless (fl) mutant at fibre initiation (0 dpa/days post anthesis), elongation (5, 10 and 15 dpa) and SCW synthesis (20 dpa) stages. Results Scanning electron microscopy study revealed the delay in the initiation of fibre cells and lack of any further development after 2 dpa in the fl mutant. Transcriptome analysis showed major down regulation of transcripts (90%) at fibre initiation and early elongation (5 dpa) stages in the fl mutant. Majority of the down regulated transcripts at fibre initiation stage in the fl mutant represent calcium and phytohormone mediated signal transduction pathways, biosynthesis of auxin and ethylene and stress responsive transcription factors (TFs). Further, transcripts involved in carbohydrate and lipid metabolisms, mitochondrial electron transport system (mETS) and cell wall loosening and elongation were highly down-regulated at fibre elongation stage (5–15 dpa) in the fl mutant. In addition, cellulose synthases and sucrose synthase C were down-regulated at SCW biosynthesis stage (15–20 dpa). Interestingly, some of the transcripts (~50%) involved in phytohormone signalling and stress responsive transcription factors that were up-regulated at fibre initiation stage in the WT were found to be up-regulated at much later stage (15 dpa) in fl mutant. Conclusions Comparative transcriptome analysis of WT and its near isogenic fl mutant revealed key genes and pathways involved at various stages of fibre development. Our data implicated the significant role of mitochondria mediated energy metabolism during fibre elongation process. The delayed expression of genes involved in phytohormone signalling and stress responsive TFs in the fl mutant suggests the need for a coordinated expression of regulatory mechanisms in fibre cell initiation and differentiation. PMID:23151214

Ovarian steroids, oxytocin, and tumor necrosis factor modulate equine oviduct function.

PubMed

Pinto-Bravo, P; Galvão, A; Rebordão, M R; Amaral, A; Ramilo, D; Silva, E; Szóstek-Mioduchowska, A; Alexandre-Pires, G; Roberto da Costa, R; Skarzynski, D J; Ferreira-Dias, G

2017-10-01

The oviduct plays important roles in the early reproductive process. The aim of this study was to evaluate gene transcription and protein expression of progesterone receptor (PGR), estrogen receptors 1 (ESR1) and 2 (ESR2); oxytocin receptor (OXTR); prostaglandin F2α synthase (AKR1C3), and prostaglandin E2 synthase (Ptges) in mare oviduct in different estrous cycle stages. Estradiol (E 2 ), progesterone (P 4 ), oxytocin (OXT), and tumor necrosis factor α (TNF) effect on in vitro PGE 2 and prostaglandin F2α (PGF 2α ) secretion by equine oviduct explants or by oviductal epithelial cells (OECs) were also assessed. During the breeding season, oviduct tissue was obtained post mortem from cyclic mares. Protein of ESR1, ESR2, PGR, AKR1C3, and Ptges was present in OECs, whereas OXTR was shown in oviduct stroma. In follicular phase, protein expression of ESR1, ESR2, PGR, and OXTR increased in oviduct explants (P < 0.05), whereas no estrous cycle effect was noted for AKR1C3 or Ptges. In follicular phase, mRNA transcription was upregulated for Pgr but downregulated for Oxtr, Ptges, and Akr1c3 (P < 0.05). Nevertheless, Esr1 and Esr2 mRNA levels did not change with the estrous cycle. In the ampulla, Esr1, Esr2, and Oxtr mRNA transcription increased, but not for Pgr or Ptges. In contrast, Akr1c3 mRNA level was upregulated in the infundibulum (P < 0.05). In follicular phase, E 2 , P 4 , and OXT downregulated PGE 2 production by OEC (P < 0.05), but no difference was observed in mid-luteal phase. Explants production of PGE 2 rose when treated with OXT in follicular phase; with TNF or OXT in early luteal phase; or with TNF, OXT, or P 4 in mid-luteal phase. PGF 2α production by OEC was downregulated by all treatments in follicular phase but upregulated in mid-luteal phase (P < 0.05). Oviduct explants PGF 2α production was stimulated by TNF or OXT in all estrous cycle phases. In conclusion, this work has shown that ESR1, ESR2, OXTR, Ptges, and AKRLC3 gene transcription and/or translation is estrous cycle dependent and varies with oviduct portion (infundibulum vs ampulla) and cell type. Ovarian steroid hormones, OXT and TNF stimulation of PGF 2α and/or PGE 2 production is also estrous cycle dependent and varies in the different portions of mare oviduct. Differential transcription level and protein localization in various portions of the oviduct throughout the estrous cycle, as well as PG production, suggest coordinated physiologic actions and mechanisms of steroid hormones, OXT, and TNF in the equine oviduct. Copyright © 2017 Elsevier Inc. All rights reserved.

Differential Regulation of cGMP Signaling in Human Melanoma Cells at Altered Gravity: Simulated Microgravity Down-Regulates Cancer-Related Gene Expression and Motility

NASA Astrophysics Data System (ADS)

Ivanova, Krassimira; Eiermann, Peter; Tsiockas, Wasiliki; Hemmersbach, Ruth; Gerzer, Rupert

2018-03-01

Altered gravity is known to affect cellular function by changes in gene expression and cellular signaling. The intracellular signaling molecule cyclic guanosine-3',5'-monophosphate (cGMP), a product of guanylyl cyclases (GC), e.g., the nitric oxide (NO)-sensitive soluble GC (sGC) or natriuretic peptide-activated GC (GC-A/GC-B), is involved in melanocyte response to environmental stress. NO-sGC-cGMP signaling is operational in human melanocytes and non-metastatic melanoma cells, whereas up-regulated expression of GC-A/GC-B and inducible NO synthase (iNOS) are found in metastatic melanoma cells, the deadliest skin cancer. Here, we investigated the effects of altered gravity on the mRNA expression of NOS isoforms, sGC, GC-A/GC-B and multidrug resistance-associated proteins 4/5 (MRP4/MRP5) as selective cGMP exporters in human melanoma cells with different metastatic potential and pigmentation. A specific centrifuge (DLR, Cologne Germany) was used to generate hypergravity (5 g for 24 h) and a fast-rotating 2-D clinostat (60 rpm) to simulate microgravity values ≤ 0.012 g for 24 h. The results demonstrate that hypergravity up-regulates the endothelial NOS-sGC-MRP4/MRP5 pathway in non-metastatic melanoma cells, but down-regulates it in simulated microgravity when compared to 1 g. Additionally, the suppression of sGC expression and activity has been suggested to correlate inversely to tumor aggressiveness. Finally, hypergravity is ineffective in highly metastatic melanoma cells, whereas simulated microgravity down-regulates predominantly the expression of the cancer-related genes iNOS and GC-A/GC-B (shown additionally on protein levels) as well as motility in comparison to 1 g. The results suggest that future studies in real microgravity can benefit from considering GC-cGMP signaling as possible factor for melanocyte transformation.

Small-Molecule Inhibition of PD-1 Transcription Is an Effective Alternative to Antibody Blockade in Cancer Therapy.

PubMed

Taylor, Alison; Rothstein, David; Rudd, Christopher E

2018-02-01

The impact of PD-1 immune checkpoint therapy prompts exploration of other strategies to downregulate PD-1 for cancer therapy. We previously showed that the serine/threonine kinase, glycogen synthase kinase, GSK-3α/β, is a central regulator of PD-1 transcription in CD8 + T cells. Here, we show that the use of small-molecule inhibitors of GSK-3α/β (GSK-3i) to reduce pcdc1 (PD-1) transcription and expression was as effective as anti-PD-1 and PD-L1-blocking antibodies in the control of B16 melanoma, or EL4 lymphoma, in primary tumor and metastatic settings. Furthermore, the conditional genetic deletion of GSK-3α/β reduced PD-1 expression on CD8 + T cells and limited B16 pulmonary metastasis to the same degree as PD-1 gene deficiency. In each model, GSK-3i inhibited PD-1 expression on tumor-infiltrating lymphocytes, while increasing Tbx21 (T-bet) transcription, and the expression of CD107a + (LAMP1) and granzyme B (GZMB) on CD8 + T cells. Finally, the adoptive transfer of T cells treated ex vivo with a GSK-3 inhibitor delayed the onset of EL4 lymphoma growth to a similar extent as anti-PD-1 pretreatment. Overall, our findings show how GSK-3 inhibitors that downregulate PD-1 expression can enhance CD8 + T-cell function in cancer therapy to a similar degree as PD-1-blocking antibodies. Significance: These findings show how GSK-3 inhibitors that downregulate PD-1 expression can enhance CD8 + T-cell function in cancer therapy to a similar degree as PD-1 blocking antibodies, offering a next-generation approach in the design of immunotherapeutic approaches for cancer management. Cancer Res; 78(3); 706-17. ©2017 AACR . ©2017 American Association for Cancer Research.

Genome-Wide Profile of Pleural Mesothelioma versus Parietal and Visceral Pleura: The Emerging Gene Portrait of the Mesothelioma Phenotype

PubMed Central

Røe, Oluf Dimitri; Anderssen, Endre; Helge, Eli; Pettersen, Caroline Hild; Olsen, Karina Standahl; Sandeck, Helmut; Haaverstad, Rune; Lundgren, Steinar; Larsson, Erik

2009-01-01

Background Malignant pleural mesothelioma is considered an almost incurable tumour with increasing incidence worldwide. It usually develops in the parietal pleura, from mesothelial lining or submesothelial cells, subsequently invading the visceral pleura. Chromosomal and genomic aberrations of mesothelioma are diverse and heterogenous. Genome-wide profiling of mesothelioma versus parietal and visceral normal pleural tissue could thus reveal novel genes and pathways explaining its aggressive phenotype. Methodology and Principal Findings Well-characterised tissue from five mesothelioma patients and normal parietal and visceral pleural samples from six non-cancer patients were profiled by Affymetrix oligoarray of 38 500 genes. The lists of differentially expressed genes tested for overrepresentation in KEGG PATHWAYS (Kyoto Encyclopedia of Genes and Genomes) and GO (gene ontology) terms revealed large differences of expression between visceral and parietal pleura, and both tissues differed from mesothelioma. Cell growth and intrinsic resistance in tumour versus parietal pleura was reflected in highly overexpressed cell cycle, mitosis, replication, DNA repair and anti-apoptosis genes. Several genes of the “salvage pathway” that recycle nucleobases were overexpressed, among them TYMS, encoding thymidylate synthase, the main target of the antifolate drug pemetrexed that is active in mesothelioma. Circadian rhythm genes were expressed in favour of tumour growth. The local invasive, non-metastatic phenotype of mesothelioma, could partly be due to overexpression of the known metastasis suppressors NME1 and NME2. Down-regulation of several tumour suppressor genes could contribute to mesothelioma progression. Genes involved in cell communication were down-regulated, indicating that mesothelioma may shield itself from the immune system. Similarly, in non-cancer parietal versus visceral pleura signal transduction, soluble transporter and adhesion genes were down-regulated. This could represent a genetical platform of the parietal pleura propensity to develop mesothelioma. Conclusions Genome-wide microarray approach using complex human tissue samples revealed novel expression patterns, reflecting some important features of mesothelioma biology that should be further explored. PMID:19662092

K(Ca)3.1 channel downregulation and impaired endothelium-derived hyperpolarization-type relaxation in pulmonary arteries from chronically hypoxic rats.

PubMed

Kroigaard, Christel; Kudryavtseva, Olga; Dalsgaard, Thomas; Wandall-Frostholm, Christine; Olesen, Søren-Peter; Simonsen, Ulf

2013-04-01

Calcium-activated potassium channels of small (K(Ca)2, SK) and intermediate (K(Ca)3.1, IK) conductance are involved in endothelium-dependent relaxation of pulmonary arteries. We hypothesized that the function and expression of K(Ca)2 and K(Ca)3.1 increase as a compensatory mechanism to counteract hypoxia-induced pulmonary hypertension in rats. For functional studies, pulmonary arteries were mounted in microvascular myographs for isometric tension recordings. The K(Ca) channel expression was evaluated by immunoblotting and quantitative PCR. Although ACh induced similar relaxations, the ACh-induced relaxations were abolished by the combined inhibition of nitric oxide synthase (by L-nitro-arginine, L-NOARG), cyclo-oxygenase (by indomethacin) and soluble guanylate cyclase (by ODQ) in pulmonary arteries from hypoxic rats, whereas 20 ± 6% (n = 8) maximal relaxation in response to ACh persisted in arteries from normoxic rats. Inhibiting Na(+),K(+)-ATPase with ouabain or blocking K(Ca)2 and K(Ca)3.1 channels reduced the persisting ACh-induced relaxation. In the presence of L-NOARG and indomethacin, a novel K(Ca)2 and K(Ca)3.1 channel activator, NS4591, induced concentration- and endothelium-dependent relaxations, which were markedly reduced in arteries from chronically hypoxic rats compared with arteries from normoxic rats. The mRNA levels of K(Ca)2.3 and K(Ca)3.1 were unaltered, whereas K(Ca)2.3 protein expression was upregulated and K(Ca)3.1 protein expression downregulated in pulmonary arteries from rats exposed to hypoxia. In conclusion, endothelium-dependent relaxation was conserved in pulmonary arteries from chronically hypoxic rats, while endothelium-derived hyperpolarization (EDH)-type relaxation was impaired in chronically hypoxic pulmonary small arteries despite upregulation of K(Ca)2.3 channels. Since impaired EDH-type relaxation was accompanied by K(Ca)3.1 channel protein downregulation, these findings suggest that K(Ca)3.1 channels are important for the maintenance of EDH-type relaxation.

Adipose differentiation-related protein regulates lipids and insulin in pancreatic islets

PubMed Central

Faleck, D. M.; Ali, K.; Roat, R.; Graham, M. J.; Crooke, R. M.; Battisti, R.; Garcia, E.; Ahima, R. S.

2010-01-01

The excess accumulation of lipids in islets is thought to contribute to the development of diabetes in obesity by impairing β-cell function. However, lipids also serve a nutrient function in islets, and fatty acids acutely increase insulin secretion. A better understanding of lipid metabolism in islets will shed light on complex effects of lipids on β-cells. Adipose differentiation-related protein (ADFP) is localized on the surface of lipid droplets in a wide range of cells and plays an important role in intracellular lipid metabolism. We found that ADFP was highly expressed in murine β-cells. Moreover, islet ADFP was increased in mice on a high-fat diet (3.5-fold of control) and after fasting (2.5-fold of control), revealing dynamic changes in ADFP in response to metabolic cues. ADFP expression was also increased by addition of fatty acids in human islets. The downregulation of ADFP in MIN6 cells by antisense oligonucleotide (ASO) suppressed the accumulation of triglycerides upon fatty acid loading (56% of control) along with a reduction in the mRNA levels of lipogenic genes such as diacylglycerol O-acyltransferase-2 and fatty acid synthase. Fatty acid uptake, oxidation, and lipolysis were also reduced by downregulation of ADFP. Moreover, the reduction of ADFP impaired the ability of palmitate to increase insulin secretion. These findings demonstrate that ADFP is important in regulation of lipid metabolism and insulin secretion in β-cells. PMID:20484013

Transcriptome and Gene Ontology (GO) Enrichment Analysis Reveals Genes Involved in Biotin Metabolism That Affect L-Lysine Production in Corynebacterium glutamicum.

PubMed

Kim, Hong-Il; Kim, Jong-Hyeon; Park, Young-Jin

2016-03-09

Corynebacterium glutamicum is widely used for amino acid production. In the present study, 543 genes showed a significant change in their mRNA expression levels in L-lysine-producing C. glutamicum ATCC21300 than that in the wild-type C. glutamicum ATCC13032. Among these 543 differentially expressed genes (DEGs), 28 genes were up- or downregulated. In addition, 454 DEGs were functionally enriched and categorized based on BLAST sequence homologies and gene ontology (GO) annotations using the Blast2GO software. Interestingly, NCgl0071 (bioB, encoding biotin synthase) was expressed at levels ~20-fold higher in the L-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain. Five other genes involved in biotin metabolism or transport--NCgl2515 (bioA, encoding adenosylmethionine-8-amino-7-oxononanoate aminotransferase), NCgl2516 (bioD, encoding dithiobiotin synthetase), NCgl1883, NCgl1884, and NCgl1885--were also expressed at significantly higher levels in the L-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain, which we determined using both next-generation RNA sequencing and quantitative real-time PCR analysis. When we disrupted the bioB gene in C. glutamicum ATCC21300, L-lysine production decreased by approximately 76%, and the three genes involved in biotin transport (NCgl1883, NCgl1884, and NCgl1885) were significantly downregulated. These results will be helpful to improve our understanding of C. glutamicum for industrial amino acid production.

Rosmanol potently inhibits lipopolysaccharide-induced iNOS and COX-2 expression through downregulating MAPK, NF-kappaB, STAT3 and C/EBP signaling pathways.

PubMed

Lai, Ching-Shu; Lee, Jong Hun; Ho, Chi-Tang; Liu, Cheng Bin; Wang, Ju-Ming; Wang, Ying-Jan; Pan, Min-Hsiung

2009-11-25

Rosmanol is a natural polyphenol from the herb rosemary (Rosmarinus officinalis L.) with high antioxidant activity. In this study, we investigated the inhibitory effects of rosmanol on the induction of NO synthase (NOS) and COX-2 in RAW 264.7 cells induced by lipopolysaccharide (LPS). Rosmanol markedly inhibited LPS-stimulated iNOS and COX-2 protein and gene expression, as well as the downstream products, NO and PGE2. Treatment with rosmanol also reduced translocation of the nuclear factor-kappaB (NF-kappaB) subunits by prevention of the degradation and phosphorylation of inhibitor kappaB (IkappaB). Western blot analysis showed that rosmanol significantly inhibited translocation and phosphorylation of NF-kappaB, signal transducer and activator of transcription-3 (STAT3), and the protein expression of C/EBPbeta and C/EBPdelta. We also found that rosmanol suppressed LPS-induced phosphorylation of ERK1/2, p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling. Our results demonstrate that rosmanol downregulates inflammatory iNOS and COX-2 gene expression by inhibiting the activation of NF-kappaB and STAT3 through interfering with the activation of PI3K/Akt and MAPK signaling. Taken together, rosmanol might contribute to the potent anti-inflammatory effect of rosemary and may have potential to be developed into an effective anti-inflammatory agent.

Phosphodiesterase 4B plays a role in benzophenone-3-induced phototoxicity in normal human keratinocytes.

PubMed

Kim, Hyoung-June; Lee, Eunyoung; Lee, Moonyoung; Ahn, Sungjin; Kim, Jungmin; Liu, Jingjing; Jin, Sun Hee; Ha, Jaehyoun; Bae, Il Hong; Lee, Tae Ryong; Noh, Minsoo

2018-01-01

Benzophenone-3 (BP-3), which is extensively used in organic sunscreen, has phototoxic potential in human skin. Phosphodiesterase 4B (PDE4B) has a well-established role in inflammatory responses in immune cells. Currently, it is unknown if PDE4B is associated with BP-3-induced phototoxicity in normal human keratinocytes (NHKs). We found that BP-3 significantly increased PDE4B expression in ultraviolet B (UVB)-irradiated NHKs. Notably, BP-8, a sunscreen agent that shares the 2-hydroxy-4-methoxyphenyl methanone moiety with BP-3, also upregulated PDE4B expression in NHKs. Upon UVB irradiation, BP-3 upregulated the expression of pro-inflammatory factors, such as prostaglandin endoperoxide synthase 2, tumor necrosis factor α, interleukin 8, and S100A7, and downregulated the level of cornified envelope associated proteins, which are important in the development of the epidermal permeability barrier. The additive effects of UVB-activated BP-3 on the expression of both pro-inflammatory mediators and cornified envelope associated proteins were antagonized by treatment with the PDE4 inhibitor rolipram. The BP-3 and UVB co-stimulation-induced PDE4B upregulation and its association with the upregulation of pro-inflammatory mediators and the downregulation of epidermal differentiation markers were confirmed in a reconstituted three dimensional human epidermis model. Therefore, PDE4B has a role in the mechanism of BP-3-induced phototoxicity. Copyright © 2017 Elsevier Inc. All rights reserved.

Downregulation of dTps1 in Drosophila melanogaster larvae confirms involvement of trehalose in redox regulation following desiccation.

PubMed

Thorat, Leena; Mani, Krishna-Priya; Thangaraj, Pradeep; Chatterjee, Suvro; Nath, Bimalendu B

2016-03-01

As a survival strategy to environmental water deficits, desiccation-tolerant organisms are commonly known for their ability to recruit stress-protective biomolecules such as trehalose. We have previously reported the pivotal role of trehalose in larval desiccation tolerance in Drosophila melanogaster. Trehalose has emerged as a versatile molecule, serving mainly as energy source in insects and also being a stress protectant. While several recent reports have revealed the unconventional role of trehalose in scavenging reactive oxygen species in yeast and plants, this aspect has not received much attention in animals. We examined the status of desiccation-induced generation of reactive oxygen species in D. melanogaster larvae and the possible involvement of trehalose in ameliorating the harmful consequences thereof. Insect trehalose synthesis is governed by the enzyme trehalose 6-phosphate synthase 1 (TPS1). Using the ubiquitous da-GAL4-driven expression of the dTps1-RNAi transgene, we generated dTps1-downregulated Drosophila larvae possessing depleted levels of dTps1 transcripts. This resulted in the inability of the larvae for trehalose synthesis, thereby allowing us to elucidate the significance of trehalose in the regulation of desiccation-responsive redox homeostasis. Furthermore, the results from molecular genetics studies, biochemical assays, electron spin resonance analyses and a simple, non-invasive method of whole larval live imaging suggested that trehalose in collaboration with superoxide dismutase (SOD) is involved in the maintenance of redox state in D. melanogaster.

Statins: novel additions to the dermatologic arsenal?

PubMed

Namazi, M R

2004-06-01

The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins), atorvastatin, cerivastatin, fluvastatin, pravastatin, lovastatin and simvastatin, reduce atherogenesis and cardiovascular morbidity. Besides, there is growing evidence that statins have immunomodulatory activities. Statins downregulate the expression of adhesion molecules, intercellular adhesion molecule-1 (ICAM-1), monocyte chemotactic protein-1 (MAC-1) and lymphocyte function-associated antigen-1 (LFA-1), on leucocytes and endothelial cells and, through binding to LFA-1, interfere with ICAM-1-LFA-1 interaction, which is crucial for activation of lymphocytes by antigen-presenting cells, ingress of leucocytes into the inflammation sites and immunologic cytotoxicity. Statins inhibit the inducible expression of major histocompatibility complex class II in several cell types including macrophages and downregulate the expression of T-helper-1 (Th1) chemokine receptors on T cells, leading further to inhibition of activation of lymphocytes and their infiltration into the inflammation sites. Statins block the induction of inducible nitric oxide synthase and the expression of several proinflammatory cytokines such as tumour necrosis factor-alpha and interferon-gamma in macrophages and possess antioxidant effects. These agents inhibit the proliferation of immunocytes and the activation of natural killer cells. Regarding the above facts and in view of their safety and inexpensiveness, statins may prove invaluable in the treatment of a multiplicity of dermatologic disorders, especially those characterized by ingress of activated leucocytes into the skin, such as alopecia areata, vitiligo, lichen planus, subacute cutaneous lupus erythematosus, erythema multiforme, psoriasis, bullous pemphigoid, systemic sclerosis, mycosis fungoides, toxic epidermal necrolysis and Behcet's disease.

Rosuvastatin Attenuates the Elevation in Blood Pressure Induced by Overexpression of Human C-Reactive Protein

PubMed Central

Li, Xuguang; Yang, Guangtian; Edin, Matthew L.; Zeldin, Darryl C.; Wang, Dao Wen

2014-01-01

Background Our previous studies demonstrated that C-reactive protein (CRP) acts as an inflammatory factor to induce endothelial dysfunction and hypertension in rats. The anti-inflammatory effects of statins suggest that they may attenuate CRP-induced endothelial dysfunction and hypertension in Sprague Dawley (SD) rats. Methods Male SD rats were injected with an adeno-associated virus (AAV) to induce overexpression of human CRP (AAV-hCRP) or GFP control (AAV-GFP). Two months after injection, rats were administered rosuvastatin by daily oral gavage (10 mg/kg) for two additional months. Blood pressure was monitored, serum hCRP concentrations were assessed by ELISA, and vascular levels of endothelial nitric oxide synthase (eNOS), PI3K/Akt, Rho kinase, angiotensin type 1 (AT1) receptor, MAPK, SOD-1, and NADPH oxidase was determined by immunoblotting. Results Rosuvastatin administration attenuated the increased blood pressure and loss of vascular eNOS expression in AAV-hCRP-treated rats. Rosuvastatin also activated PI3K/Akt, inhibited Rho kinase activity, and downregulated the AT1 receptor expression in aorta. Rosuvastatin reduced production of ROS through downregulation of NADPH oxidase subunit p22 phox and gp91 phox, and upregulation of SOD-1 expression. Conclusions Rosuvastatin attenuated the increase in blood pressure in AAV-hCRP-treated rats through endothelial protection and antioxidant effects. Our data reveals a novel mechanism through which statins may lower blood pressure and suggests the potential use of statins in the treatment of hypertension. PMID:21562509

Pure Δ9-tetrahydrocannabivarin and a Cannabis sativa extract with high content in Δ9-tetrahydrocannabivarin inhibit nitrite production in murine peritoneal macrophages.

PubMed

Romano, Barbara; Pagano, Ester; Orlando, Pierangelo; Capasso, Raffaele; Cascio, Maria Grazia; Pertwee, Roger; Marzo, Vincenzo Di; Izzo, Angelo A; Borrelli, Francesca

2016-11-01

Historical and scientific evidence suggests that Cannabis use has immunomodulatory and anti-inflammatory effects. We have here investigated the effect of the non-psychotropic phytocannabinoid Δ 9 -tetrahydrocannabivarin (THCV) and of a Cannabis sativa extract with high (64.8%) content in THCV (THCV-BDS) on nitric oxide (NO) production, and on cannabinoid and transient receptor potential (TRP) channel expression in lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages. THCV-BDS and THCV exhibited similar affinity in radioligand binding assays for CB 1 and CB 2 receptors, and inhibited, via CB 2 but not CB 1 cannabinoid receptors, nitrite production evoked by LPS in peritoneal macrophages. THCV down-regulated the over-expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and interleukin 1β (IL-1β) proteins induced by LPS. Furthermore, THCV counteracted LPS-induced up-regulation of CB 1 receptors, without affecting the changes in CB 2 , TRPV2 or TRPV4 mRNA expression caused by LPS. Other TRP channels, namely, TRPA1, TRPV1, TRPV3 and TRPM8 were poorly expressed or undetectable in both unstimulated and LPS-challenged macrophages. It is concluded that THCV - via CB 2 receptor activation - inhibits nitrite production in macrophages. The effect of this phytocannabinoid was associated with a down-regulation of CB 1 , but not CB 2 or TRP channel mRNA expression. Copyright © 2016 Elsevier Ltd. All rights reserved.

Leonurine exerts anti-inflammatory effect by regulating inflammatory signaling pathways and cytokines in LPS-induced mouse mastitis.

PubMed

Song, Xiaojing; Wang, Tiancheng; Zhang, Zecai; Jiang, Haichao; Wang, Wei; Cao, Yongguo; Zhang, Naisheng

2015-02-01

Bovine mastitis is defined as the inflammation of mammary gland and is the most multiple diseases in dairy cattle. There is still no effective treatment now. Leonurine, extracted from Leonurus cardiaca, has been proved to have anti-inflammatory effect. In the present study, we utilized a mouse mastitis model to study the effect of leonurine on LPS-induced mastitis. Leonurine was administered three times during the 24 h after inducing infection in the mammary gland. The results showed that leonurine significantly alleviated LPS-induced histopathological changes, downregulated the levels of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), upregulated the level of anti-inflammatory cytokine interleukin-10 (IL-10), and inhibited the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Further study revealed that leonurine inhibited the expression of Toll-like receptor 4 (TLR4) and the activation of nuclear factor-kappaB (NF-κB) and the phosphorylation of p38, extracellular signal-regulated kinase (ERK), and Jun N-terminal kinase (JNK). Therefore, the results demonstrated that leonurine could downregulate the expression of TNF-α, IL-6, iNOS, and COX-2 and upregulate the expression of IL-10 mainly by inhibiting the expression of TLR4 and the activation of NF-κB and the phosphorylation of p38, ERK, and JNK. Leonurine may be a potential agent for mastitis therapy.

Application of Fourier transform infrared spectroscopy to biomolecular profiling of cultured fibroblast cells from Gaucher disease patients: A preliminary investigation.

PubMed

Igci, Nasit; Sharafi, Parisa; Demiralp, Duygu Ozel; Demiralp, Cemil Ozerk; Yuce, Aysel; Emre, Serap Dokmeci

2017-10-01

Gaucher disease (GD) is defined as an autosomal recessive disorder resulting from the deficiency of glucocerebrosidase (E.C. 3.2.1.45). Glucocerebrosidase is responsible for the degradation of glucosylceramide into ceramide and glucose. The deficiency of this enzyme results in the accumulation of undegraded glucosylceramide, almost exclusively in macrophages. With Fourier transform infrared (FTIR) spectroscopy, the complete molecular diversity of the samples can be studied comparatively and the amount of the particular materials can be determined. Also, the secondary structure ratios of proteins can be determined by analysing the amide peaks. The primary aim of this study is to introduce FTIR-ATR spectroscopy technique to GD research for the first time in the literature and to assess its potential as a new molecular method. Primary fibroblast cell cultures obtained from biopsy samples were used, since this material is widely used for the diagnosis of GD. Intact cells were placed onto a FTIR-ATR crystal and dried by purging nitrogen gas. Spectra were recorded in the mid-infrared region between 4500-850 cm-1 wavenumbers. Each peak in the spectra was assigned to as organic biomolecules according to their chemical bond information. A quantitative analysis was performed using peak areas and we also used a hierarchical cluster analysis as a multivariate spectral analysis. We obtained FTIR spectra of fibroblast samples and assigned the biomolecule origins of the peaks. We observed individual heterogeneity in FTIR spectra of GD fibroblast samples, confirming the well-known phenotypic heterogeneity in GD at the molecular level. Significant alterations in protein, lipid and carbohydrate levels related to the enzyme replacement therapy were also observed, which is also supported by cluster analysis. Our results showed that the application of FTIR spectroscopy to GD research deserves more attention and detailed studies with an increased sample size in order to evaluate its potential in the diagnosis and follow-up of GD patients.

Resveratrol suppresses growth of cancer stem-like cells by inhibiting fatty acid synthase.

PubMed

Pandey, Puspa R; Okuda, Hiroshi; Watabe, Misako; Pai, Sudha K; Liu, Wen; Kobayashi, Aya; Xing, Fei; Fukuda, Koji; Hirota, Shigeru; Sugai, Tamotsu; Wakabayashi, Go; Koeda, Keisuke; Kashiwaba, Masahiro; Suzuki, Kazuyuki; Chiba, Toshimi; Endo, Masaki; Fujioka, Tomoaki; Tanji, Susumu; Mo, Yin-Yuan; Cao, Deliang; Wilber, Andrew C; Watabe, Kounosuke

2011-11-01

Resveratrol is a natural polyphenolic compound and has been shown to exhibit cardio-protective as well as anti-neoplastic effects on various types of cancers. However, the exact mechanism of its anti-tumor effect is not clearly defined. Resveratrol has been shown to have strong hypolipidemic effect on normal adipocytes and as hyper-lipogenesis is a hallmark of cancer cell physiology, the effect of resveratrol on lipid synthesis in cancer stem-like cells (CD24(-)/CD44(+)/ESA(+)) that were isolated from both ER+ and ER- breast cancer cell lines was examined. The authors found that resveratrol significantly reduced the cell viability and mammosphere formation followed by inducing apoptosis in cancer stem-like cells. This inhibitory effect of resveratrol is accompanied by a significant reduction in lipid synthesis which is caused by the down-regulation of the fatty acid synthase (FAS) gene followed by up-regulation of pro-apoptotic genes, DAPK2 and BNIP3. The activation of apoptotic pathway in the cancer stem-like cells was suppressed by TOFA and by Fumonisin B1, suggesting that resveratrol-induced apoptosis is indeed through the modulation of FAS-mediated cell survival signaling. Importantly, resveratrol was able to significantly suppress the growth of cancer stem-like cells in an animal model of xenograft without showing apparental toxicity. Taken together, the results of this study indicate that resveratrol is capable of inducing apoptosis in the cancer stem-like cells through suppression of lipogenesis by modulating FAS expression, which highlights a novel mechanism of anti-tumor effect of resveratrol.

Resveratrol suppresses growth of cancer stem-like cells by inhibiting fatty acid synthase

PubMed Central

Pandey, Puspa R.; Okuda, Hiroshi; Watabe, Misako; Pai, Sudha K.; Liu, Wen; Kobayashi, Aya; Xing, Fei; Fukuda, Koji; Hirota, Shigeru; Sugai, Tamotsu; Wakabayashi, Go; Koeda, Keisuke; Kashiwaba, Masahiro; Suzuki, Kazuyuki; Chiba, Toshimi; Endo, Masaki; Fujioka, Tomoaki; Tanji, Susumu; Mo, Yin-Yuan; Cao, Deliang; Wilber, Andrew C.; Watabe, Kounosuke

2012-01-01

Resveratrol is a natural polyphenolic compound and has been shown to exhibit cardio-protective as well as anti-neoplastic effects on various types of cancers. However, the exact mechanism of its anti-tumor effect is not clearly defined. Resveratrol has been shown to have strong hypolipidemic effect on normal adipocytes and as hyper-lipogenesis is a hallmark of cancer cell physiology, we examined the effect of resveratrol on lipid synthesis in cancer stem-like cells (CD24−/CD44+/ESA+) that were isolated from both ER+ and ER− breast cancer cell lines. We found that resveratrol significantly reduced the cell viability and mammosphere formation followed by inducing apoptosis in cancer stem-like cells. This inhibitory effect of resveratrol is accompanied by a significant reduction in lipid synthesis which is caused by the down-regulation of the fatty acid synthase (FAS) gene followed by up-regulation of pro-apoptotic genes, DAPK2 and BNIP3. The activation of apoptotic pathway in the cancer stem-like cells was suppressed by TOFA and by Fumonisin B1, suggesting that resveratrol-induced apoptosis is indeed through the modulation of FAS-mediated cell survival signaling. Importantly, resveratrol was able to significantly suppress the growth of cancer stem-like cells in an animal model of xenograft without showing apparental toxicity. Taken together, our results indicate that resveratrol is capable of inducing apoptosis in the cancer stem-like cells through suppression of lipogenesis by modulating FAS expression, which highlights a novel mechanism of anti-tumor effect of resveratrol. PMID:21188630

Focal adhesion kinase-mediated activation of glycogen synthase kinase 3β regulates IL-33 receptor internalization and IL-33 signaling.

PubMed

Zhao, Jing; Wei, Jianxin; Bowser, Rachel K; Traister, Russell S; Fan, Ming-Hui; Zhao, Yutong

2015-01-15

IL-33, a relatively new member of the IL-1 cytokine family, plays a crucial role in allergic inflammation and acute lung injury. Long form ST2 (ST2L), the receptor for IL-33, is expressed on immune effector cells and lung epithelia and plays a critical role in triggering inflammation. We have previously shown that ST2L stability is regulated by the ubiquitin-proteasome system; however, its upstream internalization has not been studied. In this study, we demonstrate that glycogen synthase kinase 3β (GSK3β) regulates ST2L internalization and IL-33 signaling. IL-33 treatment induced ST2L internalization, and an effect was attenuated by inhibition or downregulation of GSK3β. GSK3β was found to interact with ST2L on serine residue 446 in response to IL-33 treatment. GSK3β binding site mutant (ST2L(S446A)) and phosphorylation site mutant (ST2L(S442A)) are resistant to IL-33-induced ST2L internalization. We also found that IL-33 activated focal adhesion kinase (FAK). Inhibition of FAK impaired IL-33-induced GSK3β activation and ST2L internalization. Furthermore, inhibition of ST2L internalization enhanced IL-33-induced cytokine release in lung epithelial cells. These results suggest that modulation of the ST2L internalization by FAK/GSK3β might serve as a unique strategy to lessen pulmonary inflammation. Copyright © 2015 by The American Association of Immunologists, Inc.

MicroRNA and dsRNA targeting chitin synthase A reveal a great potential for pest management of the hemipteran insect Nilaparvata lugens.

PubMed

Li, Tengchao; Chen, Jie; Fan, Xiaobin; Chen, Weiwen; Zhang, Wenqing

2017-07-01

Two RNA silencing pathways in insects are known to exist that are mediated by short interfering RNAs (siRNAs) and microRNAs (miRNAs), which have been hypothesised to be promising methods for insect pest control. However, a comparison between miRNA and siRNA in pest control is still unavailable, particularly in targeting chitin synthase gene A (CHSA). The dsRNA for Nilaparvata lugens CHSA (dsNlCHSA) and the microR-2703 (miR-2703) mimic targeting NlCHSA delivered via feeding affected the development of nymphs, reduced their chitin content and led to lethal phenotypes. The protein level of NlCHSA was downregulated after female adults were injected with dsNlCHSA or the miR-2703 mimic, but there were no significant differences in vitellogenin (NlVg) expression or in total oviposition relative to the control group. However, 90.68 and 46.13% of the eggs laid by the females injected with dsNlCHSA and miR-2703 mimic were unable to hatch, respectively. In addition, a second-generation miRNA and RNAi effect on N. lugens was observed. Ingested miR-2703 seems to be a good option for killing N. lugens nymphs, while NlCHSA may be a promising target for RNAi-based pest management. These findings provide important evidence for applications of small non-coding RNAs (snRNAs) in insect pest management. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Effects of down-regulating ornithine decarboxylase upon putrescine-associated metabolism and growth in Nicotiana tabacum L.

PubMed Central

Dalton, Heidi L.; Blomstedt, Cecilia K.; Neale, Alan D.; Gleadow, Ros; DeBoer, Kathleen D.; Hamill, John D.

2016-01-01

Transgenic plants of Nicotiana tabacum L. homozygous for an RNAi construct designed to silence ornithine decarboxylase (ODC) had significantly lower concentrations of nicotine and nornicotine, but significantly higher concentrations of anatabine, compared with vector-only controls. Silencing of ODC also led to significantly reduced concentrations of polyamines (putrescine, spermidine and spermine), tyramine and phenolamides (caffeoylputrescine and dicaffeoylspermidine) with concomitant increases in concentrations of amino acids ornithine, arginine, aspartate, glutamate and glutamine. Root transcript levels of S-adenosyl methionine decarboxylase, S-adenosyl methionine synthase and spermidine synthase (polyamine synthesis enzymes) were reduced compared with vector controls, whilst transcript levels of arginine decarboxylase (putrescine synthesis), putrescine methyltransferase (nicotine production) and multi-drug and toxic compound extrusion (alkaloid transport) proteins were elevated. In contrast, expression of two other key proteins required for alkaloid synthesis, quinolinic acid phosphoribosyltransferase (nicotinic acid production) and a PIP-family oxidoreductase (nicotinic acid condensation reactions), were diminished in roots of odc-RNAi plants relative to vector-only controls. Transcriptional and biochemical differences associated with polyamine and alkaloid metabolism were exacerbated in odc-RNAi plants in response to different forms of shoot damage. In general, apex removal had a greater effect than leaf wounding alone, with a combination of these injury treatments producing synergistic responses in some cases. Reduced expression of ODC appeared to have negative effects upon plant growth and vigour with some leaves of odc-RNAi lines being brittle and bleached compared with vector-only controls. Together, results of this study demonstrate that ornithine decarboxylase has important roles in facilitating both primary and secondary metabolism in Nicotiana. PMID:27126795

Comparative transcriptomic analysis of key genes involved in flavonoid biosynthetic pathway and identification of a flavonol synthase from Artemisia annua L.

PubMed

Liu, S; Liu, L; Tang, Y; Xiong, S; Long, J; Liu, Z; Tian, N

2017-07-01

The regulatory mechanism of flavonoids, which synergise anti-malarial and anti-cancer compounds in Artemisia annua, is still unclear. In this study, an anthocyanidin-accumulating mutant callus was induced from A. annua and comparative transcriptomic analysis of wild-type and mutant calli performed, based on the next-generation Illumina/Solexa sequencing platform and de novo assembly. A total of 82,393 unigenes were obtained and 34,764 unigenes were annotated in the public database. Among these, 87 unigenes were assigned to 14 structural genes involved in the flavonoid biosynthetic pathway and 37 unigenes were assigned to 17 structural genes related to metabolism of flavonoids. More than 30 unigenes were assigned to regulatory genes, including R2R3-MYB, bHLH and WD40, which might regulate flavonoid biosynthesis. A further 29 unigenes encoding flavonoid biosynthetic enzymes or transcription factors were up-regulated in the mutant, while 19 unigenes were down-regulated, compared with the wild type. Expression levels of nine genes involved in the flavonoid pathway were compared using semi-quantitative RT-PCR, and results were consistent with comparative transcriptomic analysis. Finally, a putative flavonol synthase gene (AaFLS1) was identified from enzyme assay in vitro and in vivo through heterogeneous expression, and confirmed comparative transcriptomic analysis of wild-type and mutant callus. The present work has provided important target genes for the regulation of flavonoid biosynthesis in A. annua. © 2017 German Botanical Society and The Royal Botanical Society of the Netherlands.

New Inducible Nitric Oxide Synthase and Cyclooxygenase-2 Inhibitors, Nalidixic Acid Linked to Isatin Schiff Bases via Certain l-Amino Acid Bridges.

PubMed

Naglah, Ahmed M; Ahmed, Atallah F; Wen, Zhi-Hong; Al-Omar, Mohamed A; Amr, Abd El-Galil E; Kalmouch, Atef

2016-04-15

A series of new Schiff bases were synthesized by condensation of isatins with the nalidixic acid-l-amino acid hydrazides. Prior to hydrazide formation, a peptide linkage has been prepared via coupling of nalidixic acid with appropriate l-amino acid methyl esters to yield 3a-c. The chemical structures of the new Schiff bases (5b and 5d-h) were confirmed by means of IR, NMR, mass spectroscopic, and elemental analyses. The anti-inflammatory activity of these Schiff bases was evaluated via measurement of the expressed inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in the lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells model. The Schiff bases exhibited significant dual inhibitory effect against the induction of the pro-inflammatory iNOS and COX-2 proteins with variable potencies. However, they strongly down-regulated the iNOS expression to the level of 16.5% ± 7.4%-42.2% ± 19.6% compared to the effect on COX-2 expression (<56.4% ± 3.1% inhibition) at the same concentration (10 μM). The higher iNOS inhibition activity of the tested Schiff bases, relative to that of COX-2, seems to be a reflection of the combined suppressive effects exerted by their nalidixic acid, isatins (4a-c), and l-amino acid moieties against iNOS expression. These synthesized nalidixic acid-l-amino acid-isatin conjugates can be regarded as a novel class of anti-inflammatory antibacterial agents.

Lack of Thromboxane Synthase Prevents Hypertension and Fetal Growth Restriction after High Salt Treatment during Pregnancy.

PubMed

Pai, Chen-Hsueh; Yen, Ching-Tzu; Chen, Chie-Pein; Yu, I-Shing; Lin, Shu-Wha; Lin, Shu-Rung

2016-01-01

Preeclampsia (PE) is a potentially fatal pregnancy-related hypertensive disorder characterized by poor placenta development that can cause fetal growth restriction. PE-associated pathologies, including thrombosis, hypertension, and impaired placental development, may result from imbalances between thromboxane A2 (TXA2) and prostacyclin. Low-dose aspirin, which selectively inhibits TXA2 production, is used to prevent high-risk PE. However, the role of TXA2 in aspirin-mediated protective effects in women with PE is not understood fully. In this study, we examined the role of prostanoids in PE using human samples and an induced PE mouse model. We demonstrated that the administration of salted drinking water (2.7% NaCl) to wild-type mice resulted in elevated placental TXA2 synthase (TXAS) and plasma TXA2, but not prostacyclin, levels, which was also found in our clinical PE placenta samples. The high salt-treated wild-type pregnant mice had shown unchanged maternal body weight, hypertension (MAP increase 15 mmHg), and decreased pup weight (~50%) and size (~24%), but these adverse effects were ameliorated in TXAS knockout (KO) mice. Moreover, increased expression of interleukin-1β and downstream phosphorylated-p38-mitogen-activated protein kinase were concordant with apoptosis induction in the placentas of salt water-treated wild-type mice. These alterations were not observed in TXAS KO mice. Together, our data suggest that TXA2 depletion has anti-PE effects due to the prevention of hypertension and placental damage through downregulation of the interleukin-1β pathway.

Boswellia serrata resin extract alleviates azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colon tumorigenesis.

PubMed

Chou, Ya-Chun; Suh, Joon Hyuk; Wang, Yu; Pahwa, Manoj; Badmaev, Vladimir; Ho, Chi-Tang; Pan, Min-Hsiung

2017-09-01

Boswellia serrata (BS) resin is a popular dietary supplement for joint nourishment. In this study, we investigated the chemopreventive effects of dietary BS extract and its impact of gut microbiota on azoxymethane/dextran sulfate sodium (AOM/DSS)-induced colitis-associated colon cancer in mice. Male ICR mice were injected with AOM and 2% DSS via drinking water. The mice were fed with 0.25 or 0.5% BS extract, and colonic tissue were collected at 15 weeks. The main effective components of BS supercritical CO 2 extraction were analyzed by LC-MS/MS are boswellic acids. We found that treatment with BS extract significantly reduce the colonic tumor formation. Western blot and histological analysis revealed that dietary BS extract could markedly reduce the inflammation associated protein levels expression. Furthermore, BS extract reduced cell proliferation via inhibiting phosphorylation level of protein kinase B (Akt), glycogen synthase kinase 3β (GSK3β), and downregulation of cyclin D1. In addition, BS extract also altered the composition of gut microbiota by enhancing the proportion of Clostridiales and reducing the percentage of Bacteroidales. In summary, BS extract decreased the protein levels of inflammative enzymes such as inducible nitric oxide synthase and cyclooxygenase-2 in colonic mucosa. It also mediated Akt/GSK3β/cyclin D1 signaling pathway and altered the composition of gut microbiota to alleviate tumor growth. Taken together, this study suggests that BS extract has great potential to suppress colon tumorigenesis. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Response of Vitis vinifera cell cultures to Eutypa lata and Trichoderma atroviride culture filtrates: expression of defence-related genes and phenotypes.

