Regulation of human ß-galactoside α2,6-sialyltransferase (hST6Gal I) gene expression during differentiation of human osteoblastic MG-63 cells.

In this study, we found that gene expression of the human ß-galactoside α2,6-sialyltransferase (hST6Gal I) was specifically increased during differentiation of human MG-63 osteoblastic cells by serum starvation (SS). In parallel, a distinct increase in binding to SNA, the α2,6-sialyl-specific lectin, was observed in serum-