PubMed

Mutawila, C; Stander, C; Halleen, F; Vivier, M A; Mostert, L

2017-03-01

Cell suspension cultures of Vitis vinifera cv. Dauphine berries were used to study the response to the vascular pathogen, Eutypa lata, in comparison with a biological control agent, Trichoderma atroviride, that was previously shown to be effective in pruning wound protection. The expression of genes coding for enzymes of the phenylpropanoid pathway and pathogenesis-related (PR) proteins was profiled over a 48-h period using quantitative reverse transcriptase PCR. The cell cultures responded to elicitors of both fungi with a hypersensitive-like response that lead to a decrease in cell viability. Similar genes were triggered by both the pathogen and biocontrol agent, but the timing patterns and magnitude of expression was dependent on the specific fungal elicitor. Culture filtrates of both fungi caused upregulation of phenylalanine ammonia-lyase (PAL), 4-coumaroyl Co-A ligase (CCo-A) and stilbene synthase (STS), and a downregulation of chalcone synthase (CHS) genes. The pathogen filtrate caused a biphasic pattern in the upregulation of PAL and STS genes which was not observed in cells treated with filtrates of the biocontrol agent. Analytical assays showed significantly higher total phenolic content and chitinolytic enzyme activity in the cell cultures treated with the T. atroviride filtrate compared to the pathogen filtrate. These results corresponded well to the higher expression of PAL and chitinase class IV genes. The response of the cell cultures to T. atroviride filtrate provides support for the notion that the wound protection by the biocontrol agent at least partially relies on the induction of grapevine resistance mechanisms.

Methane rescues retinal ganglion cells and limits retinal mitochondrial dysfunction following optic nerve crush.

PubMed

Wang, Ruobing; Sun, Qinglei; Xia, Fangzhou; Chen, Zeli; Wu, Jiangchun; Zhang, Yuelu; Xu, Jiajun; Liu, Lin

2017-06-01

Secondary degeneration is a common event in traumatic central nervous system disorders, which involves neuronal apoptosis and mitochondrial dysfunction. Exogenous methane exerts the therapeutic effects in many organ injury. Our study aims to investigate the potential neuroprotection of methane in a rat model of optic nerve crush (ONC). Adult male Sprague-Dawley rats were subjected to ONC and administrated intraperitoneally with methane-saturated or normal saline (10 ml/kg) once per day for one week after ONC. The retinal ganglion cells (RGCs) density was assessed by hematoxylin and eosin staining and Fluoro-Gold retrogradely labeling. Visual function was evaluated by flash visual evoked potentials (FVEP). The retinal apoptosis was measured by terminal-deoxy-transferase-mediated dUTP nick end labeling (TUNEL) assay and the expression of apoptosis-related factors, such as phosphorylated Bcl-2-associated death promoter (pBAD), phosphorylated glycogen synthase kinase-3β (pGSK-3β), Bcl-2 associated X protein (Bax) and Bcl-2 extra large (Bcl-xL). Retinal mitochondrial function was assessed by the mRNA expressions of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM), the mitochondrial DNA (mtDNA) copy number, citrate synthase activity and ATP content. Methane treatment significantly improved the RGC loss and visual dysfunction following ONC. As expected, methane also remarkably inhibited the retinal neural apoptosis, such as the fewer TUNEL-positive cells in ganglion cell layer, accompanied by the up-regulations of anti-apoptotic factors (pGSK-3β, pBAD, Bcl-xL) and the down-regulation of pro-apoptotic factor (Bax). Furthermore, methane treatment suppressed up-regulations of critical mitochondrial components (PGC-1α, NRF1 and TFAM) mRNA and mtDNA copy number, as well as improved the reduction of functional mitochondria markers, including citrate synthase activity and ATP content, in retinas with ONC. Taken together, methane treatment promotes RGC survival and limits retinal mitochondrial dysfunction against ONC insult. Methane can be a potential neuroprotective agent for traumatic and glaucomatous neurodegeneration. Copyright © 2017. Published by Elsevier Ltd.

Oryza sativa (Rice) Hull Extract Inhibits Lipopolysaccharide-Induced Inflammatory Response in RAW264.7 Macrophages by Suppressing Extracellular Signal-regulated Kinase, c-Jun N-terminal Kinase, and Nuclear Factor-κB Activation.

PubMed

Ha, Sang Keun; Sung, Jeehye; Choi, Inwook; Kim, Yoonsook

2016-01-01

Rice ( Oryza sativa ) is a major cereal crop in many Asian countries and an important staple food source. Rice hulls have been reported to possess antioxidant activities. In this study, we evaluated the antiinflammatory effects of rice hull extract and associated signal transduction mechanisms in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. We found that rice hull extract inhibited nitric oxide (NO) and prostaglandin E 2 by suppressing the expression of inducible NO synthase and cyclooxygenase-2, respectively. The release of interleukin-1β and tumor necrosis factor-α was also reduced in a dose-dependent manner. Furthermore, rice hull extract attenuated the activation of nuclear factor-kappa B (NF-κB), as well as the phosphorylation of mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), in LPS-stimulated RAW264.7 cells. This suggests that rice hull extract decreases the production of inflammatory mediators by downregulating ERK and JNK and the NF-κB signal pathway in RAW 264.7 cells. Rice hull extract inhibits the lipopolysaccharide-induced inflammatory response in RAW264.7 macrophages.Rice hull extract inhibited nitric oxide and prostaglandin E 2 by suppressing the expression of inducible NO synthase and cyclooxygenase-2, respectively.Rice hull extract exerted anti-inflammatory effect through inhibition of nuclear factor-kappa B, extracellular signal-regulated kinase and c-Jun N-terminal kinase signaling pathways.Rice hull extract may provide a potential therapeutic approach for inflammatory diseases. Abbreviations used: COX-2: cyclooxygenase-2, ERK: extracellular signal-regulated kinase, IκB: inhibitory kappa B, IL-1β: interleukin-1β, iNOS: inducible NO synthase, JNK: c-Jun N-terminal kinase, LPS: lipopolysaccharide, MAPKs: mitogen-activated protein kinases, NF-κB: nuclear factor-κB, NO: nitric oxide, PGE2: prostaglandin E2, RHE: rice hull extract, ROS: reactive oxygen species, TNF-α: tumor necrosis factor-α.

Proteomic Profiling of Liver and Plasma in Chronic Ethanol Feeding Model of Hepatic Alcohol Dehydrogenase-Deficient Deer Mice.

PubMed

Bhopale, Kamlesh K; Amer, Samir M; Kaphalia, Lata; Soman, Kizhake V; Wiktorowicz, John E; Shakeel Ansari, Ghulam A; Kaphalia, Bhupendra S

2017-10-01

Chronic alcohol abuse, a major risk factor for such diseases as hepatitis and cirrhosis, impairs hepatic alcohol dehydrogenase (ADH; key ethanol [EtOH]-metabolizing enzyme). Therefore, differentially altered hepatic and plasma proteomes were identified in chronic EtOH feeding model of hepatic ADH-deficient (ADH - ) deer mice to understand the metabolic basis of alcoholic liver disease (ALD). ADH - deer mice were fed 3.5 g% EtOH via Lieber-DeCarli liquid diet daily for 3 months and histology of the liver assessed. Liver and plasma proteins were separated by 2-dimensional gel electrophoresis. The proteins differentially expressed were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Histology of the liver showed panlobular steatosis and infiltration of T lymphocytes. Using the criteria of ≥1.5 for fold change (p-value ≤0.05) with expectation value (E ≤10 -3 ) and protein score (≥64), 18 proteins in the livers and 5 in the plasma of EtOH-fed mice were differentially expressed and identified. Prolyl 4-hydroxylase, cytochrome b-5, endo A cytokeratin, ATP synthase, heat-shock 70 kD proteins, enoyl CoA hydratase, stress-70 protein, peroxiredoxin 1, and ornithine carbamoyl transferase were up-regulated in the livers. However, carbonic anhydrase 3, mitochondrial ATP synthase, aldolase 2, actin γ, laminin receptor, and carbamoyl phosphate synthase were down-regulated. Contrary to the increased expression of creatine kinase M-type, a decreased expression of serine protease inhibitor A3A precursor, sulfated glycoprotein-2 (clusterin), and apolipoprotein E isoforms were found in the plasma of EtOH group. Chronic EtOH feeding in ADH - deer mice causes steatosis and infiltration of T lymphocytes in the livers along with increased expression of proteins involved in endoplasmic reticulum (ER) stress, fibrosis, fatty acid β oxidation and biogenesis, and decreased expression of proteins involved in ATP synthesis, carbohydrate metabolism, in cell regulation and architecture. Reduced expression of various carrier proteins as found in the plasma of EtOH group has a biomarker potential. Copyright © 2017 by the Research Society on Alcoholism.

Priming of seeds with methyl jasmonate induced resistance to hemi-biotroph Fusarium oxysporum f.sp. lycopersici in tomato via 12-oxo-phytodienoic acid, salicylic acid, and flavonol accumulation.

PubMed

Król, P; Igielski, R; Pollmann, S; Kępczyńska, E

2015-05-01

Methyl jasmonate (MeJA) was tested by seed treatment for its ability to protect tomato seedlings against fusarium wilt caused by the soil-borne fungal pathogen Fusarium oxysporum f.sp. lycopersici. Isolated from Solanum lycopersicon L. seeds, cv. Beta fungus was identified as F. oxysporum f.sp. lycopersici Race 3 fungus by using phytopathological and molecular methods. MeJA applied at 0.01, 0.1 and 1 mM reduced spore germination and mycelial growth in vitro. Soaking of tomato seeds in MeJA solution at 0.1 mM for 1 h significantly enhanced the resistance level against the tested fungus in tomato seedlings 4 weeks after inoculation. The extracts from leaves of 15-day-old seedlings obtained from previously MeJA soaked seeds had the ability to inhibit in vitro spore germination of tested fungus. In these seedlings a significant increase in the levels phenolic compounds such as salicylic acid (SA), kaempferol and quercetin was observed. Up-regulation of phenylalanine ammonia-lyase (PAL5) and benzoic acid/salicylic acid carboxyl methyltransferase (BSMT) genes and down-regulation of the isochorysmate synthase (ICS) gene in response to exogenous MeJA application indicate that the phenylalanine ammonia-lyase (PAL), not the isochorismate (IC) pathway, is the primary route for SA production in tomato. Moreover, the increased accumulation of the flavonols quercetin and kaempferol appears closely related to the increase of PAL5, chalcone synthase (CHS) and flavonol synthase/flavanone 3-hydroxylase-like (FLS) genes. Elevated levels of salicylic acid in seedlings raised from MeJA-soaked seeds were simultaneously accompanied by a decrease of jasmonic acid, the precursor of MeJA, and an increase of 12-oxo-phytodienoic acid (OPDA), the precursor of jasmonic acid. The present results indicate that the priming of tomato seeds with 0.1mM MeJA before sowing enables the seedlings grown from these seeds to reduce the attack of the soil-borne fungal pathogen F. oxysporum f.sp. lycopersici, so it can be applied in practice. Copyright © 2015 Elsevier GmbH. All rights reserved.

Functional characterization of nine Norway Spruce TPS genes and evolution of gymnosperm terpene synthases of the TPS-d subfamily.

PubMed

Martin, Diane M; Fäldt, Jenny; Bohlmann, Jörg

2004-08-01

Constitutive and induced terpenoids are important defense compounds for many plants against potential herbivores and pathogens. In Norway spruce (Picea abies L. Karst), treatment with methyl jasmonate induces complex chemical and biochemical terpenoid defense responses associated with traumatic resin duct development in stems and volatile terpenoid emissions in needles. The cloning of (+)-3-carene synthase was the first step in characterizing this system at the molecular genetic level. Here we report the isolation and functional characterization of nine additional terpene synthase (TPS) cDNAs from Norway spruce. These cDNAs encode four monoterpene synthases, myrcene synthase, (-)-limonene synthase, (-)-alpha/beta-pinene synthase, and (-)-linalool synthase; three sesquiterpene synthases, longifolene synthase, E,E-alpha-farnesene synthase, and E-alpha-bisabolene synthase; and two diterpene synthases, isopimara-7,15-diene synthase and levopimaradiene/abietadiene synthase, each with a unique product profile. To our knowledge, genes encoding isopimara-7,15-diene synthase and longifolene synthase have not been previously described, and this linalool synthase is the first described from a gymnosperm. These functionally diverse TPS account for much of the structural diversity of constitutive and methyl jasmonate-induced terpenoids in foliage, xylem, bark, and volatile emissions from needles of Norway spruce. Phylogenetic analyses based on the inclusion of these TPS into the TPS-d subfamily revealed that functional specialization of conifer TPS occurred before speciation of Pinaceae. Furthermore, based on TPS enclaves created by distinct branching patterns, the TPS-d subfamily is divided into three groups according to sequence similarities and functional assessment. Similarities of TPS evolution in angiosperms and modeling of TPS protein structures are discussed.

Distribution of Callose Synthase, Cellulose Synthase, and Sucrose Synthase in Tobacco Pollen Tube Is Controlled in Dissimilar Ways by Actin Filaments and Microtubules1[W

PubMed Central

Cai, Giampiero; Faleri, Claudia; Del Casino, Cecilia; Emons, Anne Mie C.; Cresti, Mauro

2011-01-01

Callose and cellulose are fundamental components of the cell wall of pollen tubes and are probably synthesized by distinct enzymes, callose synthase and cellulose synthase, respectively. We examined the distribution of callose synthase and cellulose synthase in tobacco (Nicotiana tabacum) pollen tubes in relation to the dynamics of actin filaments, microtubules, and the endomembrane system using specific antibodies to highly conserved peptide sequences. The role of the cytoskeleton and membrane flow was investigated using specific inhibitors (latrunculin B, 2,3-butanedione monoxime, taxol, oryzalin, and brefeldin A). Both enzymes are associated with the plasma membrane, but cellulose synthase is present along the entire length of pollen tubes (with a higher concentration at the apex) while callose synthase is located in the apex and in distal regions. In longer pollen tubes, callose synthase accumulates consistently around callose plugs, indicating its involvement in plug synthesis. Actin filaments and endomembrane dynamics are critical for the distribution of callose synthase and cellulose synthase, showing that enzymes are transported through Golgi bodies and/or vesicles moving along actin filaments. Conversely, microtubules appear to be critical in the positioning of callose synthase in distal regions and around callose plugs. In contrast, cellulose synthases are only partially coaligned with cortical microtubules and unrelated to callose plugs. Callose synthase also comigrates with tubulin by Blue Native-polyacrylamide gel electrophoresis. Membrane sucrose synthase, which expectedly provides UDP-glucose to callose synthase and cellulose synthase, binds to actin filaments depending on sucrose concentration; its distribution is dependent on the actin cytoskeleton and the endomembrane system but not on microtubules. PMID:21205616

Mild thrombocytopenia as presenting symptom of type 1 Gauchers's disease.

PubMed

Müzes, G; Pitlik, E; Somogyi, A; Tulassay, Z

2001-06-01

A young woman was examined for a mild thrombocytopenia which was present for some months. No signs of bleeding had so far occurred. Physical examination was normal except for a moderately enlarged spleen. Laboratory investigations showed a low platelet count. There was no evidence of an autoimmune or hematologic disease. Bone narrow aspirate indicated Gaucher's-like cells raising the suspicion of Gaucher's disease. This was further supported by electron microscopic demonstration of Gaucher's bodies in crista biopsy specimens. However, the definitive diagnosis was obtained by verifying deficient lysosomal glucosylceramide-beta-D-glucosidase activity in peripheral blood leukocytes. Upon the absence of neurologic involvement the patient was typical for the adult-onset or type 1 form of Gaucher's disease.

Electromagnetic Radiation Disturbed the Photosynthesis of Microcystis aeruginosa at the Proteomics Level.

PubMed

Tang, Chao; Yang, Chuanjun; Yu, Hui; Tian, Shen; Huang, Xiaomei; Wang, Weiyi; Cai, Peng

2018-01-11

Photosynthesis of Microcystis aeruginosa under Electromagnetic Radiation (1.8 GHz, 40 V/m) was studied by using the proteomics. A total of 30 differentially expressed proteins, including 15 up-regulated and 15 down-regulated proteins, were obtained in this study. The differentially expressed proteins were significantly enriched in the photosynthesis pathway, in which the protein expression levels of photosystems II cytochrome b559 α subunit, cytochrome C550, PsbY, and F-type ATP synthase (a, b) decreased. Our results indicated that electromagnetic radiation altered the photosynthesis-related protein expression levels, and aimed at the function of photosynthetic pigments, photosystems II potential activity, photosynthetic electron transport process, and photosynthetic phosphorylation process of M. aeruginosa. Based on the above evidence, that photoreaction system may be deduced as a target of electromagnetic radiation on the photosynthesis in cyanobacteria; the photoreaction system of cyanobacteria is a hypothetical "shared target effector" that responds to light and electromagnetic radiation; moreover, electromagnetic radiation does not act on the functional proteins themselves but their expression processes.

PSD-95 promotes synaptogenesis and multiinnervated spine formation through nitric oxide signaling.

PubMed

Nikonenko, Irina; Boda, Bernadett; Steen, Sylvain; Knott, Graham; Welker, Egbert; Muller, Dominique

2008-12-15

Postsynaptic density 95 (PSD-95) is an important regulator of synaptic structure and plasticity. However, its contribution to synapse formation and organization remains unclear. Using a combined electron microscopic, genetic, and pharmacological approach, we uncover a new mechanism through which PSD-95 regulates synaptogenesis. We find that PSD-95 overexpression affected spine morphology but also promoted the formation of multiinnervated spines (MISs) contacted by up to seven presynaptic terminals. The formation of multiple contacts was specifically prevented by deletion of the PDZ(2) domain of PSD-95, which interacts with nitric oxide (NO) synthase (NOS). Similarly, PSD-95 overexpression combined with small interfering RNA-mediated down-regulation or the pharmacological blockade of NOS prevented axon differentiation into varicosities and multisynapse formation. Conversely, treatment of hippocampal slices with an NO donor or cyclic guanosine monophosphate analogue induced MISs. NOS blockade also reduced spine and synapse density in developing hippocampal cultures. These results indicate that the postsynaptic site, through an NOS-PSD-95 interaction and NO signaling, promotes synapse formation with nearby axons.

Proteomic analysis of protective effects of polysaccharides from Salvia miltiorrhiza against immunological liver injury in mice.

PubMed

Sun, Xue-Gang; Fu, Xiu-Qiong; Cai, Hong-Bing; Liu, Qiang; Li, Chun-Hua; Liu, Ya-Wei; Li, Ying-Jia; Liu, Zhi-Feng; Song, Yu-Hong; Lv, Zhi-Ping

2011-07-01

This study was designed to investigate mechanisms of the protective effects of Salvia miltiorrhiza polysaccharide (SMPS) against lipopolysaccharide (LPS)-induced immunological liver injury (ILI) in Bacille Calmette-Guérin (BCG)-primed mice. Two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis showed that three proteins are down-regulated and six proteins are up-regulated by SMPS. SMPS reduces the degree of liver injury by up-regulating the enzymes of the citric acid cycle, namely malate dehydrogenase (MDH) and 2-oxoglutarate dehydrogenase complex. LPS significantly increases nuclear factor kappa B (NF-κB) activation, inducible nitric oxide synthase (iNOS) expression and MDA level in BCG primed mice liver, whereas SMPS treatment protects against the immunological liver injury through inhibition of the NF-κB activation by up-regulation of PRDX6 and the subsequent attenuation of lipid peroxidation, iNOS expression and inflammation. Copyright © 2011 John Wiley & Sons, Ltd.

Spatial and Temporal Localization of Flavonoid Metabolites in Strawberry Fruit (Fragaria × ananassa).

PubMed

Crecelius, Anna C; Hölscher, Dirk; Hoffmann, Thomas; Schneider, Bernd; Fischer, Thilo C; Hanke, Magda-Viola; Flachowsky, Henryk; Schwab, Wilfried; Schubert, Ulrich S

2017-05-03

Flavonoids are important metabolites in strawberries (Fragaria × ananassa) because they accomplish an extensive collection of physiological functions and are valuable for human health. However, their localization within the fruit tissue has not been extensively explored. Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) was employed to shed light on the spatial distribution of flavonoids during fruit development. One wild-type (WT) and two transgenic lines were compared, wherein the transgenic enzymes anthocyanidin reductase (ANRi) and flavonol synthase (FLSi), respectively, were down-regulated using an RNAi-based silencing approach. In most cases, fruit development led to a reduction of the investigated flavonoids in the fruit tissue; as a consequence, they were exclusively present in the skin of mature red fruits. In the case of (epi)catechin dimer, both the ANRi and the WT phenotypes revealed low levels in mature red fruits, whereas the ANRi line bore the lowest relative concentration, as analyzed by liquid chromatography-electrospray ionization multiple-step mass spectrometry (LC-ESI-MS n ).

Multiple ketolases involved in light regulation of canthaxanthin biosynthesis in Nostoc punctiforme PCC 73102.

PubMed

Schöpf, Lotte; Mautz, Jürgen; Sandmann, Gerhard

2013-05-01

In the genome of Nostoc punctiforme PCC 73102, three functional β-carotene ketolase genes exist, one of the crtO and two of the crtW type. They were all expressed and their corresponding enzymes were functional inserting 4-keto groups into β-carotene as shown by functional pathway complementation in Escherichia coli. They all synthesized canthaxanthin but with different efficiencies. Canthaxanthin is the photoprotective carotenoid of N. punctiforme PCC 73102. Under high-light stress, its synthesis was enhanced. This was caused by up-regulation of the transcripts of two genes in combination. The first crtB-encoding phytoene synthase is the gate way enzyme of carotenogenesis resulting in an increased inflow into the pathway. The second was the ketolase gene crtW148 which in high light takes over β-carotene conversion into canthaxanthin from the other ketolases. The other ketolases were down-regulated under high-light conditions. CrtW148 was also exclusively responsible for the last step in 4-keto-myxoxanthophyll synthesis.

Crosstalk between EET and HO-1 downregulates Bach1 and adipogenic marker expression in mesenchymal stem cell derived adipocytes

PubMed Central

Vanella, Luca; Kim, Dong Hyun; Sodhi, Komal; Barbagallo, Ignazio; Burgess, Angela P.; Falck, John R.; Schwartzman, Michal L.; Abraham, Nader G.

2013-01-01

Epoxygenase activity and synthesis of epoxyeicosatrienoic acids (EETs) have emerged as important modulators of obesity and diabetes. We examined the effect of the EET-agonist 12-(3-hexylureido)dodec-8(2) enoic acid on mesenchymal stem cell (MSC) derived adipocytes proliferation and differentiation. MSCs expressed substantial levels of EETs and inhibition of soluble epoxide hydrolase (sEH) increased the level of EETs and decreased adipogenesis. EET agonist treatment increased HO-1 expression by inhibiting a negative regulator of HO-1 expression, Bach-1. EET treatment also increased βcatenin and pACC levels while decreasing PPARγ C/EBPα and fatty acid synthase levels. These changes were manifested by a decrease in the number of large inflammatory adipocytes, TNFα, IFNγ and IL-1α, but an increase in small adipocytes and in adiponectin levels. In summary, EET agonist treatment inhibits adipogenesis and decreases the levels of inflammatory cytokines suggesting the potential action of EETs as intracellular lipid signaling modulators of adipogenesis and adiponectin. PMID:21821145

β-arrestin is critical for early shear stress-induced Akt/eNOS activation in human vascular endothelial cells.

PubMed

Carneiro, Ana Paula; Fonseca-Alaniz, Miriam Helena; Dallan, Luís Alberto Oliveira; Miyakawa, Ayumi Aurea; Krieger, Jose Eduardo

2017-01-29

Recent evidence suggests that β-arrestins, which are involved in G protein-coupled receptors desensitization, may influence mechanotransduction. Here, we observed that nitric oxide (NO) production was abrogated in human saphenous vein endothelial cells (SVECs) transfected with siRNA against β-arrestin 1 and 2 subjected to shear stress (SS, 15 dynes/cm 2 , 10 min). The downregulation of β-arrestins 1/2 in SVECs cells also prevented the SS-induced rise in levels of phosphorylation of Akt and endothelial nitric oxide synthase (eNOS, Serine 1177). Interestingly, immunoprecipitation revealed that β-arrestin interacts with Akt, eNOS and caveolin-1 and these interactions are not influenced by SS. Our data indicate that β-arrestins and Akt/eNOS downstream signaling are required for early SS-induced NO production in SVECs, which is consistent with the idea that β-arrestins and caveolin-1 are part of a pre-assembled complex associated with the cellular mechanotransduction machinery. Copyright © 2017 Elsevier Inc. All rights reserved.

Transcriptomic biomarkers of altered erythropoiesis to detect autologous blood transfusion.

PubMed

Salamin, Olivier; Mignot, Jonathan; Kuuranne, Tiia; Saugy, Martial; Leuenberger, Nicolas

2018-03-01

Autologous blood transfusion is a powerful means of improving performance and remains one of the most challenging methods to detect. Recent investigations have identified 3 candidate reticulocytes genes whose expression was significantly influenced by blood transfusion. Using quantitative reverse transcription polymerase chain reaction as an alternative quantitative method, the present study supports that delta-aminolevulinate synthase 2 (ALAS2), carbonic anhydrase (CA1), and solute carrier family 4 member 1 (SLC4A1) genes are down-regulated post-transfusion. The expression of these genes exhibited stronger correlation with immature reticulocyte fraction than with reticulocytes percentage. Moreover, the repression of reticulocytes' gene expression was more pronounced than the diminution of immature reticulocyte fraction and reticulocyte percentage following blood transfusion. It suggests that the 3 candidate genes are reliable predictors of bone marrow's response to blood transfusion and that they represent potential biomarkers for the detection of this method prohibited in sports. Copyright © 2017 John Wiley & Sons, Ltd.

Anti-inflammatory effects of an ethanolic extract of guava (Psidium guajava L.) leaves in vitro and in vivo.

PubMed

Jang, Mi; Jeong, Seung-Weon; Cho, Somi K; Ahn, Kwang Seok; Lee, Jong Hyun; Yang, Deok Chun; Kim, Jong-Chan

2014-06-01

Plant extracts have been used as a source of medicines for a wide variety of human ailments. Among the numerous traditional medicinal herbs, Psidium guajava L. (Myrtaceae), commonly known as guava, has long been used in folk medicines as a therapeutic agent for the treatment of numerous diseases in East Asian and other countries. The aim of this study was to investigate the anti-inflammatory activity of an ethanolic leaf extract of P. guajava (guava) in vitro and in vivo. Our results demonstrated that guava leaf extract (GLE) significantly inhibited lipopolysaccharide (LPS)-induced production of nitric oxide and prostaglandin E2 in a dose-dependent manner. GLE suppressed the expression and activity of both inducible nitric oxide synthase and cyclooxygenase-2 in part through the downregulation of ERK1/2 activation in RAW264.7 macrophages. Furthermore, GLE exhibited significant anti-inflammatory activity in 2 different animal models-Freund's complete adjuvant-induced hyperalgesia in the rat and LPS-induced endotoxic shock in mice.

2-Furaldehyde diethyl acetal from tender coconut water (Cocos nucifera) attenuates biofilm formation and quorum sensing-mediated virulence of Chromobacterium violaceum and Pseudomonas aeruginosa.

PubMed

Sethupathy, Sivasamy; Nithya, Chari; Pandian, Shunmugiah Karutha

2015-01-01

The aim of this study was to evaluate the anti-biofilm and quorum sensing inhibitory (QSI) potential of tender coconut water (TCW) against Chromobacterium violaceum and Pseudomonas aeruginosa. TCW significantly inhibited the QS regulated violacein, virulence factors and biofilm production without affecting their growth. qRT-PCR analysis revealed the down-regulation of autoinducer synthase, transcriptional regulator and virulence genes. Mass-spectrometric analysis of a petroleum ether extract of the TCW hydrolyte revealed that 2-furaldehyde diethyl acetal (2FDA) and palmitic acid (PA) are the major compounds. In vitro bioassays confirmed the ability of 2FDA to inhibit the biofilm formation and virulence factors. In addition, the combination of PA with 2FDA resulted in potent inhibition of biofilm formation and virulence factors. The results obtained strongly suggest that TCW can be exploited as a base for designing a novel antipathogenic drug formulation to treat biofilm mediated infections caused by P. aeruginosa.

Transcriptional regulation of lycopene metabolism mediated by rootstock during the ripening of grafted watermelons.

PubMed

Kong, Qiusheng; Yuan, Jingxian; Gao, Lingyun; Liu, Peng; Cao, Lei; Huang, Yuan; Zhao, Liqiang; Lv, Huifang; Bie, Zhilong

2017-01-01

Rootstocks have comprehensive effects on lycopene accumulation in grafted watermelon fruits. However, little is known about lycopene metabolic regulation in grafted watermelon. To address this problem, parallel changes in lycopene contents and the expression of its metabolic genes were analyzed during the fruit ripening of nongrafted watermelon and watermelon grafted onto bottle gourd, pumpkin, and wild watermelon. Results showed that rootstocks mediated the transcriptional regulations of lycopene accumulation in different ways. Bottle gourd and wild watermelon promoted lycopene accumulation in grafted watermelon fruits by upregulating the biosynthetic genes phytoene synthase (PSY) and ζ-carotene desaturase (ZDS), and downregulating the catabolic genes β-carotene hydroxylase (CHYB), zeaxanthin epoxidase (ZEP), 9-cis-epoxycarotenoid dioxygenase (NCED), and carotenoid cleavage dioxygenase (CCD). However, pumpkin did not affect lycopene accumulation by upregulating both biosynthetic and catabolic genes. The rootstock-dependent characteristic of lycopene accumulation in grafted watermelon fruits provided an alternative model for investigating lycopene metabolic regulation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Hydrogen Sulfide as a Potential Therapeutic Target in Fibrosis

PubMed Central

Zhang, Shufang; Pan, Chuli; Zhou, Feifei; Yuan, Zhi; Wang, Huiying; Cui, Wei; Zhang, Gensheng

2015-01-01

Hydrogen sulfide (H2S), produced endogenously by the activation of two major H2S-generating enzymes (cystathionine β-synthase and cystathionine γ-lyase), plays important regulatory roles in different physiologic and pathologic conditions. The abnormal metabolism of H2S is associated with fibrosis pathogenesis, causing damage in structure and function of different organs. A number of in vivo and in vitro studies have shown that both endogenous H2S level and the expressions of H2S-generating enzymes in plasma and tissues are significantly downregulated during fibrosis. Supplement with exogenous H2S mitigates the severity of fibrosis in various experimental animal models. The protective role of H2S in the development of fibrosis is primarily attributed to its antioxidation, antiapoptosis, anti-inflammation, proangiogenesis, and inhibition of fibroblasts activities. Future studies might focus on the potential to intervene fibrosis by targeting the pathway of endogenous H2S-producing enzymes and H2S itself. PMID:26078809

Isoprene synthase genes form a monophyletic clade of acyclic terpene synthases in the TPS-B terpene synthase family.

PubMed

Sharkey, Thomas D; Gray, Dennis W; Pell, Heather K; Breneman, Steven R; Topper, Lauren

2013-04-01

Many plants emit significant amounts of isoprene, which is hypothesized to help leaves tolerate short episodes of high temperature. Isoprene emission is found in all major groups of land plants including mosses, ferns, gymnosperms, and angiosperms; however, within these groups isoprene emission is variable. The patchy distribution of isoprene emission implies an evolutionary pattern characterized by many origins or many losses. To better understand the evolution of isoprene emission, we examine the phylogenetic relationships among isoprene synthase and monoterpene synthase genes in the angiosperms. In this study we identify nine new isoprene synthases within the rosid angiosperms. We also document the capacity of a myrcene synthase in Humulus lupulus to produce isoprene. Isoprene synthases and (E)-β-ocimene synthases form a monophyletic group within the Tps-b clade of terpene synthases. No asterid genes fall within this clade. The chemistry of isoprene synthase and ocimene synthase is similar and likely affects the apparent relationships among Tps-b enzymes. The chronology of rosid evolution suggests a Cretaceous origin followed by many losses of isoprene synthase over the course of evolutionary history. The phylogenetic pattern of Tps-b genes indicates that isoprene emission from non-rosid angiosperms likely arose independently. © 2012 The Author(s). Evolution© 2012 The Society for the Study of Evolution.

Modulations in primary and secondary metabolic pathways and adjustment in physiological behaviour of Withania somnifera under drought stress.

PubMed

Singh, Ruchi; Gupta, Pankhuri; Khan, Furqan; Singh, Susheel Kumar; Sanchita; Mishra, Tripti; Kumar, Anil; Dhawan, Sunita Singh; Shirke, Pramod Arvind

2018-07-01

In general medicinal plants grown under water limiting conditions show much higher concentrations of secondary metabolites in comparison to control plants. In the present study, Withania somnifera plants were subjected to water stress and data related to drought tolerance phenomenon was collected and a putative mechanistic concept considering growth responses, physiological behaviour, and metabolite content and gene expression aspects is presented. Drought induced metabolic and physiological responses as well as drastic decrease in CO 2 uptake due to stomatal limitations. As a result, the consumption of reduction equivalents (NADPH 2+ ) for CO 2 assimilation via the calvin cycle declines significantly resulting in the generation of a large oxidative stress and an oversupply of antioxidant enzymes. Drought also results in the shifting of metabolic processes towards biosynthetic activities that consume reduction equivalents. Thus, biosynthesis of reduced compounds (isoprenoids, phenols and alkaloids) is enhanced. The dynamics of various metabolites have been discussed in the light of gene expression analysis of control and drought treated leaves. Gene encoding enzymes of pathways leading to glucose, fructose and fructan production, conversion of triose phosphates to hexoses and hexose phosphorylation were up-regulated in the drought stressed leaves. The down-regulated Calvin cycle genes were co-ordinately regulated with the down-regulation of chloroplast triosephosphate/phosphate translocator, cytoplasmic fructose-1,6-bisphosphate aldolase and fructose bisphosphatase. Expression of gene encoding Squalene Synthase (SQS) was highly upregulated under drought stress which is responsible for the diversion of carbon flux towards withanolides biosynthesis from isoprenoid pathway. Copyright © 2018 Elsevier B.V. All rights reserved.

Rb-Raf-1 interaction disruptor RRD-251 induces apoptosis in metastatic melanoma cells and synergizes with dacarbazine

PubMed Central

Singh, Sandeep; Davis, Rebecca; Alamanda, Vignesh; Pireddu, Roberta; Pernazza, Daniel; Sebti, Said; Lawrence, Nicholas; Chellappan, Srikumar

2010-01-01

Metastatic melanoma is an aggressive cancer with very low response rate against conventional chemotherapeutic agents such as dacarbazine (DTIC). Inhibitor of Rb-Raf-1 interaction (RRD-251) was tested against the melanoma cell lines SK-MEL-28, SK-MEL-5 and SK-MEL-2. RRD-251 was found to be a potent inhibitor of melanoma cell proliferation, irrespective of V600E B-Raf mutation status of the cell lines. In a SK-MEL-28 xenograft experiment, RRD-251 exerted a significant suppression of tumor growth compared to vehicle (p=0.003). Similar to in vitro effects, tumors from RRD-251 treated animals showed decreased Rb-Raf-1 interaction in vivo. Growth suppressive effects of RRD-251 were associated with induction of apoptosis as well as a G1 arrest, with an accompanying decrease in S-phase cells. RRD-251 inhibited Rb phosphorylation, and downregulated E2F1 protein levels in these cells. Real-time PCR analysis showed that RRD-251 caused downregulation of cell cycle regulatory genes thymidylate synthase (TS) and cdc6 as well as anti-apoptotic gene Mcl-1. Combinatorial treatment of RRD-251 and DTIC resulted in a significantly higher apoptosis in DTIC resistant cell lines SK-MEL-28 and SK-MEL-5, as revealed by increased Caspase-3 activity and PARP cleavage. Since aberrant Rb/E2F pathway is associated with melanoma progression and resistance to apoptosis, these results suggest that the Rb-Raf-1 inhibitor could be an effective agent for melanoma treatment, either alone or in combination with DTIC. PMID:21139044

Rb-Raf-1 interaction disruptor RRD-251 induces apoptosis in metastatic melanoma cells and synergizes with dacarbazine.

PubMed

Singh, Sandeep; Davis, Rebecca; Alamanda, Vignesh; Pireddu, Roberta; Pernazza, Daniel; Sebti, Said; Lawrence, Nicholas; Chellappan, Srikumar

2010-12-01

Metastatic melanoma is an aggressive cancer with very low response rate against conventional chemotherapeutic agents such as dacarbazine (DTIC). Inhibitor of Rb-Raf-1 interaction RRD-251 was tested against the melanoma cell lines SK-MEL-28, SK-MEL-5, and SK-MEL-2. RRD-251 was found to be a potent inhibitor of melanoma cell proliferation, irrespective of V600E B-Raf mutation status of the cell lines. In a SK-MEL-28 xenograft experiment, RRD-251 exerted a significant suppression of tumor growth compared with vehicle (P = 0.003). Similar to in vitro effects, tumors from RRD-251-treated animals showed decreased Rb-Raf-1 interaction in vivo. Growth suppressive effects of RRD-251 were associated with induction of apoptosis as well as a G(1) arrest, with an accompanying decrease in S-phase cells. RRD-251 inhibited Rb phosphorylation and downregulated E2F1 protein levels in these cells. Real-time PCR analysis showed that RRD-251 caused downregulation of cell-cycle regulatory genes thymidylate synthase (TS) and cdc6 as well as the antiapoptotic gene Mcl-1. Combinatorial treatment of RRD-251 and DTIC resulted in a significantly higher apoptosis in DTIC resistant cell lines SK-MEL-28 and SK-MEL-5, as revealed by increased caspase-3 activity and PARP cleavage. Because aberrant Rb/E2F pathway is associated with melanoma progression and resistance to apoptosis, these results suggest that the Rb-Raf-1 inhibitor could be an effective agent for melanoma treatment, either alone or in combination with DTIC. ©2010 AACR.

Withaferin A inhibits iNOS expression and nitric oxide production by Akt inactivation and down-regulating LPS-induced activity of NF-kappaB in RAW 264.7 cells.

PubMed

Oh, Jung Hwa; Lee, Tae-Jin; Park, Jong-Wook; Kwon, Taeg Kyu

2008-12-03

Induction of inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production is thought to have beneficial immunomodulatory effects in acute and chronic inflammatory disorders. In Raw 264.7 cells stimulated with lipopolysaccharide (LPS) to mimic inflammation, withaferin A inhibited LPS-induced expression of both iNOS protein and mRNA in a dose-dependent manner. To investigate the mechanism by which withaferin A inhibits iNOS gene expression, we examined activation of mitogen-activated protein kinases (MAPKs) and Akt in Raw 264.7 cells. We did not observe any significant changes in the phosphorylation of p38 MAPK in cells treated with LPS alone or LPS plus withaferin A. However, LPS-induced Akt phosphorylation was markedly inhibited by withaferin A, while the phosphorylation of p42/p44 extracellular signal-regulated kinases (ERKs) was slightly inhibited by withaferin A treatment. Withaferin A prevented IkappaB phosphorylation, blocking the subsequent nuclear translocation of nuclear factor-kappaB (NF-kappaB) and inhibiting its DNA binding activity. LPS-induced p65 phosphorylation, which is mediated by extracellular signal-regulated kinase (ERK) and Akt pathways, was attenuated by withaferin A treatment. Moreover, LPS-induced NO production and NF-kappaB activation were inhibited by SH-6, a specific inhibitor of Akt. Taken together, these results suggest that withaferin A inhibits inflammation through inhibition of NO production and iNOS expression, at least in part, by blocking Akt and subsequently down-regulating NF-kappaB activity.

Expression of Glycogen synthase kinase 3-β (GSK3-β) gene in azoospermic men.

PubMed

Nazarian, Hamid; Ghaffari Novin, Marefat; Jalili, Mohammad Reza; Mirfakhraie, Reza; Heidari, Mohammad Hassan; Hosseini, Seyed Jalil; Norouzian, Mohsen; Ehsani, Nahid

2014-05-01

The Wnt/β- The Wnt/β-catenin signaling pathway is involved in many developmental processes in both fetal and adult life; its abnormalities can lead to disorders including several types of cancers and malfunction of specific cells and tissues in both animals and humans. Its role in reproductive processes has been proven. This study was designed to evaluate the expression of the key regulator of this signaling pathway GSK3-β and its presumed role in azoospermia. WNT3a protein concentration and GSK3-β gene expression levels were measured and compared between two groups of infertile men. The test groups consisted of 10 patients with obstructive and 10 non-obstructive azoospermia. The control group was selected among healthy men after vasectomies that were willing to conceive a child using a testicular biopsy technique. Samples were obtained by testicular biopsy and screened for the most common mutations (84, 86 and 255) in the SRY region before analyzing. GSK3-β gene expression was assessed quantitatively by real time-PCR. The WNT3a protein concentration had no significant difference between the two test groups and controls. Expression of GSK3-β was down-regulated in non-obstructive azoospermia (3.10±0.19) compared with normal (7.12±0.39) and obstructive azoospermia (6.32±0.42) groups (p=0.001). Down-regulation of GSK-3β may cause to non-obstructive azoospermia. Regulation and modification of GSK-3β gene expression by drugs could be used as a therapeutic solution.

In vitro anticancer effects of insect tea in TCA8113 cells.

PubMed

Qian, Yu; Li, Gui-Jie; Wang, Rui; Zhou, Ya-Lin; Sun, Peng; Zhao, Xin

2014-01-01

Insect tea is widely used a traditional drink or traditional Chinese medicine in China. This study was conducted with an aim to determine the in vitro anticancer effect of Insect tea in cancer cells. The anticancer effects of Insect tea were evaluated in human tongue carcinoma TCA8113 cells using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry analysis, nuclear staining with 4,6-diamidino-2-phenylindole (DAPI), reverse transcription-polymerase chain reaction (RT-PCR) analysis, and western bolt assay. At 200 μg/mL, Insect tea inhibited the growth of TCA8113 cells by 80.7%, which was higher than the inhibition caused by 100 μg/mL Insect tea but lower than that of 200 μg/mL green tea. Compared to the control cancer cells, Insect tea significantly (P<0.05) induced apoptosis as determined by DAPI staining and flow cytometry analysis results. Insect tea significantly induced apoptosis in cancer cells by upregulating BAX, CASP3, CASP9 and downregulating BCL2. Genes encoding nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) were significantly downregulated by Insect tea, demonstrating its anti-inflammatory properties. Insect tea also exerted a great anti-metastasis effect on cancer cells as demonstrated by decreased expression of matrix metalloproteinase (MMP) genes and increased expression of tissue inhibitors of metalloproteinases (TIMPs). The results showed that Insect tea has good in vitro anticancer effects in TCA8113 cells, like green tea.

Immobilized heavy chain-hyaluronic acid polarizes lipopolysaccharide-activated macrophages toward M2 phenotype.

PubMed

He, Hua; Zhang, Suzhen; Tighe, Sean; Son, Ji; Tseng, Scheffer C G

2013-09-06

Despite the known anti-inflammatory effect of amniotic membrane, its action mechanism remains largely unknown. HC-HA complex (HC-HA) purified from human amniotic membrane consists of high molecular weight hyaluronic acid (HA) covalently linked to the heavy chain (HC) 1 of inter-α-trypsin inhibitor. In this study, we show that soluble HC-HA also contained pentraxin 3 and induced the apoptosis of both formyl-Met-Leu-Phe or LPS-activated neutrophils and LPS-activated macrophages while not affecting the resting cells. This enhanced apoptosis was caused by the inhibition of cell adhesion, spreading, and proliferation caused by HC-HA binding of LPS-activated macrophages and preventing adhesion to the plastic surface. Preferentially, soluble HC-HA promoted phagocytosis of apoptotic neutrophils in resting macrophages, whereas immobilized HC-HA promoted phagocytosis in LPS-activated macrophages. Upon concomitant LPS stimulation, immobilized HC-HA but not HA polarized macrophages toward the M2 phenotype by down-regulating IRF5 protein and preventing its nuclear localization and by down-regulating IL-12, TNF-α, and NO synthase 2. Additionally, IL-10, TGF-β1, peroxisome proliferator-activated receptor γ, LIGHT (TNF superfamily 14), and sphingosine kinase-1 were up-regulated, and such M2 polarization was dependent on TLR ligation. Collectively, these data suggest that HC-HA is a unique matrix component different from HA and uses multiple mechanisms to suppress M1 while promoting M2 phenotype. This anti-inflammatory action of HC-HA is highly desirable to promote wound healing in diseases heightened by unsuccessful transition from M1 to M2 phenotypes.

Skeletal muscle proteomic signature and metabolic impairment in pulmonary hypertension.

PubMed

Malenfant, Simon; Potus, François; Fournier, Frédéric; Breuils-Bonnet, Sandra; Pflieger, Aude; Bourassa, Sylvie; Tremblay, Ève; Nehmé, Benjamin; Droit, Arnaud; Bonnet, Sébastien; Provencher, Steeve

2015-05-01

Exercise limitation comes from a close interaction between cardiovascular and skeletal muscle impairments. To better understand the implication of possible peripheral oxidative metabolism dysfunction, we studied the proteomic signature of skeletal muscle in pulmonary arterial hypertension (PAH). Eight idiopathic PAH patients and eight matched healthy sedentary subjects were evaluated for exercise capacity, skeletal muscle proteomic profile, metabolism, and mitochondrial function. Skeletal muscle proteins were extracted, and fractioned peptides were tagged using an iTRAQ protocol. Proteomic analyses have documented a total of 9 downregulated proteins in PAH skeletal muscles and 10 upregulated proteins compared to healthy subjects. Most of the downregulated proteins were related to mitochondrial structure and function. Focusing on skeletal muscle metabolism and mitochondrial health, PAH patients presented a decreased expression of oxidative enzymes (pyruvate dehydrogenase, p < 0.01) and an increased expression of glycolytic enzymes (lactate dehydrogenase activity, p < 0.05). These findings were supported by abnormal mitochondrial morphology on electronic microscopy, lower citrate synthase activity (p < 0.01) and lower expression of the transcription factor A of the mitochondria (p < 0.05), confirming a more glycolytic metabolism in PAH skeletal muscles. We provide evidences that impaired mitochondrial and metabolic functions found in the lungs and the right ventricle are also present in skeletal muscles of patients. • Proteomic and metabolic analysis show abnormal oxidative metabolism in PAH skeletal muscle. • EM of PAH patients reveals abnormal mitochondrial structure and distribution. • Abnormal mitochondrial health and function contribute to exercise impairments of PAH. • PAH may be considered a vascular affliction of heart and lungs with major impact on peripheral muscles.

[Effects of parabolic flight on redox status in SH-SY5Y cells].

PubMed

Bi, Lei; Qu, Li-Na; Huang, Zeng-Ming; Wang, Chun-Yan; Li, Qi; Tan, Ying-Jun; Li, Ying-Hui

2009-10-25

Space flight is known to produce a number of neurological disturbances. The etiology is unknown, but it may involve increased oxidative stress. A line of experimental evidence indicates that space flight may disrupt antioxidant defense system and result in increased oxidative stress. In vitro studies found that abundant of NO was produced in rat pheochromocytoma (PC12) cells, SHSY5Y neuroblastoma cells, and protein nitration was increased in PC12 cells within a simulated microgravity rotating wall bioreactor high aspect ratio vessel system or clinostat system. In the present study, we observed the change of redox status in SH-SY5Y cells after parabolic flight, and studied the effects of key redox molecule, thioredoxin (TRX), during the altered gravity. SH-SY5Y cells were divided into four groups: control cells, control cells transfected with TRX, flight cells and flight cells transfected with TRX. The expression levels of 3-nitrotyrosine (3-NT), inducible nitric oxide synthase (iNOS), TRX and thioredoxin reductase (TRXR) were observed by immunocytochemical method. It was shown that after parabolic flight, the staining of 3-NT and TRX were enhanced, while the expression level of TRXR was down-regulated compared with control. As for flight cells transfected with TRX, the staining of 3-NT and iNOS were weakened compared with flight cells. These results obtained suggest that altered gravity may increase protein nitration, down-regulate TRXR and elicit oxidative stress in SH-SY5Y cells, while TRX transfection could partly protect cells against oxidative stress induced by parabolic flight.

Differential gene expression induced by exposure of captive mink to fuel oil: A model for the sea otter

USGS Publications Warehouse

Bowen, Lizabeth; Riva, F.; Mohr, C.; Aldridge, B.; Schwartz, J.; Miles, A. Keith; Stott, J.L.

2007-01-01

Free-ranging sea otters are subject to hydrocarbon exposure from a variety of sources, both natural and anthropogenic. Effects of direct exposure to unrefined crude oil, such as that associated with the Exxon Valdez oil spill, are readily apparent. However, the impact of subtle but pathophysiologically relevant concentrations of crude oil on sea otters is difficult to assess. The present study was directed at developing a model for assessing the impact of low concentrations of fuel oil on sea otters. Quantitative PCR was used to identify differential gene expression in American mink that were exposed to low concentrations of bunker C fuel oil. A total of 23 genes, representing 10 different physiological systems, were analyzed for perturbation. Six genes with immunological relevance were differentially expressed in oil-fed mink. Interleukin-18 (IL-18), IL-10, inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), and complement cytolysis inhibitor (CLI) were down-regulated while IL-2 was up-regulated. Expression of two additional genes was affected; heat shock protein 70 (HSP70) was up-regulated and thyroid hormone receptor (THR) was down-regulated. While the significance of each perturbation is not immediately evident, we identified differential expression of genes that would be consistent with the presence of immune system-modifying and endocrine-disrupting compounds in fuel oil. Application of this approach to identify effects of petroleum contamination on sea otters should be possible following expansion of this mink model to identify a greater number of affected genes in peripheral blood leukocytes.

Lycium barbarum Polysaccharides Protect against Trimethyltin Chloride-Induced Apoptosis via Sonic Hedgehog and PI3K/Akt Signaling Pathways in Mouse Neuro-2a Cells.

PubMed

Zhao, Wanyun; Pan, Xiaoqi; Li, Tao; Zhang, Changchun; Shi, Nian

2016-01-01

Trimethyltin chloride (TMT) is a classic neurotoxicant that can cause severe neurodegenerative diseases. Some signaling pathways involving cell death play pivotal roles in the central nervous system. In this study, the role of Sonic Hedgehog (Shh) and PI3K/Akt pathways in TMT-induced apoptosis and protective effect of Lycium barbarum polysaccharides (LBP) on mouse neuro-2a (N2a) cells were investigated. Results showed that TMT treatment significantly enhanced apoptosis, upregulated proapoptotic Bax, downregulated antiapoptotic Bcl-2 expression, and increased caspase-3 activity in a dose-dependent manner in N2a cells. TMT induced oxidative stress in cells, performing reactive oxygen species (ROS) and malondialdehyde (MDA) excessive generation, and superoxide dismutase (SOD) activity reduction. TMT significantly decreased phosphorylated glycogen synthase kinase-3β (GSK-3β) and inhibited Shh and PI3K/Akt pathways. However, the addition of LBP upregulated GSK-3β phosphorylation, activated Shh and PI3K/Akt pathways, and eventually reduced apoptosis and oxidative stress caused by TMT. The interaction between Shh and PI3K/Akt pathways was clarified by specific PI3K inhibitor LY294002 or Shh inhibitor GDC-0449. Moreover, LY294002 and GDC-0449 pretreatment both induced phosphorylated GSK-3β downregulation and significantly promoted apoptosis induced by TMT. These results suggest that LBP could reduce TMT-induced N2a cells apoptosis by regulating GSK-3β phosphorylation, Shh, and PI3K/Akt signaling pathways.

Lycium barbarum Polysaccharides Protect against Trimethyltin Chloride-Induced Apoptosis via Sonic Hedgehog and PI3K/Akt Signaling Pathways in Mouse Neuro-2a Cells

PubMed Central

Zhao, Wanyun; Pan, Xiaoqi; Li, Tao; Zhang, Changchun; Shi, Nian

2016-01-01

Trimethyltin chloride (TMT) is a classic neurotoxicant that can cause severe neurodegenerative diseases. Some signaling pathways involving cell death play pivotal roles in the central nervous system. In this study, the role of Sonic Hedgehog (Shh) and PI3K/Akt pathways in TMT-induced apoptosis and protective effect of Lycium barbarum polysaccharides (LBP) on mouse neuro-2a (N2a) cells were investigated. Results showed that TMT treatment significantly enhanced apoptosis, upregulated proapoptotic Bax, downregulated antiapoptotic Bcl-2 expression, and increased caspase-3 activity in a dose-dependent manner in N2a cells. TMT induced oxidative stress in cells, performing reactive oxygen species (ROS) and malondialdehyde (MDA) excessive generation, and superoxide dismutase (SOD) activity reduction. TMT significantly decreased phosphorylated glycogen synthase kinase-3β (GSK-3β) and inhibited Shh and PI3K/Akt pathways. However, the addition of LBP upregulated GSK-3β phosphorylation, activated Shh and PI3K/Akt pathways, and eventually reduced apoptosis and oxidative stress caused by TMT. The interaction between Shh and PI3K/Akt pathways was clarified by specific PI3K inhibitor LY294002 or Shh inhibitor GDC-0449. Moreover, LY294002 and GDC-0449 pretreatment both induced phosphorylated GSK-3β downregulation and significantly promoted apoptosis induced by TMT. These results suggest that LBP could reduce TMT-induced N2a cells apoptosis by regulating GSK-3β phosphorylation, Shh, and PI3K/Akt signaling pathways. PMID:27143997

Comparative Proteomic Analysis of the Graft Unions in Hickory (Carya cathayensis) Provides Insights into Response Mechanisms to Grafting Process

PubMed Central

Xu, Dongbin; Yuan, Huwei; Tong, Yafei; Zhao, Liang; Qiu, Lingling; Guo, Wenbin; Shen, Chenjia; Liu, Hongjia; Yan, Daoliang; Zheng, Bingsong

2017-01-01

Hickory (Carya cathayensis), a tree with high nutritional and economic value, is widely cultivated in China. Grafting greatly reduces the juvenile phase length and makes the large scale cultivation of hickory possible. To reveal the response mechanisms of this species to grafting, we employed a proteomics-based approach to identify differentially expressed proteins in the graft unions during the grafting process. Our study identified 3723 proteins, of which 2518 were quantified. A total of 710 differentially expressed proteins (DEPs) were quantified and these were involved in various molecular functional and biological processes. Among these DEPs, 341 were up-regulated and 369 were down-regulated at 7 days after grafting compared with the control. Four auxin-related proteins were down-regulated, which was in agreement with the transcription levels of their encoding genes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the ‘Flavonoid biosynthesis’ pathway and ‘starch and sucrose metabolism’ were both significantly up-regulated. Interestingly, five flavonoid biosynthesis-related proteins, a flavanone 3-hyfroxylase, a cinnamate 4-hydroxylase, a dihydroflavonol-4-reductase, a chalcone synthase, and a chalcone isomerase, were significantly up-regulated. Further experiments verified a significant increase in the total flavonoid contents in scions, which suggests that graft union formation may activate flavonoid biosynthesis to increase the content of a series of downstream secondary metabolites. This comprehensive analysis provides fundamental information on the candidate proteins and secondary metabolism pathways involved in the grafting process for hickory. PMID:28496455

Role of dietary polyphenols in the management of peptic ulcer

PubMed Central

Farzaei, Mohammad Hosein; Abdollahi, Mohammad; Rahimi, Roja

2015-01-01

Peptic ulcer disease is a multifactorial and complex disease involving gastric and duodenal ulcers. Despite medical advances, the management of peptic ulcer and its complications remains a challenge, with high morbidity and death rates for the disease. An accumulating body of evidence suggests that, among a broad reach of natural molecules, dietary polyphenols with multiple biological mechanisms of action play a pivotal part in the management of gastric and duodenal ulcers. The current review confirmed that dietary polyphenols possess protective and therapeutic potential in peptic ulcer mediated by: improving cytoprotection, re-epithelialization, neovascularization, and angiogenesis; up-regulating tissue growth factors and prostaglandins; down-regulating anti-angiogenic factors; enhancing endothelial nitric oxide synthase-derived NO; suppressing oxidative mucosal damage; amplifying antioxidant performance, antacid, and anti-secretory activity; increasing endogenous mucosal defensive agents; and blocking Helicobacter pylori colonization associated gastric morphological changes and gastroduodenal inflammation and ulceration. In addition, anti-inflammatory activity due to down-regulation of proinflammatory cytokines and cellular and intercellular adhesion agents, suppressing leukocyte-endothelium interaction, inhibiting nuclear signaling pathways of inflammatory process, and modulating intracellular transduction and transcription pathways have key roles in the anti-ulcer action of dietary polyphenols. In conclusion, administration of a significant amount of dietary polyphenols in the human diet or as part of dietary supplementation along with conventional treatment can result in perfect security and treatment of peptic ulcer. Further well-designed preclinical and clinical tests are recommended in order to recognize higher levels of evidence for the confirmation of bioefficacy and safety of dietary polyphenols in the management of peptic ulcer. PMID:26074689

Activation of SIRT1 Attenuates Klotho Deficiency-induced Arterial Stiffness and Hypertension by Enhancing AMPKα Activity

PubMed Central

Gao, Diansa; Zuo, Zhong; Tian, Jing; Ali, Quaisar; Lin, Yi; Lei, Han; Sun, Zhongjie

2016-01-01

Arterial stiffness is an independent risk factor for stroke and myocardial infarction. This study was designed to investigate the role of SIRT1, an important deacetylase, and its relationship with Klotho, a kidney-derived aging-suppressor protein, in the pathogenesis of arterial stiffness and hypertension. We found that the serum level of Klotho was decreased by nearly 45% in patients with arterial stiffness and hypertension. Interestingly, Klotho haplodeficiency caused arterial stiffening and hypertension, as evidenced by significant increases in pulse wave velocity (PWV) and blood pressure (BP) in Klotho-haplodeficient (KL+/−) mice. Notably, the expression and activity of SIRT1 were decreased significantly in aortic endothelial and smooth muscle cells in KL+/− mice, suggesting that Klotho deficiency downregulates SIRT1. Treatment with SRT1720 (15 mg/kg/day, IP), a specific SIRT1 activator, abolished Klotho deficiency-induced arterial stiffness and hypertension in KL+/− mice. Klotho deficiency was associated with significant decreases in activities of AMP-activated protein kinase alpha (AMPKα) and endothelial nitric oxide synthase (eNOS) in aortas, which were abolished by SRT1720. Furthermore, Klotho deficiency upregulated NADPH oxidase activity and superoxide production, increased collagen expression, and enhanced elastin fragmentation in the media of aortas. These Klotho deficiency-associated changes were blocked by SRT1720. In conclusion, this study provides the first evidence that Klotho deficiency downregulates SIRT1 activity in arterial endothelial and smooth muscle cells. Pharmacological activation of SIRT1 may be an effective therapeutic strategy for arterial stiffness and hypertension. PMID:27620389

CD147 modulates androgen receptor activity through the Akt/Gsk-3β/β-catenin/AR pathway in prostate cancer cells.

PubMed

Fang, Fang; Qin, Yingxin; Hao, Feng; Li, Qiang; Zhang, Wei; Zhao, Chen; Chen, Shuang; Zhao, Liangzhong; Wang, Liguo; Cai, Jianhui

2016-08-01

The androgen signaling pathway serves an important role in the development of prostate cancer. β-Catenin is an androgen receptor (AR) cofactor and augments AR signaling. Glycogen synthase kinase-3β (GSK-3β), a target of phosphorylated serine/threonine protein kinase B (p-Akt), regulates β-catenin stability. In addition, β-catenin, a coregulator of AR, physically interacts with AR and enhances AR-mediated target gene transcription. The multifunctional glycoprotein cluster of differentiation (CD) 147 is highly expressed on the cell surface of the majority of cancer cells, and it promotes tumor invasion, metastasis and growth. In the present study, the molecular effects of CD147 on the Akt/GSK-3β/β-catenin/AR signaling network were investigated in LNCaP cells. Using short hairpin-mediated RNA knockdown of CD147 in LNCaP cells, it was demonstrated that downregulation of CD147 resulted in inhibitory phosphorylation of GSK-3β, and then promoted degeneration of β-catenin and reduced nuclear accumulation of β-catenin. In addition, immunoprecipitation studies demonstrated that CD147 downregulation decreased the formation of a complex between β-catenin and AR. It was shown that CD147 knockdown suppressed the expression of the AR target gene prostate-specific antigen and the growth of AR-positive LNCaP cells. Furthermore, inhibition of PI3K/Akt with LY294002 augmented CD147-mediated function. The present study indicates that the PI3K/Akt pathway may facilitate CD147-mediated activation of the AR pathway.

Role of dietary polyphenols in the management of peptic ulcer.

PubMed

Farzaei, Mohammad Hosein; Abdollahi, Mohammad; Rahimi, Roja

2015-06-07

Peptic ulcer disease is a multifactorial and complex disease involving gastric and duodenal ulcers. Despite medical advances, the management of peptic ulcer and its complications remains a challenge, with high morbidity and death rates for the disease. An accumulating body of evidence suggests that, among a broad reach of natural molecules, dietary polyphenols with multiple biological mechanisms of action play a pivotal part in the management of gastric and duodenal ulcers. The current review confirmed that dietary polyphenols possess protective and therapeutic potential in peptic ulcer mediated by: improving cytoprotection, re-epithelialization, neovascularization, and angiogenesis; up-regulating tissue growth factors and prostaglandins; down-regulating anti-angiogenic factors; enhancing endothelial nitric oxide synthase-derived NO; suppressing oxidative mucosal damage; amplifying antioxidant performance, antacid, and anti-secretory activity; increasing endogenous mucosal defensive agents; and blocking Helicobacter pylori colonization associated gastric morphological changes and gastroduodenal inflammation and ulceration. In addition, anti-inflammatory activity due to down-regulation of proinflammatory cytokines and cellular and intercellular adhesion agents, suppressing leukocyte-endothelium interaction, inhibiting nuclear signaling pathways of inflammatory process, and modulating intracellular transduction and transcription pathways have key roles in the anti-ulcer action of dietary polyphenols. In conclusion, administration of a significant amount of dietary polyphenols in the human diet or as part of dietary supplementation along with conventional treatment can result in perfect security and treatment of peptic ulcer. Further well-designed preclinical and clinical tests are recommended in order to recognize higher levels of evidence for the confirmation of bioefficacy and safety of dietary polyphenols in the management of peptic ulcer.

The Redox-sensitive Induction of the Local Angiotensin System Promotes Both Premature and Replicative Endothelial Senescence: Preventive Effect of a Standardized Crataegus Extract.

PubMed

Khemais-Benkhiat, Sonia; Idris-Khodja, Noureddine; Ribeiro, Thais Porto; Silva, Grazielle Caroline; Abbas, Malak; Kheloufi, Marouane; Lee, Jung-Ok; Toti, Florence; Auger, Cyril; Schini-Kerth, Valérie B

2016-12-01

Endothelial senescence, characterized by an irreversible cell cycle arrest, oxidative stress, and downregulation of endothelial nitric oxide synthase (eNOS), has been shown to promote endothelial dysfunction leading to the development of age-related vascular disorders. This study has assessed the possibility that the local angiotensin system promotes endothelial senescence in coronary artery endothelial cells and also the protective effect of the Crataegus extract WS1442, a quantified hawthorn extract. Serial passaging from P1 to P4 (replicative senescence) and treatment of P1 endothelial cells with the eNOS inhibitor L-NAME (premature senescence) promoted acquisition of markers of senescence, enhanced ROS formation, decreased eNOS expression, and upregulation of angiotensin-converting enzyme (ACE) and AT1 receptors. Increased SA-β-gal activity and the upregulation of ACE and AT1R in senescent cells were prevented by antioxidants, an ACE inhibitor, and by an AT1 receptor blocker. WS1442 prevented SA-β-gal activity, the downregulation of eNOS, and oxidative stress in P3 cells. These findings indicate that the impairment of eNOS-derived nitric oxide formation favors a pro-oxidant response triggering the local angiotensin system, which, in turn, promotes endothelial senescence. Such a sequence of events can be effectively inhibited by a standardized polyphenol-rich extract mainly by targeting the oxidative stress. © The Author 2015. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

An agonist to the A3 adenosine receptor inhibits colon carcinoma growth in mice via modulation of GSK-3 beta and NF-kappa B.

PubMed

Fishman, Pnina; Bar-Yehuda, Sara; Ohana, Gil; Barer, Faina; Ochaion, Avivit; Erlanger, Abigail; Madi, Lea

2004-04-01

A(3) adenosine receptor (A(3)AR) activation with the specific agonist CF101 has been shown to inhibit the development of colon carcinoma growth in syngeneic and xenograft murine models. In the present study, we looked into the effect of CF101 on the molecular mechanisms involved in the inhibition of HCT-116 colon carcinoma in mice. In tumor lesions derived from CF101-treated mice, a decrease in the expression level of protein kinase A (PKA) and an increase in glycogen synthase kinase-3 beta (GSK-3 beta) was observed. This gave rise to downregulation of beta-catenin and its transcriptional gene products cyclin D1 and c-Myc. Further mechanistic studies in vitro revealed that these responses were counteracted by the selective A(3)AR antagonist MRS 1523 and by the GSK-3 beta inhibitors lithium and SB216763, confirming that the observed effects were A(3)AR and GSK-3 beta mediated. CF101 downregulated PKB/Akt expression level, resulting in a decrease in the level and DNA-binding capacity of NF-kappa B, both in vivo and in vitro. Furthermore, the PKA and PKB/Akt inhibitors H89 and Worthmannin mimicked the effect of CF101, supporting their involvement in mediating the response to the agonist. This is the first demonstration that A(3)AR activation induces colon carcinoma growth inhibition via the modulation of the key proteins GSK-3 beta and NF-kappa B.

Fenofibrate Decreases Insulin Clearance and Insulin Secretion to Maintain Insulin Sensitivity*

PubMed Central

Ramakrishnan, Sadeesh K.; Russo, Lucia; Ghanem, Simona S.; Patel, Payal R.; Oyarce, Ana Maria; Heinrich, Garrett; Najjar, Sonia M.

2016-01-01

High fat diet reduces the expression of CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1), a transmembrane glycoprotein that promotes insulin clearance and down-regulates fatty acid synthase activity in the liver upon its phosphorylation by the insulin receptor. Because peroxisome proliferator-activated receptor α (PPARα) transcriptionally suppresses CEACAM1 expression, we herein examined whether high fat down-regulates CEACAM1 expression in a PPARα-dependent mechanism. By activating PPARα, the lipid-lowering drug fenofibrate reverses dyslipidemia and improves insulin sensitivity in type 2 diabetes in part by promoting fatty acid oxidation. Despite reducing glucose-stimulated insulin secretion, fenofibrate treatment does not result in insulin insufficiency. To examine whether this is mediated by a parallel decrease in CEACAM1-dependent hepatic insulin clearance pathways, we fed wild-type and Pparα−/− null mice a high fat diet supplemented with either fenofibrate or Wy14643, a selective PPARα agonist, and examined their effect on insulin metabolism and action. We demonstrated that the decrease in insulin secretion by fenofibrate and Wy14643 is accompanied by reduction in insulin clearance in wild-type but not Pparα−/− mice, thereby maintaining normoinsulinemia and insulin sensitivity despite continuous high fat intake. Intact insulin secretion in L-CC1 mice with protected hepatic insulin clearance and CEACAM1 levels provides in vivo evidence that insulin secretion responds to changes in insulin clearance to maintain physiologic insulin and glucose homeostasis. These results also emphasize the relevant role of hepatic insulin extraction in regulating insulin sensitivity. PMID:27662905

Activation of SIRT1 Attenuates Klotho Deficiency-Induced Arterial Stiffness and Hypertension by Enhancing AMP-Activated Protein Kinase Activity.

PubMed

Gao, Diansa; Zuo, Zhong; Tian, Jing; Ali, Quaisar; Lin, Yi; Lei, Han; Sun, Zhongjie

2016-11-01

Arterial stiffness is an independent risk factor for stroke and myocardial infarction. This study was designed to investigate the role of SIRT1, an important deacetylase, and its relationship with Klotho, a kidney-derived aging-suppressor protein, in the pathogenesis of arterial stiffness and hypertension. We found that the serum level of Klotho was decreased by ≈45% in patients with arterial stiffness and hypertension. Interestingly, Klotho haplodeficiency caused arterial stiffening and hypertension, as evidenced by significant increases in pulse wave velocity and blood pressure in Klotho-haplodeficient (KL +/- ) mice. Notably, the expression and activity of SIRT1 were decreased significantly in aortic endothelial and smooth muscle cells in KL +/- mice, suggesting that Klotho deficiency downregulates SIRT1. Treatment with SRT1720 (15 mg/kg/d, IP), a specific SIRT1 activator, abolished Klotho deficiency-induced arterial stiffness and hypertension in KL +/- mice. Klotho deficiency was associated with significant decreases in activities of AMP-activated protein kinase α (AMPKα) and endothelial NO synthase (eNOS) in aortas, which were abolished by SRT1720. Furthermore, Klotho deficiency upregulated NADPH oxidase activity and superoxide production, increased collagen expression, and enhanced elastin fragmentation in the media of aortas. These Klotho deficiency-associated changes were blocked by SRT1720. In conclusion, this study provides the first evidence that Klotho deficiency downregulates SIRT1 activity in arterial endothelial and smooth muscle cells. Pharmacological activation of SIRT1 may be an effective therapeutic strategy for arterial stiffness and hypertension. © 2016 American Heart Association, Inc.

High-calorie diet exacerbates prostate neoplasia in mice with haploinsufficiency of Pten tumor suppressor gene.

PubMed

Liu, Jehnan; Ramakrishnan, Sadeesh K; Khuder, Saja S; Kaw, Meenakshi K; Muturi, Harrison T; Lester, Sumona Ghosh; Lee, Sang Jun; Fedorova, Larisa V; Kim, Andrea J; Mohamed, Iman E; Gatto-Weis, Cara; Eisenmann, Kathryn M; Conran, Philip B; Najjar, Sonia M

2015-03-01

Association between prostate cancer and obesity remains controversial. Allelic deletions of PTEN, a tumor suppressor gene, are common in prostate cancer in men. Monoallelic Pten deletion in mice causes low prostatic intraepithelial neoplasia (mPIN). This study tested the effect of a hypercaloric diet on prostate cancer in Pten (+/-) mice. 1-month old mice were fed a high-calorie diet deriving 45% calories from fat for 3 and 6 months before prostate was analyzed histologically and biochemically for mPIN progression. Because Pten (+/-) mice are protected against diet-induced insulin resistance, we tested the role of insulin on cell growth in RWPE-1 normal human prostatic epithelial cells with siRNA knockdown of PTEN. In addition to activating PI3 kinase/Akt and Ras/MAPkinase pathways, high-calorie diet causes neoplastic progression, angiogenesis, inflammation and epithelial-mesenchymal transition. It also elevates the expression of fatty acid synthase (FAS), a lipogenic gene commonly elevated in progressive cancer. SiRNA-mediated downregulation of PTEN demonstrates increased cell growth and motility, and soft agar clonicity in addition to elevation in FAS in response to insulin in RWPE-1 normal human prostatic cells. Downregulating FAS in addition to PTEN, blunted the proliferative effect of insulin (and IL-6) in RWPE-1 cells. High-calorie diet promotes prostate cancer progression in the genetically susceptible Pten haploinsufficient mouse while preserving insulin sensitivity. This appears to be partly due to increased inflammatory response to high-caloric intake in addition to increased ability of insulin to promote lipogenesis.

HSF1 phosphorylation by ERK/GSK3 suppresses RNF126 to sustain IGF-IIR expression for hypertension-induced cardiomyocyte hypertrophy.

PubMed

Huang, Chih-Yang; Lee, Fa-Lun; Peng, Shu-Fen; Lin, Kuan-Ho; Chen, Ray-Jade; Ho, Tsung-Jung; Tsai, Fu-Jen; Padma, Vijaya V; Kuo, Wei-Wen; Huang, Chih-Yang

2018-02-01

Hypertension-induced cardiac hypertrophy and apoptosis are major characteristics of early-stage heart failure (HF). Inhibition of extracellular signal-regulated kinases (ERK) efficaciously suppressed angiotensin II (ANG II)-induced cardiomyocyte hypertrophy and apoptosis by blocking insulin-like growth factor II receptor (IGF-IIR) signaling. However, the detailed mechanism by which ANG II induces ERK-mediated IGF-IIR signaling remains elusive. Here, we found that ANG II activated ERK to upregulate IGF-IIR expression via the angiotensin II type I receptor (AT 1 R). ERK activation subsequently phosphorylates HSF1 at serine 307, leading to a secondary phosphorylation by glycogen synthase kinase III (GSK3) at serine 303. Moreover, we found that ANG II mediated ERK/GSK3-induced IGF-IIR protein stability by downregulating the E3 ubiquitin ligase of IGF-IIR RING finger protein CXXVI (RNF126). The expression of RNF126 decreased following ANG II-induced HSF1 S303 phosphorylation, resulting in IGF-IIR protein stability and increased cardiomyocyte injury. Inhibition of GSK3 significantly alleviated ANG II-induced cardiac hypertrophy in vivo and in vitro. Taken together, these results suggest that HSF1 phosphorylation stabilizes IGF-IIR protein stability by downregulating RNF126 during cardiac hypertrophy. ANG II activates ERK/GSK3 to phosphorylate HSF1, resulting in RNF126 degradation, which stabilizes IGF-IIR protein expression and eventually results in cardiac hypertrophy. HSF1 could be a valuable therapeutic target for cardiac diseases among hypertensive patients. © 2017 Wiley Periodicals, Inc.

Immune and inflammatory gene signature in rat cerebrum in subarachnoid hemorrhage with microarray analysis.

PubMed

Lee, Chu-I; Chou, An-Kuo; Lin, Ching-Chih; Chou, Chia-Hua; Loh, Joon-Khim; Lieu, Ann-Shung; Wang, Chih-Jen; Huang, Chi-Ying F; Howng, Shen-Long; Hong, Yi-Ren

2012-01-01

Cerebral vasospasm following subarachnoid hemorrhage (SAH) has been studied in terms of a contraction of the major cerebral arteries, but the effect of cerebrum tissue in SAH is not yet well understood. To gain insight into the biology of SAH-expressing cerebrum, we employed oligonucleotide microarrays to characterize the gene expression profiles of cerebrum tissue at the early stage of SAH. Functional gene expression in the cerebrum was analyzed 2 h following stage 1-hemorrhage in Sprague-Dawley rats. mRNA was investigated by performing microarray and quantitative real-time PCR analyses, and protein expression was determined by Western blot analysis. In this study, 18 upregulated and 18 downregulated genes displayed at least a 1.5-fold change. Five genes were verified by real-time PCR, including three upregulated genes [prostaglandin E synthase (PGES), CD14 antigen, and tissue inhibitor of metalloproteinase 1 (TIMP1)] as well as two downregulated genes [KRAB-zinc finger protein-2 (KZF-2) and γ-aminobutyric acid B receptor 1 (GABA B receptor)]. Notably, there were functional implications for the three upregulated genes involved in the inflammatory SAH process. However, the mechanisms leading to decreased KZF-2 and GABA B receptor expression in SAH have never been characterized. We conclude that oligonucleotide microarrays have the potential for use as a method to identify candidate genes associated with SAH and to provide novel investigational targets, including genes involved in the immune and inflammatory response. Furthermore, understanding the regulation of MMP9/TIMP1 during the early stages of SAH may elucidate the pathophysiological mechanisms in SAH rats.

Comparative transcriptome investigation of global gene expression changes caused by miR156 overexpression in Medicago sativa.

PubMed

Gao, Ruimin; Austin, Ryan S; Amyot, Lisa; Hannoufa, Abdelali

2016-08-19

Medicago sativa (alfalfa) is a low-input forage and potential bioenergy crop, and improving its yield and quality has always been a focus of the alfalfa breeding industry. Transgenic alfalfa plants overexpressing a precursor of alfalfa microRNA156 (MsmiR156) were recently generated by our group. These plants (miR156OE) showed enhanced biomass yield, reduced internodal length, increased shoot branching and trichome density, and a delay in flowering time. Transcripts of three SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) genes (MsSPL6, MsSPL12, and MsSPL13) were found to be targeted for cleavage by MsmiR156 in alfalfa. To further illustrate the molecular mechanisms underlying the effects of miR156 in alfalfa, two miR156OE genotypes (A11a and A17) were subjected to Next Generation RNA Sequencing with Illumina HiSeq. More than 1.11 billion clean reads were obtained from our available sequenced samples. A total of 160,472 transcripts were generated using Trinity de novo assembly and 4,985 significantly differentially expressed genes were detected in miR156OE plants A11a and A17 using the Medicago truncatula genome as reference. A total of 17 genes (including upregulated, downregulated, and unchanged) were selected for quantitative real-time PCR (qRT-PCR) validation, which showed that gene expression levels were largely consistent between qRT-PCR and RNA-Seq data. In addition to the established SPL genes MsSPL6, MsSPL12 and MsSPL13, four new SPLs; MsSPL2, MsSPL3, MsSPL4 and MsSPL9 were also down-regulated significantly in both miR156OE plants. These seven SPL genes belong to genes phylogeny clades VI, IV, VIII, V and VII, which have been reported to be targeted by miR156 in Arabidopsis thaliana. The gene ontology terms characterized electron transporter, starch synthase activity, sucrose transport, sucrose-phosphate synthase activity, chitin binding, sexual reproduction, flavonoid biosynthesis and lignin catabolism correlate well to the phenotypes of miR156OE alfalfa plants. This is the first report of changes in global gene expression in response to miR156 overexpression in alfalfa. The discovered miR156-targeted SPL genes belonging to different clades indicate miR156 plays fundamental and multifunctional roles in regulating alfalfa plant development.

Biochemical Characterization and Homology Modeling of Methylbutenol Synthase and Implications for Understanding Hemiterpene Synthase Evolution in Plants*

PubMed Central

Gray, Dennis W.; Breneman, Steven R.; Topper, Lauren A.; Sharkey, Thomas D.

2011-01-01

2-Methyl-3-buten-2-ol (MBO) is a five-carbon alcohol produced and emitted in large quantities by many species of pine native to western North America. MBO is structurally and biosynthetically related to isoprene and can have an important impact on regional atmospheric chemistry. The gene for MBO synthase was identified from Pinus sabiniana, and the protein encoded was functionally characterized. MBO synthase is a bifunctional enzyme that produces both MBO and isoprene in a ratio of ∼90:1. Divalent cations are required for activity, whereas monovalent cations are not. MBO production is enhanced by K+, whereas isoprene production is inhibited by K+ such that, at physiologically relevant [K+], little or no isoprene emission should be detected from MBO-emitting trees. The Km of MBO synthase for dimethylallyl diphosphate (20 mm) is comparable with that observed for angiosperm isoprene synthases and 3 orders of magnitude higher than that observed for monoterpene and sesquiterpene synthases. Phylogenetic analysis showed that MBO synthase falls into the TPS-d1 group (gymnosperm monoterpene synthases) and is most closely related to linalool synthase from Picea abies. Structural modeling showed that up to three phenylalanine residues restrict the size of the active site and may be responsible for making this a hemiterpene synthase rather than a monoterpene synthase. One of these residues is homologous to a Phe residue found in the active site of isoprene synthases. The remaining two Phe residues do not have homologs in isoprene synthases but occupy the same space as a second Phe residue that closes off the isoprene synthase active site. PMID:21504898

Engineered living blood vessels: functional endothelia generated from human umbilical cord-derived progenitors.

PubMed

Schmidt, Dörthe; Asmis, Lars M; Odermatt, Bernhard; Kelm, Jens; Breymann, Christian; Gössi, Matthias; Genoni, Michele; Zund, Gregor; Hoerstrup, Simon P

2006-10-01

Tissue-engineered living blood vessels (TEBV) with growth capacity represent a promising new option for the repair of congenital malformations. We investigate the functionality of TEBV with endothelia generated from human umbilical cord blood-derived endothelial progenitor cells. Tissue-engineered living blood vessels were generated from human umbilical cord-derived myofibroblasts seeded on biodegradable vascular scaffolds, followed by endothelialization with differentiated cord blood-derived endothelial progenitor cells. During in vitro maturation the TEBV were exposed to physiologic conditioning in a flow bioreactor. For functional assessment, a subgroup of TEBV was stimulated with tumor necrosis factor-alpha. Control vessels endothelialized with standard vascular endothelial cells were treated in parallel. Analysis of the TEBV included histology, immunohistochemistry, biochemistry (extracellular matrix analysis, DNA), and biomechanical testing. Endothelia were analyzed by flow cytometry and immunohistochemistry (CD31, von Willebrand factor, thrombomodulin, tissue factor, endothelial nitric oxide synthase). Histologically, a three-layered tissue organization of the TEBV analogous to native vessels was observed, and biochemistry revealed the major matrix constituents (collagen, proteoglycans) of blood vessels. Biomechanical properties (Young's modulus, 2.03 +/- 0.65 MPa) showed profiles resembling those of native tissue. Endothelial progenitor cells expressed typical endothelial cell markers CD31, von Willebrand factor, and endothelial nitric oxide synthase comparable to standard vascular endothelial cells. Stimulation with tumor necrosis factor-alpha resulted in physiologic upregulation of tissue factor and downregulation of thrombomodulin expression. These results indicate that TEBV with tissue architecture and functional endothelia similar to native blood vessels can be successfully generated from human umbilical cord progenitor cells. Thus, blood-derived progenitor cells obtained before or at birth may enable the clinical realization of tissue engineering constructs for pediatric applications.

Structural and functional basis of phospholipid oxygenase activity of bacterial lipoxygenase from Pseudomonas aeruginosa.

PubMed

Banthiya, Swathi; Kalms, Jacqueline; Galemou Yoga, Etienne; Ivanov, Igor; Carpena, Xavi; Hamberg, Mats; Kuhn, Hartmut; Scheerer, Patrick

2016-11-01

Pseudomonas aeruginosa expresses a secreted LOX-isoform (PA-LOX, LoxA) capable of oxidizing polyenoic fatty acids to hydroperoxy derivatives. Here we report high-level expression of this enzyme in E. coli and its structural and functional characterization. Recombinant PA-LOX oxygenates polyenoic fatty acids including eicosapentaenoic acid and docosahexaenoic acid to the corresponding (n-6)S-hydroperoxy derivatives. This reaction involves abstraction of the proS-hydrogen from the n-8 bisallylic methylene. PA-LOX lacks major leukotriene synthase activity but converts 5S-HETE and 5S,6R/S-DiHETE to anti-inflammatory and pro-resolving lipoxins. It also exhibits phospholipid oxygenase activity as indicated by the formation of a specific pattern of oxygenation products from different phospholipid subspecies. Multiple mutagenesis studies revealed that PA-LOX does not follow classical concepts explaining the reaction specificity of mammalian LOXs. The crystal structure of PA-LOX was solved with resolutions of up to 1.48Å and its polypeptide chain is folded as single domain. The substrate-binding pocket consists of two fatty acid binding subcavities and lobby. Subcavity-1 contains the catalytic non-heme iron. A phosphatidylethanolamine molecule occupies the substrate-binding pocket and its sn1 fatty acid is located close to the catalytic non-heme iron. His377, His382, His555, Asn559 and the C-terminal Ile685 function as direct iron ligands and a water molecule (hydroxyl) completes the octahedral ligand sphere. Although the biological relevance of PA-LOX is still unknown its functional characteristics (lipoxin synthase activity) implicate this enzyme in a bacterial evasion strategy aimed at downregulating the hosts' immune system. Copyright © 2016 Elsevier B.V. All rights reserved.

Silencing of flavanone-3-hydroxylase in apple (Malus × domestica Borkh.) leads to accumulation of flavanones, but not to reduced fire blight susceptibility.

PubMed

Flachowsky, Henryk; Halbwirth, Heidi; Treutter, Dieter; Richter, Klaus; Hanke, Magda-Viola; Szankowski, Iris; Gosch, Christian; Stich, Karl; Fischer, Thilo C

2012-02-01

Transgenic antisense flavanone-3-hydroxylase apple plants were produced to mimic the effect of the agrochemical prohexadione-Ca on apple leaves. This enzyme inhibitor for 2-oxoglutarate dependent dioxygenases is used as a growth retardant and for control of secondary fire blight of leaves. Like using the agent, silencing of flavanone-3-hydroxylase leads to an accumulation of flavanones in leaves, but in contrast not to the formation of 3-deoxyflavonoids. In prohexadione-Ca treated leaves the 3-deoxyflavonoid luteoforol is formed from accumulating flavanones, acting as an antimicrobial compound against the fire blight pathogen Erwinia amylovora. Seemingly, the silencing of just one of the 2-oxoglutarate dependent dioxygenases (in apple also flavonol synthase and anthocyanidin synthase take part downstream in the pathway) does not provide a sufficiently high ratio of flavanones to dihydroflavonols. This seems to be needed to let the dihydroflavonol-4-reductase/flavanone-4-reductase enzyme reduce flavanones to luteoforol, and to let this be reduced by the leucoanthocyanidin-4-reductase/3-deoxyleucoanthocyanidin-4-reductase, each acting with their respective weak secondary activities. Accordingly, also the intended inducible resistance to fire blight by prohexadione-Ca is not observed with the antisense flavanone-3-hydroxylase apple plants. On the other hand, for most transgenic lines with strong flavanone-4-reductase down-regulation, up-regulation of gene expression for the other flavonoid genes was found. This provides further evidence for the feedback regulation of flavonoid gene expression having been previously reported for the prohexadione-Ca inhibited apple plants. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Physiological performance and differential expression profiling of genes associated with drought tolerance in contrasting varieties of two Gossypium species.

PubMed

Singh, Ruchi; Pandey, Neha; Naskar, Jishnu; Shirke, Pramod A

2015-03-01

Cotton is mostly cultivated under rain-fed conditions in India, thus faces frequent drought conditions during its life cycle. Drought being a major stress factor responsible for yield penalty, there has always been a high priority to generate knowledge on adaptation and tolerance of cotton. In the present study, four cotton varieties, JKC-770 and KC-2 (Gossypium hirsutum), and JKC-717 and RAHS-187(Gossypium herbaceum), were imposed to drought. Under drought condition, differential changes in physiological characters like net photosynthesis, transpiration, stomatal conductance, chlorophyll fluorescence, relative water content (RWC), and predawn water potential (ψ 0) showed a change. While proline, malondialdehyde (MDA), and glutathione-S-transferase (GST) content increased along with a concomitant change in the expression of their associated genes. Under moderate stress, tolerant varieties maintain lower ψ 0 probably due to higher proline content as compared to sensitive varieties. Cyclic electron flow (CEF) also plays an important role in tolerance under mild water stress in G. hirsutum varieties. CEF not only activates at high light but also initiates at a very low light intensity. Expression analysis of genes reveals that drought-tolerant varieties showed enhanced detoxifying mechanism by up-regulation of asparagine synthase (AS), glutathione-S-transferase (GST), and methyl glyoxalase (GlyI) genes under drought stress. Up-regulation of Δ(1)-pyrroline-5-carboxylase synthase (Δ(1)P5CS) enhanced accumulation of proline, an osmolyte, under drought in tolerant varieties. While the drought-sensitive varieties showed up-regulation of ethylene responsive factor (ERF) and down-regulation of WRKY70 responsible for senescence of the leaf which correlated well with the high rate of leaf fall in sensitive varieties under water stress.

Enhanced anti-inflammatory potential of cinnamate-zinc layered hydroxide in lipopolysaccharide-stimulated RAW 264.7 macrophages

PubMed Central

Adewoyin, Malik; Mohsin, Sumaiyah Megat Nabil; Arulselvan, Palanisamy; Hussein, Mohd Zobir; Fakurazi, Sharida

2015-01-01

Background Cinnamic acid (CA) is a phytochemical originally derived from Cinnamomum cassia, a plant with numerous pharmacological properties. The intercalation of CA with a nanocarrier, zinc layered hydroxide, produces cinnamate-zinc layered hydroxide (ZCA), which has been previously characterized. Intercalation is expected to improve the solubility and cell specificity of CA. The nanocarrier will also protect CA from degradation and sustain its release. The aim of this study was to assess the effect of intercalation on the anti-inflammatory capacity of CA. Methods In this study, the anti-inflammatory activity of ZCA was investigated and compared with that of nonintercalated CA. Evaluations were based on the capacity of ZCA and CA to modulate the release of nitric oxide, prostaglandin E2, interleukin (IL)-6, tumor necrosis factor alpha (TNF-α), IL-1β, and IL-10 in lipopolysaccharide-induced RAW 264.7 cells. Additionally, the expression of proinflammatory enzymes, ie, cyclooxygenase-2, inducible nitric oxide synthase, and nuclear factor kappa B (NF-κB), were examined. Results Although both ZCA and CA downregulated nitric oxide, prostaglandin E2, tumor necrosis factor alpha, IL-1β, and IL-6, ZCA clearly displayed better activity. Similarly, expression of cyclooxygenase-2 and inducible nitric oxide synthase were inhibited in samples treated with ZCA and CA. The two compounds effectively inactivated the transcription factor NF-κB, but the anti-inflammatory cytokine, IL-10, was significantly upregulated by ZCA only. Conclusion The present findings suggest that ZCA possesses better anti-inflammatory potential than CA, while zinc layered hydroxide had little or no effect, and these results were comparable with the positive control. PMID:25995619

Possible mechanisms of action of Caesalpinia pyramidalis against ethanol-induced gastric damage.

PubMed

Diniz, Polyana B F; Ribeiro, Ana Roseli S; Estevam, Charles S; Bani, Cristiane C; Thomazzi, Sara M

2015-06-20

Caesalpinia pyramidalis Tul. (Fabaceae), known as "catingueira", is an endemic tree of the Northeast region of Brazil. This plant, mainly inner bark and flowers, has been used in traditional medicine to treat gastritis, heartburn, indigestion, stomachache, dysenteries, and diarrheas. The ethanol extract of C. pyramidalis inner bark was used in rats via oral route, at the doses of 30, 100, and 300 mg/kg, in the ethanol-induced ulcer model and some of the mechanisms underlying to the gastroprotective effect of this plant investigated. The ethanol extract of C. pyramidalis inner bark (100 mg/kg) produced reduction (P < 0.001) on the total lesion area in the ethanol-induced gastric damage. The gastroprotective response caused by the ethanol extract (100 mg/kg) was significantly attenuated (P < 0.05) by intraperitoneal treatment of rats with DL-Propargylglycine (PAG, a cystathionine-γ-lyase inhibitor; 25 mg/kg), but not by Nw-nitro-L-arginine methyl ester hydrochloride (L-NAME, an inhibitor of nitric oxide synthase; 70 mg/kg), and confirmed by microscopic evidence. The ethanol extract significantly decreased the number of mucosal mast cells compared to vehicle-treated group. The inflammatory cells of the ethanol extract (100 mg/kg)-treated ulcerated rats exhibited an upregulation of interleukin (IL)-4 protein expression and downregulation of inducible nitric oxide synthase (iNOS) expression, observed by immunohistochemistry and flow cytometer. The present results suggest that the ethanol extract of C. pyramidalis produced dose-related gastroprotective response on ethanol-induce ulcer in rats through mechanisms that involved an interaction with endogenous hydrogen sulfide and reduction of inflammatory process with imbalance between pro-inflammatory and anti-inflammatory mediators, supporting the popular usage of this plant. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

HMG-CoA synthase isoenzymes 1 and 2 localize to satellite glial cells in dorsal root ganglia and are differentially regulated by peripheral nerve injury.

PubMed

Wang, Fei; Xiang, Hongfei; Fischer, Gregory; Liu, Zhen; Dupont, Matthew J; Hogan, Quinn H; Yu, Hongwei

2016-12-01

In dorsal root ganglia (DRG), satellite glial cells (SGCs) tightly ensheathe the somata of primary sensory neurons to form functional sensory units. SGCs are identified by their flattened and irregular morphology and expression of a variety of specific marker proteins. In this report, we present evidence that the 3-hydroxy-3-methylglutaryl coenzyme A synthase isoenzymes 1 and 2 (HMGCS1 and HMGCS2) are abundantly expressed in SGCs. Immunolabeling with the validated antibodies revealed that both HMGCS1 and HMGCS2 are highly colabeled with a selection of SGC markers, including GS, GFAP, K ir 4.1, GLAST1, GDNF, and S100 but not with microglial cell marker Iba1, myelin sheath marker MBP, and neuronal marker β3-tubulin or phosphorylated CaMKII. HMGCS1 but not HMGCS2 immunoreactivity in SGCs is reduced in the fifth lumbar (L5) DRGs that contain axotomized neurons following L5 spinal nerve ligation (SNL) in rats. Western blot showed that HMGCS1 protein level in axotomized L5 DRGs is reduced after SNL to 66±8% at 3 days (p<0.01, n=4 animals in each group) and 58±13% at 28 days (p<0.001, n=9 animals in each group) of its level in control samples, whereas HMGCS2 protein was comparable between injured and control DRGs. These results identify HMGCSs as the alternative markers for SGCs in DRGs. Downregulated HMGCS1 expression in DRGs after spinal nerve injury may reflect a potential role of abnormal sterol metabolism of SGCs in the nerve injured-induced neuropathic pain. Copyright © 2016 Elsevier B.V. All rights reserved.

mPGES-1-derived PGE2 mediates dehydration natriuresis

PubMed Central

Jia, Zhanjun; Liu, Gang; Sun, Ying; Kakizoe, Yutaka; Guan, Guangju; Zhang, Aihua; Zhou, Shu-Feng

2013-01-01

PGE2 is a natriuretic factor whose production is elevated after water deprivation (WD) but its role in dehydration natriuresis is not well-defined. The goal of the present study was to investigate the role of microsomal prostaglandin E synthase-1 (mPGES-1) in dehydration natriuresis. After 24-h WD, wild-type (WT) mice exhibited a significant increase in 24-h urinary Na+ excretion accompanied with normal plasma Na+ concentration and osmolality. In contrast, WD-induced elevation of urinary Na+ excretion was completely abolished in mPGES-1 knockout (KO) mice in parallel with increased plasma Na+ concentration and a trend increase in plasma osmolality. WD induced a 1.8-fold increase in urinary PGE2 output and a 1.6-fold increase in PGE2 content in the renal medulla of WT mice, both of which were completely abolished by mPGES-1 deletion. Similar patterns of changes were observed for urinary nitrate/nitrite and cGMP. The natriuresis in dehydrated WT mice was associated with a significant downregulation of renal medullary epithelial Na channel-α mRNA and protein, contrasting to unaltered expressions in dehydrated KO mice. By quantitative RT-PCR, WD increased the endothelial nitric oxide synthase (eNOS), inducible NOS, and neuronal NOS expressions in the renal medulla of WT mice by 3.9-, 1.48-, and 2.6-fold, respectively, all of which were significantly blocked in mPGES-1 KO mice. The regulation of eNOS expression was further confirmed by immunoblotting. Taken together, our results suggest that mPGES-1-derived PGE2 contributes to dehydration natriuresis likely via NO/cGMP. PMID:23171554

Down-regulation of a chitin synthase a gene by RNA interference enhances pathogenicity of Beauveria bassiana ANU1 against Spodoptera exigua (HÜBNER).

PubMed

Lee, Jung-Bok; Kim, Hyun Soo; Park, Youngjin

2017-02-01

Chitin synthase (CHS) is an important enzymatic component, which is required for chitin formation in the cuticles and cuticular linings of other tissues in insects. CHSs have been divided into two classes, classes A and B, based on their amino acid sequence similarities and functions. Class A CHS (CHS-A) is specifically expressed in the epidermis and related ectodermal cells such as tracheal cells, while class B CHS (CHS-B) is expressed in gut epithelial cells that produce peritrophic matrices. In this study, we cloned the CHS-A gene from the beet armyworm, Spodoptera exigua (SeCHS-A). The SeCHS-A contains an open reading frame of 4,698 nucleotides, encoding a protein of 1,565 amino acids with a predicted molecular mass of approximately 177.8 kDa. The SeCHS-A mRNA was expressed in all developmental stages and specifically in the epidermis and tracheae tissue by quantitative real-time-PCR analysis. Expression of SeCHS-A gene was suppressed by feeding double-stranded RNA (dsCHS-A, 400 ng/larva) in the third instar larvae of S. exigua. Suppression of the SeCHS-A gene expression significantly increased 35% of mortality on pupation of S. exigua. Also, the third instar larvae fed with dsCHS-A significantly increased susceptibility to entomopathogenic fungi, Beauveria bassiana ANU1 at 3 days after treatment. These results suggest that the SeCHS-A gene plays an important role in development of S. exigua and RNA interference may apply to effective pest control with B. bassiana. © 2017 Wiley Periodicals, Inc.

Specific glycogen synthase kinase-3 inhibition reduces neuroendocrine markers and suppresses neuroblastoma cell growth.

PubMed

Carter, Yvette M; Kunnimalaiyaan, Selvi; Chen, Herbert; Gamblin, T Clark; Kunnimalaiyaan, Muthusamy

2014-05-01

Neuroblastoma is a common neuroendocrine (NE) tumor that presents in early childhood, with a high incidence of malignancy and recurrence. The glycogen synthase kinase-3 (GSK-3) pathway is a potential therapeutic target, as this pathway has been shown to be crucial in the management of other NE tumors. However, it is not known which isoform is necessary for growth inhibition. In this study, we investigated the effect of the GSK-3 inhibitor AR-A014418 on the different GSK-3 isoforms in neuroblastoma. NGP and SH-5Y-SY cells were treated with 0-20 μM of AR-A014418 and cell viability was measured by MTT assay. Expression levels of NE markers CgA and ASCL1, GSK-3 isoforms, and apoptotic markers were analyzed by western blot. Neuroblastoma cells treated with AR-A014418 had a significant reduction in growth at all doses and time points (P<0.001). A reduction in growth was noted in cell lines on day 6, with 10 μM (NGP-53% vs. 0% and SH-5Y-SY-38% vs. 0%, P<0.001) treatment compared to control, corresponding with a noticeable reduction in tumor marker ASCL1 and CgA expression. Treatment of neuroblastoma cell lines with AR-A014418 reduced the level of GSK-3α phosphorylation at Tyr279 compared to GSK-3β phosphorylation at Tyr216, and attenuated growth via the maintenance of apoptosis. This study supports further investigation to elucidate the mechanism(s) by which GSK-3α inhibition downregulates the expression of NE tumor markers and growth of neuroblastoma.

Small-interfering RNAs from natural antisense transcripts derived from a cellulose synthase gene modulate cell wall biosynthesis in barley

PubMed Central

Held, Michael A.; Penning, Bryan; Brandt, Amanda S.; Kessans, Sarah A.; Yong, Weidong; Scofield, Steven R.; Carpita, Nicholas C.

2008-01-01

Small-interfering RNAs (siRNAs) from natural cis-antisense pairs derived from the 3′-coding region of the barley (Hordeum vulgare) CesA6 cellulose synthase gene substantially increase in abundance during leaf elongation. Strand-specific RT-PCR confirmed the presence of an antisense transcript of HvCesA6 that extends ≥1230 bp from the 3′ end of the CesA-coding sequence. The increases in abundance of the CesA6 antisense transcript and the 21-nt and 24-nt siRNAs derived from the transcript are coincident with the down-regulation of primary wall CesAs, several Csl genes, and GT8 glycosyl transferase genes, and are correlated with the reduction in rates of cellulose and (1 → 3),(1 → 4)-β-D-glucan synthesis. Virus induced gene silencing using unique target sequences derived from HvCesA genes attenuated expression not only of the HvCesA6 gene, but also of numerous nontarget Csls and the distantly related GT8 genes and reduced the incorporation of D-14C-Glc into cellulose and into mixed-linkage (1 → 3),(1 → 4)-β-D-glucans of the developing leaves. Unique target sequences for CslF and CslH conversely silenced the same genes and lowered rates of cellulose and (1 → 3),(1 → 4)-β-D-glucan synthesis. Our results indicate that the expression of individual members of the CesA/Csl superfamily and glycosyl transferases share common regulatory control points, and siRNAs from natural cis-antisense pairs derived from the CesA/Csl superfamily could function in this global regulation of cell-wall synthesis. PMID:19075248

Expression Patterns of Genes Involved in Sugar Metabolism and Accumulation during Apple Fruit Development

PubMed Central

Cheng, Lailiang

2012-01-01

Both sorbitol and sucrose are imported into apple fruit from leaves. The metabolism of sorbitol and sucrose fuels fruit growth and development, and accumulation of sugars in fruit is central to the edible quality of apple. However, our understanding of the mechanisms controlling sugar metabolism and accumulation in apple remains quite limited. We identified members of various gene families encoding key enzymes or transporters involved in sugar metabolism and accumulation in apple fruit using homology searches and comparison of their expression patterns in different tissues, and analyzed the relationship of their transcripts with enzyme activities and sugar accumulation during fruit development. At the early stage of fruit development, the transcript levels of sorbitol dehydrogenase, cell wall invertase, neutral invertase, sucrose synthase, fructokinase and hexokinase are high, and the resulting high enzyme activities are responsible for the rapid utilization of the imported sorbitol and sucrose for fruit growth, with low levels of sugar accumulation. As the fruit continues to grow due to cell expansion, the transcript levels and activities of these enzymes are down-regulated, with concomitant accumulation of fructose and elevated transcript levels of tonoplast monosaccharide transporters (TMTs), MdTMT1 and MdTMT2; the excess carbon is converted into starch. At the late stage of fruit development, sucrose accumulation is enhanced, consistent with the elevated expression of sucrose-phosphate synthase (SPS), MdSPS5 and MdSPS6, and an increase in its total activity. Our data indicate that sugar metabolism and accumulation in apple fruit is developmentally regulated. This represents a comprehensive analysis of the genes involved in sugar metabolism and accumulation in apple, which will serve as a platform for further studies on the functions of these genes and subsequent manipulation of sugar metabolism and fruit quality traits related to carbohydrates. PMID:22412983

Graft reconditioning with nitric oxide gas in rat liver transplantation from cardiac death donors.

PubMed

Kageyama, Shoichi; Yagi, Shintaro; Tanaka, Hirokazu; Saito, Shunichi; Nagai, Kazuyuki; Hata, Koichiro; Fujimoto, Yasuhiro; Ogura, Yasuhiro; Tolba, Rene; Shinji, Uemoto

2014-03-27

Liver transplant outcomes using grafts donated after cardiac death (DCD) remain poor. We investigated the effects of ex vivo reconditioning of DCD grafts with venous systemic oxygen persufflation using nitric oxide gas (VSOP-NO) in rat liver transplants. Orthotopic liver transplants were performed in Lewis rats, using DCD grafts prepared using static cold storage alone (group-control) or reconditioning using VSOP-NO during cold storage (group-VSOP-NO). Experiment I: In a 30-min warm ischemia model, graft damage and hepatic expression of inflammatory cytokines, endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and endothelin-1 (ET-1) were examined, and histologic analysis was performed 2, 6, 24, and 72 hr after transplantation. Experiment II: In a 60-min warm ischemia model, grafts were evaluated 2 hr after transplantation (6 rats/group), and survival was assessed (7 rats/group). Experiment I: Group-VSOP-NO had lower alanine aminotransferase (ALT) (P<0.001), hyaluronic acid (P<0.05), and malondialdehyde (MDA) (P<0.001), hepatic interleukin-6 expression (IL-6) (P<0.05), and hepatic tumor necrosis factor-alpha (TNF-α) expression (P<0.001). Hepatic eNOS expression (P<0.001) was upregulated, whereas hepatic iNOS (P<0.01) and ET-1 (P<0.001) expressions were downregulated. The damage of hepatocyte and sinusoidal endothelial cells (SECs) were lower in group-VSOP-NO.Experiment II: VSOP-NO decreased ET-1 and 8-hydroxy-2'deoxyguanosine (8-OHdG) expression and improved survival after transplantation by 71.4% (P<0.01). These results suggest that VSOP-NO effectively reconditions warm ischemia-damaged grafts, presumably by decreasing ET-1 upregulation and oxidative damage.

Hyperforin is a modulator of inducible nitric oxide synthase and phagocytosis in microglia and macrophages.

PubMed

Kraus, Birgit; Wolff, Horst; Elstner, Erich F; Heilmann, Jörg

2010-06-01

Upon activation, microglia, the immunocompetent cells in the brain, get highly phagocytic and release pro-inflammatory mediators like nitric oxide (NO). Excessive NO production is pivotal in neurodegenerative disorders, and there is evidence that abnormalities in NO production and inflammatory responses may at least support a range of neuropsychiatric disorders, including depression. Although extracts of St. John's wort (Hypericum perforatum L.) have been used for centuries in traditional medicine, notably for the treatment of depression, there is still considerable lack in scientific knowledge about the impact on microglia. We used N11 and BV2 mouse microglia, as well as RAW 264.7 macrophages to investigate the effects of St. John's wort extract and constituents thereof on NO production Moreover, flow cytometry and fluorescence microscopy were employed to analyze the influence on phagocytosis, transcription factor activation states, and cell motility. We found that extracts of St. John's wort efficiently suppress lipopolysaccharide-induced NO release and identified hyperforin as the responsible compound, being effective at concentrations between 0.25 and 0.75 microM. The reduced NO production was mediated by diminished inducible nitric oxide synthase expression on the mRNA and protein level. In addition, at similar concentrations, hyperforin reduced zymosan phygocytosis to 20-40% and putatively acted by downregulating the CD206 macrophage mannose receptor and modulation of cell motility. We found that the observed effects correlate with a suppression of the activated state of Nf-kappaB and phospho-CREB, while c-JUN, STAT1, and HIF-1alpha activity and cyclooxygenase-2 expression remained unaffected by hyperforin. These results reveal that hyperforin influences pro-inflammatory and immunological responses of microglia that are involved in the progression of neuropathologic disorders.

Transcriptional regulation of host NH₄⁺ transporters and GS/GOGAT pathway in arbuscular mycorrhizal rice roots.

PubMed

Pérez-Tienda, Jacob; Corrêa, Ana; Azcón-Aguilar, Concepción; Ferrol, Nuria

2014-02-01

Arbuscular mycorrhizal (AM) fungi play a key role in the nutrition of many land plants. AM roots have two pathways for nutrient uptake, directly through the root epidermis and root hairs and via AM fungal hyphae into root cortical cells, where arbuscules or hyphal coils provide symbiotic interfaces. Recent studies demonstrated that the AM symbiosis modifies the expression of plant transporter genes and that NH₄⁺ is the main form of N transported in the symbiosis. The aim of the present work was to get insights into the mycorrhizal N uptake pathway in Oryza sativa by analysing the expression of genes encoding ammonium transporters (AMTs), glutamine synthase (GS) and glutamate synthase (GOGAT) in roots colonized by the AM fungus Rhizophagus irregularis and grown under two N regimes. We found that the AM symbiosis down-regulated OsAMT1;1 and OsAMT1;3 expression at low-N, but not at high-N conditions, and induced, independently of the N status of the plant, a strong up-regulation of OsAMT3;1 expression. The AM-inducible NH₄⁺ transporter OsAMT3;1 belongs to the family 2 of plant AMTs and is phylogenetically related to the AM-inducible AMTs of other plant species. Moreover, for the first time we provide evidence of the specific induction of a GOGAT gene upon colonization with an AM fungus. These data suggest that OsAMT3;1 is likely involved in the mycorrhizal N uptake pathway in rice roots and that OsGOGAT2 plays a role in the assimilation of the NH₄⁺ supplied via the OsAMT3;1 AM-inducible transporter. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Reduced fat mass in rats fed a high oleic acid-rich safflower oil diet is associated with changes in expression of hepatic PPARalpha and adipose SREBP-1c-regulated genes.

PubMed

Hsu, Shan-Ching; Huang, Ching-Jang

2006-07-01

PPARs and sterol regulatory element-binding protein-1c (SREPB-1c) are fatty acid-regulated transcription factors that control lipid metabolism at the level of gene expression. This study compared a high oleic acid-rich safflower oil (ORSO) diet and a high-butter diet for their effect on adipose mass and expressions of genes regulated by PPAR and SREPB-1c in rats. Four groups of Wistar rats were fed 30S (30% ORSO), 5S (5% ORSO), 30B (29% butter + 1% ORSO), or 5B (4% butter plus 1% ORSO) diets for 15 wk. Compared with the 30B group, the 30S group had less retroperitoneal white adipose tissue (RWAT) mass and lower mRNA expressions of lipoprotein lipase, adipocyte fatty acid-binding protein, fatty acid synthase, acetyl CoA carboxylase, and SREBP-1c in the RWAT, higher mRNA expressions of acyl CoA oxidase, carnitine palmitoyl-transferase 1A, fatty acid binding protein, and mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in the liver (P < 0.05). The 18:2(n-6) and 20:4(n-6) contents in the liver and RWAT of the 30S group were >2 fold those of the 30B group (P < 0.05). These results suggested that the smaller RWAT mass in rats fed the high-ORSO diet might be related to the higher tissue 18:2(n-6) and 20:4(n-6). This in turn could upregulate the expressions of fatty acid catabolic genes through the activation of PPARalpha in the liver and downregulate the expressions of lipid storage and lipogenic gene through the suppression of SREBP-1c in the RWAT.

Tudor-staphylococcal nuclease regulates the expression and biological function of alkylglycerone phosphate synthase via nuclear factor-κB and microRNA-127 in human glioma U87MG cells.

PubMed

Zhang, Yongqiang; Jia, Jun; Li, Ying; Chen, Yan-Ge; Huang, Huan; Qiao, Yang; Zhu, Yu

2018-06-01

Glioma is one of the malignant tumor types detrimental to human health; therefore, it is important to find novel targets and therapeutics for this tumor. The downregulated expression of Tudor-staphylococcal nuclease (SN) and alkylglycerone phosphate synthase (AGPS) can decrease cancer malignancy, and the overexpression of them can the increase viability and migration potential of various tumor cell types; however, the role of AGPS in the proliferation and migration of glioma, and the association of Tudor-SN and AGPS in human glioma is not clear. In the present study, it was determined that AGPS silencing suppressed the proliferation and migration potential of glioma U87MG cells, and suppressed the expression of the circular RNAs circ-ubiquitin-associated protein 2, circ-zinc finger protein 292 and circ-homeodomain-interacting protein kinase 3, and the long non-coding RNAs H19 imprinted maternally expressed transcript (non-protein coding), colon cancer-associated transcript 1 (non-protein coding) and hepatocellular carcinoma upregulated long non-coding RNA. Furthermore, Tudor-SN silencing suppressed the expression of AGPS; however, nuclear factor (NF)-κB and microRNA (miR)-127 retrieval experiments partially reduced the expression of AGPS. Additionally, it was determined that Tudor-SN silencing suppressed the activity of the mechanistic target of rapamycin (mTOR) signaling pathway, and NF-κB and miR-127 retrieval experiments partially reduced the activity of mTOR. Therefore, it was considered that NF-κB and miR-127 may be the mediators of Tudor-SN-regulated AGPS via the mTOR signaling pathway. These results improve on our knowledge of the mechanisms underlying Tudor-SN and AGPS in human glioma.

Lysophosphatidylcholine up-regulates human endothelial nitric oxide synthase gene transactivity by c-Jun N-terminal kinase signalling pathway

PubMed Central

Xing, Feiyue; Liu, Jing; Mo, Yongyan; Liu, Zhifeng; Qin, Qinghe; Wang, Jingzhen; Fan, Zhenhua; Long, Yutian; Liu, Na; Zhao, Kesen; Jiang, Yong

2009-01-01

Human endothelial nitric oxide synthase (eNOS) plays a pivotal role in maintaining blood pressure homeostasis and vascular integrity. It has recently been reported that mitogen-activated protein kinases (MAPKs) are intimately implicated in expression of eNOS. However detailed mechanism mediated by them remains to be clarified. In this study, eNOS gene transactivity in human umbilical vein endothelial cells was up-regulated by stimulation of lysophosphatidylcholine (LPC). The stimulation of LPC highly activated both extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK), with differences in the dynamic processes of activation between them. Unexpectedly, p38 MAPK could not be activated by the stimulation of LPC. The activation of JNK signalling pathway by overexpression of JNK or its upstream kinase active mutant up-regulated the transactivity of eNOS significantly, but the activation of p38 signalling pathway down-regulated it largely. The inhibition of either ERK1/2 or JNK signalling pathway by kinase-selective inhibitors could markedly block the induction of the transactivity by LPC. It was observed by electrophoretic mobility shift assay that LPC stimulated both SP1 and AP1 DNA binding activity to go up. Additionally using decoy oligonucleotides proved that SP1 was necessary for maintaining the basal or stimulated transactivity, whereas AP1 contributed mainly to the increase of the stimulated transactivity. These findings indicate that the up-regulation of the eNOS gene transactivity by LPC involves the enhancement of SP1 transcription factor by the activation of JNK and ERK1/2 signalling pathways and AP1 transcription factor by the activation of JNK signalling pathway. PMID:18624763

The Selective Glucocorticoid Receptor Modulator Cort 113176 Reduces Neurodegeneration and Neuroinflammation in Wobbler Mice Spinal Cord.

PubMed

Meyer, Maria; Lara, Agustina; Hunt, Hazel; Belanoff, Joseph; de Kloet, E Ronald; Gonzalez Deniselle, Maria Claudia; De Nicola, Alejandro F

2018-06-08

Wobbler mice are experimental models for amyotrophic lateral sclerosis. As such they show motoneuron degeneration, motor deficits, and astrogliosis and microgliosis of the spinal cord. Additionally, Wobbler mice show increased plasma, spinal cord and brain corticosterone levels and focal adrenocortical hyperplasia, suggesting a pathogenic role for glucocorticoids in this disorder. Considering this endocrine background, we examined whether the glucocorticoid receptor (GR) modulator CORT 113176 prevents spinal cord neuropathology of Wobblers. CORT 113176 shows high affinity for the GR, with low or null affinity for other steroid receptors. We employed five-month-old genotyped Wobbler mice that received s.c. vehicle or 30 mg/kg/day for 4 days of CORT 113176 dissolved in sesame oil. The mice were used on the 4th day, 2 h after the last dose of CORT 113176. Vehicle-treated Wobbler mice presented vacuolated motoneurons, increased glial fibrillary acidic protein (GFAP)+ astrocytes and decreased glutamine synthase (GS)+ cells. There was strong neuroinflammation, shown by increased staining for IBA1+ microglia and CD11b mRNA, enhanced expression of tumor necrosis factor-α, its cognate receptor TNFR1, toll-like receptor 4, the inducible nitric oxide synthase, NFkB and the high-mobility group box 1 protein (HMGB1). Treatment of Wobbler mice with CORT 113176 reversed the abnormalities of motoneurons and down-regulated proinflammatory mediators and glial reactivity. Expression of glutamate transporters GLT1 and GLAST mRNAs and GLT1 protein was significantly enhanced over untreated Wobblers. In summary, antagonism of GR with CORT 113176 prevented neuropathology and showed anti-inflammatory and anti-glutamatergic effects in the spinal cord of Wobbler mice. Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.

The novel thymidylate synthase inhibitor trifluorothymidine (TFT) and TRAIL synergistically eradicate non-small cell lung cancer cells.

PubMed

Azijli, Kaamar; van Roosmalen, Ingrid A M; Smit, Jorn; Pillai, Saravanan; Fukushima, Masakazu; de Jong, Steven; Peters, Godefridus J; Bijnsdorp, Irene V; Kruyt, Frank A E

2014-06-01

TRAIL, a tumor selective anticancer agent, may be used for the treatment of non-small cell lung cancer (NSCLC). However, TRAIL resistance is frequently encountered. Here, the combined use of TRAIL with trifluorothymidine (TFT), a thymidylate synthase inhibitor, was examined for sensitizing NSCLC cells to TRAIL. Interactions between TRAIL and TFT were studied in NSCLC cells using growth inhibition and apoptosis assays. Western blotting and flow cytometry were used to investigate underlying mechanisms. The combined treatment of TFT and TRAIL showed synergistic cytotoxicity in A549, H292, H322 and H460 cells. For synergistic activity, the sequence of administration was important; TFT treatment followed by TRAIL exposure did not show sensitization. Combined TFT and TRAIL treatment for 24 h followed by 48 h of TFT alone was synergistic in all cell lines, with combination index values below 0.9. The treatments affected cell cycle progression, with TRAIL inducing a G1 arrest and TFT, a G2/M arrest. TFT activated Chk2 and reduced Cdc25c levels known to cause G2/M arrest. TRAIL-induced caspase-dependent apoptosis was enhanced by TFT, whereas TFT alone mainly induced caspase-independent death. TFT increased the expression of p53 and p21/WAF1, and p53 was involved in the increase of TRAIL-R2 surface expression. TFT also caused downregulation of cFLIP and XIAP and increased Bax expression. TFT enhances TRAIL-induced apoptosis in NSCLC cells by sensitizing the apoptotic machinery at different levels in the TRAIL pathway. Our findings suggest a possible therapeutic benefit of the combined use of TFT and TRAIL in NSCLC.

Downregulation of putative UDP-glucose: flavonoid 3-O-glucosyltransferase gene alters flower coloring in Phalaenopsis.

PubMed

Chen, Wen-Huei; Hsu, Chi-Yin; Cheng, Hao-Yun; Chang, Hsiang; Chen, Hong-Hwa; Ger, Mang-Jye

2011-06-01

Anthocyanin is the primary pigment contributing to red, violet, and blue flower color formation. The solubility of anthocyanins is enhanced by UDP glucose: flavonoid 3-O-glucosyltransferase (UFGT) through transfer of the glucosyl moiety from UDP-glucose to 3-hydroxyl group to produce the first stable pigments. To assess the possibility that UFGT is involved in the flower color formation in Phalaenopsis, the transcriptional activities of PeUFGT3, and other flower color-related genes in developing red or white flower buds were examined using RT-PCR analysis. In contrast with chalcone synthase, chalcone isomerase, and anthocyanidin synthase genes, PeUFGT3 transcriptional activity was higher expressed in the red color of Phalaenopsis cultivars. In the red labellum of Phalaenopsis 'Luchia Lady', PeUFGT3 also showed higher expression levels than that in the white perianth. PeUFGT3 was predominantly expressed in the red region of flower among various Phalaenopsis cultivars. To investigate the role of PeUFGT3 in red flower color formation, PeUFGT3 was specifically knocked down using RNA interference technology via virus inducing gene silencing in Phalaenopsis. The PeUFGT3-suppressed Phalaenopsis exhibited various levels of flower color fading that was well correlated with the extent of reduced level of PeUFGT3 transcriptional activity. Furthermore, there was a significant decrease in anthocyanin content in the PeUFGT3-suppressed Phalaenopsis flowers. The decrease of anthocyanin content due to PeUFGT3 gene silencing possibly caused the faded flower color in PeUFGT3-suppressed Phalaenopsis. Consequently, these results suggested that the glycosylation-related gene PeUFGT3 plays a critical role in red color formation in Phalaenopsis.

Transcriptome Analysis of Dendrobium officinale and its Application to the Identification of Genes Associated with Polysaccharide Synthesis

PubMed Central

Zhang, Jianxia; He, Chunmei; Wu, Kunlin; Teixeira da Silva, Jaime A.; Zeng, Songjun; Zhang, Xinhua; Yu, Zhenming; Xia, Haoqiang; Duan, Jun

2016-01-01

Dendrobium officinale is one of the most important Chinese medicinal herbs. Polysaccharides are one of the main active ingredients of D. officinale. To identify the genes that maybe related to polysaccharides synthesis, two cDNA libraries were prepared from juvenile and adult D. officinale, and were named Dendrobium-1 and Dendrobium-2, respectively. Illumina sequencing for Dendrobium-1 generated 102 million high quality reads that were assembled into 93,881 unigenes with an average sequence length of 790 base pairs. The sequencing for Dendrobium-2 generated 86 million reads that were assembled into 114,098 unigenes with an average sequence length of 695 base pairs. Two transcriptome databases were integrated and assembled into a total of 145,791 unigenes. Among them, 17,281 unigenes were assigned to 126 KEGG pathways while 135 unigenes were involved in fructose and mannose metabolism. Gene Ontology analysis revealed that the majority of genes were associated with metabolic and cellular processes. Furthermore, 430 glycosyltransferase and 89 cellulose synthase genes were identified. Comparative analysis of both transcriptome databases revealed a total of 32,794 differential expression genes (DEGs), including 22,051 up-regulated and 10,743 down-regulated genes in Dendrobium-2 compared to Dendrobium-1. Furthermore, a total of 1142 and 7918 unigenes showed unique expression in Dendrobium-1 and Dendrobium-2, respectively. These DEGs were mainly correlated with metabolic pathways and the biosynthesis of secondary metabolites. In addition, 170 DEGs belonged to glycosyltransferase genes, 37 DEGs were related to cellulose synthase genes and 627 DEGs encoded transcription factors. This study substantially expands the transcriptome information for D. officinale and provides valuable clues for identifying candidate genes involved in polysaccharide biosynthesis and elucidating the mechanism of polysaccharide biosynthesis. PMID:26904032

Intracellular acidification reduces l-arginine transport via system y+L but not via system y+/CATs and nitric oxide synthase activity in human umbilical vein endothelial cells.

PubMed

Ramírez, Marco A; Morales, Jorge; Cornejo, Marcelo; Blanco, Elias H; Mancilla-Sierpe, Edgardo; Toledo, Fernando; Beltrán, Ana R; Sobrevia, Luis

2018-04-01

l-Arginine is taken up via the cationic amino acid transporters (system y + /CATs) and system y + L in human umbilical vein endothelial cells (HUVECs). l-Arginine is the substrate for endothelial NO synthase (eNOS) which is activated by intracellular alkalization, but nothing is known regarding modulation of system y + /CATs and system y + L activity, and eNOS activity by the pHi in HUVECs. We studied whether an acidic pHi modulates l-arginine transport and eNOS activity in HUVECs. Cells loaded with a pH-sensitive probe were subjected to 0.1-20 mmol/L NH 4 Cl pulse assay to generate pHi 7.13-6.55. Before pHi started to recover, l-arginine transport (0-20 or 0-1000 μmol/L, 10 s, 37 °C) in the absence or presence of 200 μmol/L N-ethylmaleimide (NEM) (system y + /CATs inhibitor) or 2 mmol/L l-leucine (systemy + L substrate) was measured. Protein abundance for eNOS and serine 1177 or threonine 495 phosphorylated eNOS was determined. The results show that intracellular acidification reduced system y + L but not system y + /CATs mediated l-arginine maximal transport capacity due to reduced maximal velocity. Acidic pHi reduced NO synthesis and eNOS serine 1177 phosphorylation. Thus, system y + L activity is downregulated by an acidic pHi, a phenomenon that may result in reduced NO synthesis in HUVECs. Copyright © 2018 Elsevier B.V. All rights reserved.

Expression of the nuclear gene TaF(A)d is under mitochondrial retrograde regulation in anthers of male sterile wheat plants with timopheevii cytoplasm.

PubMed

Xu, Pei; Yang, Yuwen; Zhang, Zhengzhi; Chen, Weihua; Zhang, Caiqin; Zhang, Lixia; Zou, Sixiang; Ma, Zhengqiang

2008-01-01

Alterations of mitochondrial-encoded subunits of the F(o)F(1)-ATP synthase are frequently associated with cytoplasmic male sterility (CMS) in plants; however, little is known about the relationship of the nuclear encoded subunits of this enzyme with CMS. In the present study, the full cDNA of the gene TaF(A)d that encodes the putative F(A)d subunit of the F(o)F(1)-ATP synthase was isolated from the wheat (Triticum aestivum) fertility restorer '2114' for timopheevii cytoplasm-based CMS. The deduced 238 amino acid polypeptide is highly similar to its counterparts in dicots and other monocots but has low homology to its mammalian equivalents. TaF(A)d is a single copy gene in wheat and maps to the short arm of the group 6 chromosomes. Transient expression of the TaF(A)d-GFP fusion in onion epidermal cells demonstrated TaF(A)d's mitochondrial location. TaF(A)d was expressed abundantly in stem, leaf, anther, and ovary tissues of 2114. Nevertheless, its expression was repressed in anthers of CMS plants with timopheevii cytoplasm. Genic male sterility did not affect its expression in anthers. The expression of the nuclear gene encoding the 20 kDa subunit of F(o) was down-regulated in a manner similar to TaF(A)d in the T-CMS anthers while that of genes encoding the 6 kDa subunit of F(o) and the gamma subunit of F(1) was unaffected. These observations implied that TaF(A)d is under mitochondrial retrograde regulation in the anthers of CMS plants with timopheevii cytoplasm.

Metabolism of organic acids, nitrogen and amino acids in chlorotic leaves of 'Honeycrisp' apple (Malus domestica Borkh) with excessive accumulation of carbohydrates.

PubMed

Wang, Huicong; Ma, Fangfang; Cheng, Lailiang

2010-07-01

Metabolite profiles and activities of key enzymes in the metabolism of organic acids, nitrogen and amino acids were compared between chlorotic leaves and normal leaves of 'Honeycrisp' apple to understand how accumulation of non-structural carbohydrates affects the metabolism of organic acids, nitrogen and amino acids. Excessive accumulation of non-structural carbohydrates and much lower CO(2) assimilation were found in chlorotic leaves than in normal leaves, confirming feedback inhibition of photosynthesis in chlorotic leaves. Dark respiration and activities of several key enzymes in glycolysis and tricarboxylic acid (TCA) cycle, ATP-phosphofructokinase, pyruvate kinase, citrate synthase, aconitase and isocitrate dehydrogenase were significantly higher in chlorotic leaves than in normal leaves. However, concentrations of most organic acids including phosphoenolpyruvate (PEP), pyruvate, oxaloacetate, 2-oxoglutarate, malate and fumarate, and activities of key enzymes involved in the anapleurotic pathway including PEP carboxylase, NAD-malate dehydrogenase and NAD-malic enzyme were significantly lower in chlorotic leaves than in normal leaves. Concentrations of soluble proteins and most free amino acids were significantly lower in chlorotic leaves than in normal leaves. Activities of key enzymes in nitrogen assimilation and amino acid synthesis, including nitrate reductase, glutamine synthetase, ferredoxin and NADH-dependent glutamate synthase, and glutamate pyruvate transaminase were significantly lower in chlorotic leaves than in normal leaves. It was concluded that, in response to excessive accumulation of non-structural carbohydrates, glycolysis and TCA cycle were up-regulated to "consume" the excess carbon available, whereas the anapleurotic pathway, nitrogen assimilation and amino acid synthesis were down-regulated to reduce the overall rate of amino acid and protein synthesis.

Chromosome doubling to overcome the chrysanthemum cross barrier based on insight from transcriptomic and proteomic analyses.

PubMed

Zhang, Fengjiao; Hua, Lichun; Fei, Jiangsong; Wang, Fan; Liao, Yuan; Fang, Weimin; Chen, Fadi; Teng, Nianjun

2016-08-09

Cross breeding is the most commonly used method in chrysanthemum (Chrysanthemum morifolium) breeding; however, cross barriers always exist in these combinations. Many studies have shown that paternal chromosome doubling can often overcome hybridization barriers during cross breeding, although the underlying mechanism has seldom been investigated. In this study, we performed two crosses: C. morifolium (pollen receptor) × diploid C. nankingense (pollen donor) and C. morifolium × tetraploid C. nankingense. Seeds were obtained only from the latter cross. RNA-Seq and isobaric tags for relative and absolute quantitation (iTRAQ) were used to investigate differentially expressed genes and proteins during key embryo development stages in the latter cross. A previously performed cross, C. morifolium × diploid C. nankingense, was compared to our results and revealed that transcription factors (i.e., the agamous-like MADS-box protein AGL80 and the leucine-rich repeat receptor protein kinase EXS), hormone-responsive genes (auxin-binding protein 1), genes and proteins related to metabolism (ATP-citrate synthase, citrate synthase and malate dehydrogenase) and other genes reported to contribute to embryo development (i.e., LEA, elongation factor and tubulin) had higher expression levels in the C. morifolium × tetraploid C. nankingense cross. In contrast, genes related to senescence and cell death were down-regulated in the C. morifolium × tetraploid C. nankingense cross. The data resources helped elucidate the gene and protein expression profiles and identify functional genes during different development stages. When the chromosomes from the male parent are doubled, the genes contributing to normal embryo developmentare more abundant. However, genes with negative functions were suppressed, suggesting that chromosome doubling may epigenetically inhibit the expression of these genes and allow the embryo to develop normally.

Overexpression of hyaluronan synthase 2 and gonadotropin receptors in cumulus cells of goats subjected to one-shot eCG/FSH hormonal treatment for ovarian stimulation.

PubMed

Santos, Juliana D R; Batista, Ribrio I T P; Magalhães, Livia C; Paula, Alexandre R; Souza, Samara S; Salamone, Daniel F; Bhat, Maajid H; Teixeira, Dárcio I A; Freitas, Vicente J F; Melo, Luciana M

2016-07-01

Hormonal ovarian stimulation may affect transcripts in somatic cells of cumulus-oocyte complexes (COCs) and affect the resulting oocyte quality. Here, in parallel with morphological classification and in vitro maturation (IVM) rate analysis, we investigated the expression of hyaluronan synthase 2 (HAS2), gonadotropic receptors (FSHR and LHR) and connexin 43 (GJA1) in cumulus cells (CCs) from goat COCs after multi-dose FSH (MD) or one-shot FSH/eCG (OS) treatments, using bovine COCs as control groups. The MD treatment produced more large follicles, and the resulting COCs had a better morphology and IVM rate than were obtained with OS. The OS treatment produced COCs with increased HAS2, FSHR, LHR and GJA1 expression. This gene expression pattern was also observed in the CCs of COCs that showed poor morphological characteristics. On the other hand, the mRNA levels were more similar between groups after IVM; FSHR and LHR were the main genes that showed decreased expression. Some events that occurred in bovine CCs during IVM, such as cell expansion, increased HAS2 expression and decreased GJA1 expression, were less evident or did not occur in goat COCs. In conclusion, increasing HAS2, FSHR, LHR and GJA1 expression in goat COCs does not confer greater meiotic competence to oocytes. Instead, it may result from poor regulation of gene expression in CCs by lower quality oocytes. Finally, cumulus expansion, together with HAS2 upregulation and GJA1 downregulation, seems to be more important for bovine COCs than for goat COCs. Additional studies are needed to investigate the importance of other HAS isoforms and connexins in goat COCs. Copyright © 2016 Elsevier B.V. All rights reserved.

Inhibition of neutral sphingomyelinase decreases elevated levels of inducible nitric oxide synthase and apoptotic cell death in ocular hypertensive rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aslan, Mutay, E-mail: mutayaslan@akdeniz.edu.tr; Basaranlar, Goksun; Unal, Mustafa

Endoplasmic reticulum (ER) stress and excessive nitric oxide production via induction of inducible nitric oxide synthase (NOS2) have been implicated in the pathogenesis of neuronal retinal cell death in ocular hypertension. Neutral sphingomyelinase (N-SMase)/ceramide pathway can regulate NOS2 expression, hence this study determined the role of selective neutral sphingomyelinase (N-SMase) inhibition on retinal NOS2 levels, ER stress, apoptosis and visual evoked potentials (VEPs) in a rat model of elevated intraocular pressure (EIOP). NOS2 expression and retinal protein nitration were significantly greater in EIOP and significantly decreased with N-SMase inhibition. A significant increase was observed in retinal ER stress markers pPERK,more » CHOP and GRP78 in EIOP, which were not significantly altered by N-SMase inhibition. Retinal TUNEL staining showed increased apoptosis in all EIOP groups; however N-SMase inhibition significantly decreased the percent of apoptotic cells in EIOP. Caspase-3, -8 and -9 activities were significantly increased in EIOP and returned to baseline levels following N-SMase inhibition. Latencies of all VEP components were significantly prolonged in EIOP and shortened following N-SMase inhibition. Data confirm the role of nitrative injury in EIOP and highlight the protective effect of N-SMase inhibition in EIOP via down-regulation of NOS2 levels and nitrative stress. - Highlights: • Inhibition of N-SMase decreases NOS2 levels in ocular hypertension. • Inhibition of N-SMase decreases protein nitration in ocular hypertension. • Inhibition of N-SMase decreases caspase activation in ocular hypertension. • Inhibition of N-SMase decreases apoptosis in ocular hypertension.« less

Bauhinia championii flavone inhibits apoptosis and autophagy via the PI3K/Akt pathway in myocardial ischemia/reperfusion injury in rats

PubMed Central

Jian, Jie; Xuan, Feifei; Qin, Feizhang; Huang, Renbin

2015-01-01

This study aimed to determine the effects of Bauhinia championii flavone (BCF) on myocardial ischemia/reperfusion injury (MI/RI) in rats and to explore potential mechanisms. The MI/RI model in rats was established by ligating the left anterior descending coronary artery for 30 minutes, then reperfusing for 3 hours. BCF at 20 mg/kg was given 20 minutes prior to ischemia via sublingual intravenous injection, with 24 μg/kg phosphoinositide 3-kinase inhibitor (PI3K; wortmannin) as a control. The creatine kinase-MB and nitric oxide content were assessed by colorimetry. The levels of mitochondrial permeability transition pores and tumor necrosis factor alpha were determined by an enzyme-linked immunosorbent assay. Cardiomyocyte apoptosis was detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Additionally, the expression of PI3K, endothelial nitric oxide synthase, caspase-3, and Beclin1 was analyzed by fluorescence quantitative polymerase chain reaction and Western blotting, respectively. Akt and microtubule-associated protein 1 light chain 3-II protein levels were also evaluated. Pretreatment with BCF significantly decreased the levels of creatine kinase-MB, tumor necrosis factor alpha, and mitochondrial permeability transition pores, but increased the nitric oxide content. Furthermore, BCF inhibited apoptosis, downregulated caspase-3, Beclin1, and microtubule-associated protein 1 light chain 3-II, upregulated PI3K, and increased the protein levels of phosphorylated Akt and endothelial nitric oxide synthase. However, all of the previously mentioned effects of BCF were blocked when BCF was coadministered with wortmannin. In conclusion, these observations indicated that BCF has cardioprotective effects against MI/RI by reducing cell apoptosis and excessive autophagy, which might be related to the activation of the PI3K/Akt signaling pathway. PMID:26604691

Bauhinia championii flavone inhibits apoptosis and autophagy via the PI3K/Akt pathway in myocardial ischemia/reperfusion injury in rats.

PubMed

Jian, Jie; Xuan, Feifei; Qin, Feizhang; Huang, Renbin

2015-01-01

This study aimed to determine the effects of Bauhinia championii flavone (BCF) on myocardial ischemia/reperfusion injury (MI/RI) in rats and to explore potential mechanisms. The MI/RI model in rats was established by ligating the left anterior descending coronary artery for 30 minutes, then reperfusing for 3 hours. BCF at 20 mg/kg was given 20 minutes prior to ischemia via sublingual intravenous injection, with 24 μg/kg phosphoinositide 3-kinase inhibitor (PI3K; wortmannin) as a control. The creatine kinase-MB and nitric oxide content were assessed by colorimetry. The levels of mitochondrial permeability transition pores and tumor necrosis factor alpha were determined by an enzyme-linked immunosorbent assay. Cardiomyocyte apoptosis was detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Additionally, the expression of PI3K, endothelial nitric oxide synthase, caspase-3, and Beclin1 was analyzed by fluorescence quantitative polymerase chain reaction and Western blotting, respectively. Akt and microtubule-associated protein 1 light chain 3-II protein levels were also evaluated. Pretreatment with BCF significantly decreased the levels of creatine kinase-MB, tumor necrosis factor alpha, and mitochondrial permeability transition pores, but increased the nitric oxide content. Furthermore, BCF inhibited apoptosis, downregulated caspase-3, Beclin1, and microtubule-associated protein 1 light chain 3-II, upregulated PI3K, and increased the protein levels of phosphorylated Akt and endothelial nitric oxide synthase. However, all of the previously mentioned effects of BCF were blocked when BCF was coadministered with wortmannin. In conclusion, these observations indicated that BCF has cardioprotective effects against MI/RI by reducing cell apoptosis and excessive autophagy, which might be related to the activation of the PI3K/Akt signaling pathway.

Changes in the expression of prostaglandin family members in bovine corpus luteum during the oestrous cycle and pregnancy.

PubMed

Berisha, Bajram; Schams, Dieter; Rodler, Daniela; Sinowatz, Fred; Pfaffl, Michael W

2018-06-06

The aim of this study was to characterize certain prostaglandin family members in the bovine corpus luteum (CL) during the oestrous cycle and pregnancy. The CL tissue was assigned to the following stages of the oestrous cycle: 1-2, 3-4, 5-7, 8-12, 13-16, >18 days (after regression) and of pregnancy: 1-2, 3-4, 6-7 and >8 months. In these samples, we investigated prostaglandin F2alpha (PTGF), prostaglandin E2 (PTGE) and their receptors (PTGFR, PTGER2, PTGER4), cyclooxygenase 2 (COX-2), PTGF synthase (PTGFS) and PTGE synthase (PTGES). The expression of mRNA was measured by RT-qPCR, hormones by EIA and localization by immunohistochemistry. The mRNA expression of COX-2, PTGFS and PTGES in CL during the early luteal phase was high followed by a continuous and significant downregulation afterwards, as well as during all phases of pregnancy. The concentration of PTGF in CL tissue was high during the early luteal phase, decreased significantly in the mid-luteal phase, and increased again afterwards. In contrast, the concentration of PTGE increased significantly during late luteal phase followed by a decrease during regression. The PTGE level increased again during late pregnancy. Immunohistochemically, the large granulose-luteal cells show strong staining for COX-2 and PTGES during the early luteal stage followed by lower activity afterwards. During pregnancy, most of the luteal cells were only weakly positive or negative. In conclusion, our results indicate that the examined prostaglandin family members are involved in the local mechanisms that regulate luteal function, specifically during CL formation, function and regression and during pregnancy in the cow. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Geranyl diphosphate synthase large subunit, and methods of use

DOEpatents

Croteau, Rodney B.; Burke, Charles C.; Wildung, Mark R.

2001-10-16

A cDNA encoding geranyl diphosphate synthase large subunit from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase large subunit). In another aspect, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase large subunit. In yet another aspect, the present invention provides isolated, recombinant geranyl diphosphate synthase protein comprising an isolated, recombinant geranyl diphosphate synthase large subunit protein and an isolated, recombinant geranyl diphosphate synthase small subunit protein. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase.

Molecular architectures of benzoic acid-specific type III polyketide synthases

PubMed Central

Stewart, Charles; Woods, Kate; Macias, Greg; Allan, Andrew C.; Noel, Joseph P.

2017-01-01

Biphenyl synthase and benzophenone synthase constitute an evolutionarily distinct clade of type III polyketide synthases (PKSs) that use benzoic acid-derived substrates to produce defense metabolites in plants. The use of benzoyl-CoA as an endogenous substrate is unusual for type III PKSs. Moreover, sequence analyses indicate that the residues responsible for the functional diversification of type III PKSs are mutated in benzoic acid-specific type III PKSs. In order to gain a better understanding of structure–function relationships within the type III PKS family, the crystal structures of biphenyl synthase from Malus × domestica and benzophenone synthase from Hypericum androsaemum were compared with the structure of an archetypal type III PKS: chalcone synthase from Malus × domestica. Both biphenyl synthase and benzophenone synthase contain mutations that reshape their active-site cavities to prevent the binding of 4-coumaroyl-CoA and to favor the binding of small hydrophobic substrates. The active-site cavities of biphenyl synthase and benzophenone synthase also contain a novel pocket associated with their chain-elongation and cyclization reactions. Collectively, these results illuminate structural determinants of benzoic acid-specific type III PKSs and expand the understanding of the evolution of specialized metabolic pathways in plants. PMID:29199980

Suites of Terpene Synthases Explain Differential Terpenoid Production in Ginger and Turmeric Tissues

PubMed Central

Koo, Hyun Jo; Gang, David R.

2012-01-01

The essential oils of ginger (Zingiber officinale) and turmeric (Curcuma longa) contain a large variety of terpenoids, some of which possess anticancer, antiulcer, and antioxidant properties. Despite their importance, only four terpene synthases have been identified from the Zingiberaceae family: (+)-germacrene D synthase and (S)-β-bisabolene synthase from ginger rhizome, and α-humulene synthase and β-eudesmol synthase from shampoo ginger (Zingiber zerumbet) rhizome. We report the identification of 25 mono- and 18 sesquiterpene synthases from ginger and turmeric, with 13 and 11, respectively, being functionally characterized. Novel terpene synthases, (−)-caryolan-1-ol synthase and α-zingiberene/β-sesquiphellandrene synthase, which is responsible for formation of the major sesquiterpenoids in ginger and turmeric rhizomes, were also discovered. These suites of enzymes are responsible for formation of the majority of the terpenoids present in these two plants. Structures of several were modeled, and a comparison of sets of paralogs suggests how the terpene synthases in ginger and turmeric evolved. The most abundant and most important sesquiterpenoids in turmeric rhizomes, (+)-α-turmerone and (+)-β-turmerone, are produced from (−)-α-zingiberene and (−)-β-sesquiphellandrene, respectively, via α-zingiberene/β-sesquiphellandrene oxidase and a still unidentified dehydrogenase. PMID:23272109

Geranyl diphosphate synthase from mint

DOEpatents

Croteau, Rodney Bruce; Wildung, Mark Raymond; Burke, Charles Cullen; Gershenzon, Jonathan

1999-01-01

A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate.

Geranyl diphosphate synthase from mint

DOEpatents

Croteau, R.B.; Wildung, M.R.; Burke, C.C.; Gershenzon, J.

1999-03-02

A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate. 5 figs.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsai, Jie-Heng; Hsu, Li-Sung; Clinical Laboratory, Chung Shan Medical University Hospital, Taichung 402, Taiwan, ROC

The molecular basis of epithelial–mesenchymal transition (EMT) functions as a potential therapeutic target for breast cancer because EMT may endow breast tumor-initiating cells with stem-like characteristics and enable the dissemination of breast cancer cells. We have recently verified the antitumor activity of 3,5,4′-trimethoxystilbene (MR-3), a naturally methoxylated derivative of resveratrol, in colorectal cancer xenografts via an induction of apoptosis. The effect of MR-3 on EMT and the invasiveness of human MCF-7 breast adenocarcinoma cell line were also explored. We found that MR-3 significantly increased epithelial marker E-cadherin expression and triggered a cobblestone-like morphology of MCF-7 cells, while reciprocally decreasing themore » expression of mesenchymal markers, such as snail, slug, and vimentin. In parallel with EMT reversal, MR-3 downregulated the invasion and migration of MCF-7 cells. Exploring the action mechanism of MR-3 on the suppression of EMT and invasion indicates that MR-3 markedly reduced the expression and nuclear translocation of β-catenin, accompanied with the downregulation of β-catenin target genes and the increment of membrane-bound β-catenin. These results suggest the involvement of Wnt/β-catenin signaling in the MR-3-induced EMT reversion of MCF-7 cells. Notably, MR-3 restored glycogen synthase kinase-3β activity by inhibiting the phosphorylation of Akt, the event required for β-catenin destruction via a proteasome-mediated system. Overall, these findings indicate that the anti-invasive activity of MR-3 on MCF-7 cells may result from the suppression of EMT via down-regulating phosphatidylinositol 3-kinase (PI3K)/AKT signaling, and consequently, β-catenin nuclear translocation. These occurrences ultimately lead to the blockage of EMT and the invasion of breast cancer cells. - Highlights: • MR-3 blocked MCF-7 cell invasion by inducing a reversal of EMT. • Wnt/β-catenin signaling is involved in MR-3-induced EMT reversion of MCF-7 cells. • Knockdown of β-catenin was sufficient to restore epithelial marker E-cadherin levels. • MR-3 recovered the function of GSK-3β that inhibits β-catenin nuclear translocation.« less

Ursolic acid inhibits breast cancer growth by inhibiting proliferation, inducing autophagy and apoptosis, and suppressing inflammatory responses via the PI3K/AKT and NF-κB signaling pathways in vitro

PubMed Central

Luo, Juan; Hu, Yan-Ling; Wang, Hong

2017-01-01

Breast cancer, which is the second leading cause of cancer-associated mortality in women worldwide, develops from breast tissue. Chemotherapy is the most commonly used therapy to treat breast cancer. However, a number of natural plant-derived products have been suggested as alternative therapies to treat different types of cancer, such as breast cancer. The aim of the present study was to determine the anti-tumor effects of ursolic acid and its effect on apoptosis and inflammation in breast cancer cells. The anti-cancer effects of ursolic acid were evaluated in vitro using flow cytometry, western blotting and reverse transcription-quantitative polymerase chain reaction. The results suggest that ursolic acid inhibits the viability of breast cancer cells by inducing autophagy and apoptosis without inducing cell death. Cellular migration assays demonstrated that ursolic acid was able to suppress the invasive ability of breast cancer cells (P<0.05). In addition, the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway was downregulated following ursolic acid administration (P<0.05), leading to an upregulation of glycogen synthase kinase activity (P<0.05) and downregulation of B-cell lymphoma 2 (P<0.05), subsequently causing autophagy and apoptosis via cyclin-D1 inhibition and caspase-3 stimulation (P<0.05). Furthermore, the inflammatory response of breast cancer cells was assessed by measuring levels of nuclear factor (NF)-κB. Ursolic acid was found to downregulate NF-κB in breast cancer cells, thus inhibiting inflammation and preventing the progression of breast cancer (P<0.05). To the best of our knowledge, the present study is the first to assess the effect of ursolic acid on breast cancer cells through PI3K/AKT-regulated GSK and caspase-3 accompanied by NF-κB signaling pathways. The results of the present study regarding the potential underlying molecular mechanisms of ursolic acid may be used to develop novel therapeutic strategies for breast cancer treatment. PMID:29042957

[An adult form of type-I. Gaucher's disease].

PubMed

Múzes, G; Pitlik, E; Gohér, A; Somogyi, A; Tulassay, Z

2000-03-26

A young woman with no previous history of any diseases was admitted for further evaluation of a mild thrombocytopenia she has had for some months. No signs of bleeding have so far occurred. Physical examination was normal except for a moderately enlarged spleen. Routine investigations showed lower platelet count. There was no laboratory evidence of disease conditions with autoimmune/inflammatory or hematologic origin. Bone marrow aspirate indicated Gaucher's-like cells raising the suspicion of Gaucher's disease. This was further supported by electron microscopic demonstration of Gaucher's bodies (with the characteristic tubular structures) in crista biopsy specimens. However, definitive diagnosis was obtained by verifying deficient lysosomal glucosylceramide-beta-D-glucosidase activity in peripheral blood leukocytes. Upon the absence of neurologic involvement the patient was typical for the adult form or type-1 Gaucher's disease.

Inhibition of glycogen-synthase kinase 3 stimulates glycogen synthase and glucose transport by distinct mechanisms in 3T3-L1 adipocytes.

PubMed

Oreña, S J; Torchia, A J; Garofalo, R S

2000-05-26

The role of glycogen-synthase kinase 3 (GSK3) in insulin-stimulated glucose transport and glycogen synthase activation was investigated in 3T3-L1 adipocytes. GSK3 protein was clearly present in adipocytes and was found to be more abundant than in muscle and liver cell lines. The selective GSK3 inhibitor, LiCl, stimulated glucose transport and glycogen synthase activity (20 and 65%, respectively, of the maximal (1 microm) insulin response) and potentiated the responses to a submaximal concentration (1 nm) of insulin. LiCl- and insulin-stimulated glucose transport were abolished by the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, wortmannin; however, LiCl stimulation of glycogen synthase was not. In contrast to the rapid stimulation of glucose transport by insulin, transport stimulated by LiCl increased gradually over 3-5 h reaching 40% of the maximal insulin-stimulated level. Both LiCl- and insulin-stimulated glycogen synthase activity were maximal at 25 min. However, insulin-stimulated glycogen synthase activity returned to basal after 2 h, coincident with reactivation of GSK3. After a 2-h exposure to insulin, glycogen synthase was refractory to restimulation with insulin, indicating selective desensitization of this pathway. However, LiCl could partially stimulate glycogen synthase in desensitized cells. Furthermore, coincubation with LiCl during the 2 h exposure to insulin completely blocked desensitization of glycogen synthase activity. In summary, inhibition of GSK3 by LiCl: 1) stimulated glycogen synthase activity directly and independently of PI3-kinase, 2) stimulated glucose transport at a point upstream of PI3-kinase, 3) stimulated glycogen synthase activity in desensitized cells, and 4) prevented desensitization of glycogen synthase due to chronic insulin treatment. These data are consistent with GSK3 playing a central role in the regulation of glycogen synthase activity and a contributing factor in the regulation of glucose transport in 3T3-L1 adipocytes.

Sandalwood Fragrance Biosynthesis Involves Sesquiterpene Synthases of Both the Terpene Synthase (TPS)-a and TPS-b Subfamilies, including Santalene Synthases*

PubMed Central

Jones, Christopher G.; Moniodis, Jessie; Zulak, Katherine G.; Scaffidi, Adrian; Plummer, Julie A.; Ghisalberti, Emilio L.; Barbour, Elizabeth L.; Bohlmann, Jörg

2011-01-01

Sandalwood oil is one of the worlds most highly prized fragrances. To identify the genes and encoded enzymes responsible for santalene biosynthesis, we cloned and characterized three orthologous terpene synthase (TPS) genes SaSSy, SauSSy, and SspiSSy from three divergent sandalwood species; Santalum album, S. austrocaledonicum, and S. spicatum, respectively. The encoded enzymes catalyze the formation of α-, β-, epi-β-santalene, and α-exo-bergamotene from (E,E)-farnesyl diphosphate (E,E-FPP). Recombinant SaSSy was additionally tested with (Z,Z)-farnesyl diphosphate (Z,Z-FPP) and remarkably, found to produce a mixture of α-endo-bergamotene, α-santalene, (Z)-β-farnesene, epi-β-santalene, and β-santalene. Additional cDNAs that encode bisabolene/bisabolol synthases were also cloned and functionally characterized from these three species. Both the santalene synthases and the bisabolene/bisabolol synthases reside in the TPS-b phylogenetic clade, which is more commonly associated with angiosperm monoterpene synthases. An orthologous set of TPS-a synthases responsible for formation of macrocyclic and bicyclic sesquiterpenes were characterized. Strict functionality and limited sequence divergence in the santalene and bisabolene synthases are in contrast to the TPS-a synthases, suggesting these compounds have played a significant role in the evolution of the Santalum genus. PMID:21454632

[BIOINFORMATIC SEARCH AND PHYLOGENETIC ANALYSIS OF THE CELLULOSE SYNTHASE GENES OF FLAX (LINUM USITATISSIMUM)].

PubMed

Pydiura, N A; Bayer, G Ya; Galinousky, D V; Yemets, A I; Pirko, Ya V; Podvitski, T A; Anisimova, N V; Khotyleva, L V; Kilchevsky, A V; Blume, Ya B

2015-01-01

A bioinformatic search of sequences encoding cellulose synthase genes in the flax genome, and their comparison to dicots orthologs was carried out. The analysis revealed 32 cellulose synthase gene candidates, 16 of which are highly likely to encode cellulose synthases, and the remaining 16--cellulose synthase-like proteins (Csl). Phylogenetic analysis of gene products of cellulose synthase genes allowed distinguishing 6 groups of cellulose synthase genes of different classes: CesA1/10, CesA3, CesA4, CesA5/6/2/9, CesA7 and CesA8. Paralogous sequences within classes CesA1/10 and CesA5/6/2/9 which are associated with the primary cell wall formation are characterized by a greater similarity within these classes than orthologous sequences. Whereas the genes controlling the biosynthesis of secondary cell wall cellulose form distinct clades: CesA4, CesA7, and CesA8. The analysis of 16 identified flax cellulose synthase gene candidates shows the presence of at least 12 different cellulose synthase gene variants in flax genome which are represented in all six clades of cellulose synthase genes. Thus, at this point genes of all ten known cellulose synthase classes are identify in flax genome, but their correct classification requires additional research.

Molecular cloning and functional expression of geranylgeranyl pyrophosphate synthase from Coleus forskohlii Briq

PubMed Central

Engprasert, Surang; Taura, Futoshi; Kawamukai, Makoto; Shoyama, Yukihiro

2004-01-01

Background Isopentenyl diphosphate (IPP), a common biosynthetic precursor to the labdane diterpene forskolin, has been biosynthesised via a non-mevalonate pathway. Geranylgeranyl diphosphate (GGPP) synthase is an important branch point enzyme in terpenoid biosynthesis. Therefore, GGPP synthase is thought to be a key enzyme in biosynthesis of forskolin. Herein we report the first confirmation of the GGPP synthase gene in Coleus forskohlii Briq. Results The open reading frame for full-length GGPP synthase encodes a protein of 359 amino acids, in which 1,077 nucleotides long with calculated molecular mass of 39.3 kDa. Alignments of C. forskohlii GGPP synthase amino acid sequences revealed high homologies with other plant GGPP synthases. Several highly conserved regions, including two aspartate-rich motifs were identified. Transient expression of the N-terminal region of C. forskohlii GGPP synthase-GFP fusion protein in tobacco cells demonstrated subcellular localization in the chloroplast. Carotenoid production was observed in Escherichia coli harboring pACCAR25ΔcrtE from Erwinia uredovora and plasmid carrying C. forskohlii GGPP synthase. These results suggested that cDNA encoded functional GGPP synthase. Furthermore, C. forskohlii GGPP synthase expression was strong in leaves, decreased in stems and very little expression was observed in roots. Conclusion This investigation proposed that forskolin was synthesised via a non-mevalonate pathway. GGPP synthase is thought to be involved in the biosynthesis of forskolin, which is primarily synthesised in the leaves and subsequently accumulates in the stems and roots. PMID:15550168

[Advances in isoprene synthase research].

PubMed

Gou, Yan; Liu, Zhongchuan; Wang, Ganggang

2017-11-25

Isoprene emission can lead to significant consequence for atmospheric chemistry. In addition, isoprene is a chemical compound for various industrial applications. In the organisms, isoprene is produced by isoprene synthase that eliminates the pyrophosphate from the dimethylallyl diphosphate. As a key enzyme of isoprene formation, isoprene synthase plays an important role in the process of natural emission and artificial synthesis of isoprene. So far, isoprene synthase has been found in various plants. Isoprene synthases from different sources are of conservative structural and similar biochemical properties. In this review, the biochemical and structural characteristics of isoprene synthases from different sources were compared, the catalytic mechanism of isoprene synthase was discussed, and the perspective application of the enzyme in bioengineering was proposed.

ESR studies on reactivity of protein-derived tyrosyl radicals formed by prostaglandin H synthase and ribonucleotide reductase.

PubMed

Lassmann, G; Curtis, J; Liermann, B; Mason, R P; Eling, T E

1993-01-01

Using ESR spectroscopy, the ability of enzyme inhibitors to quench protein-derived tyrosyl radicals was studied in two different enzymes, prostaglandin H synthase and ribonucleotide reductase. The prostaglandin H synthase inhibitors indomethacin, eugenol, and MK-410 effectively prevent the formation of tyrosyl radicals during the oxidation of arachidonic acid by prostaglandin H synthase from ram seminal vesicles. A direct reaction with preformed tyrosyl radicals was observed only with eugenol. The other prostaglandin H synthase inhibitors were ineffective. The ribonucleotide reductase inhibitors hydroxyurea and 4-hydroxyanisole, which effectively inactivate the tyrosyl radical in the active site of ribonucleotide reductase present in tumor cells, exhibit a different reactivity with tyrosyl radicals formed by prostaglandin H synthase. Hydroxyurea quenches preformed tyrosyl radicals in prostaglandin H synthase weakly, whereas 4-hydroxyanisole does not quench tyrosyl radicals in prostaglandin H synthase at all. Eugenol, which quenches preformed prostaglandin H synthase-derived tyrosyl radicals, also quenches the tyrosyl radical in ribonucleotide reductase. The results suggest that the reactivity of protein-linked tyrosyl radicals in ribonucleotide reductase and those formed during prostaglandin H synthase catalysis are very different and have unrelated roles in enzyme catalysis.

Parkinson's disease: acid-glucocerebrosidase activity and alpha-synuclein clearance.

PubMed

Blanz, Judith; Saftig, Paul

2016-10-01

The role of mutations in the gene GBA1 encoding the lysosomal hydrolase β-glucocerebrosidase for the development of synucleinopathies, such as Parkinson's disease and dementia with Lewy bodies, was only very recently uncovered. The knowledge obtained from the study of carriers or patients suffering from Gaucher disease (a common lysosomal storage disorder because of GBA1 mutations) is of particular importance for understanding the role of the enzyme and its catabolic pathway in the development of synucleinopathies. Decreased activity of β-glucocerebrosidase leads to lysosomal dysfunction and the accumulation of its substrate glucosylceramide and related lipid derivatives. Glucosylceramide is suggested to stabilize toxic oligomeric forms of α-synuclein that negatively influence the activity of β-glucocerebrosidase and to partially block export of newly synthesized β-glucocerebrosidase from the endoplasmic reticulum to late endocytic compartments, amplifying the pathological effects of α-synuclein and ultimately resulting in neuronal cell death. This pathogenic molecular feedback loop and most likely other factors (such as impaired endoplasmic reticulum-associated degradation, activation of the unfolded protein response and dysregulation of calcium homeostasis induced by misfolded GC mutants) are involved in shifting the cellular homeostasis from monomeric α-synuclein towards oligomeric neurotoxic and aggregated forms, which contribute to Parkinson's disease progression. From a therapeutic point of view, strategies aiming to increase either the expression, stability or delivery of the β-glucocerebrosidase to lysosomes are likely to decrease the α-synuclein burden and may be useful for an in depth evaluation at the organismal level. Lysosomes are critical for protein and lipid homeostasis. Recent research revealed that dysfunction of this organelle contributes to the development of neurodegenerative diseases such as Parkinson's disease (PD). Mutations in the lysosomal hydrolase β-glucocerebrosidase (GBA1) are a major risk factor for the development of PD and the molecular events linked to the reduced activity of GBA1 and the pathological accumulation of lipids and α-synuclein are just at the beginning to be understood. New therapeutic concepts in regards to how to increase the expression, stability, or delivery of β-glucocerebrosidase to lysosomes are currently developed. This article is part of a special issue on Parkinson disease. © 2016 International Society for Neurochemistry.

Sterylglucoside catabolism in Cryptococcus neoformans with endoglycoceramidase-related protein 2 (EGCrP2), the first steryl-β-glucosidase identified in fungi.

PubMed

Watanabe, Takashi; Ito, Tomoharu; Goda, Hatsumi M; Ishibashi, Yohei; Miyamoto, Tomofumi; Ikeda, Kazutaka; Taguchi, Ryo; Okino, Nozomu; Ito, Makoto

2015-01-09

Cryptococcosis is an infectious disease caused by pathogenic fungi, such as Cryptococcus neoformans and Cryptococcus gattii. The ceramide structure (methyl-d18:2/h18:0) of C. neoformans glucosylceramide (GlcCer) is characteristic and strongly related to its pathogenicity. We recently identified endoglycoceramidase-related protein 1 (EGCrP1) as a glucocerebrosidase in C. neoformans and showed that it was involved in the quality control of GlcCer by eliminating immature GlcCer during the synthesis of GlcCer (Ishibashi, Y., Ikeda, K., Sakaguchi, K., Okino, N., Taguchi, R., and Ito, M. (2012) Quality control of fungus-specific glucosylceramide in Cryptococcus neoformans by endoglycoceramidase-related protein 1 (EGCrP1). J. Biol. Chem. 287, 368-381). We herein identified and characterized EGCrP2, a homologue of EGCrP1, as the enzyme responsible for sterylglucoside catabolism in C. neoformans. In contrast to EGCrP1, which is specific to GlcCer, EGCrP2 hydrolyzed various β-glucosides, including GlcCer, cholesteryl-β-glucoside, ergosteryl-β-glucoside, sitosteryl-β-glucoside, and para-nitrophenyl-β-glucoside, but not α-glucosides or β-galactosides, under acidic conditions. Disruption of the EGCrP2 gene (egcrp2) resulted in the accumulation of a glycolipid, the structure of which was determined following purification to ergosteryl-3β-glucoside, a major sterylglucoside in fungi, by mass spectrometric and two-dimensional nuclear magnetic resonance analyses. This glycolipid accumulated in vacuoles and EGCrP2 was detected in vacuole-enriched fraction. These results indicated that EGCrP2 was involved in the catabolism of ergosteryl-β-glucoside in the vacuoles of C. neoformans. Distinct growth arrest, a dysfunction in cell budding, and an abnormal vacuole morphology were detected in the egcrp2-disrupted mutants, suggesting that EGCrP2 may be a promising target for anti-cryptococcal drugs. EGCrP2, classified into glycohydrolase family 5, is the first steryl-β-glucosidase identified as well as a missing link in sterylglucoside metabolism in fungi. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Nitric oxide donors rescue diabetic nephropathy through oxidative-stress-and nitrosative-stress-mediated Wnt signaling pathways

PubMed Central

Hsu, Yung-Chien; Lee, Pei-Hsien; Lei, Chen-Chou; Ho, Cheng; Shih, Ya-Hsueh; Lin, Chun-Liang

2015-01-01

Aims/Introduction The role of the renal nitric oxide (NO) system in the pathophysiology of diabetic nephropathy constitutes a very challenging and fertile field for future investigation. The purpose of the present study was to investigate whether NO donors can attenuate diabetic renal fibrosis and apoptosis through modulating oxidative-and nitrosative-stress, and Wnt signaling using in vivo diabetic models. Materials and Methods Diabetic rat was induced by a single intraperitoneal injection of streptozotocin. Rats in each group were intraperitoneally given 2,2′-(hydroxynitrosohydrazino)bis-ethanamine (1 U/kg/day) and vehicle for 28 and 56 consecutive days. Expression of the oxidative-and nitrosative-stress, and Wnt signaling components were examined in kidneys from diabetic animals by quantitative reverse transcription polymerase chain reaction, western blot analysis and immunohistochemical staining. Results NO donor treatment significantly reduced the ratio of kidney weight to bodyweight and proteinuria. This treatment also significantly restored the suppressive effect of diabetes on urinary NO2 + NO3 levels. Immunohistochemistry showed that NO donor treatment significantly reduced transforming growth factor (TGF)-β1, fibronectin, cleaved caspase-3 and triphosphate-biotin nick end-labeling expression in the glomeruli of diabetic rats. We found that diabetes promoted 8-hydroxy-2′-deoxyguanosine, and peroxynitrite expression coincided with reduced endothelial NO synthase expression in glomeruli. Interestingly, NO donor treatment completely removed oxidative stress and nitrosative stress, and restored endothelial NO synthase expression in diabetic renal glomeruli. Immunohistomorphometry results showed that NO donor treatment significantly restored suppressed Wnt5a expression and β-catenin immunoreactivities in glomeruli. Based on laser-captured microdissection for quantitative reverse transcription polymerase chain reaction, diabetes significantly increased TGF-β1, and fibronectin expression coincided with depressed Wnt5a expression. NO donor treatment reduced TGF-β1, fibronectin activation, and the suppressing effect of diabetes on Wnt5a and β-catenin expression in renal glomeruli. Conclusions NO donor treatment alleviates extracellular matrix accumulation and apoptosis in diabetic nephropathy in vivo by not only preventing the diabetes-mediated oxidative and nitrostative stress, but also restoring downregulation of endothelial NO synthase expression and Wnt/β-catenin signaling. These findings suggest that modulation of NO is a viable alternative strategy for rescuing diabetic renal injury. PMID:25621130

Sandalwood fragrance biosynthesis involves sesquiterpene synthases of both the terpene synthase (TPS)-a and TPS-b subfamilies, including santalene synthases.

PubMed

Jones, Christopher G; Moniodis, Jessie; Zulak, Katherine G; Scaffidi, Adrian; Plummer, Julie A; Ghisalberti, Emilio L; Barbour, Elizabeth L; Bohlmann, Jörg

2011-05-20

Sandalwood oil is one of the worlds most highly prized fragrances. To identify the genes and encoded enzymes responsible for santalene biosynthesis, we cloned and characterized three orthologous terpene synthase (TPS) genes SaSSy, SauSSy, and SspiSSy from three divergent sandalwood species; Santalum album, S. austrocaledonicum, and S. spicatum, respectively. The encoded enzymes catalyze the formation of α-, β-, epi-β-santalene, and α-exo-bergamotene from (E,E)-farnesyl diphosphate (E,E-FPP). Recombinant SaSSy was additionally tested with (Z,Z)-farnesyl diphosphate (Z,Z-FPP) and remarkably, found to produce a mixture of α-endo-bergamotene, α-santalene, (Z)-β-farnesene, epi-β-santalene, and β-santalene. Additional cDNAs that encode bisabolene/bisabolol synthases were also cloned and functionally characterized from these three species. Both the santalene synthases and the bisabolene/bisabolol synthases reside in the TPS-b phylogenetic clade, which is more commonly associated with angiosperm monoterpene synthases. An orthologous set of TPS-a synthases responsible for formation of macrocyclic and bicyclic sesquiterpenes were characterized. Strict functionality and limited sequence divergence in the santalene and bisabolene synthases are in contrast to the TPS-a synthases, suggesting these compounds have played a significant role in the evolution of the Santalum genus. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Aromatic Polyketide Synthases (Purification, Characterization, and Antibody Development to Benzalacetone Synthase from Raspberry Fruits).

PubMed Central

Borejsza-Wysocki, W.; Hrazdina, G.

1996-01-01

p-Hydroxyphenylbutan-2-one, the characteristic aroma compound of raspberries (Rubus idaeus L.), is synthesized from p-coumaryl-coenzyme A and malonyl-coenzyme A in a two-step reaction sequence that is catalyzed by benzalacetone synthase and benzalacetone reductase (W. Borejsza-Wysocki and G. Hrazdina [1994] Phytochemistry 35: 623-628). Benzalacetone synthase condenses one malonate with p-coumarate to form the pathway intermediate p-hydroxyphenylbut-3-ene-2-one (p-hydroxybenzalacetone) in a reaction that is similar to those catalyzed by chalcone and stilbene synthases. We have obtained an enzyme preparation from ripe raspberries that was preferentially enriched in benzalacetone synthase (approximately 170-fold) over chalcone synthase (approximately 14-fold) activity. This preparation was used to characterize benzalacetone synthase and to develop polyclonal antibodies in rabbits. Benzalacetone synthase showed similarity in its molecular properties to chalcone synthase but differed distinctly in its substrate specificity, response to 2-mercaptoethanol and ethylene glycol, and induction in cell-suspension cultures. The product of the enzyme, p-hydroxybenzalacetone, inhibited mycelial growth of the raspberry pathogen Phytophthora fragariae var rubi at 250 [mu]M. We do not know whether the dual activity in the benzalacetone synthase preparation is the result of a bifunctional enzyme or is caused by contamination with chalcone synthase that was also present. The rapid induction of the enzyme in cell-suspension cultures upon addition of yeast extract and the toxicity of its product, p-hydroxybenzalacetone, to phytopathogenic fungi also suggest that the pathway may be part of a plant defense response. PMID:12226219

Mitochondrial F1Fo-ATP synthase translocates to cell surface in hepatocytes and has high activity in tumor-like acidic and hypoxic environment.

PubMed

Ma, Zhan; Cao, Manlin; Liu, Yiwen; He, Yiqing; Wang, Yingzhi; Yang, Cuixia; Wang, Wenjuan; Du, Yan; Zhou, Muqing; Gao, Feng

2010-08-01

F1Fo-ATP synthase was originally thought to exclusively locate in the inner membrane of the mitochondria. However, recent studies prove the existence of ectopic F1Fo-ATP synthase on the outside of the cell membrane. Ectopic ATP synthase was proposed as a marker for tumor target therapy. Nevertheless, the protein transport mechanism of the ectopic ATP synthase is still unclear. The specificity of the ectopic ATP synthase, with regard to tumors, is questioned because of its widespread expression. In the current study, we constructed green fluorescent protein-ATP5B fusion protein and introduced it into HepG2 cells to study the localization of the ATP synthase. The expression of ATP5B was analyzed in six cell lines with different 'malignancies'. These cells were cultured in both normal and tumor-like acidic and hypoxic conditions. The results suggested that the ectopic expression of ATP synthase is a consequence of translocation from the mitochondria. The expression and catalytic activity of ectopic ATP synthase were similar on the surface of malignant cells as on the surface of less malignant cells. Interestingly, the expression of ectopic ATP synthase was not up-regulated in tumor-like acidic and hypoxic microenvironments. However, the catalytic activity of ectopic ATP synthase was up-regulated in tumor-like microenvironments. Therefore, the specificity of ectopic ATP synthase for tumor target therapy relies on the high level of catalytic activity that is observed in acidic and hypoxic microenvironments in tumor tissues.

Characterization of a monoterpene synthase from Paeonia lactiflora producing α-pinene as its single product.

PubMed

Ma, Xiaohui; Guo, Juan; Ma, Ying; Jin, Baolong; Zhan, Zhilai; Yuan, Yuan; Huang, Luqi

2016-07-01

To identify a terpene synthase that catalyzes the conversion of geranyl pyrophosphate (GPP) to α-pinene and is involved in the biosynthesis of paeoniflorin. Two new terpene synthase genes were isolated from the transcriptome data of Peaonia lactiflora. Phylogenetic analysis and sequence characterization revealed that one gene, named PlPIN, encoded a monoterpene synthase that might be involved in the biosynthesis of paeoniflorin. In vitro enzyme assay showed that, in contrast to most monoterpene synthases, PlPIN encoded an α-pinene synthase which converted GPP into α-pinene as a single product. This newly identified α-pinene synthase could be used for improving paeoniflorin accumulation by metabolic engineering or for producing α-pinene via synthetic biology.

Effects of Cadmium on Lipid Storage and Metabolism in the Freshwater Crab Sinopotamon henanense

PubMed Central

Yang, Jian; Liu, Dongmei; Jing, Weixin; Dahms, Hans-Uwe; Wang, Lan

2013-01-01

Since environmental effects of molecular traits are often questioned we analyze here the molecular effects of cadmium (Cd) on lipid pathways and their effects on tissues development. Lipids are an important energy source for the developing embryo, and accumulate in the ovary and hepatopancreas of decapod crustaceans. The extend of Cd affecting lipid storage and metabolism, is studied here with the freshwater crabs Sinopotamon henanense. Crabs were exposed to water-born Cd at 1.45, 2.9, 5.8 mg/l for 10, 15, and 20 days. With significantly increased Cd accumulation in exposed crabs, lipid content in hepatopancreas and ovary showed a time-dependent and concentration-dependent reduction, being at least one of the reasons for a lower ovarian index (OI) and hepatopancreatic index (HI). After 10-day exposure increased triglyceride (TG) level in hemolymph and up-regulation of pancreatic lipase (PL) activity in the hepatopancreas suggested an increased nutritional lipid uptake. However, two processes led to lower lipid levels upon Cd exposure: an increased utilization of lipids and a down-regulated lipoprotein lipase (LPL) led to insufficient lipid transport. 10-day Cd exposure also triggered the production of β-nicotinamide adenine dinucleotide 2'-phosphate reduced tetrasodium salt hydrate (NADPH), as well as to the synthesis of adenosine triphosphate (ATP) and fatty acids. With increasing exposure time, the crabs at 15 and 20-day exposure contained less lipid and TG, suggesting that more energy was consumed during the exposure time. Meanwhile, the level of NADPH, ATP and the activity of PL, LPL, fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC) activity was down-regulated suggesting an impairment of the crab metabolism by Cd in addition to causing a lower lipid level. PMID:24130894

Regulation of Cadmium-Induced Proteomic and Metabolic Changes by 5-Aminolevulinic Acid in Leaves of Brassica napus L.

PubMed Central

Ali, Basharat; Gill, Rafaqat A.; Yang, Su; Gill, Muhammad B.; Farooq, Muhammad A.; Liu, Dan; Daud, Muhammad K.; Ali, Shafaqat; Zhou, Weijun

2015-01-01

It is evident from previous reports that 5-aminolevulinic acid (ALA), like other known plant growth regulators, is effective in countering the injurious effects of heavy metal-stress in oilseed rape (Brassica napus L.). The present study was carried out to explore the capability of ALA to improve cadmium (Cd2+) tolerance in B. napus through physiological, molecular, and proteomic analytical approaches. Results showed that application of ALA helped the plants to adjust Cd2+-induced metabolic and photosynthetic fluorescence changes in the leaves of B. napus under Cd2+ stress. The data revealed that ALA treatment enhanced the gene expressions of antioxidant enzyme activities substantially and could increase the expression to a certain degree under Cd2+ stress conditions. In the present study, 34 protein spots were identified that differentially regulated due to Cd2+ and/or ALA treatments. Among them, 18 proteins were significantly regulated by ALA, including the proteins associated with stress related, carbohydrate metabolism, catalysis, dehydration of damaged protein, CO2 assimilation/photosynthesis and protein synthesis/regulation. From these 18 ALA-regulated proteins, 12 proteins were significantly down-regulated and 6 proteins were up-regulated. Interestingly, it was observed that ALA-induced the up-regulation of dihydrolipoyl dehydrogenase, light harvesting complex photo-system II subunit 6 and 30S ribosomal proteins in the presence of Cd2+ stress. In addition, it was also observed that ALA-induced the down-regulation in thioredoxin-like protein, 2, 3-bisphosphoglycerate, proteasome and thiamine thiazole synthase proteins under Cd2+ stress. Taken together, the present study sheds light on molecular mechanisms involved in ALA-induced Cd2+ tolerance in B. napus leaves and suggests a more active involvement of ALA in plant physiological processes than previously proposed. PMID:25909456

Phosphoproteomics reveals the effect of ethylene in soybean root under flooding stress.

PubMed

Yin, Xiaojian; Sakata, Katsumi; Komatsu, Setsuko

2014-12-05

Flooding has severe negative effects on soybean growth. To explore the flooding-responsive mechanisms in early-stage soybean, a phosphoproteomic approach was used. Two-day-old soybean plants were treated without or with flooding for 3, 6, 12, and 24 h, and root tip proteins were then extracted and analyzed at each time point. After 3 h of flooding exposure, the fresh weight of soybeans increased, whereas the ATP content of soybean root tips decreased. Using a gel-free proteomic technique, a total of 114 phosphoproteins were identified in the root tip samples, and 34 of the phosphoproteins were significantly changed with respect to phosphorylation status after 3 h of flooding stress. Among these phosphoproteins, eukaryotic translation initiation factors were dephosphorylated, whereas several protein synthesis-related proteins were phosphorylated. The mRNA expression levels of sucrose phosphate synthase 1F and eukaryotic translation initiation factor 4 G were down-regulated, whereas UDP-glucose 6-dehydrogenase mRNA expression was up-regulated during growth but down-regulated under flooding stress. Furthermore, bioinformatic protein interaction analysis of flooding-responsive proteins based on temporal phosphorylation patterns indicated that eukaryotic translation initiation factor 4 G was located in the center of the network during flooding. Soybean eukaryotic translation initiation factor 4 G has homology to programmed cell death 4 protein and is implicated in ethylene signaling. The weight of soybeans was increased with treatment by an ethylene-releasing agent under flooding condition, but it was decreased when plants were exposed to an ethylene receptor antagonist. These results suggest that the ethylene signaling pathway plays an important role, via the protein phosphorylation, in mechanisms of plant tolerance to the initial stages of flooding stress in soybean root tips.

GSK-3β and vitamin D receptor are involved in β-catenin and snail signaling in high glucose-induced epithelial-mesenchymal transition of mouse podocytes.

PubMed

Guo, Jia; Xia, Nannan; Yang, Lili; Zhou, Sijie; Zhang, Qian; Qiao, Yingjin; Liu, Zhangsuo

2014-01-01

Epithelial-mesenchymal transition (EMT) is recognized to play an important role in diabetic nephropathy (DN). To analyze the roles of glycogen synthase kinase 3β (GSK-3β), β-catenin and Snail signaling in high glucose (HG)-induced mouse podocytes EMT. Differentiated podocytes were divided into: the normal glucose group (NG: glucose 5.6mM), the HG groups (12.5HG: 12.5mM; 25HG: 25mM; and 50HG: 50mM of glucose), and the osmotic control group (NG+M: glucose 5.6mM and mannitol 44.4mM). GSK-3β, β-catenin and Snail were assessed using semi-quantitative RT-PCR, western blot and immunofluorescence. β-catenin and Snail pathways were assessed after down-regulating GSK-3β expression using an inhibitor (LiCl) or a small-interfering RNA (siRNA). HG increased GSK-3β, β-catenin and Snail expressions, and promoted EMT, as shown by decreased nephrin expression (epithelial marker), and increased α-SMA expression (mesenchymal marker). GSK-3β inhibitor and GSK-3β siRNA decreased β-catenin and Snail expressions, and reversed HG-induced EMT. Immunofluorescence showed that GSK-3β and β-catenin did not completely overlap; β-catenin was transferred to the nucleus in the 25HG group. VDR seems to be involved in HG-induced β-catenin nuclear translocation. Down-regulating GSK-3β expression decreased β-catenin and Snail expression and reversed HG-induced podocytes EMT. Thus, modulating GSK-3β might be a target to slow or prevent DN. © 2014 S. Karger AG, Basel. © 2014 S. Karger AG, Basel.

Silencing of p53 RNA through transarterial delivery ameliorates renal tubular injury and downregulates GSK-3β expression after ischemia-reperfusion injury.

PubMed

Fujino, Takayuki; Muhib, Sharifi; Sato, Nobuyuki; Hasebe, Naoyuki

2013-12-01

p53, a pivotal protein in the apoptotic pathway, has been identified as a mediator of transcriptional responses to ischemia-reperfusion (IR) injury. The characteristics and functional significance of the p53 response in vivo are largely unknown in IR-induced kidney injury. Therapeutic opportunities of delivering small interfering RNA (siRNA) via venous injection have gained recognition; however, systemic adverse effects of siRNA therapy should be considered. To prevent IR-induced kidney injury, we tested the efficacy of transarterial administration of siRNA targeting p53 (p53 siRNA). Female C57BL/6 mice underwent unilateral renal artery ischemia for 30 min, followed by reperfusion. siRNA experiments utilized short hairpin (sh) RNA plasmid-based approaches. Transfection of shRNA was performed using cationic polymer transfection reagent. Injection of synthetic p53 shRNA into the left renal artery just after ischemia improved tubular injury, apoptosis, and the swelling of mitochondria in cells of the thick ascending limb of Henle (mTALH) at the outer medullary regions. Staining of upregulated p53 was colocalized with the inducible expression of glycogen synthase kinase-3β (GSK-3β) at mTALH after IR injury. p53 shRNA inhibited GSK-3β expression and restored β-catenin expression at mTALH. For IR-induced kidney injury, transarterial delivery of p53 siRNA is an effective pharmacological intervention. Targeting siRNA to p53 leads to an attenuation of apoptosis and mitochondrial damage through the downregulation of GSK-3β expression and upregulation of β-catenin. Local delivery of vectors such as p53 siRNA through a transaortic catheter is clinically useful in reducing the adverse effect of siRNA-related therapy.

α-Linolenic acid (ALA) is an anti-inflammatory agent in inflammatory bowel disease.

PubMed

Reifen, Ram; Karlinsky, Anna; Stark, Aliza H; Berkovich, Zipi; Nyska, Abraham

2015-12-01

Studies suggest that consumption of omega-3 (n-3) polyunsaturated fatty acids (PUFA) plays a protective role in inflammatory bowel disease; however, the use of plant-derived oils rich in α-linolenic acid (ALA) has not been widely investigated. The aims of this study were to test the effects of two different sources of (n-3) PUFA, fish and plant-derived oils, in two animal models of experimental colitis and to determine whether the (n-3) PUFA-enriched diets could ameliorate the inflammatory status. Rats were fed diets rich in corn, fish or sage oil with or without vitamin A supplementation for 3weeks then colitis was induced by adding dextran sodium sulfate to the drinking water or by injecting 2,4,6-trinitrobenzene sulfonic acid. We show that colitic rats fed the sage oil diets had a lower inflammatory response, improved histological repair and had less necrotic damage in the mucosa when compared to the corn and fish oil groups. Colonic damage and myeloperoxidase activity were significantly lower. Colonic mRNA levels of pro-inflammatory genes including interleukin IL-6, cyclooxygenase 2 and tumor necrosis factor α were markedly down-regulated in rats fed fish and sage oils compared to control. These results were supported by experiments in the human colonic epithelial cell line Caco-2, where ALA supplementation was shown to be effective in inhibiting inflammation induced by IL-1β by down-regulating mRNA levels of pro-inflammatory genes including IL-8, COX2 and inducible nitric oxide synthase. Taken together, these results suggest that plant-derived oil rich in ALA could ameliorate the inflammatory damage in colitis. Copyright © 2015 Elsevier Inc. All rights reserved.

Immunoprotective responses of T helper type 1 stimulatory protein-S-adenosyl-L-homocysteine hydrolase against experimental visceral leishmaniasis.

PubMed

Khare, P; Jaiswal, A K; Tripathi, C D P; Sundar, S; Dube, A

2016-08-01

It is well known that a patient in clinical remission of visceral leishmaniasis (VL) remains immune to reinfection, which provides a rationale for the feasibility of a vaccine against this deadly disease. In earlier studies, observation of significant cellular responses in treated Leishmania patients as well as in hamsters against leishmanial antigens from different fractions led to its further proteomic characterization, wherein S-adenosyl-L-homocysteine hydrolase (AdoHcy) was identified as a helper type 1 (Th1) stimulatory protein. The present study includes immunological characterization of this protein, its cellular responses [lymphoproliferation, nitric oxide (NO) production and cytokine responses] in treated Leishmania-infected hamsters and patients as well as prophylactic efficacy against Leishmania challenge in hamsters and the immune responses generated thereof. Significantly higher cellular responses were noticed against recombinant L. donovani S-adenosyl-L-homocysteine hydrolase (rLdAdoHcy) compared to soluble L. donovani antigen in treated samples. Moreover, stimulation of peripheral blood mononuclear cells with rLdAdoHcy up-regulated the levels of interferon (IFN)-γ, interleukin (IL)-12 and down-regulated IL-10. Furthermore, vaccination with rLdAdoHcy generated perceptible delayed-type hypersensitivity response and exerted considerably good prophylactic efficacy (∼70% inhibition) against L. donovani challenge. The efficacy was confirmed by the increased expression levels of inducible NO synthase and Th1-type cytokines, IFN-γ and IL-12 and down-regulation of IL-4, IL-10 and transforming growth factor (TGF)-β. The results indicate the potentiality of rLdAdoHcy protein as a suitable vaccine candidate against VL. © 2016 British Society for Immunology.

Effects of ZEB1 on regulating osteosarcoma cells via NF-κB/iNOS.

PubMed

Xu, X-M; Liu, W; Cao, Z-H; Liu, M-X

2017-03-01

Osteosarcoma is one common malignant bone tumors, as it frequently has invasion, metastasis and recurrence, causing unfavorable prognosis of patients. Osteosarcoma has complicated pathogenesis, which has not been elucidated fully. Therefore, the identification of effective molecular target of osteosarcoma onset can help to improve treatment efficacy and prognosis of osteosarcoma. Zinc finger E-box binding homeobox 1 (ZEB1) protein is one member of zinc finger E-box binding protein family, and participates in embryonic genesis and development. A recent study found the participation of ZEB1 in mediating multiple tumor onset and its up-regulation of osteosarcoma. The regulatory mechanism of ZEB1 in osteosarcoma has not been illustrated yet. In vitro cultured osteosarcoma MG-63 cells were transfected with ZEB1 siRNA. Real-time PCR and Western blot were tested for ZEB1 mRNA/protein expression. MTT was used to test MG-63 cell proliferation, whilst cell invasion was used to describe the effect of ZEB1 on MG-63 cells. Caspase-3 activity assay was employed to test MG-63 cell apoptosis. Western blot was employed to detect nuclear factor kappa B (NF-kB) and inducible nitric oxide synthase (iNOS) protein expression. After transfecting with ZEB1 siRNA, MG-63 cell proliferation or invasion was inhibited accompanied with lower ZEB1 mRNA/protein expression. Caspase3 activity was also increased after transfection (p < 0.05), along with down-regulation of NF-kB and iNOS proteins in MG-63 cells (p < 0.05). Inhibition of ZEB1 can facilitate osteosarcoma cell apoptosis and inhibit cell proliferation or invasion via down-regulating NF-kB/iNOS signal pathway.

Modulation of NF-κB and Nrf2 control of inflammatory responses in FHs 74 Int cell line is tocopherol isoform-specific

PubMed Central

Elisia, Ingrid

2013-01-01

The present study investigates the relative ability of α-, γ-, and δ-tocopherol (Toc) to modulate cell signaling events that are associated with inflammatory responses in fetal-derived intestinal (FHs 74 Int) cells. Secretion of the proinflammatory cytokine IL-8 in FHs 74 Int cells was stimulated in the following order: α-Toc < γ-Toc < δ-Toc. A similar proinflammatory response was observed when inflammation was induced in FHs 74 Int cells. Modulation of IL-8 expression by Toc corresponded to an isoform-specific modulation of NF-κB and nuclear factor-erythroid 2-related factor 2 (Nrf2) cell signaling pathways involved in expression of proinflammatory cytokines and antioxidant enzymes, respectively. δ-Toc and, to a lesser extent, γ-Toc activated NF-κB and Nrf2 signaling, as indicated by the greater nuclear translocation of transcription factors. Activation of NF-κB signaling by γ- and δ-Toc was accompanied by upregulation of NF-κB target genes, such as IL-8 and prostaglandin-endoperoxide synthase 2, with and without a prior IFNγ-PMA challenge. Nevertheless, γ- and δ-Toc, particularly δ-Toc, concurrently downregulated glutamate-cysteine ligase, a Nrf2 target gene that encodes for glutathione biosynthesis. This observation was substantiated by confirmation that γ- and δ-Toc were effective at decreasing glutamate-cysteine ligase protein expression and cellular glutathione content. Downregulation of glutathione content in fetal intestinal cells corresponded to induction of apoptosis-mediated cytotoxicity. In conclusion, γ- and δ-Toc are biologically active isoforms of vitamin E and show superior bioactivity to α-Toc in modulating cell signaling events that contribute to a proinflammatory response in fetal-derived intestinal cells. PMID:24136788

Inhibitory effects of diallyl disulfide on the production of inflammatory mediators and cytokines in lipopolysaccharide-activated BV2 microglia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Hye Young; Department of Pharmacy, Pusan National University, Busan 609-735; Kim, Nam Deuk

2012-07-15

Diallyl disulfide (DADS), a main organosulfur component responsible for the diverse biological effects of garlic, displays a wide variety of internal biological activities. However, the cellular and molecular mechanisms underlying DADS' anti-inflammatory activity remain poorly understood. In this study, therefore, the anti-inflammatory effects of DADS were studied to investigate its potential therapeutic effects in lipopolysaccharide (LPS)-stimulated BV2 microglia. We found that pretreatment with DADS prior to treatment with LPS significantly inhibited excessive production of nitric oxide (NO) and prostaglandin E{sub 2} (PGE{sub 2}) in a dose-dependent manner. The inhibition was associated with down-regulation of inducible nitric oxide synthase (iNOS) andmore » cyclooxygenase-2 (COX-2) expression. DADS also attenuated the production of pro-inflammatory cytokines and chemokines, including interleukin-1β (IL-1β), tumor necrosis factor (TNF)-α, and monocyte chemoattractant protein-1 (MCP-1) by suppressing the expression of mRNAs for these proteins. The mechanism underlying this protective effect might be related to the inhibition of nuclear factor-kappaB, Akt and mitogen-activated protein kinase signaling pathway activation in LPS-stimulated microglial cells. These findings indicated that DADS is potentially a novel therapeutic candidate for the treatment of various neurodegenerative diseases. -- Highlights: ► DADS attenuates production of NO and PGE2 in LPS-activated BV2 microglia. ► DADS downregulates levels of iNOS and COX-2. ► DADS inhibits production and expression of inflammatory cytokines and chemokine. ► DADS exhibits these effects by suppression of NF-κB, PI3K/Akt and MAPKs pathways.« less

Differential effects of fluoxetine and venlafaxine on memory recognition: possible mechanisms of action.

PubMed

Carlini, Valeria Paola; Poretti, María Belén; Rask-Andersen, Mathias; Chavan, Rohit A; Ponzio, Marina F; Sawant, Rahul S; de Barioglio, Susana Rubiales; Schiöth, Helgi B; de Cuneo, Marta Fiol

2012-08-07

Serotonin-specific reuptake inhibitors (SSRI) and serotonin-norepinephrine reuptake inhibitors (SNRI) are antidepressant drugs commonly used to treat a wide spectrum of mood disorders (Wong and Licinio, 2001). Although they have been clinically used for more than 50 years, the molecular and cellular basis for the action of SSRIs and SNRIs is not clear. Considering that the changes in gene expression involved in the action of antidepressant drugs on memory have not been identified, in this study we investigated the impact of chronic treatment with a SSRI (fluoxetine) and a SNRI (venlafaxine) on the mRNA expression of genes related to memory cascade in the mouse hippocampus, namely, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), nitric oxide synthase 1 (NOS1), neurotrophic tyrosine kinase receptor type 2 (TrKB), mitogen-activated protein kinases (MAPK/ERK) and serotonin transporter (SERT). Animals treated with fluoxetine 10 mg/Kg/day for 28 days showed a significant decrease in the percentage of time spent in the novel object recognition test (p≤0.005) and induced MAPK1/ERK2 down-regulation (p=0.005). Our results suggest that the effect on cognition could probably be explained by fluoxetine interference in the MAPK/ERK memory pathway. In contrast, chronic treatment with venlafaxine did not reduce MAPK1/ERK2 expression, suggesting that MAPK1/ERK2 down-regulation is not a common effect of all antidepressant drugs. Further studies are needed to examine the effect of chronic fluoxetine treatment on the ERK-CREB system, and to determine whether there is a causal relationship between the disruption of the ERK-CREB system and the effect of this antidepressant on memory performance. Copyright © 2012 Elsevier Inc. All rights reserved.

[Change of chart genes expression in small intestines of mouse induced by electromagnetic pulse irradiation].

PubMed

Ren, Dongqing; Jin, Juan; Li, Xiaojuan; Zeng, Guiying

2008-01-01

To explore the bio-effects of electromagnetic pulse(EMP) on mouse small intestines induced by means of gene chip. Twelve BALB/c mice were randomly assigned to the normal control group and the EMP group with 6 in each group. The EMP group was irradiated with 200 kV/m, 200 pulses EMP. 18 hours after the irradiation, the mice were sacrificed and their jejunum of small intestines were eviscerated. The fluorescent cDNA probes labeled with Cy3 and Cy5 were prepared from RNA extracted from the intestines of the two groups. Probes of the two groups were then hybridized against cDNA gene chip, the fluorescent signals were scanned with a scanner and the results were analyzed by computer. Compared with the control, 56 genes in gene expression profile were altered. The expression levels of 37 genes were up-regulated distinctly while 19 genes were down-regulated significantly. Among the 56 genes, 19 were reported with known or inferred functions, 12 up-regulated genes were catenin alpha 1 (alpha-catenin), ly-6 alloantigen(Ly-6E), fructose-6-phosphate transaminase (GF6P), ribosomal protein S17 (rpS17), small proline-rich protein 2A (Sprr2a), glandular kallikrein27 (GK27), lipoxygenase-3, aldo-keto reductase (Akr1c12), GSG1, amylase 2 (Amy2),elastase 2, p6-5 gene and 7 down-regulated genes were junctional adhesion molecule (Jam), protein arginine methyltransferase (Carm1),NNP-1, 2-5 A synthetase L2,Mlark gene, ATP synthase alpha subunit, uncoupling protein-2 (Ucp2) gene; the other 37 were reported with unknown functions. EMP irradiation could induce specific expressions of some genes in mouse small intestines and most of these genes were up-regulated ones.

Comparison of anti-inflammatory activities of an anthocyanin-rich fraction from Portuguese blueberries (Vaccinium corymbosum L.) and 5-aminosalicylic acid in a TNBS-induced colitis rat model

PubMed Central

Pereira, Sónia R.; Pereira, Rita; Figueiredo, Isabel; Freitas, Victor; Dinis, Teresa C. P.; Almeida, Leonor M.

2017-01-01

Despite the actual therapeutic approaches for inflammatory bowel disease (IBD), efficient and secure alternative options remain a research focus. In this context, anthocyanins seem promising natural anti-inflammatory agents, but their action mechanisms and efficacy as compared with established drugs still require more clarification. The main aim of this study was to compare the anti-inflammatory action of a chemically characterized anthocyanin-rich fraction (ARF), obtained from Portuguese blueberries (Vaccinium corymbosum L.), with that of 5-aminosalicylic acid (5-ASA), a first-line drug in IBD, in a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis rat model. Such fraction showed a high content and great molecular diversity of anthocyanins, with malvidin-3-galactoside and petunidin-3-arabinoside in the highest concentrations. After daily administration by intragastric infusion for 8 days, ARF, at a molar anthocyanin concentration about 30 times lower than 5-ASA, showed a higher effectiveness in counteracting the intestinal inflammation, as assessed by i) body weight variation and colon damage score, ii) reduction in leukocyte infiltration, iii) increase in antioxidant defenses and iv) by downregulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in colon tissue homogenates. The strong inhibition of COX-2 expression seems to be a crucial anti-inflammatory mechanism common to both ARF and 5-ASA, but the additional higher abilities of anthocyanins to downregulate iNOS and to decrease leukocytes infiltration and to increase antioxidant defenses in colon may account for the much higher anti-inflammatory action of anthocyanins. These data may contribute to the development of a promising natural approach in IBD management. PMID:28329021

The A3 adenosine receptor agonist CF502 inhibits the PI3K, PKB/Akt and NF-kappaB signaling pathway in synoviocytes from rheumatoid arthritis patients and in adjuvant-induced arthritis rats.

PubMed

Ochaion, A; Bar-Yehuda, S; Cohen, S; Amital, H; Jacobson, K A; Joshi, B V; Gao, Z G; Barer, F; Patoka, R; Del Valle, L; Perez-Liz, G; Fishman, P

2008-08-15

The A(3) adenosine receptor (A(3)AR) is over-expressed in inflammatory cells and was defined as a target to combat inflammation. Synthetic agonists to this receptor, such as IB-MECA and Cl-IB-MECA, exert an anti-inflammatory effect in experimental animal models of adjuvant- and collagen-induced arthritis. In this study we present a novel A(3)AR agonist, CF502, with high affinity and selectivity at the human A(3)AR. CF502 induced a dose dependent inhibitory effect on the proliferation of fibroblast-like synoviocytes (FLS) via de-regulation of the nuclear factor-kappa B (NF-kappaB) signaling pathway. Furthermore, CF502 markedly suppressed the clinical and pathological manifestations of adjuvant-induced arthritis (AIA) in a rat experimental model when given orally at a low dose (100 microg/kg). As is typical of other G-protein coupled receptors, the A(3)AR expression level was down-regulated shortly after treatment with agonist CF502 in paw and in peripheral blood mononuclear cells (PBMCs) derived from treated AIA animals. Subsequently, a decrease in the expression levels of protein kinase B/Akt (PKB/Akt), IkappaB kinase (IKK), I kappa B (IkappaB), NF-kappaB and tumor necrosis factor-alpha (TNF-alpha) took place. In addition, the expression levels of glycogen synthase kinase-3 beta (GSK-3beta), beta-catenin, and poly(ADP-ribose)polymerase (PARP), known to control the level and activity of NF-kappaB, were down-regulated upon treatment with CF502. Taken together, CF502 inhibits FLS growth and the inflammatory manifestations of arthritis, supporting the development of A(3)AR agonists for the treatment of rheumatoid arthritis.

Enhanced endothelial cell senescence by lithium-induced matrix metalloproteinase-1 expression.

PubMed

Struewing, Ian T; Durham, Samuel N; Barnett, Corey D; Mao, Catherine D

2009-06-26

Endothelial cell (EC) senescence and dysfunction occurring after chronic injury and inflammation are highly associated with the development and progression of cardiovascular diseases. However, the factors involved in the establishment of EC senescence remain poorly understood. We have previously shown that lithium, an inhibitor of glycogen synthase kinase (GSK)-3beta and activator of the Wnt/beta-catenin signaling pathway, induces an EC senescent-like phenotype. Herein, we show that lithium induces a rapid and pronounced up-regulation of the matrix metalloproteinase (MMP)-1, an inflammation and senescent cell marker, at the mRNA and protein levels, whereas the induction of two other senescent cell markers is either weak (interleukin-8) or delayed (plasminogen activator inhibitor-1). Lithium effect on MMP-1 expression is also specific among other MMPs and not mediated by GSK3beta inhibition. Lithium affects MMP-1 expression mainly at the transcriptional level but neither the AP1/Ets regulatory sites nor the redox sensitive (-1607/2G) site in MMP-1 promoter are involved in lithium-dependent MMP-1 regulation. However, down-regulation of p53, a target of lithium in EC, dampens both basal and lithium-induced MMP-1 expression, which further links MMP-1 up-regulation with the establishment of cell senescence. Although increased MMP-1 levels are usually associated with angiogenesis in enabled proliferative EC, the exogenous addition of activated MMP-1 on lithium- arrested EC increases the number of EC positive for the senescent-associated-beta-galactosidase marker. Conversely, down-regulation of MMP-1 expression by small interfering RNAs blunts the lithium-dependent increase in senescent-associated-beta-galactosidase positive cells. Altogether our data indicate that lithium-induced MMP-1 may participate in the reinforcement of EC senescence and reveal a novel mechanism for lithium-induced tissue remodeling.

Dynamics of biomass partitioning, stem gene expression, cell wall biosynthesis, and sucrose accumulation during development of Sorghum bicolor.

PubMed

McKinley, Brian; Rooney, William; Wilkerson, Curtis; Mullet, John

2016-11-01

Biomass accumulated preferentially in leaves of the sweet sorghum Della until floral initiation, then stems until anthesis, followed by panicles until grain maturity, and apical tillers. Sorghum stem RNA-seq transcriptome profiles and composition data were collected for approximately 100 days of development beginning at floral initiation. The analysis identified >200 differentially expressed genes involved in stem growth, cell wall biology, and sucrose accumulation. Genes encoding expansins and xyloglucan endotransglucosylase/hydrolases were differentially expressed in growing stem internodes. Genes encoding enzymes involved in the synthesis of cellulose, lignin, and glucuronoarabinoxylan were expressed at elevated levels in stems until approximately 7 days before anthesis and then down-regulated. CESA genes involved in primary and secondary cell wall synthesis showed different temporal patterns of expression. Following floral initiation, the level of sucrose and other non-structural carbohydrates increased to approximately 50% of the stem's dry weight. Stem sucrose accumulation was inversely correlated with >100-fold down-regulation of SbVIN1, a gene encoding a vacuolar invertase. Accumulation of stem sucrose was also correlated with cessation of leaf and stem growth at anthesis, decreased expression of genes involved in stem cell wall synthesis, and approximately 10-fold lower expression of SbSUS4, a gene encoding sucrose synthase that generates UDP-glucose from sucrose for cell wall biosynthesis. Genes for mixed linkage glucan synthesis (CSLF) and turnover were expressed at high levels in stems throughout development. Overall, the stem transcription profile resource and the genes and regulatory dynamics identified in this study will be useful for engineering sorghum stem composition for improved conversion to biofuels and bio-products. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

MDA-MB-231 breast cancer cell viability, motility and matrix adhesion are regulated by a complex interplay of heparan sulfate, chondroitin-/dermatan sulfate and hyaluronan biosynthesis.

PubMed

Viola, Manuela; Brüggemann, Kathrin; Karousou, Evgenia; Caon, Ilaria; Caravà, Elena; Vigetti, Davide; Greve, Burkhard; Stock, Christian; De Luca, Giancarlo; Passi, Alberto; Götte, Martin

2017-06-01

Proteoglycans and glycosaminoglycans modulate numerous cellular processes relevant to tumour progression, including cell proliferation, cell-matrix interactions, cell motility and invasive growth. Among the glycosaminoglycans with a well-documented role in tumour progression are heparan sulphate, chondroitin/dermatan sulphate and hyaluronic acid/hyaluronan. While the mode of biosynthesis differs for sulphated glycosaminoglycans, which are synthesised in the ER and Golgi compartments, and hyaluronan, which is synthesized at the plasma membrane, these polysaccharides partially compete for common substrates. In this study, we employed a siRNA knockdown approach for heparan sulphate (EXT1) and heparan/chondroitin/dermatan sulphate-biosynthetic enzymes (β4GalT7) in the aggressive human breast cancer cell line MDA-MB-231 to study the impact on cell behaviour and hyaluronan biosynthesis. Knockdown of β4GalT7 expression resulted in a decrease in cell viability, motility and adhesion to fibronectin, while these parameters were unchanged in EXT1-silenced cells. Importantly, these changes were associated with a decreased expression of syndecan-1, decreased signalling response to HGF and an increase in the synthesis of hyaluronan, due to an upregulation of the hyaluronan synthases HAS2 and HAS3. Interestingly, EXT1-depleted cells showed a downregulation of the UDP-sugar transporter SLC35D1, whereas SLC35D2 was downregulated in β4GalT7-depleted cells, indicating an intricate regulatory network that connects all glycosaminoglycans synthesis. The results of our in vitro study suggest that a modulation of breast cancer cell behaviour via interference with heparan sulphate biosynthesis may result in a compensatory upregulation of hyaluronan biosynthesis. These findings have important implications for the development of glycosaminoglycan-targeted therapeutic approaches for malignant diseases.

RNAi-mediated endogene silencing in strawberry fruit: detection of primary and secondary siRNAs by deep sequencing.

PubMed

Härtl, Katja; Kalinowski, Gregor; Hoffmann, Thomas; Preuss, Anja; Schwab, Wilfried

2017-05-01

RNA interference (RNAi) has been exploited as a reverse genetic tool for functional genomics in the nonmodel species strawberry (Fragaria × ananassa) since 2006. Here, we analysed for the first time different but overlapping nucleotide sections (>200 nt) of two endogenous genes, FaCHS (chalcone synthase) and FaOMT (O-methyltransferase), as inducer sequences and a transitive vector system to compare their gene silencing efficiencies. In total, ten vectors were assembled each containing the nucleotide sequence of one fragment in sense and corresponding antisense orientation separated by an intron (inverted hairpin construct, ihp). All sequence fragments along the full lengths of both target genes resulted in a significant down-regulation of the respective gene expression and related metabolite levels. Quantitative PCR data and successful application of a transitive vector system coinciding with a phenotypic change suggested propagation of the silencing signal. The spreading of the signal in strawberry fruit in the 3' direction was shown for the first time by the detection of secondary small interfering RNAs (siRNAs) outside of the primary targets by deep sequencing. Down-regulation of endogenes by the transitive method was less effective than silencing by ihp constructs probably because the numbers of primary siRNAs exceeded the quantity of secondary siRNAs by three orders of magnitude. Besides, we observed consistent hotspots of primary and secondary siRNA formation along the target sequence which fall within a distance of less than 200 nt. Thus, ihp vectors seem to be superior over the transitive vector system for functional genomics in strawberry fruit. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Detecting genome-wide gene transcription profiles associated with high pollution burden in the critically endangered European eel.

PubMed

Pujolar, J M; Milan, M; Marino, I A M; Capoccioni, F; Ciccotti, E; Belpaire, C; Covaci, A; Malarvannan, G; Patarnello, T; Bargelloni, L; Zane, L; Maes, G E

2013-05-15

The European eel illustrates an example of a critically endangered fish species strongly affected by human stressors throughout its life cycle, in which pollution is considered to be one of the factors responsible for the decline of the stock. The objective of our study was to better understand the transcriptional response of European eels chronically exposed to pollutants in their natural environment. A total of 42 pre-migrating (silver) female eels from lowly, highly and extremely polluted environments in Belgium and, for comparative purposes, a lowly polluted habitat in Italy were measured for polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs) and brominated flame retardants (BFRs). Multipollutant level of bioaccumulation was linked to their genome-wide gene transcription using an eel-specific array of 14,913 annotated cDNAs. Shared responses to pollutant exposure were observed when comparing the highly polluted site in Belgium with the relatively clean sites in Belgium and Italy. First, an altered pattern of transcription of genes was associated with detoxification, with a novel European eel CYP3A gene and gluthatione S-transferase transcriptionally up-regulated. Second, an altered pattern of transcription of genes associated with the oxidative phosphorylation pathway, with the following genes involved in the generation of ATP being transcriptionally down-regulated in individuals from the highly polluted site: NADH dehydrogenase, succinate dehydrogenase, ubiquinol-cytochrome c reductase, cytochrome c oxidase and ATP synthase. Although we did not measure metabolism directly, seeing that the transcription level of many genes encoding enzymes involved in the mitochondrial respiratory chain and oxidative phosphorylation were down-regulated in the highly polluted site suggests that pollutants may have a significant effect on energy metabolism in these fish. Copyright © 2013 Elsevier B.V. All rights reserved.

Astaxanthin alleviates oxidative stress insults-related derangements in human vascular endothelial cells exposed to glucose fluctuations.

PubMed

Abdelzaher, Lobna A; Imaizumi, Takahiro; Suzuki, Tokiko; Tomita, Kengo; Takashina, Michinori; Hattori, Yuichi

2016-04-01

Glycemic fluctuations may play a critical role in the pathogenesis of diabetic complications, such as cardiovascular disease. We investigated whether the oxycarotenoid astaxanthin can reduce the detrimental effects of fluctuating glucose on vascular endothelial cells. Human umbilical venous endothelial cells were incubated for 3 days in media containing 5.5mM glucose, 22 mM glucose, or 5.5mM glucose alternating with 22 mM glucose in the absence or presence of astaxanthin or N-acetyl-L-cysteine (NAC). Constant high glucose increased reactive oxygen species (ROS) generation, but such an effect was more pronounced in fluctuating glucose. This was associated with up-regulated p22(phox) expression and down-regulated peroxisome proliferator activated receptor-γ coactivator (PGC-1α) expression. Astaxanthin inhibited ROS generation, p22(phox) up-regulation, and PGC-1α down-regulation by the stimuli of glucose fluctuation. Fluctuating glucose, but not constant high glucose, significantly decreased the endothelial nitric oxide synthase (eNOS) phosphorylation level at Ser-1177 without affecting total eNOS expression, which was prevented by astaxanthin as well as by the anti-oxidant NAC. Transferase-mediated dUTP nick end labeling (TUNEL) showed increased cell apoptosis in fluctuating glucose. Glucose fluctuation also resulted in up-regulating gene expression of pro-inflammatory mediators, interleukin-6 and intercellular adhesion molecule-1. These adverse changes were subdued by astaxanthin. The phosphorylation levels of c-Jun N-terminal kinase (JNK) and p38 were significantly increased by glucose fluctuations, and astaxanthin significantly inhibited the increase in JNK and p38 phosphorylation. Taken together, our results suggest that astaxanthin can protect vascular endothelial cells against glucose fluctuation by reducing ROS generation. Copyright © 2016 Elsevier Inc. All rights reserved.

Celastrol Ameliorates Ulcerative Colitis-Related Colorectal Cancer in Mice via Suppressing Inflammatory Responses and Epithelial-Mesenchymal Transition

PubMed Central

Lin, Lianjie; Sun, Yan; Wang, Dongxu; Zheng, Shihang; Zhang, Jing; Zheng, Changqing

2016-01-01

Celastrol, also named as tripterine, is a pharmacologically active ingredient extracted from the root of traditional Chinese herb Tripterygium wilfordii Hook F with potent anti-inflammatory and anti-tumor activities. In the present study, we investigated the effects of celastrol on ulcerative colitis-related colorectal cancer (UC-CRC) as well as CRC in vivo and in vitro and explored its underlying mechanisms. UC-CRC model was induced in C57BL/6 mice by administration of azoxymethane (AOM) and dextran sodium sulfate (DSS). Colonic tumor xenograft models were developed in BALB/c-nu mice by subcutaneous injection with HCT116 and HT-29 cells. Intragastric administration of celastrol (2 mg/kg/d) for 14 weeks significantly increased the survival ratio and reduced the multiplicity of colonic neoplasms compared with AOM/DSS model mice. Mechanically, celastrol treatment significantly prevented AOM/DSS-induced up-regulation of expression levels of oncologic markers including mutated p53 and phospho-p53, β-catenin and proliferating cell nuclear antigen (PCNA). In addition, treatment with celastrol inhibited inflammatory responses, as indicated by the decrease of serum tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6, down-regulation of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), and inactivation of nuclear factor κB (NF-κB). Moreover, celastrol obviously suppressed epithelial-mesenchymal transition (EMT) through up-regulating E-cadherin and down-regulating N-cadherin, Vimentin and Snail. Additionally, we also demonstrated that celastrol inhibited human CRC cell proliferation and attenuated colonic xenograft tumor growth via reversing EMT. Taken together, celastrol could effectively ameliorate UC-CRC by suppressing inflammatory responses and EMT, suggesting a potential drug candidate for UC-CRC therapy. PMID:26793111

Antiaging Gene Klotho Deficiency Promoted High-Fat Diet-Induced Arterial Stiffening via Inactivation of AMP-Activated Protein Kinase.

PubMed

Lin, Yi; Chen, Jianglei; Sun, Zhongjie

2016-03-01

Klotho was originally discovered as an aging-suppressor gene. The objective of this study is to investigate whether klotho gene deficiency affects high-fat diet (HFD)-induced arterial stiffening. Heterozygous Klotho-deficient (KL(+/-)) mice and WT littermates were fed on HFD or normal diet. HFD increased pulse wave velocity within 5 weeks in KL(+/-) mice but not in wild-type mice, indicating that klotho deficiency accelerates and exacerbates HFD-induced arterial stiffening. A greater increase in blood pressure was found in KL(+/-) mice fed on HFD. Protein expressions of phosphorylated AMP-activated protein kinase-α (AMPKα), phosphorylated endothelial nitric oxide synthase (eNOS), and manganese-dependent superoxide dismutase (Mn-SOD) were decreased, whereas protein expressions of collagen I, transforming growth factor-β1, and Runx2 were increased in aortas of KL(+/-) mice fed on HFD. Interestingly, daily injections of an AMPKα activator, 5-aminoimidazole-4-carboxamide-3-ribonucleoside, abolished the increases in pulse wave velocity, blood pressure, and blood glucose in KL(+/-) mice fed on HFD. Treatment with 5-aminoimidazole-4-carboxamide-3-ribonucleoside for 2 weeks not only abolished the downregulation of phosphorylated AMPKα, phosphorylated eNOS, and Mn-SOD levels but also attenuated the increased levels of collagen I, transforming growth factor-β1, Runx2, superoxide, elastic lamellae breaks, and calcification in aortas of KL(+/-) mice fed on HFD. In cultured mouse aortic smooth muscle cells, cholesterol plus KL-deficient serum decreased phosphorylation levels of AMPKα and LKB1 (an important upstream regulator of AMPKα activity) but increased collagen I synthesis, which can be eliminated by activation of AMPKα by 5-aminoimidazole-4-carboxamide-3-ribonucleoside. In conclusions, Klotho deficiency promoted HFD-induced arterial stiffening and hypertension via downregulation of AMPKα activity. © 2016 American Heart Association, Inc.

Gender hormones and the progression of experimental polycystic kidney disease.

PubMed

Stringer, Kenneth D; Komers, Radko; Osman, Shukri A; Oyama, Terry T; Lindsley, Jessie N; Anderson, Sharon

2005-10-01

Male gender is a risk factor for progression of autosomal-dominant polycystic kidney disease (ADPKD), clinically and in the Han:SPRD rat model. Orchiectomy limits progression, but mechanisms of the detrimental effect of androgen, and/or beneficial effects of estrogen, are not known. This protocol tested the hypothesis that male gender (intact androgen status) promotes progression, while female gender (intact estrogen status) is protective; and that these disease-modifying effects are due to changes in expression of known fibrotic mediators. Studies were performed in male and female noncystic control (+/+) and cystic (+/-) rats subjected to orchiectomy, ovariectomy, or sham operation. At 12 weeks of age, renal function was measured. Blood and kidneys were taken for measurement of plasma and renal renin, endothelin (ET-1), endothelial nitric oxide synthase (eNOS), and vascular endothelial growth factor (VEGF), using biochemical, protein expression, and immunohistochemical methods. Cystic male rats exhibited significantly reduced glomerular filtration (GFR) and effective renal plasma flow (ERPF) rates, with suppression of plasma and renal renin, up-regulation of renal ET-1 and eNOS, and down-regulation of renal VEGF expression. Orchiectomy attenuated the fall in GFR and ERPF, while numerically limiting changes in eNOS and VEGF. Female rats exhibited less cystic growth, with normal renin status, lesser elevation of renal ET-1, and proportionately lesser changes in VEGF and eNOS. Ovariectomy led to higher blood pressure and reduced GFR and ERPF, with a trend toward upregulation of ET-1, and significant down-regulation of VEGF and eNOS. Female gender is protective, but ovariectomy attenuates the protective effect of female gender, in association with changes in renal expression of ET-1, VEGF, and eNOS. The accelerated disease in male rats can be attenuated by orchiectomy and consequent changes in expression of disease mediators.

Adaptation of Mouse Skeletal Muscle to Long-Term Microgravity in the MDS Mission

PubMed Central

Camerino, Giulia M.; Bianchini, Elisa; Ciciliot, Stefano; Danieli-Betto, Daniela; Dobrowolny, Gabriella; Furlan, Sandra; Germinario, Elena; Goto, Katsumasa; Gutsmann, Martina; Kawano, Fuminori; Nakai, Naoya; Ohira, Takashi; Ohno, Yoshitaka; Picard, Anne; Salanova, Michele; Schiffl, Gudrun; Blottner, Dieter; Musarò, Antonio; Ohira, Yoshinobu; Betto, Romeo; Conte, Diana; Schiaffino, Stefano

2012-01-01

The effect of microgravity on skeletal muscles has so far been examined in rat and mice only after short-term (5–20 day) spaceflights. The mice drawer system (MDS) program, sponsored by Italian Space Agency, for the first time aimed to investigate the consequences of long-term (91 days) exposure to microgravity in mice within the International Space Station. Muscle atrophy was present indistinctly in all fiber types of the slow-twitch soleus muscle, but was only slightly greater than that observed after 20 days of spaceflight. Myosin heavy chain analysis indicated a concomitant slow-to-fast transition of soleus. In addition, spaceflight induced translocation of sarcolemmal nitric oxide synthase-1 (NOS1) into the cytosol in soleus but not in the fast-twitch extensor digitorum longus (EDL) muscle. Most of the sarcolemmal ion channel subunits were up-regulated, more in soleus than EDL, whereas Ca2+-activated K+ channels were down-regulated, consistent with the phenotype transition. Gene expression of the atrophy-related ubiquitin-ligases was up-regulated in both spaceflown soleus and EDL muscles, whereas autophagy genes were in the control range. Muscle-specific IGF-1 and interleukin-6 were down-regulated in soleus but up-regulated in EDL. Also, various stress-related genes were up-regulated in spaceflown EDL, not in soleus. Altogether, these results suggest that EDL muscle may resist to microgravity-induced atrophy by activating compensatory and protective pathways. Our study shows the extended sensitivity of antigravity soleus muscle after prolonged exposition to microgravity, suggests possible mechanisms accounting for the resistance of EDL, and individuates some molecular targets for the development of countermeasures. PMID:22470446

Host lung immunity is severely compromised during tropical pulmonary eosinophilia: role of lung eosinophils and macrophages.

PubMed

Sharma, Pankaj; Sharma, Aditi; Vishwakarma, Achchhe Lal; Agnihotri, Promod Kumar; Sharma, Sharad; Srivastava, Mrigank

2016-04-01

Eosinophils play a central role in the pathogenesis of tropical pulmonary eosinophilia, a rare, but fatal, manifestation of filariasis. However, no exhaustive study has been done to identify the genes and proteins of eosinophils involved in the pathogenesis of tropical pulmonary eosinophilia. In the present study, we established a mouse model of tropical pulmonary eosinophilia that mimicked filarial manifestations of human tropical pulmonary eosinophilia pathogenesis and used flow cytometry-assisted cell sorting and real-time RT-PCR to study the gene expression profile of flow-sorted, lung eosinophils and lung macrophages during tropical pulmonary eosinophilia pathogenesis. Our results show that tropical pulmonary eosinophilia mice exhibited increased levels of IL-4, IL-5, CCL5, and CCL11 in the bronchoalveolar lavage fluid and lung parenchyma along with elevated titers of IgE and IgG subtypes in the serum. Alveolar macrophages from tropical pulmonary eosinophilia mice displayed decreased phagocytosis, attenuated nitric oxide production, and reduced T-cell proliferation capacity, and FACS-sorted lung eosinophils from tropical pulmonary eosinophilia mice upregulated transcript levels of ficolin A and anti-apoptotic gene Bcl2,but proapoptotic genes Bim and Bax were downregulated. Similarly, flow-sorted lung macrophages upregulated transcript levels of TLR-2, TLR-6, arginase-1, Ym-1, and FIZZ-1 but downregulated nitric oxide synthase-2 levels, signifying their alternative activation. Taken together, we show that the pathogenesis of tropical pulmonary eosinophilia is marked by functional impairment of alveolar macrophages, alternative activation of lung macrophages, and upregulation of anti-apoptotic genes by eosinophils. These events combine together to cause severe lung inflammation and compromised lung immunity. Therapeutic interventions that can boost host immune response in the lungs might thus provide relief to patients with tropical pulmonary eosinophilia. © Society for Leukocyte Biology.

Secoisolariciresinol diglucoside in high-fat diet and streptozotocin-induced diabetic nephropathy in rats: a possible renoprotective effect.

PubMed

Sherif, Iman O

2014-12-01

Due to substantial morbidity and high complication rate of diabetes mellitus, which is considered as the third killer in the world, a search for the effective blockade of the progression of diabetic nephropathy (DN) remains a therapeutic challenge. Alternative antidiabetic drugs from natural plants are highly demanded nowadays. The aim of this study was to investigate the renoprotective effect of secoisolariciresinol diglucoside (SDG) on DN induced in rats. Diabetes was induced in male Sprague-Dawley rats by a high-fat diet (HFD) and an intraperitoneal 35 mg/kg streptozotocin (STZ) injection. Rats were divided into four groups: normal control rats, diabetic control rats, diabetic rats treated with SDG at 10 mg/kg/day for 4 weeks, and diabetic rats treated with SDG at 20 mg/kg/day for 4 weeks. At the end of the treatment, blood and renal tissue samples were collected for biochemical examination. The results revealed that SDG treatment significantly increased insulin level and decreased blood glucose, fructosamine, creatinine, and blood urea nitrogen levels in diabetic rats. Also, SDG significantly increased renal reduced glutathione, superoxide dismutase and decreased malondialdehyde and nitric oxide levels. In addition, SDG downregulated the renal nuclear factor kappa-B (NF-κB), tumor necrosis factor (TNF)-α, and inducible nitric oxide synthase (iNOS) and upregulated renal survivin and B-cell lymphoma-2 (Bcl-2) expressions when compared with untreated diabetic control rats. This study demonstrated, for the first time, the renoprotective effects of SDG in HFD/STZ-induced DN in rats through correction of hyperglycemia; attenuation of oxidative/nitrosative stress markers; downregulation of renal expressions of inflammatory markers NF-κB, TNF-α, and iNOS; along with upregulation of renal expressions of antiapoptotic markers survivin and Bcl-2.

Comparison of anti-inflammatory activities of an anthocyanin-rich fraction from Portuguese blueberries (Vaccinium corymbosum L.) and 5-aminosalicylic acid in a TNBS-induced colitis rat model.

PubMed

Pereira, Sónia R; Pereira, Rita; Figueiredo, Isabel; Freitas, Victor; Dinis, Teresa C P; Almeida, Leonor M

2017-01-01

Despite the actual therapeutic approaches for inflammatory bowel disease (IBD), efficient and secure alternative options remain a research focus. In this context, anthocyanins seem promising natural anti-inflammatory agents, but their action mechanisms and efficacy as compared with established drugs still require more clarification. The main aim of this study was to compare the anti-inflammatory action of a chemically characterized anthocyanin-rich fraction (ARF), obtained from Portuguese blueberries (Vaccinium corymbosum L.), with that of 5-aminosalicylic acid (5-ASA), a first-line drug in IBD, in a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis rat model. Such fraction showed a high content and great molecular diversity of anthocyanins, with malvidin-3-galactoside and petunidin-3-arabinoside in the highest concentrations. After daily administration by intragastric infusion for 8 days, ARF, at a molar anthocyanin concentration about 30 times lower than 5-ASA, showed a higher effectiveness in counteracting the intestinal inflammation, as assessed by i) body weight variation and colon damage score, ii) reduction in leukocyte infiltration, iii) increase in antioxidant defenses and iv) by downregulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in colon tissue homogenates. The strong inhibition of COX-2 expression seems to be a crucial anti-inflammatory mechanism common to both ARF and 5-ASA, but the additional higher abilities of anthocyanins to downregulate iNOS and to decrease leukocytes infiltration and to increase antioxidant defenses in colon may account for the much higher anti-inflammatory action of anthocyanins. These data may contribute to the development of a promising natural approach in IBD management.

Relaxation Response Induces Temporal Transcriptome Changes in Energy Metabolism, Insulin Secretion and Inflammatory Pathways

PubMed Central

Joseph, Marie G.; Denninger, John W.; Fricchione, Gregory L.; Benson, Herbert; Libermann, Towia A.

2013-01-01

The relaxation response (RR) is the counterpart of the stress response. Millennia-old practices evoking the RR include meditation, yoga and repetitive prayer. Although RR elicitation is an effective therapeutic intervention that counteracts the adverse clinical effects of stress in disorders including hypertension, anxiety, insomnia and aging, the underlying molecular mechanisms that explain these clinical benefits remain undetermined. To assess rapid time-dependent (temporal) genomic changes during one session of RR practice among healthy practitioners with years of RR practice and also in novices before and after 8 weeks of RR training, we measured the transcriptome in peripheral blood prior to, immediately after, and 15 minutes after listening to an RR-eliciting or a health education CD. Both short-term and long-term practitioners evoked significant temporal gene expression changes with greater significance in the latter as compared to novices. RR practice enhanced expression of genes associated with energy metabolism, mitochondrial function, insulin secretion and telomere maintenance, and reduced expression of genes linked to inflammatory response and stress-related pathways. Interactive network analyses of RR-affected pathways identified mitochondrial ATP synthase and insulin (INS) as top upregulated critical molecules (focus hubs) and NF-κB pathway genes as top downregulated focus hubs. Our results for the first time indicate that RR elicitation, particularly after long-term practice, may evoke its downstream health benefits by improving mitochondrial energy production and utilization and thus promoting mitochondrial resiliency through upregulation of ATPase and insulin function. Mitochondrial resiliency might also be promoted by RR-induced downregulation of NF-κB-associated upstream and downstream targets that mitigates stress. PMID:23650531

Cellular and Molecular Responses of Dunaliella tertiolecta by Expression of a Plant Medium Chain Length Fatty Acid Specific Acyl-ACP Thioesterase

PubMed Central

Lin, Huixin; Shen, Hui; Lee, Yuan K.

2018-01-01

Metabolic engineering of microalgae to accumulate high levels of medium chain length fatty acids (MCFAs) has met with limited success. Traditional approaches employ single introduction of MCFA specific acyl-ACP thioesterases (TEs), but our current research in transgenic Dunaliella tertiolecta line has highlighted that, there is no single rate-limiting approach that can effectively increase MCFA levels. Here, we explore the accumulation of MCFAs in D. tertiolecta after transgenic expression of myristic acid biased TE (C14TE). We observe that the MCFA levels were negatively correlated to the fatty acid (FA) synthesis genes, ketoacyl-ACP synthase II (KASII), stearoyl-CoA-9-desaturase (Δ9D), and oleoyl-CoA-12-desaturase (Δ12D). To further examine the molecular mechanism of MCFA accumulation in microalgae, we investigate the transcriptomic dynamics of the MCFA producing strain of D. tertiolecta. At the transcript level, enhanced MCFA accumulation primarily involved up-regulation of photosynthetic genes and down-regulation of genes from central carbon metabolic processes, resulting in an overall decrease in carbon precursors for FA synthesis. We additionally observe that MCFA specific peroxisomal β-oxidation gene (ACX3) was greatly enhanced to prevent excessive build-up of unusual MCFA levels. Besides, long chain acyl-CoA synthetase gene (LACS) was down-regulated, likely in attempt to control fatty acyl supply flux to FA synthesis cycle. This article provides a spatial regulation model of unusual FA accumulation in microalgae and a platform for additional metabolic engineering targeting pathways from FA synthesis, FA transport, and peroxisomal β-oxidation to achieve microalgae oils with higher levels of MCFAs. PMID:29670594

Transcriptional profile of Paracoccidioides induced by oenothein B, a potential antifungal agent from the Brazilian Cerrado plant Eugenia uniflora

PubMed Central

2013-01-01

Background The compound oenothein B (OenB), which is isolated from the leaves of Eugenia uniflora, a Brazilian Cerrado plant, interferes with Paracoccidioides yeast cell morphology and inhibits 1,3-β-D-glucan synthase (PbFKS1) transcript accumulation, which is involved in cell wall synthesis. In this work we examined the gene expression changes in Paracoccidioides yeast cells following OenB treatment in order to investigate the adaptive cellular responses to drug stress. Results We constructed differential gene expression libraries using Representational Difference Analysis (RDA) of Paracoccidioides yeast cells treated with OenB for 90 and 180 min. Treatment for 90 min resulted in the identification of 463 up-regulated expressed sequences tags (ESTs) and 104 down-regulated ESTs. For the 180 min treatment 301 up-regulated ESTs and 143 down-regulated were identified. Genes involved in the cell wall biosynthesis, such as GLN1, KRE6 and FKS1, were found to be regulated by OenB. Infection experiments in macrophages corroborated the in vitro results. Fluorescence microscopy showed increased levels of chitin in cells treated with OenB. The carbohydrate polymer content of the cell wall of the fungus was also evaluated, and the results corroborated with the transcriptional data. Several other genes, such as those involved in a variety of important cellular processes (i.e., membrane maintenance, stress and virulence) were found to be up-regulated in response to OenB treatment. Conclusions The exposure of Paracoccidioides to OenB resulted in a complex altered gene expression profile. Some of the changes may represent specific adaptive responses to this compound in this important pathogenic fungus. PMID:24119145

Effects of clopidogrel on inflammatory cytokines and adhesion molecules in human endothelial cells: Role of nitric oxide mediating pleiotropic effects.

PubMed

Cerda, Alvaro; Pavez, Monica; Manriquez, Victor; Luchessi, Andre Ducati; Leal, Pamela; Benavente, Felipe; Fajardo, Cristina Moreno; Salazar, Luis; Hirata, Mario Hiroyuki; Hirata, Rosario Dominguez Crespo

2017-08-01

Clopidogrel is commonly used in prevention and treatment of atherothrombosis. Some previous studies have suggested a pleiotropic effect of clopidogrel; however, when this drug causes platelet-independent effects on endothelial function remains unclear. To evaluate the influence of clopidogrel on inflammatory biomarkers and adhesion molecules in human endothelial cells and the role of nitric oxide (NO) in this process. TNF-α-induced human umbilical vein endothelial cells (HUVEC) were exposed to clopidogrel. Gene expression and protein expression of ICAM-1, P-selectin, IL-8, IL-6, and MCP-1 were evaluated by qPCR, flux cytometry, or milliplex technology. Expression of endothelial nitric oxide synthase (NOS3) and NO release were also evaluated. Influence of clopidogrel was further evaluated in NOS3 downregulated HUVEC by RNAi. Clopidogrel at 20 μmol/L induced NO release in HUVEC after 24-hours treatment. Gene expressions of inflammatory markers IL-8 and MCP1 were reduced after clopidogrel treatment (P<.05); however, only MCP-1 remained reduced at protein level. IL-6 was not modified by clopidogrel treatment. Gene expression and protein expression of ICAM-1 were diminished by 24-hours clopidogrel exposure, whereas P-selectin was not modified. NOS3 downregulated HUVEC model revealed that ICAM-1 modification by clopidogrel is dependent of this via, whereas MCP-1 is modulated in an NO-independent form. Our results support new evidence for pleiotropic effects of clopidogrel on inflammation and endothelial function. Reduction in ICAM-1 and MCP-1 in human endothelium is an important extent of the use of this drug for treatment of cardiovascular diseases, and NO has an important role in this process. © 2017 John Wiley & Sons Ltd.

Dietary DHA/EPA ratio affected tissue fatty acid profiles, antioxidant capacity, hematological characteristics and expression of lipid-related genes but not growth in juvenile black seabream (Acanthopagrus schlegelii)

PubMed Central

Monroig, Óscar; Lu, You; Yuan, Ye; Li, Yi; Ding, Liyun; Tocher, Douglas R.; Zhou, Qicun

2017-01-01

An 8-week feeding trial was conducted to investigate the effects of dietary docosahexaenoic to eicosapentaenoic acid ratio (DHA/EPA) on growth performance, fatty acid profiles, antioxidant capacity, hematological characteristics and expression of some lipid metabolism related genes of juvenile black seabream (Acanthopagrus schlegelii) of initial weight 9.47 ± 0.03 g. Five isonitrogenous and isolipidic diets (45% crude protein and 14% crude lipid) were formulated to contain graded DHA/EPA ratios of 0.65, 1.16, 1.60, 2.03 and 2.67. There were no differences in growth performance and feed utilization among treatments. Fish fed higher DHA/EPA ratios had higher malondialdehyde (MDA) contents in serum than lower ratios. Serum triacylglycerol (TAG) content was significantly higher in fish fed the lowest DHA/EPA ratio. Tissue fatty acid profiles reflected the diets despite down-regulation of LC-PUFA biosynthesis genes, fatty acyl desaturase 2 (fads2) and elongase of very long-chain fatty acids 5 (elovl5), by high DHA/EPA ratios. Expression of acetyl-CoA carboxylase alpha (accα) and carnitine palmitoyl transferase 1A (cpt1a) were up-regulated by high DHA/EPA ratio, whereas sterol regulatory element-binding protein-1 (srebp-1) and hormone-sensitive lipase (hsl) were down-regulated. Fatty acid synthase (fas), 6-phosphogluconate dehydrogenase (6pgd) and peroxisome proliferator-activated receptor alpha (pparα) showed highest expression in fish fed intermediate (1.16) DHA/EPA ratio. Overall, this study indicated that dietary DHA/EPA ratio affected fatty acid profiles and significantly influenced lipid metabolism including LC-PUFA biosynthesis and other anabolic and catabolic pathways, and also had impacts on antioxidant capacity and hematological characteristics. PMID:28430821

Dietary DHA/EPA ratio affected tissue fatty acid profiles, antioxidant capacity, hematological characteristics and expression of lipid-related genes but not growth in juvenile black seabream (Acanthopagrus schlegelii).

PubMed

Jin, Min; Monroig, Óscar; Lu, You; Yuan, Ye; Li, Yi; Ding, Liyun; Tocher, Douglas R; Zhou, Qicun

2017-01-01

An 8-week feeding trial was conducted to investigate the effects of dietary docosahexaenoic to eicosapentaenoic acid ratio (DHA/EPA) on growth performance, fatty acid profiles, antioxidant capacity, hematological characteristics and expression of some lipid metabolism related genes of juvenile black seabream (Acanthopagrus schlegelii) of initial weight 9.47 ± 0.03 g. Five isonitrogenous and isolipidic diets (45% crude protein and 14% crude lipid) were formulated to contain graded DHA/EPA ratios of 0.65, 1.16, 1.60, 2.03 and 2.67. There were no differences in growth performance and feed utilization among treatments. Fish fed higher DHA/EPA ratios had higher malondialdehyde (MDA) contents in serum than lower ratios. Serum triacylglycerol (TAG) content was significantly higher in fish fed the lowest DHA/EPA ratio. Tissue fatty acid profiles reflected the diets despite down-regulation of LC-PUFA biosynthesis genes, fatty acyl desaturase 2 (fads2) and elongase of very long-chain fatty acids 5 (elovl5), by high DHA/EPA ratios. Expression of acetyl-CoA carboxylase alpha (accα) and carnitine palmitoyl transferase 1A (cpt1a) were up-regulated by high DHA/EPA ratio, whereas sterol regulatory element-binding protein-1 (srebp-1) and hormone-sensitive lipase (hsl) were down-regulated. Fatty acid synthase (fas), 6-phosphogluconate dehydrogenase (6pgd) and peroxisome proliferator-activated receptor alpha (pparα) showed highest expression in fish fed intermediate (1.16) DHA/EPA ratio. Overall, this study indicated that dietary DHA/EPA ratio affected fatty acid profiles and significantly influenced lipid metabolism including LC-PUFA biosynthesis and other anabolic and catabolic pathways, and also had impacts on antioxidant capacity and hematological characteristics.

Anti-inflammatory effects of Zea mays L. husk extracts.

PubMed

Roh, Kyung-Baeg; Kim, Hyoyoung; Shin, Seungwoo; Kim, Young-Soo; Lee, Jung-A; Kim, Mi Ok; Jung, Eunsun; Lee, Jongsung; Park, Deokhoon

2016-08-19

Zea mays L. (Z. mays) has been used for human consumption in the various forms of meal, cooking oil, thickener in sauces and puddings, sweetener in processed food and beverage products, bio-disel. However, especially, in case of husk extract of Z. mays, little is known about its anti-inflammatory effects. Therefore, in this study, the anti-inflammatory effects of Z. mays husk extract (ZMHE) and its mechanisms of action were investigated. The husks of Z. Mays were harvested in kangwondo, Korea. To assess the anti-inflammatory activities of ZMHE, we examined effects of ZMHE on nitric oxide (NO) production, and release of soluble intercellular adhesion molecule-1 (sICAM-1) and eotaxin-1. The expression level of inducible nitric oxide synthase (iNOS) gene was also determined by Western blot and luciferase reporter assays. To determine its mechanisms of action, a luciferase reporter assay for nuclear factor kappa B (NF-kB) and activator protein-1 (AP-1) was introduced. ZMHE inhibited lipopolysaccharide (LPS)-induced production of NO in RAW264.7 cells. In addition, expression of iNOS gene was reduced, as confirmed by Western blot and luciferase reporter assays. Effects of ZMHE on the AP-1 and NF-kB promoters were examined to elucidate the mechanism of its anti-inflammatory activity. Activation of AP-1 and NF-kB promoters induced by LPS was significantly reduced by ZMHE treatment. In addition, LPS-induced production of sICAM-1 and IL-4-induced production of eotaxin-1 were all reduced by ZMHE. Our results indicate that ZMHE has anti-inflammatory effects by downregulating the expression of iNOS gene and its downregulation is mediated by inhibiting NF-kB and AP-1 signaling.

Osteopontin inhibits osteoblast responsiveness through the down-regulation of focal adhesion kinase mediated by the induction of low-molecular weight protein tyrosine phosphatase.

PubMed

Kusuyama, Joji; Bandow, Kenjiro; Ohnishi, Tomokazu; Hisadome, Mitsuhiro; Shima, Kaori; Semba, Ichiro; Matsuguchi, Tetsuya

2017-05-15

Osteopontin (OPN) is an osteogenic marker protein. Osteoblast functions are affected by inflammatory cytokines and pathological conditions. OPN is highly expressed in bone lesions such as those in rheumatoid arthritis. However, local regulatory effects of OPN on osteoblasts remain ambiguous. Here we examined how OPN influences osteoblast responses to mechanical stress and growth factors. Expression of NO synthase 1 ( Nos1 ) and Nos2 was increased by low-intensity pulsed ultrasound (LIPUS) in MC3T3-E1 cells and primary osteoblasts. The increase of Nos1/2 expression was abrogated by both exogenous OPN overexpression and recombinant OPN treatment, whereas it was promoted by OPN-specific siRNA and OPN antibody. Moreover, LIPUS-induced phosphorylation of focal adhesion kinase (FAK), a crucial regulator of mechanoresponses, was down-regulated by OPN treatments. OPN also attenuated hepatocyte growth factor-induced vitamin D receptor ( Vdr ) expression and platelet-derived growth factor-induced cell mobility through the repression of FAK activity. Of note, the expression of low-molecular weight protein tyrosine phosphatase (LMW-PTP), a FAK phosphatase, was increased in both OPN-treated and differentiated osteoblasts. CD44 was a specific OPN receptor for LWW-PTP induction. Consistently, the suppressive influence of OPN on osteoblast responsiveness was abrogated by LMW-PTP knockdown. Taken together, these results reveal novel functions of OPN in osteoblast physiology. © 2017 Kusuyama et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Molecular Diversity of Terpene Synthases in the Liverwort Marchantia polymorpha[OPEN

PubMed Central

Zhuang, Xun; Jiang, Zuodong; Jia, Qidong; Babbitt, Patricia C.

2016-01-01

Marchantia polymorpha is a basal terrestrial land plant, which like most liverworts accumulates structurally diverse terpenes believed to serve in deterring disease and herbivory. Previous studies have suggested that the mevalonate and methylerythritol phosphate pathways, present in evolutionarily diverged plants, are also operative in liverworts. However, the genes and enzymes responsible for the chemical diversity of terpenes have yet to be described. In this study, we resorted to a HMMER search tool to identify 17 putative terpene synthase genes from M. polymorpha transcriptomes. Functional characterization identified four diterpene synthase genes phylogenetically related to those found in diverged plants and nine rather unusual monoterpene and sesquiterpene synthase-like genes. The presence of separate monofunctional diterpene synthases for ent-copalyl diphosphate and ent-kaurene biosynthesis is similar to orthologs found in vascular plants, pushing the date of the underlying gene duplication and neofunctionalization of the ancestral diterpene synthase gene family to >400 million years ago. By contrast, the mono- and sesquiterpene synthases represent a distinct class of enzymes, not related to previously described plant terpene synthases and only distantly so to microbial-type terpene synthases. The absence of a Mg2+ binding, aspartate-rich, DDXXD motif places these enzymes in a noncanonical family of terpene synthases. PMID:27650333

Interactions of citrate synthases from osmoconforming and osmoregulating animals with salt: possible signs of molecular eco-adaptation?

PubMed

Sarkissian, I V

1977-01-01

This study considers differential sensitivity of citrate synthase (citrate oxaloacetatelyase [CoA acetylating]) EC 4.1.3.7. from an osmoconforming animal (sea anemone) and an osmoregulating animal (the pig) to salt. Attention is drawn to the fact that the osmoconforming sea anemone is in essence a sessile creature while the pig is readily mobile and able to change its ionic environment at will. It had been shown earlier that citrate synthase from another osmoconformer (oyster) is also not sensitive to ionic strength while citrate synthase from osmoregulating white shrimp is sensitive to increasing levels of salt. However, these enzymes are characteristically regulated by ATP and alpha-ketoglutarate. Both forms of citrate synthase are denatured by 6 M guanidine hydrochloride and are aided by salt levels in their refolding but the rate and extent of refolding of the osmoconformer citrate synthase are greater than those of the osmoregulator citrate synthase. Catalytic activity of both forms of citrate synthase is inhibited by incubation in distilled water; osmoconformer citrate synthase was inhibited completely in 7 h while osmoregulator citrate synthase was inhibited only 60% in this time and 80% after 22 h in distilled water. The eco-adaptive and evolutionary implications of these findings are discussed.

PSD-95 promotes synaptogenesis and multiinnervated spine formation through nitric oxide signaling

PubMed Central

Nikonenko, Irina; Boda, Bernadett; Steen, Sylvain; Knott, Graham; Welker, Egbert; Muller, Dominique

2008-01-01

Postsynaptic density 95 (PSD-95) is an important regulator of synaptic structure and plasticity. However, its contribution to synapse formation and organization remains unclear. Using a combined electron microscopic, genetic, and pharmacological approach, we uncover a new mechanism through which PSD-95 regulates synaptogenesis. We find that PSD-95 overexpression affected spine morphology but also promoted the formation of multiinnervated spines (MISs) contacted by up to seven presynaptic terminals. The formation of multiple contacts was specifically prevented by deletion of the PDZ2 domain of PSD-95, which interacts with nitric oxide (NO) synthase (NOS). Similarly, PSD-95 overexpression combined with small interfering RNA–mediated down-regulation or the pharmacological blockade of NOS prevented axon differentiation into varicosities and multisynapse formation. Conversely, treatment of hippocampal slices with an NO donor or cyclic guanosine monophosphate analogue induced MISs. NOS blockade also reduced spine and synapse density in developing hippocampal cultures. These results indicate that the postsynaptic site, through an NOS–PSD-95 interaction and NO signaling, promotes synapse formation with nearby axons. PMID:19075115

Exploiting Natural Variation to Uncover an Alkene Biosynthetic Enzyme in Poplar

DOE PAGES

Gonzales-Vigil, Eliana; Hefer, Charles A.; von Loessl, Michelle E.; ...

2017-07-20

Alkenes are linear hydrocarbons with one or more double bonds. Despite their potential as biofuels and precursors for specialty chemicals, the underlying biochemistry and genetics of alkene biosynthesis in plants remain elusive. Here, we report on a screen of natural accessions of poplar (Populus trichocarpa), revealing that the leaf cuticular waxes are predominantly composed of alkanes and alkenes. Interestingly, the accumulation of alkenes increases with leaf development, is limited to the abaxial side of the leaf, and is impaired in a few accessions. Among other genes, a b-ketoacyl CoA synthase gene (PotriKCS1) was downregulated in leaves from non-alkene-producing accessions. Wemore » demonstrated biochemically that PotriKCS1 elongates monounsaturated fatty acids and is responsible for the recruitment of unsaturated substrates to the cuticular wax. Moreover, we found significant associations between the presence of alkenes and tree growth and resistance to leaf spot. These findings highlight the crucial role of cuticular waxes as the first point of contact with the environment, and they provide a foundation for engineering long-chain monounsaturated oils in other species.« less

Mondo complexes regulate TFEB via TOR inhibition to promote longevity in response to gonadal signals

PubMed Central

Nakamura, Shuhei; Karalay, Özlem; Jäger, Philipp S.; Horikawa, Makoto; Klein, Corinna; Nakamura, Kayo; Latza, Christian; Templer, Sven E.; Dieterich, Christoph; Antebi, Adam

2016-01-01

Germline removal provokes longevity in several species and shifts resources towards survival and repair. Several Caenorhabditis elegans transcription factors regulate longevity arising from germline removal; yet, how they work together is unknown. Here we identify a Myc-like HLH transcription factor network comprised of Mondo/Max-like complex (MML-1/MXL-2) to be required for longevity induced by germline removal, as well as by reduced TOR, insulin/IGF signalling and mitochondrial function. Germline removal increases MML-1 nuclear accumulation and activity. Surprisingly, MML-1 regulates nuclear localization and activity of HLH-30/TFEB, a convergent regulator of autophagy, lysosome biogenesis and longevity, by downregulating TOR signalling via LARS-1/leucyl-transfer RNA synthase. HLH-30 also upregulates MML-1 upon germline removal. Mammalian MondoA/B and TFEB show similar mutual regulation. MML-1/MXL-2 and HLH-30 transcriptomes show both shared and preferential outputs including MDL-1/MAD-like HLH factor required for longevity. These studies reveal how an extensive interdependent HLH transcription factor network distributes responsibility and mutually enforces states geared towards reproduction or survival. PMID:27001890

Methylphenidate administration determines enduring changes in neuroglial network in rats.

PubMed

Cavaliere, Carlo; Cirillo, Giovanni; Bianco, Maria Rosaria; Adriani, Walter; De Simone, Antonietta; Leo, Damiana; Perrone-Capano, Carla; Papa, Michele

2012-01-01

Repeated exposure to psychostimulant drugs induces complex molecular and structural modifications in discrete brain regions of the meso-cortico-limbic system. This structural remodeling is thought to underlie neurobehavioral adaptive responses. Administration to adolescent rats of methylphenidate (MPH), commonly used in attention deficit and hyperactivity disorder (ADHD), triggers alterations of reward-based behavior paralleled by persistent and plastic synaptic changes of neuronal and glial markers within key areas of the reward circuits. By immunohistochemistry, we observe a marked increase of glial fibrillary acidic protein (GFAP) and neuronal nitric oxide synthase (nNOS) expression and a down-regulation of glial glutamate transporter GLAST in dorso-lateral and ventro-medial striatum. Using electron microscopy, we find in the prefrontal cortex a significant reduction of the synaptic active zone length, paralleled by an increase of dendritic spines. We demonstrate that in limbic areas the MPH-induced reactive astrocytosis affects the glial glutamatergic uptake system that in turn could determine glutamate receptor sensitization. These processes could be sustained by NO production and synaptic rearrangement and contribute to MPH neuroglial induced rewiring. Copyright © 2011. Published by Elsevier B.V.

Therapeutic ultrasound plus pulsed electromagnetic field improves recovery from peripheral arterial disease in hypertension

PubMed Central

Lu, Zhao-Yang; Zhou, Hong-Sheng; Su, Zhi-Xiao; Qi, Jia; Zhang, Jian; Xue, Guan-Hua; Li, Yue; Hao, Chang-Ning; Shi, Yi-Qin; Duan, Jun-Li

2017-01-01

The objective of this investigation was to evaluate the therapy effect of combined therapeutic ultrasound (TUS) treatment and pulsed electromagnetic field (PEMF) therapy on angiogenesis in hypertension-related hindlimb ischemia. After subjecting excision of the left femoral artery, spontaneously hypertensive rats (SHRs) were randomly distributed to one of four groups: SHR; TUS treated SHR (SHR-TUS); PEMF treated SHR (PEMF-TUS); and TUS plus PEMF treated SHR (SHR-TUS-PEMF). Wistar-Kyoto rats (WKYs) with femoral artery excision were regarded as a control group. At day 14 after surgery, the TUS plus PEMF united administration had the greatest blood perfusion accompanied by elevated capillary density and the lowest TUNEL index. Interestingly, the united administration up-regulated the angiogenic factors expression of phosphorylated Akt (p-Akt), phosphorylated endothelial nitric oxide synthase (p-eNOS), vascular endothelial growth factor (VEGF), anti-apoptotic protein of Bcl-2 and down-regulated pro-apoptotic protein levels of Bax and Cleaved caspase-3 in vivo. Our results demonstrated that the united administration could significantly rescue hypertension-related inhibition of ischemia-induced neovascularization partly by promoting angiogenesis and inhibiting apoptosis. PMID:28979692

Polyamines Attenuate Ethylene-Mediated Defense Responses to Abrogate Resistance to Botrytis cinerea in Tomato1[C][W][OA

PubMed Central

Nambeesan, Savithri; AbuQamar, Synan; Laluk, Kristin; Mattoo, Autar K.; Mickelbart, Michael V.; Ferruzzi, Mario G.; Mengiste, Tesfaye; Handa, Avtar K.

2012-01-01

Transgenic tomato (Solanum lycopersicum) lines overexpressing yeast spermidine synthase (ySpdSyn), an enzyme involved in polyamine (PA) biosynthesis, were developed. These transgenic lines accumulate higher levels of spermidine (Spd) than the wild-type plants and were examined for responses to the fungal necrotrophs Botrytis cinerea and Alternaria solani, bacterial pathogen Pseudomonas syringae pv tomato DC3000, and larvae of the chewing insect tobacco hornworm (Manduca sexta). The Spd-accumulating transgenic tomato lines were more susceptible to B. cinerea than the wild-type plants; however, responses to A. solani, P. syringae, or M. sexta were similar to the wild-type plants. Exogenous application of ethylene precursors, S-adenosyl-Met and 1-aminocyclopropane-1-carboxylic acid, or PA biosynthesis inhibitors reversed the response of the transgenic plants to B. cinerea. The increased susceptibility of the ySpdSyn transgenic tomato to B. cinerea was associated with down-regulation of gene transcripts involved in ethylene biosynthesis and signaling. These data suggest that PA-mediated susceptibility to B. cinerea is linked to interference with the functions of ethylene in plant defense. PMID:22128140

Precise precursor rebalancing for isoprenoids production by fine control of gapA expression in Escherichia coli.

PubMed

Jung, Juyoung; Lim, Jae Hyung; Kim, Se Yeon; Im, Dae-Kyun; Seok, Joo Yeon; Lee, Seung-Jae V; Oh, Min-Kyu; Jung, Gyoo Yeol

2016-11-01

Biosynthesis of isoprenoids via the 1-deoxy-D-xylulose-5-phosphate (DXP) pathway requires equimolar glyceraldehyde 3-phosphate and pyruvate to divert carbon flux toward the products of interest. Here, we demonstrate that precursor balancing is one of the critical steps for the production of isoprenoids in Escherichia coli. First, the implementation of the synthetic lycopene production pathway as a model system and the amplification of the native DXP pathway were accomplished using synthetic constitutive promoters and redesigned 5'-untranslated regions (5'-UTRs). Next, fine-controlled precursor balancing was investigated by tuning phosphoenolpyruvate synthase (PpsA) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The results showed that tuning-down of gapA improved the specific lycopene content by 45% compared to the overexpression of ppsA. The specific lycopene content in the strains with down-regulated gapA increased by 97% compared to that in the parental strain. Our results indicate that gapA is the best target for precursor balancing to increase biosynthesis of isoprenoids. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Argininosuccinate Synthase 1-Deficiency Enhances the Cell Sensitivity to Arginine through Decreased DEPTOR Expression in Endometrial Cancer

PubMed Central

Ohshima, Kenji; Nojima, Satoshi; Tahara, Shinichiro; Kurashige, Masako; Hori, Yumiko; Hagiwara, Kohei; Okuzaki, Daisuke; Oki, Shinya; Wada, Naoki; Ikeda, Jun-ichiro; Kanai, Yoshikatsu; Morii, Eiichi

2017-01-01

Argininosuccinate synthetase 1 (ASS1) is a rate-limiting enzyme in arginine biosynthesis. Although ASS1 expression levels are often reduced in several tumors and low ASS1 expression can be a poor prognostic factor, the underlying mechanism has not been elucidated. In this study, we reveal a novel association between ASS1 and migration/invasion of endometrial tumors via regulation of mechanistic target of rapamycin complex (mTORC) 1 signaling. ASS1-knockout cells showed enhanced migration and invasion in response to arginine following arginine starvation. In ASS1-knockout cells, DEPTOR, an inhibitor of mTORC1 signal, was downregulated and mTORC1 signaling was more activated in response to arginine. ASS1 epigenetically enhanced DEPTOR expression by altering the histone methylation. Consistent with these findings, tumor cells at the invasive front of endometrioid carcinoma cases showed lower ASS1 and DEPTOR expression. Our findings suggest that ASS1 levels in each tumor cell are associated with invasion capability in response to arginine within the tumor microenvironment through mTORC1 signal regulation. PMID:28358054

Neuroprotective effect of resveratrol against brain ischemia reperfusion injury in rats entails reduction of DJ-1 protein expression and activation of PI3K/Akt/GSK3b survival pathway.

PubMed

Abdel-Aleem, Ghada A; Khaleel, Eman F; Mostafa, Dalia G; Elberier, Lydia K

2016-10-01

In the current study, we aimed to investigate the mechanistic role of DJ-1/PI3K/Akt survival pathway in ischemia/reperfusion (I/R) induced cerebral damage and to investigate if the resveratrol (RES) mediates its ischemic neuroptotection through this pathway. RES administration to Sham rats boosted glutathione level and superoxide dismutase activity and downregulated inducible nitric oxide synthase expression without affecting redox levels of DJ-1 forms or components of PI3K/Akt pathway including PTEN, p-Akt or p/p-GSK3b. However, RES pre-administration to I/R rats reduced infarction area, oxidative stress, inflammation and apoptosis. Concomitantly, RES ameliorated the decreased levels of oxidized forms of DJ-1 and enhancing its reduction, increased the nuclear protein expression of Nfr-2 and led to activation of PI3K/Akt survival pathway. In conclusion, overoxidation of DJ-1 is a major factor that contributes to post-I/R cerebral damage and its reduction by RES could explain the neuroprotection offered by RES.

Light-dark regulation of carotenoid biosynthesis in pepper (Capsicum annuum) leaves.

PubMed

Simkin, Andrew J; Zhu, Changfu; Kuntz, Marcel; Sandmann, Gerhard

2003-05-01

The carotenoid content in photosynthetic plant tissue reflects a steady state value resulting from permanent biosynthesis and concurrent photo-oxidation. The contributions of both reactions were determined in illuminated pepper leaves. The amount of carotenoids provided by biosynthesis were quantified by the accumulation of the colourless carotenoid phytoene in the presence of the inhibitor norflurazon. When applied, substantial amounts of this rather photo-stable intermediate were formed in the light. However, carotenoid biosynthesis was completely stalled in darkness. This switch off in the absence of light is related to the presence of very low messenger levels of the phytoene synthase gene, psy and the phytoene desaturase gene, pds. Other carotenogenic genes, such as zds, ptox and Icy-b also were shown to be down-regulated to some extent. By comparison of the carotenoid concentration before and after transfer of plants to increasing light intensities and accounting for the contribution of biosynthesis, the rate of photo-oxidation was estimated for pepper leaves. It could be demonstrated that light-independent degradation or conversion of carotenoids e.g. to abscisic acid is a minor process.

Valproic acid attenuates nitric oxide and interleukin-1β production in lipopolysaccharide-stimulated iron-rich microglia.

PubMed

Mairuae, Nootchanat; Cheepsunthorn, Poonlarp

2018-04-01

Iron accumulation in activated microglia has been consistently reported in neurodegenerative diseases. Previous results suggest that these cells facilitate neuroinflammation leading to neuronal cell death. Therefore, chemical compounds that alleviate the activation of iron-rich microglia may result in neuroprotection. In the present study, the effect of valproic acid (VPA) on microglial activation under iron-rich conditions was investigated. BV-2 microglial cells were exposed to lipopolysaccharide (LPS; 1 µg/ml) and iron (300 µg/ml) with or without VPA (1.6 mM). The results demonstrated that VPA attenuated the activation of iron-rich BV2 cells induced by LPS by down-regulating the mRNA expression of inducible nitric oxide (NO) synthase and interleukin 1β (IL-1β; P<0.01), to ultimately reduce the production of NO and IL-1β (P<0.01). These events were accompanied by an attenuation in the nuclear translocation of nuclear factor-κB p65 subunit (P<0.01). These findings suggest that VPA may be therapeutically useful for attenuating the activation of iron-rich microglia.

Possible Contribution of Zerumbone-Induced Proteo-Stress to Its Anti-Inflammatory Functions via the Activation of Heat Shock Factor 1.

PubMed

Igarashi, Yoko; Ohnishi, Kohta; Irie, Kazuhiro; Murakami, Akira

2016-01-01

Zerumbone is a sesquiterpene present in Zinger zerumbet. Many studies have demonstrated its marked anti-inflammatory and anti-carcinogenesis activities. Recently, we showed that zerumbone binds to numerous proteins with scant selectivity and induces the expression of heat shock proteins (HSPs) in hepatocytes. To dampen proteo-toxic stress, organisms have a stress-responsive molecular machinery, known as heat shock response. Heat shock factor 1 (HSF1) plays a key role in this protein quality control system by promoting activation of HSPs. In this study, we investigated whether zerumbone-induced HSF1 activation contributes to its anti-inflammatory functions in stimulated macrophages. Our findings showed that zerumbone increased cellular protein aggregates and promoted nuclear translocation of HSF1 for HSP expression. Interestingly, HSF1 down-regulation attenuated the suppressive effects of zerumbone on mRNA and protein expressions of pro-inflammatory genes, including inducible nitric oxide synthase and interlukin-1β. These results suggest that proteo-stress induced by zerumbone activates HSF1 for exhibiting its anti-inflammatory functions.

Differential effects of genetically distinct mechanisms of elevating amylose on barley starch characteristics.

PubMed

Regina, Ahmed; Blazek, Jaroslav; Gilbert, Elliot; Flanagan, Bernadine M; Gidley, Michael J; Cavanagh, Colin; Ral, Jean-Philippe; Larroque, Oscar; Bird, Anthony R; Li, Zhongyi; Morell, Matthew K

2012-07-01

The relationships between starch structure and functionality are important in underpinning the industrial and nutritional utilisation of starches. In this work, the relationships between the biosynthesis, structure, molecular organisation and functionality have been examined using a series of defined genotypes in barley with low (<20%), standard (20-30%), elevated (30-50%) and high (>50%) amylose starches. A range of techniques have been employed to determine starch physical features, higher order structure and functionality. The two genetic mechanisms for generating high amylose contents (down-regulation of branching enzymes and starch synthases, respectively) yielded starches with very different amylopectin structures but similar gelatinisation and viscosity properties driven by reduced granular order and increased amylose content. Principal components analysis (PCA) was used to elucidate the relationships between genotypes and starch molecular structure and functionality. Parameters associated with granule order (PC1) accounted for a large percentage of the variance (57%) and were closely related to amylose content. Parameters associated with amylopectin fine structure accounted for 18% of the variance but were less closely aligned to functionality parameters. Copyright © 2012 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoon, Sarah; Jung, Jaesung; Kim, Taeyeung

In this study, HepG2-hepatitis B virus (HBV)-stable cells that did not overexpress HBx and HBx-deficient mutant-transfected cells were analyzed for their expression of HBV-induced, upregulated adipogenic and lipogenic genes. The mRNAs of CCAAT enhancer binding protein {alpha} (C/EBP{alpha}), peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), adiponectin, liver X receptor {alpha} (LXR{alpha}), sterol regulatory element binding protein 1c (SREBP1c), and fatty acid synthase (FAS) were expressed at higher levels in HepG2-HBV and lamivudine-treated stable cells and HBx-deficient mutant-transfected cells than in the HepG2 cells. Lamivudine treatment reduced the mRNA levels of PPAR{gamma} and C/EBP{alpha}. Conversely, HBV replication was upregulated by adiponectin and PPAR{gamma}more » agonist rosiglitazone treatments and was downregulated by adiponectin siRNAs. Collectively, our results demonstrate that HBV replication and/or protein expression, even in the absence of HBx, upregulated adipogenic or lipogenic genes, and that the control of adiponectin might prove useful as a therapeutic modality for the treatment of chronic hepatitis B.« less

Regulation of apoptosis by somatostatin and substance P in peritoneal macrophages.

PubMed

Kang, B N; Jeong, K S; Park, S J; Kim, S J; Kim, T H; Kim, H J; Ryu, S Y

2001-09-15

Recent studies have shown that somatostatin (SOM) inhibits interleukin 6 (IL-6) and interferon gamma (IFNgamma) production by lymphocytes and peritoneal macrophages, whereas substance P (SP) enhances these cytokines production. To define the mechanism of the cytokine production enhancements and inhibitions by SOM and SP, we examined the expression of apoptosis modulator, p53, Bcl-2, Bax, inducible nitric oxide synthase (iNOS), Fas, caspase-8 and nitric oxide (NO) in thioglycolate-elicited peritoneal macrophages. SOM caused up-regulation of p53, Bcl-2, Fas and caspase-8 activities, and down-regulation of iNOS expression and NO production. On the other hand, SP slightly induces p53 and highly induces Bcl-2, iNOS expression and NO production. These data suggest that apoptosis by SOM may occur by a Bax- and NO-independent p53 accumulation, and through Fas and caspase-8 activation pathways, and that the inducible expression of Bcl-2 and NO production by SP may contribute to prevent the signals of apoptosis by Bax, and via Fas and caspase-8 activation.

Exploiting Natural Variation to Uncover an Alkene Biosynthetic Enzyme in Poplar

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gonzales-Vigil, Eliana; Hefer, Charles A.; von Loessl, Michelle E.

Alkenes are linear hydrocarbons with one or more double bonds. Despite their potential as biofuels and precursors for specialty chemicals, the underlying biochemistry and genetics of alkene biosynthesis in plants remain elusive. Here, we report on a screen of natural accessions of poplar (Populus trichocarpa), revealing that the leaf cuticular waxes are predominantly composed of alkanes and alkenes. Interestingly, the accumulation of alkenes increases with leaf development, is limited to the abaxial side of the leaf, and is impaired in a few accessions. Among other genes, a b-ketoacyl CoA synthase gene (PotriKCS1) was downregulated in leaves from non-alkene-producing accessions. Wemore » demonstrated biochemically that PotriKCS1 elongates monounsaturated fatty acids and is responsible for the recruitment of unsaturated substrates to the cuticular wax. Moreover, we found significant associations between the presence of alkenes and tree growth and resistance to leaf spot. These findings highlight the crucial role of cuticular waxes as the first point of contact with the environment, and they provide a foundation for engineering long-chain monounsaturated oils in other species.« less

Cardioprotective effect of sulphonated formononetin on acute myocardial infarction in rats.

PubMed

Zhang, Shumin; Tang, Xuexi; Tian, Jingwei; Li, Chunmei; Zhang, Guanbo; Jiang, Wanglin; Zhang, Zunting

2011-06-01

This study was designed to investigate the therapeutic effect of sodium formononetin-3'-sulphonate (Sul-F), a water-soluble derivate of formononetin, on acute myocardial infarction in rats. The results showed that treatment with Sul-F significantly prevented the elevation of ST-segment level, decreased the contents of creatine kinase-MB, lactate dehydrogenase, alanine aminotransferase and cardiac troponin T in serum and reduced the myocardium necrosis scores. The number of apoptosis cardiocytes is well accordance with the up-regulated expression of Bcl-2 and the down-regulated expression of Bax. Meanwhile, Sul-F significantly increased the cardiac mitochondrial ATP content, improved ATP synthase activity, decreased thiobarbituric acid-reactive substances content and attenuated the decrease in superoxide dismutase and glutathione peroxidase activities. These findings indicate that Sul-F has a protective potential against myocardial infarction injury. A possible mechanism for the protective effect is the elevated expression of endogenous antioxidant defence enzymes degraded lipid peroxidation products and improved energy metholism of cardiac mitochondrial, thus attenuating cardiocyte apoptosis. © 2011 The Authors. Basic & Clinical Pharmacology & Toxicology © 2011 Nordic Pharmacological Society.

Immunomodulatory Activities of a Fungal Protein Extracted from Hericium erinaceus through Regulating the Gut Microbiota.

PubMed

Diling, Chen; Chaoqun, Zheng; Jian, Yang; Jian, Li; Jiyan, Su; Yizhen, Xie; Guoxiao, Lai

2017-01-01

A single-band protein (HEP3) was isolated from Hericium erinaceus using a chemical separation combined with pharmacodynamic evaluation methods. This protein exhibited immunomodulatory activity in lipopolysaccharide-activated RAW 264.7 macrophages by decreasing the overproduction of tumor necrosis factor-α, interleukin (IL)-1β, and IL-6, and downregulating the expression of inducible nitric oxide synthase and nuclear factor-κB p65. Further researches revealed that HEP3 could improve the immune system via regulating the composition and metabolism of gut microbiota to activate the proliferation and differentiation of T cells, stimulate the intestinal antigen-presenting cells in high-dose cyclophosphamide-induced immunotoxicity in mice, and play a prebiotic role in the case of excessive antibiotics in inflammatory bowel disease model mice. Aided experiments also showed that HEP3 could be used as an antitumor immune inhibitor in tumor-burdened mice. The results of the present study suggested that fungal protein from H. erinaceus could be used as a drug or functional food ingredient for immunotherapy because of its immunomodulatory activities.

Immunomodulatory Activities of a Fungal Protein Extracted from Hericium erinaceus through Regulating the Gut Microbiota

PubMed Central

Diling, Chen; Chaoqun, Zheng; Jian, Yang; Jian, Li; Jiyan, Su; Yizhen, Xie; Guoxiao, Lai

2017-01-01

A single-band protein (HEP3) was isolated from Hericium erinaceus using a chemical separation combined with pharmacodynamic evaluation methods. This protein exhibited immunomodulatory activity in lipopolysaccharide-activated RAW 264.7 macrophages by decreasing the overproduction of tumor necrosis factor-α, interleukin (IL)-1β, and IL-6, and downregulating the expression of inducible nitric oxide synthase and nuclear factor-κB p65. Further researches revealed that HEP3 could improve the immune system via regulating the composition and metabolism of gut microbiota to activate the proliferation and differentiation of T cells, stimulate the intestinal antigen-presenting cells in high-dose cyclophosphamide-induced immunotoxicity in mice, and play a prebiotic role in the case of excessive antibiotics in inflammatory bowel disease model mice. Aided experiments also showed that HEP3 could be used as an antitumor immune inhibitor in tumor-burdened mice. The results of the present study suggested that fungal protein from H. erinaceus could be used as a drug or functional food ingredient for immunotherapy because of its immunomodulatory activities. PMID:28713364

The role of prostacyclin synthase and thromboxane synthase signaling in the development and progression of cancer.

PubMed

Cathcart, Mary-Clare; Reynolds, John V; O'Byrne, Kenneth J; Pidgeon, Graham P

2010-04-01

Prostacyclin synthase and thromboxane synthase signaling via arachidonic acid metabolism affects a number of tumor cell survival pathways such as cell proliferation, apoptosis, tumor cell invasion and metastasis, and angiogenesis. However, the effects of these respective synthases differ considerably with respect to the pathways described. While prostacyclin synthase is generally believed to be anti-tumor, a pro-carcinogenic role for thromboxane synthase has been demonstrated in a variety of cancers. The balance of oppositely-acting COX-derived prostanoids influences many processes throughout the body, such as blood pressure regulation, clotting, and inflammation. The PGI(2)/TXA(2) ratio is of particular interest in-vivo, with the corresponding synthases shown to be differentially regulated in a variety of disease states. Pharmacological inhibition of thromboxane synthase has been shown to significantly inhibit tumor cell growth, invasion, metastasis and angiogenesis in a range of experimental models. In direct contrast, prostacyclin synthase overexpression has been shown to be chemopreventive in a murine model of the disease, suggesting that the expression and activity of this enzyme may protect against tumor development. In this review, we discuss the aberrant expression and known functions of both prostacyclin synthase and thromboxane synthase in cancer. We discuss the effects of these enzymes on a range of tumor cell survival pathways, such as tumor cell proliferation, induction of apoptosis, invasion and metastasis, and tumor cell angiogenesis. As downstream signaling pathways of these enzymes have also been implicated in cancer states, we examine the role of downstream effectors of PGIS and TXS activity in tumor growth and progression. Finally, we discuss current therapeutic strategies aimed at targeting these enzymes for the prevention/treatment of cancer.

mTORC2 Promotes Tumorigenesis via Lipid Synthesis.

PubMed

Guri, Yakir; Colombi, Marco; Dazert, Eva; Hindupur, Sravanth K; Roszik, Jason; Moes, Suzette; Jenoe, Paul; Heim, Markus H; Riezman, Isabelle; Riezman, Howard; Hall, Michael N

2017-12-11

Dysregulated mammalian target of rapamycin (mTOR) promotes cancer, but underlying mechanisms are poorly understood. We describe an mTOR-driven mouse model that displays hepatosteatosis progressing to hepatocellular carcinoma (HCC). Longitudinal proteomic, lipidomics, and metabolomic analyses revealed that hepatic mTORC2 promotes de novo fatty acid and lipid synthesis, leading to steatosis and tumor development. In particular, mTORC2 stimulated sphingolipid (glucosylceramide) and glycerophospholipid (cardiolipin) synthesis. Inhibition of fatty acid or sphingolipid synthesis prevented tumor development, indicating a causal effect in tumorigenesis. Increased levels of cardiolipin were associated with tubular mitochondria and enhanced oxidative phosphorylation. Furthermore, increased lipogenesis correlated with elevated mTORC2 activity and HCC in human patients. Thus, mTORC2 promotes cancer via formation of lipids essential for growth and energy production. Copyright © 2017 Elsevier Inc. All rights reserved.

Domain analysis of 3 Keto Acyl-CoA synthase for structural variations in Vitis vinifera and Oryza brachyantha using comparative modelling.

PubMed

Sagar, Mamta; Pandey, Neetesh; Qamar, Naseha; Singh, Brijendra; Shukla, Akanksha

2015-03-01

The long chain fatty acids incorporated into plant lipids are derived from the iterative addition of C2 units which is provided by malonyl-CoA to an acyl-CoA after interactions with 3-ketoacyl-CoA synthase (KCS), found in several plants. This study provides functional characterization of three 3 ketoacyl CoA synthase like proteins in Vitis vinifera (one) and Oryza brachyantha (two proteins). Sequence analysis reveals that protein of Oryza brachyantha shows 96% similarity to a hypothetical protein in Sorghum bicolor; total 11 homologs were predicted in Sorghum bicolor. Conserved domain prediction confirm the presence of FAE1/Type III polyketide synthase-like protein, Thiolase-like, subgroup; Thiolase-like and 3-Oxoacyl-ACP synthase III, C-terminal and chalcone synthase like domain but very long chain 3-keto acyl CoA domain is absent. All three proteins were found to have Chalcone and stilbene synthases C terminal domain which is similar to domain of thiolase and β keto acyl synthase. Its N terminal domain is absent in J3M9Z7 protein of Oryza brachyantha and F6HH63 protein of Vitis vinifera. Differences in N-terminal domain is responsible for distinguish activity. The J3MF16 protein of Oryza brachyantha contains N terminal domain and C terminal domain and characterized using annotation of these domains. Domains Gcs (streptomyces coelicolor) and Chalcone-stilbene synthases (KAS) in 2-pyrone synthase (Gerbera hybrid) and chalcone synthase 2 (Medicago sativa) were found to be present in three proteins. This similarity points toward anthocyanin biosynthetic process. Similarity to chalcone synthase 2 reveals its possible role in Naringenine and Chalcone synthase like activity. In 3 keto acyl CoA synthase of Oryza brachyantha. Active site residues C-240, H-407, N-447 are present in J3MF16 protein that are common in these three protein at different positions. Structural variations among dimer interface, product binding site, malonyl-CoA binding sites, were predicted in localized combination of conserved residues.

Isolation and bacterial expression of a sesquiterpene synthase CDNA clone from peppermint(mentha .chi. piperita, L.) that produces the aphid alarm pheromone (E)-.beta.-farnesene

DOEpatents

Croteau, Rodney Bruce; Wildung, Mark Raymond; Crock, John E.

1999-01-01

A cDNA encoding (E)-.beta.-farnesene synthase from peppermint (Mentha piperita) has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID NO:1) is provided which codes for the expression of (E)-.beta.-farnesene synthase (SEQ ID NO:2), from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for (E)-.beta.-farnesene synthase, or for a base sequence sufficiently complementary to at least a portion of (E)-.beta.-farnesene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (E)-.beta.-farnesene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant (E)-.beta.-farnesene synthase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant (E)-.beta.-farnesene synthase may be used to obtain expression or enhanced expression of (E)-.beta.-farnesene synthase in plants in order to enhance the production of (E)-.beta.-farnesene, or may be otherwise employed for the regulation or expression of (E)-.beta.-farnesene synthase, or the production of its product.

Isolation and bacterial expression of a sesquiterpene synthase cDNA clone from peppermint (Mentha x piperita, L.) that produces the aphid alarm pheromone (E)-.beta.-farnesene

DOEpatents

Croteau, Rodney Bruce; Crock, John E.

2005-01-25

A cDNA encoding (E)-.beta.-farnesene synthase from peppermint (Mentha piperita) has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID NO:1) is provided which codes for the expression of (E)-.beta.-farnesene synthase (SEQ ID NO:2), from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for (E)-.beta.-farnesene synthase, or for a base sequence sufficiently complementary to at least a portion of (E)-.beta.-farnesene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (E)-.beta.-farnesene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant (E)-.beta.-famesene synthase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant (E)-.beta.-farnesene synthase may be used to obtain expression or enhanced expression of (E)-.beta.-famesene synthase in plants in order to enhance the production of (E)-.beta.-farnesene, or may be otherwise employed for the regulation or expression of (E)-.beta.-farnesene synthase, or the production of its product.

CTP synthase forms cytoophidia in the cytoplasm and nucleus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gou, Ke-Mian; State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193; Chang, Chia-Chun

2014-04-15

CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthasemore » 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus.« less

Evolution of glutamine amidotransferase genes. Nucleotide sequences of the pabA genes from Salmonella typhimurium, Klebsiella aerogenes and Serratia marcescens.

PubMed

Kaplan, J B; Merkel, W K; Nichols, B P

1985-06-05

The amide group of glutamine is a source of nitrogen in the biosynthesis of a variety of compounds. These reactions are catalyzed by a group of enzymes known as glutamine amidotransferases; two of these, the glutamine amidotransferase subunits of p-aminobenzoate synthase and anthranilate synthase have been studied in detail and have been shown to be structurally and functionally related. In some micro-organisms, p-aminobenzoate synthase and anthranilate synthase share a common glutamine amidotransferase subunit. We report here the primary DNA and deduced amino acid sequences of the p-aminobenzoate synthase glutamine amidotransferase subunits from Salmonella typhimurium, Klebsiella aerogenes and Serratia marcescens. A comparison of these glutamine amidotransferase sequences to the sequences of ten others, including some that function specifically in either the p-aminobenzoate synthase or anthranilate synthase complexes and some that are shared by both synthase complexes, has revealed several interesting features of the structure and organization of these genes, and has allowed us to speculate as to the evolutionary history of this family of enzymes. We propose a model for the evolution of the p-aminobenzoate synthase and anthranilate synthase glutamine amidotransferase subunits in which the duplication and subsequent divergence of the genetic information encoding a shared glutamine amidotransferase subunit led to the evolution of two new pathway-specific enzymes.

Molecular docking studies to map the binding site of squalene synthase inhibitors on dehydrosqualene synthase of Staphylococcus aureus.

PubMed

Kahlon, Amandeep Kaur; Roy, Sudeep; Sharma, Ashok

2010-10-01

Dehydrosqualene synthase of Staphylococcus aureus is involved in the synthesis of golden carotenoid pigment staphyloxanthin. This pigment of S. aureus provides the antioxidant property to this bacterium to survive inside the host cell. Dehydrosqualene synthase (CrtM) is having structural similarity with the human squalene synthase enzyme which is involved in the cholesterol synthesis pathway in humans (Liu et al., 2008). Cholesterol lowering drugs were found to have inhibitory effect on dehydrosqualene synthase enzyme of S. aureus. The present study attempts to focus on squalene synthase inhibitors, lapaquistat acetate and squalestatins reported as cholesterol lowering agents in vitro and in vivo but not studied in context to dehydrosqualene synthase of S. aureus. Mode of binding of lapaquistat acetate and squalestatin analogs on dehydrosqualene synthase (CrtM) enzyme of S. aureus was identified by performing docking analysis with Scigress Explorer Ultra 7.7 docking software. Based on the molecular docking analysis, it was found that the His18, Arg45, Asp48, Asp52, Tyr129, Gln165, Asn168 and Asp172 residues interacted with comparatively high frequency with the inhibitors studied. Comparative docking study with Discovery studio 2.0 also confirmed the involvement of these residues of dehydrosqualene synthase enzyme with the inhibitors studied. This further confirms the importance of these residues in the enzyme function. In silico ADMET analysis was done to predict the ADMET properties of the standard drugs and test compounds. This might provide insights to develop new drugs to target the virulence factor, dehydrosqualene synthase of S. aureus.

In Planta Recapitulation of Isoprene Synthase Evolution from Ocimene Synthases

PubMed Central

Li, Mingai; Xu, Jia; Algarra Alarcon, Alberto; Carlin, Silvia; Barbaro, Enrico; Cappellin, Luca; Velikova, Violeta; Vrhovsek, Urska; Loreto, Francesco; Varotto, Claudio

2017-01-01

Abstract Isoprene is the most abundant biogenic volatile hydrocarbon compound naturally emitted by plants and plays a major role in atmospheric chemistry. It has been proposed that isoprene synthases (IspS) may readily evolve from other terpene synthases, but this hypothesis has not been experimentally investigated. We isolated and functionally validated in Arabidopsis the first isoprene synthase gene, AdoIspS, from a monocotyledonous species (Arundo donax L., Poaceae). Phylogenetic reconstruction indicates that AdoIspS and dicots isoprene synthases most likely originated by parallel evolution from TPS-b monoterpene synthases. Site-directed mutagenesis demonstrated invivo the functional and evolutionary relevance of the residues considered diagnostic for IspS function. One of these positions was identified by saturating mutagenesis as a major determinant of substrate specificity in AdoIspS able to cause invivo a dramatic change in total volatile emission from hemi- to monoterpenes and supporting evolution of isoprene synthases from ocimene synthases. The mechanism responsible for IspS neofunctionalization by active site size modulation by a single amino acid mutation demonstrated in this study might be general, as the very same amino acidic position is implicated in the parallel evolution of different short-chain terpene synthases from both angiosperms and gymnosperms. Based on these results, we present a model reconciling in a unified conceptual framework the apparently contrasting patterns previously observed for isoprene synthase evolution in plants. These results indicate that parallel evolution may be driven by relatively simple biophysical constraints, and illustrate the intimate molecular evolutionary links between the structural and functional bases of traits with global relevance. PMID:28637270

Accumulation of prenyl alcohols by terpenoid biosynthesis inhibitors in various microorganisms.

PubMed

Muramatsu, Masayoshi; Ohto, Chikara; Obata, Shusei; Sakuradani, Eiji; Shimizu, Sakayu

2008-09-01

Squalene synthase inhibitors significantly accelerate the production of farnesol by various microorganisms. However, farnesol production by Saccharomyces cerevisiae ATCC 64031, in which the squalene synthase gene is deleted, was not affected by the inhibitors, indicating that farnesol accumulation is enhanced in the absence of squalene synthase activity. The combination of diphenylamine as an inhibitor of carotenoid biosynthesis and a squalene synthase inhibitor increases geranylgeraniol production by a yeast, Rhodotorula rubra NBRC 0870. An ent-kauren synthase inhibitor also enhances the production of farnesol and geranylgeraniol by a filamentous fungus, Gibberella fujikuroi NBRC 30336. These results indicate that the inhibition of downstream enzymes from prenyl diphosphate synthase leads to the production of farnesol and geranylgeraniol.

Structural Basis for a Unique ATP Synthase Core Complex from Nanoarcheaum equitans*

PubMed Central

Mohanty, Soumya; Jobichen, Chacko; Chichili, Vishnu Priyanka Reddy; Velázquez-Campoy, Adrián; Low, Boon Chuan; Hogue, Christopher W. V.; Sivaraman, J.

2015-01-01

ATP synthesis is a critical and universal life process carried out by ATP synthases. Whereas eukaryotic and prokaryotic ATP synthases are well characterized, archaeal ATP synthases are relatively poorly understood. The hyperthermophilic archaeal parasite, Nanoarcheaum equitans, lacks several subunits of the ATP synthase and is suspected to be energetically dependent on its host, Ignicoccus hospitalis. This suggests that this ATP synthase might be a rudimentary machine. Here, we report the crystal structures and biophysical studies of the regulatory subunit, NeqB, the apo-NeqAB, and NeqAB in complex with nucleotides, ADP, and adenylyl-imidodiphosphate (non-hydrolysable analog of ATP). NeqB is ∼20 amino acids shorter at its C terminus than its homologs, but this does not impede its binding with NeqA to form the complex. The heterodimeric NeqAB complex assumes a closed, rigid conformation irrespective of nucleotide binding; this differs from its homologs, which require conformational changes for catalytic activity. Thus, although N. equitans possesses an ATP synthase core A3B3 hexameric complex, it might not function as a bona fide ATP synthase. PMID:26370083

Interaction of Constitutive Nitric Oxide Synthases with Cyclooxygenases in Regulation of Bicarbonate Secretion in the Gastric Mucosa.

PubMed

Zolotarev, V A; Andreeva, Yu V; Vershinina, E; Khropycheva, R P

2017-05-01

Neuronal NO synthase blocker 7-nitroindazole suppressed bicarbonate secretion in rat gastric mucosa induced by mild local irritation with 1 M NaCl (pH 2.0). Non-selective blocker of neuronal and endothelial synthases, Nω-nitro-L-arginine (L-NNA), did not affect HCO 3 - production, but inhibited secretion after pretreatment with omeprazole. Non-selective cyclooxygenase blocker indomethacin inhibited HCO 3 - production under conditions of normal synthase activity and in the presence of L-NNA, but was ineffective when co-administered with 7-nitroindazole. It was concluded that neuronal and endothelial synthases are involved in different mechanisms of regulation of HCO 3 - secretion in the gastric mucosa induced by mild irritation. Activation of neuronal synthase stimulated HCO 3 - production, which is mediated mainly through activation of cyclooxygenase. Theoretically, activation of endothelial synthase should suppress HCO 3 - production. The effect of endothelial synthase depends on acid secretion in the stomach and bicarbonate concentration in the submucosa, as it was demonstrated in experiments with intravenous NaHCO 3 infusion.

Nitric Oxide Synthase and Neuronal NADPH Diaphorase are Identical in Brain and Peripheral Tissues

NASA Astrophysics Data System (ADS)

Dawson, Ted M.; Bredt, David S.; Fotuhi, Majid; Hwang, Paul M.; Snyder, Solomon H.

1991-09-01

NADPH diaphorase staining neurons, uniquely resistant to toxic insults and neurodegenerative disorders, have been colocalized with neurons in the brain and peripheral tissue containing nitric oxide synthase (EC 1.14.23.-), which generates nitric oxide (NO), a recently identified neuronal messenger molecule. In the corpus striatum and cerebral cortex, NO synthase immunoreactivity and NADPH diaphorase staining are colocalized in medium to large aspiny neurons. These same neurons colocalize with somatostatin and neuropeptide Y immunoreactivity. NO synthase immunoreactivity and NADPH diaphorase staining are colocalized in the pedunculopontine nucleus with choline acetyltransferase-containing cells and are also colocalized in amacrine cells of the inner nuclear layer and ganglion cells of the retina, myenteric plexus neurons of the intestine, and ganglion cells of the adrenal medulla. Transfection of human kidney cells with NO synthase cDNA elicits NADPH diaphorase staining. The ratio of NO synthase to NADPH diaphorase staining in the transfected cells is the same as in neurons, indicating that NO synthase fully accounts for observed NADPH staining. The identity of neuronal NO synthase and NADPH diaphorase suggests a role for NO in modulating neurotoxicity.

Isolation and functional effects of monoclonal antibodies binding to thymidylate synthase.

PubMed

Jastreboff, M M; Todd, M B; Malech, H L; Bertino, J R

1985-01-29

Monoclonal antibodies against electrophoretically pure thymidylate synthase from HeLa cells have been produced. Antibodies (M-TS-4 and M-TS-9) from hybridoma clones were shown by enzyme-linked immunoassay to recognize thymidylate synthase from a variety of human cell lines, but they did not bind to thymidylate synthase from mouse cell lines. The strongest binding of antibodies was observed to enzyme from HeLa cells. These two monoclonal antibodies bind simultaneously to different antigenic sites on thymidylate synthase purified from HeLa cells, as reflected by a high additivity index and results of cross-linked radioimmunoassay. Both monoclonal antibodies inhibit the activity of thymidylate synthase from human cell lines. The strongest inhibition was observed with thymidylate synthase from HeLa cells. Monoclonal antibody M-TS-9 (IgM subclass) decreased the rate of binding of [3H]FdUMP to thymidylate synthase in the presence of 5,10-methylenetetrahydrofolate while M-TS-4 (IgG1) did not change the rate of ternary complex formation. These data indicate that the antibodies recognize different epitopes on the enzyme molecule.

Friedelin Synthase from Maytenus ilicifolia: Leucine 482 Plays an Essential Role in the Production of the Most Rearranged Pentacyclic Triterpene

PubMed Central

Souza-Moreira, Tatiana M.; Alves, Thaís B.; Pinheiro, Karina A.; Felippe, Lidiane G.; De Lima, Gustavo M. A.; Watanabe, Tatiana F.; Barbosa, Cristina C.; Santos, Vânia A. F. F. M.; Lopes, Norberto P.; Valentini, Sandro R.; Guido, Rafael V. C.; Furlan, Maysa; Zanelli, Cleslei F.

2016-01-01

Among the biologically active triterpenes, friedelin has the most-rearranged structure produced by the oxidosqualene cyclases and is the only one containing a cetonic group. In this study, we cloned and functionally characterized friedelin synthase and one cycloartenol synthase from Maytenus ilicifolia (Celastraceae). The complete coding sequences of these 2 genes were cloned from leaf mRNA, and their functions were characterized by heterologous expression in yeast. The cycloartenol synthase sequence is very similar to other known OSCs of this type (approximately 80% identity), although the M. ilicifolia friedelin synthase amino acid sequence is more related to β-amyrin synthases (65–74% identity), which is similar to the friedelin synthase cloned from Kalanchoe daigremontiana. Multiple sequence alignments demonstrated the presence of a leucine residue two positions upstream of the friedelin synthase Asp-Cys-Thr-Ala-Glu (DCTAE) active site motif, while the vast majority of OSCs identified so far have a valine or isoleucine residue at the same position. The substitution of the leucine residue with valine, threonine or isoleucine in M. ilicifolia friedelin synthase interfered with substrate recognition and lead to the production of different pentacyclic triterpenes. Hence, our data indicate a key role for the leucine residue in the structure and function of this oxidosqualene cyclase. PMID:27874020

Friedelin Synthase from Maytenus ilicifolia: Leucine 482 Plays an Essential Role in the Production of the Most Rearranged Pentacyclic Triterpene

NASA Astrophysics Data System (ADS)

Souza-Moreira, Tatiana M.; Alves, Thaís B.; Pinheiro, Karina A.; Felippe, Lidiane G.; de Lima, Gustavo M. A.; Watanabe, Tatiana F.; Barbosa, Cristina C.; Santos, Vânia A. F. F. M.; Lopes, Norberto P.; Valentini, Sandro R.; Guido, Rafael V. C.; Furlan, Maysa; Zanelli, Cleslei F.

2016-11-01

Among the biologically active triterpenes, friedelin has the most-rearranged structure produced by the oxidosqualene cyclases and is the only one containing a cetonic group. In this study, we cloned and functionally characterized friedelin synthase and one cycloartenol synthase from Maytenus ilicifolia (Celastraceae). The complete coding sequences of these 2 genes were cloned from leaf mRNA, and their functions were characterized by heterologous expression in yeast. The cycloartenol synthase sequence is very similar to other known OSCs of this type (approximately 80% identity), although the M. ilicifolia friedelin synthase amino acid sequence is more related to β-amyrin synthases (65-74% identity), which is similar to the friedelin synthase cloned from Kalanchoe daigremontiana. Multiple sequence alignments demonstrated the presence of a leucine residue two positions upstream of the friedelin synthase Asp-Cys-Thr-Ala-Glu (DCTAE) active site motif, while the vast majority of OSCs identified so far have a valine or isoleucine residue at the same position. The substitution of the leucine residue with valine, threonine or isoleucine in M. ilicifolia friedelin synthase interfered with substrate recognition and lead to the production of different pentacyclic triterpenes. Hence, our data indicate a key role for the leucine residue in the structure and function of this oxidosqualene cyclase.

Multiple defects in muscle glycogen synthase activity contribute to reduced glycogen synthesis in non-insulin dependent diabetes mellitus.

PubMed Central

Thorburn, A W; Gumbiner, B; Bulacan, F; Brechtel, G; Henry, R R

1991-01-01

To define the mechanisms of impaired muscle glycogen synthase and reduced glycogen formation in non-insulin dependent diabetes mellitus (NIDDM), glycogen synthase activity was kinetically analyzed during the basal state and three glucose clamp studies (insulin approximately equal to 300, 700, and 33,400 pmol/liter) in eight matched nonobese NIDDM and eight control subjects. Muscle glycogen content was measured in the basal state and following clamps at insulin levels of 33,400 pmol/liter. NIDDM subjects had glucose uptake matched to controls in each clamp by raising serum glucose to 15-20 mmol/liter. The insulin concentration required to half-maximally activate glycogen synthase (ED50) was approximately fourfold greater for NIDDM than control subjects (1,004 +/- 264 vs. 257 +/- 110 pmol/liter, P less than 0.02) but the maximal insulin effect was similar. Total glycogen synthase activity was reduced approximately 38% and glycogen content was approximately 30% lower in NIDDM. A positive correlation was present between glycogen content and glycogen synthase activity (r = 0.51, P less than 0.01). In summary, defects in muscle glycogen synthase activity and reduced glycogen content are present in NIDDM. NIDDM subjects also have less total glycogen synthase activity consistent with reduced functional mass of the enzyme. These findings and the correlation between glycogen synthase activity and glycogen content support the theory that multiple defects in glycogen synthase activity combine to cause reduced glycogen formation in NIDDM. PMID:1899428

Glycogen synthase activation by sugars in isolated hepatocytes.

PubMed

Ciudad, C J; Carabaza, A; Bosch, F; Gòmez I Foix, A M; Guinovart, J J

1988-07-01

We have investigated the activation by sugars of glycogen synthase in relation to (i) phosphorylase a activity and (ii) changes in the intracellular concentration of glucose 6-phosphate and adenine nucleotides. All the sugars tested in this work present the common denominator of activating glycogen synthase. On the other hand, phosphorylase a activity is decreased by mannose and glucose, unchanged by galactose and xylitol, and increased by tagatose, glyceraldehyde, and fructose. Dihydroxyacetone exerts a biphasic effect on phosphorylase. These findings provide additional evidence proving that glycogen synthase can be activated regardless of the levels of phosphorylase a, clearly establishing that a nonsequential mechanism for the activation of glycogen synthase occurs in liver cells. The glycogen synthase activation state is related to the concentrations of glucose 6-phosphate and adenine nucleotides. In this respect, tagatose, glyceraldehyde, and fructose deplete ATP and increase AMP contents, whereas glucose, mannose, galactose, xylitol, and dihydroxyacetone do not alter the concentration of these nucleotides. In addition, all these sugars, except glyceraldehyde, increase the intracellular content of glucose 6-phosphate. The activation of glycogen synthase by sugars is reflected in decreases on both kinetic constants of the enzyme, M0.5 (for glucose 6-phosphate) and S0.5 (for UDP-glucose). We propose that hepatocyte glycogen synthase is activated by monosaccharides by a mechanism triggered by changes in glucose 6-phosphate and adenine nucleotide concentrations which have been described to modify glycogen synthase phosphatase activity. This mechanism represents a metabolite control of the sugar-induced activation of hepatocyte glycogen synthase.

In Planta Recapitulation of Isoprene Synthase Evolution from Ocimene Synthases.

PubMed

Li, Mingai; Xu, Jia; Algarra Alarcon, Alberto; Carlin, Silvia; Barbaro, Enrico; Cappellin, Luca; Velikova, Violeta; Vrhovsek, Urska; Loreto, Francesco; Varotto, Claudio

2017-10-01

Isoprene is the most abundant biogenic volatile hydrocarbon compound naturally emitted by plants and plays a major role in atmospheric chemistry. It has been proposed that isoprene synthases (IspS) may readily evolve from other terpene synthases, but this hypothesis has not been experimentally investigated. We isolated and functionally validated in Arabidopsis the first isoprene synthase gene, AdoIspS, from a monocotyledonous species (Arundo donax L., Poaceae). Phylogenetic reconstruction indicates that AdoIspS and dicots isoprene synthases most likely originated by parallel evolution from TPS-b monoterpene synthases. Site-directed mutagenesis demonstrated invivo the functional and evolutionary relevance of the residues considered diagnostic for IspS function. One of these positions was identified by saturating mutagenesis as a major determinant of substrate specificity in AdoIspS able to cause invivo a dramatic change in total volatile emission from hemi- to monoterpenes and supporting evolution of isoprene synthases from ocimene synthases. The mechanism responsible for IspS neofunctionalization by active site size modulation by a single amino acid mutation demonstrated in this study might be general, as the very same amino acidic position is implicated in the parallel evolution of different short-chain terpene synthases from both angiosperms and gymnosperms. Based on these results, we present a model reconciling in a unified conceptual framework the apparently contrasting patterns previously observed for isoprene synthase evolution in plants. These results indicate that parallel evolution may be driven by relatively simple biophysical constraints, and illustrate the intimate molecular evolutionary links between the structural and functional bases of traits with global relevance. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Polyester synthases: natural catalysts for plastics.

PubMed Central

Rehm, Bernd H A

2003-01-01

Polyhydroxyalkanoates (PHAs) are biopolyesters composed of hydroxy fatty acids, which represent a complex class of storage polyesters. They are synthesized by a wide range of different Gram-positive and Gram-negative bacteria, as well as by some Archaea, and are deposited as insoluble cytoplasmic inclusions. Polyester synthases are the key enzymes of polyester biosynthesis and catalyse the conversion of (R)-hydroxyacyl-CoA thioesters to polyesters with the concomitant release of CoA. These soluble enzymes turn into amphipathic enzymes upon covalent catalysis of polyester-chain formation. A self-assembly process is initiated resulting in the formation of insoluble cytoplasmic inclusions with a phospholipid monolayer and covalently attached polyester synthases at the surface. Surface-attached polyester synthases show a marked increase in enzyme activity. These polyester synthases have only recently been biochemically characterized. An overview of these recent findings is provided. At present, 59 polyester synthase structural genes from 45 different bacteria have been cloned and the nucleotide sequences have been obtained. The multiple alignment of the primary structures of these polyester synthases show an overall identity of 8-96% with only eight strictly conserved amino acid residues. Polyester synthases can been assigned to four classes based on their substrate specificity and subunit composition. The current knowledge on the organization of the polyester synthase genes, and other genes encoding proteins related to PHA metabolism, is compiled. In addition, the primary structures of the 59 PHA synthases are aligned and analysed with respect to highly conserved amino acids, and biochemical features of polyester synthases are described. The proposed catalytic mechanism based on similarities to alpha/beta-hydrolases and mutational analysis is discussed. Different threading algorithms suggest that polyester synthases belong to the alpha/beta-hydrolase superfamily, with a conserved cysteine residue as catalytic nucleophile. This review provides a survey of the known biochemical features of these unique enzymes and their proposed catalytic mechanism. PMID:12954080

Sucrose synthase in wild tomato Lycopersicon chmielewskii and tomato fruit sink strength

Treesearch

Shi-Jean S. Sung; T. Loboda; S.S. Sung; C.C. Black

1992-01-01

Here it is reported that sucrose synthase can be readily measured in growing wild tomato fruits (Lycopersicon chmielewskii) when suitable methods are adopted during fruit extraction. The enzyme also was present in fruit pericarp tissues, in seeds, and in flowers.In mature, nongrowing fruits, sucrose synthase activities approached nil values.Therefore, sucrose synthase...

HAEM SYNTHASE AND COBALT PORPHYRIN SYNTHASE IN VARIOUS MICRO-ORGANISMS.

PubMed

PORRA, R J; ROSS, B D

1965-03-01

1. The preparation of a crude extract of Clostridium tetanomorphum containing cobalt porphyrin synthase but little haem-synthase activity is described. 2. The properties of cobalt porphyrin synthase in the clostridial extracts is compared with the properties of a haem synthase present in crude extracts of the yeast Torulopsis utilis. 3. Cobalt porphyrin synthase in extracts of C. tetanomorphum inserts Co(2+) ions into the following dicarboxylic porphyrins in descending order of rate of insertion: meso-, deutero- and proto-porphyrins. Esterification renders meso- and deutero-porphyrins inactive as substrates. Neither the tetracarboxylic (coproporphyrin III) nor the octacarboxylic (uroporphyrin III) compounds are converted into cobalt porphyrins by the extract, but the non-enzymic incorporation of Co(2+) ions into these two porphyrins is rapid. These extracts are unable to insert Mn(2+), Zn(2+), Mg(2+) or Cu(2+) ions into mesoporphyrin. 4. Crude extracts of T. utilis readily insert both Co(2+) and Fe(2+) ions into deutero-, meso, and proto-porphyrins. Unlike the extracts of C. tetanomorphum, these preparations catalyse the insertion of Co(2+) ions into deuteroporphyrin more rapidly than into mesoporphyrin. This parallels the formation of haems by the T. utilis extract. 5. Cobalt porphyrin synthase is present in the particulate fraction of the extracts of C. tetanomorphum but requires a heat-stable factor present in the soluble fraction. This soluble factor can be replaced by GSH. 6. Cobalt porphyrin synthase in the clostridial extract is inhibited by iodoacetamide and to a smaller extent by p-chloromercuribenzoate and N-ethylmaleimide. The haem synthases of T. utilis and Micrococcus denitrificans are also inhibited by various thiol reagents.

Insights into the Prostanoid Pathway in the Ovary Development of the Penaeid Shrimp Penaeus monodon

PubMed Central

Wimuttisuk, Wananit; Tobwor, Punsa; Deenarn, Pacharawan; Danwisetkanjana, Kannawat; Pinkaew, Decha; Kirtikara, Kanyawim; Vichai, Vanicha

2013-01-01

The prostanoid pathway converts polyunsaturated fatty acids (PUFAs) into bioactive lipid mediators, including prostaglandins, thromboxanes and prostacyclins, all of which play vital roles in the immune and reproductive systems in most animal phyla. In crustaceans, PUFAs and prostaglandins have been detected and often associated with female reproductive maturation. However, the presence of prostanoid biosynthesis genes remained in question in these species. In this study, we outlined the prostanoid pathway in the black tiger shrimp Penaeus monodon based on the amplification of nine prostanoid biosynthesis genes: cytosolic phospholipase A2, hematopoietic prostaglandin D synthase, glutathione-dependent prostaglandin D synthase, prostaglandin E synthase 1, prostaglandin E synthase 2, prostaglandin E synthase 3, prostaglandin F synthase, thromboxane A synthase and cyclooxygenase. TBLASTX analysis confirmed the identities of these genes with 51-99% sequence identities to their closest homologs. In addition, prostaglandin F2α (PGF2α), which is a product of the prostaglandin F synthase enzyme, was detected for the first time in P. monodon ovaries along with the previously identified PUFAs and prostaglandin E2 (PGE2) using RP-HPLC and mass-spectrometry. The prostaglandin synthase activity was also observed in shrimp ovary homogenates using in vitro activity assay. When prostaglandin biosynthesis was examined in different stages of shrimp ovaries, we found that the amounts of prostaglandin F synthase gene transcripts and PGF2α decreased as the ovaries matured. These findings not only indicate the presence of a functional prostanoid pathway in penaeid shrimp, but also suggest a possible role of the PGF2α biosynthesis in shrimp ovarian development. PMID:24116186

TALEN mediated targeted editing of GM2/GD2-synthase gene modulates anchorage independent growth by reducing anoikis resistance in mouse tumor cells

PubMed Central

Mahata, Barun; Banerjee, Avisek; Kundu, Manjari; Bandyopadhyay, Uday; Biswas, Kaushik

2015-01-01

Complex ganglioside expression is highly deregulated in several tumors which is further dependent on specific ganglioside synthase genes. Here, we designed and constructed a pair of highly specific transcription-activator like effector endonuclease (TALENs) to disrupt a particular genomic locus of mouse GM2-synthase, a region conserved in coding sequence of all four transcript variants of mouse GM2-synthase. Our designed TALENs effectively work in different mouse cell lines and TALEN induced mutation rate is over 45%. Clonal selection strategy is undertaken to generate stable GM2-synthase knockout cell line. We have also demonstrated non-homologous end joining (NHEJ) mediated integration of neomycin cassette into the TALEN targeted GM2-synthase locus. Functionally, clonally selected GM2-synthase knockout clones show reduced anchorage-independent growth (AIG), reduction in tumor growth and higher cellular adhesion as compared to wild type Renca-v cells. Insight into the mechanism shows that, reduced AIG is due to loss in anoikis resistance, as both knockout clones show increased sensitivity to detachment induced apoptosis. Therefore, TALEN mediated precise genome editing at GM2-synthase locus not only helps us in understanding the function of GM2-synthase gene and complex gangliosides in tumorigenicity but also holds tremendous potential to use TALENs in translational cancer research and therapeutics. PMID:25762467

TALEN mediated targeted editing of GM2/GD2-synthase gene modulates anchorage independent growth by reducing anoikis resistance in mouse tumor cells.

PubMed

Mahata, Barun; Banerjee, Avisek; Kundu, Manjari; Bandyopadhyay, Uday; Biswas, Kaushik

2015-03-12

Complex ganglioside expression is highly deregulated in several tumors which is further dependent on specific ganglioside synthase genes. Here, we designed and constructed a pair of highly specific transcription-activator like effector endonuclease (TALENs) to disrupt a particular genomic locus of mouse GM2-synthase, a region conserved in coding sequence of all four transcript variants of mouse GM2-synthase. Our designed TALENs effectively work in different mouse cell lines and TALEN induced mutation rate is over 45%. Clonal selection strategy is undertaken to generate stable GM2-synthase knockout cell line. We have also demonstrated non-homologous end joining (NHEJ) mediated integration of neomycin cassette into the TALEN targeted GM2-synthase locus. Functionally, clonally selected GM2-synthase knockout clones show reduced anchorage-independent growth (AIG), reduction in tumor growth and higher cellular adhesion as compared to wild type Renca-v cells. Insight into the mechanism shows that, reduced AIG is due to loss in anoikis resistance, as both knockout clones show increased sensitivity to detachment induced apoptosis. Therefore, TALEN mediated precise genome editing at GM2-synthase locus not only helps us in understanding the function of GM2-synthase gene and complex gangliosides in tumorigenicity but also holds tremendous potential to use TALENs in translational cancer research and therapeutics.

ATP Synthase: A Molecular Therapeutic Drug Target for Antimicrobial and Antitumor Peptides

PubMed Central

Ahmad, Zulfiqar; Okafor, Florence; Azim, Sofiya; Laughlin, Thomas F.

2015-01-01

In this review we discuss the role of ATP synthase as a molecular drug target for natural and synthetic antimi-crobial/antitumor peptides. We start with an introduction of the universal nature of the ATP synthase enzyme and its role as a biological nanomotor. Significant structural features required for catalytic activity and motor functions of ATP synthase are described. Relevant details regarding the presence of ATP synthase on the surface of several animal cell types, where it is associated with multiple cellular processes making it a potential drug target with respect to antimicrobial peptides and other inhibitors such as dietary polyphenols, is also reviewed. ATP synthase is known to have about twelve discrete inhibitor binding sites including peptides and other inhibitors located at the interface of α/β subunits on the F1 sector of the enzyme. Molecular interaction of peptides at the β DEELSEED site on ATP synthase is discussed with specific examples. An inhibitory effect of other natural/synthetic inhibitors on ATP is highlighted to explore the therapeutic roles played by peptides and other inhibitors. Lastly, the effect of peptides on the inhibition of the Escherichia coli model system through their action on ATP synthase is presented. PMID:23432591

Identification and characterization of two bisabolene synthases from linear glandular trichomes of sunflower (Helianthus annuus L., Asteraceae).

PubMed

Aschenbrenner, Anna-Katharina; Kwon, Moonhyuk; Conrad, Jürgen; Ro, Dae-Kyun; Spring, Otmar

2016-04-01

Sunflower is known to produce a variety of bisabolene-type sesquiterpenes and accumulates these substances in trichomes of leaves, stems and flowering parts. A bioinformatics approach was used to identify the enzyme responsible for the initial step in the biosynthesis of these compounds from its precursor farnesyl pyrophosphate. Based on sequence similarity with a known bisabolene synthases from Arabidopsis thaliana AtTPS12, candidate genes of Helianthus were searched in EST-database and used to design specific primers. PCR experiments identified two candidates in the RNA pool of linear glandular trichomes of sunflower. Their sequences contained the typical motifs of sesquiterpene synthases and their expression in yeast functionally characterized them as bisabolene synthases. Spectroscopic analysis identified the stereochemistry of the product of both enzymes as (Z)-γ-bisabolene. The origin of the two sunflower bisabolene synthase genes from the transcripts of linear trichomes indicates that they may be involved in the synthesis of sesquiterpenes produced in these trichomes. Comparison of the amino acid sequences of the sunflower bisabolene synthases showed high similarity with sesquiterpene synthases from other Asteracean species and indicated putative evolutionary origin from a β-farnesene synthase. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Sucrose Synthase Gene Family in Chinese Pear (Pyrus bretschneideri Rehd.): Structure, Expression, and Evolution.

PubMed

Abdullah, Muhammad; Cao, Yungpeng; Cheng, Xi; Meng, Dandan; Chen, Yu; Shakoor, Awais; Gao, Junshan; Cai, Yongping

2018-05-11

Sucrose synthase (SS) is a key enzyme involved in sucrose metabolism that is critical in plant growth and development, and particularly quality of the fruit. Sucrose synthase gene families have been identified and characterized in plants various plants such as tobacco, grape, rice, and Arabidopsis . However, there is still lack of detailed information about sucrose synthase gene in pear. In the present study, we performed a systematic analysis of the pear ( Pyrus bretschneideri Rehd.) genome and reported 30 sucrose synthase genes. Subsequently, gene structure, phylogenetic relationship, chromosomal localization, gene duplications, promoter regions, collinearity, RNA-Seq data and qRT-PCR were conducted on these sucrose synthase genes. The transcript analysis revealed that 10 PbSSs genes (30%) were especially expressed in pear fruit development. Additionally, qRT-PCR analysis verified the RNA-seq data and shown that PbSS30 , PbSS24 , and PbSS15 have a potential role in the pear fruit development stages. This study provides important insights into the evolution of sucrose synthase gene family in pear and will provide assistance for further investigation of sucrose synthase genes functions in the process of fruit development, fruit quality and resistance to environmental stresses.

Producing aglycons of ginsenosides in bakers' yeast

PubMed Central

Dai, Zhubo; Wang, Beibei; Liu, Yi; Shi, Mingyu; Wang, Dong; Zhang, Xianan; Liu, Tao; Huang, Luqi; Zhang, Xueli

2014-01-01

Ginsenosides are the primary bioactive components of ginseng, which is a popular medicinal plant that exhibits diverse pharmacological activities. Protopanaxadiol, protopanaxatriol and oleanolic acid are three basic aglycons of ginsenosides. Producing aglycons of ginsenosides in Saccharomyces cerevisiae was realized in this work and provides an alternative route compared to traditional extraction methods. Synthetic pathways of these three aglycons were constructed in S. cerevisiae by introducing β-amyrin synthase, oleanolic acid synthase, dammarenediol-II synthase, protopanaxadiol synthase, protopanaxatriol synthase and NADPH-cytochrome P450 reductase from different plants. In addition, a truncated 3-hydroxy-3-methylglutaryl-CoA reductase, squalene synthase and 2,3-oxidosqualene synthase genes were overexpressed to increase the precursor supply for improving aglycon production. Strain GY-1 was obtained, which produced 17.2 mg/L protopanaxadiol, 15.9 mg/L protopanaxatriol and 21.4 mg/L oleanolic acid. The yeast strains engineered in this work can serve as the basis for creating an alternative way for producing ginsenosides in place of extractions from plant sources. PMID:24424342

Unusual features of a recombinant apple alpha-farnesene synthase.

PubMed

Green, Sol; Friel, Ellen N; Matich, Adam; Beuning, Lesley L; Cooney, Janine M; Rowan, Daryl D; MacRae, Elspeth

2007-01-01

A recombinant alpha-farnesene synthase from apple (Malus x domestica), expressed in Escherichia coli, showed features not previously reported. Activity was enhanced 5-fold by K(+) and all four isomers of alpha-farnesene, as well as beta-farnesene, were produced from an isomeric mixture of farnesyl diphosphate (FDP). Monoterpenes, linalool, (Z)- and (E)-beta-ocimene and beta-myrcene, were synthesised from geranyl diphosphate (GDP), but at 18% of the optimised rate for alpha-farnesene synthesis from FDP. Addition of K(+) reduced monoterpene synthase activity. The enzyme also produced alpha-farnesene by a reaction involving coupling of GDP and isoprenyl diphosphate but at <1% of the rate with FDP. Mutagenesis of active site aspartate residues removed sesquiterpene, monoterpene and prenyltransferase activities suggesting catalysis through the same active site. Phylogenetic analysis clusters this enzyme with isoprene synthases rather than with other sesquiterpene synthases, suggesting that it has evolved differently from other plant sesquiterpene synthases. This is the first demonstration of a sesquiterpene synthase possessing prenyltransferase activity.

Medicinal Chemistry of ATP Synthase: A Potential Drug Target of Dietary Polyphenols and Amphibian Antimicrobial Peptides

PubMed Central

Ahmad, Zulfiqar; Laughlin, Thomas F.

2015-01-01

In this review we discuss the inhibitory effects of dietary polyphenols and amphibian antimicrobial/antitumor peptides on ATP synthase. In the beginning general structural features highlighting catalytic and motor functions of ATP synthase will be described. Some details on the presence of ATP synthase on the surface of several animal cell types, where it is associated with multiple cellular processes making it an interesting drug target with respect to dietary polyphenols and amphibian antimicrobial peptides will also be reviewed. ATP synthase is known to have distinct polyphenol and peptide binding sites at the interface of α/β subunits. Molecular interaction of polyphenols and peptides with ATP synthase at their respective binding sites will be discussed. Binding and inhibition of other proteins or enzymes will also be covered so as to understand the therapeutic roles of both types of molecules. Lastly, the effects of polyphenols and peptides on the inhibition of Escherichia coli cell growth through their action on ATP synthase will also be presented. PMID:20586714

Natural and engineered polyhydroxyalkanoate (PHA) synthase: key enzyme in biopolyester production.

PubMed

Zou, Huibin; Shi, Mengxun; Zhang, Tongtong; Li, Lei; Li, Liangzhi; Xian, Mo

2017-10-01

With the finite supply of petroleum and increasing concern with environmental issues associated with their harvest and processing, the development of more eco-friendly, sustainable alternative biopolymers that can effectively fill the role of petro-polymers has become a major focus. Polyhydroxyalkanoate (PHA) can be naturally produced by many species of bacteria and the PHA synthase is believed to be key enzyme in this natural pathway. Natural PHA synthases are diverse and can affect the properties of the produced PHAs, such as monomer composition, molecular weights, and material properties. Moreover, recent studies have led to major advances in the searching of PHA synthases that display specific properties, as well as engineering efforts that offer more efficient PHA synthases, increased PHA compound production, or even novel biopolyesters which cannot be naturally produced. In this article, we review the updated information of natural PHA synthases and their engineering strategies for improved performance in polyester production. We also speculate future trends on the development of robust PHA synthases and their application in biopolyester production.

Native granule associated short chain length polyhydroxyalkanoate synthase from a marine derived Bacillus sp. NQ-11/A2.

PubMed

Prabhu, Nimali N; Santimano, Maria Celisa; Mavinkurve, Suneela; Bhosle, Saroj N; Garg, Sandeep

2010-01-01

A rapidly growing marine derived Bacillus sp. strain NQ-11/A2, identified as Bacillus megaterium, accumulated 61% polyhydroxyalkanoate by weight. Diverse carbon sources served as substrates for the accumulation of short chain length polyhydroxyalkanoate. Three to nine granules either single or attached as buds could be isolated intact from each cell. Maximum activity of polyhydroxyalkanoate synthase was associated with the granules. Granule-bound polyhydroxyalkanoate synthase had a K(m) of 7.1 x 10(-5) M for DL-beta-hydroxybutyryl-CoA. Temperature and pH optima for maximum activity were 30 degrees C and 7.0, respectively. Sodium ions were required for granule-bound polyhydroxyalkanoate synthase activity and inhibited by potassium. Granule-bound polyhydroxyalkanoate synthase was apparently covalently bound to the polyhydroxyalkanoate-core of the granules and affected by the chaotropic reagent urea. Detergents inhibited the granule-bound polyhydroxyalkanoate synthase drastically whilst glycerol and bovine serum albumin stabilized the synthase.

Isolation and characterization of beta-glucan synthase: A potential biochemical regulator of gravistimulated differential cell wall loosening

NASA Technical Reports Server (NTRS)

Kuzmanoff, K. M.

1984-01-01

In plants, gravity stimulates differential growth in the upper and lower halves of horizontally oriented organs. Auxin regulation of cell wall loosening and elongation is the basis for most models of this phenomenon. Auxin treatment of pea stem tissue rapidly increases the activity of Golgi-localized Beta-1,4-glucan synthase, an enzyme involved in biosynthesis of wall xyloglucan which apparently constitutes the substrate for the wall loosening process. The primary objective is to determine if auxin induces de novo formation of Golgi glucan synthase and increases the level of this glucan synthase mRNA. This shall be accomplished by (a) preparation of a monoclonal antibody to the synthase, (b) isolation, and characterization of the glucan synthase, and (c) examination for cross reactivity between the antibody and translation products of auxin induced mRNAs in pea tissue. The antibody will also be used to localize the glucan synthase in upper and lower halves of pea stem tissue before, during and after the response to gravity.

The chloroplast ATP synthase features the characteristic redox regulation machinery.

PubMed

Hisabori, Toru; Sunamura, Ei-Ichiro; Kim, Yusung; Konno, Hiroki

2013-11-20

Regulation of the activity of the chloroplast ATP synthase is largely accomplished by the chloroplast thioredoxin system, the main redox regulation system in chloroplasts, which is directly coupled to the photosynthetic reaction. We review the current understanding of the redox regulation system of the chloroplast ATP synthase. The thioredoxin-targeted portion of the ATP synthase consists of two cysteines located on the central axis subunit γ. The redox state of these two cysteines is under the influence of chloroplast thioredoxin, which directly controls rotation during catalysis by inducing a conformational change in this subunit. The molecular mechanism of redox regulation of the chloroplast ATP synthase has recently been determined. Regulation of the activity of the chloroplast ATP synthase is critical in driving efficiency into the ATP synthesis reaction in chloroplasts. The molecular architecture of the chloroplast ATP synthase, which confers redox regulatory properties requires further investigation, in light of the molecular structure of the enzyme complex as well as the physiological significance of the regulation system.

Mir-338-3p Mediates Tnf-A-Induced Hepatic Insulin Resistance by Targeting PP4r1 to Regulate PP4 Expression.

PubMed

Dou, Lin; Wang, Shuyue; Sun, Libo; Huang, Xiuqing; Zhang, Yang; Shen, Tao; Guo, Jun; Man, Yong; Tang, Weiqing; Li, Jian

2017-01-01

Insulin resistance is a critical factor contributing to the pathogenesis of type 2 diabetes and other metabolic diseases. Recent studies have indicated that miR-338-3p plays an important role in cancer. Here, we investigated whether miR-338-3p mediates tumour necrosis factor-α (TNF-α)-induced hepatic insulin resistance. The activation of the insulin signalling pathway and the level of glycogenesis were examined in the livers of the db/db and high fat diet (HFD)-fed mice and in HEP1-6 cells transfected with miR-338-3p mimic or inhibitor. Computational prediction of microRNA target, luciferase assay and Western blot were used to assess the miR-338-3p target. Chromatin immunoprecipitation (ChIP) assay was used to determine the transcriptional regulator of miR-338-3p. miR-338-3p was down-regulated in the livers of the db/db, HFD-fed and TNF-α-treated C57BL/6J mice, as well as in mouse HEP1-6 hepatocytes treated with TNF-α. Importantly the down-regulation of miR-338-3p induced insulin resistance, as indicated by impaired glucose tolerance and insulin tolerance. Further research showed that the down-regulated miR-338-3p resulted in the impaired AKT/ glycogen synthase kinase 3 beta (GSl·Gβ) signalling pathway and glycogen synthesis. In contrast, hepatic over-expression of miR-338-3p rescued the TNF-α-induced insulin resistance. Moreover, protein phosphatase 4 regulator subunit 1 (PP4R1) was identified as a direct target of miR-338-3p that mediated hepatic insulin signalling by regulating protein phosphatase 4 (PP4). Finally we identified hepatic nuclear factor 4 alpha (HNF-4α) as the transcriptional regulator of miRNA-338-3p. Our studies provide novel insight into the critical role and molecular mechanism by which miR-338-3p is involved in TNF-α-induced hepatic insulin resistance. miR-338-3p might mediate TNF-α-induced hepatic insulin resistance by targeting PP4R1 to regulate PP4 expression. © 2017 The Author(s). Published by S. Karger AG, Basel.

Immunological Cells and Functions in Gaucher Disease

PubMed Central

Pandey, Manoj Kumar; Grabowski, Gregory A.

2013-01-01

The macrophage (MΦ) has been the focus of causality, research, and therapy of Gaucher disease, but recent evidence casts doubt its solitary role in the disease pathogenesis. The excess of glucosylceramide (GC) in such cells accounts for some of the disease manifestations. Evidence of increased expression of C-C and C-X-C chemokines (i.e., CCL2,CXCL1, CXCL8) in Gaucher disease could be critical for monocytes (MOs) transformation to inflammatory subsets of (MΦs) and dendritic cells (DCs) as well as neutrophil (PMNs) recruitment to visceral organs. These immune responses could be essential for activation of T- and B-cell subsets, and the induction of numerous cytokines and chemokines that participate in the initiation and propagation of the molecular pathogenesis of Gaucher disease. The association of Gaucher disease with a variety of cellular and humoral immune responses is reviewed here to provide a potential foundation for expanding the complex pathophysiology of Gaucher disease. PMID:23510064

The type I fatty acid and polyketide synthases: a tale of two megasynthases

PubMed Central

Tsai, Shiou-Chuan

2008-01-01

This review chronicles the synergistic growth of the fields of fatty acid and polyketide synthesis over the last century. In both animal fatty acid synthases and modular polyketide synthases, similar catalytic elements are covalently linked in the same order in megasynthases. Whereas in fatty acid synthases the basic elements of the design remain immutable, guaranteeing the faithful production of saturated fatty acids, in the modular polyketide synthases, the potential of the basic design has been exploited to the full for the elaboration of a wide range of secondary metabolites of extraordinary structural diversity. PMID:17898897

Divinyl ether synthase gene and protein, and uses thereof

DOEpatents

Howe, Gregg A [East Lansing, MI; Itoh, Aya [Tsuruoka, JP

2011-09-13

The present invention relates to divinyl ether synthase genes, proteins, and methods of their use. The present invention encompasses both native and recombinant wild-type forms of the synthase, as well as mutants and variant forms, some of which possess altered characteristics relative to the wild-type synthase. The present invention also relates to methods of using divinyl ether synthase genes and proteins, including in their expression in transgenic organisms and in the production of divinyl ether fatty acids, and to methods of suing divinyl ether fatty acids, including in the protection of plants from pathogens.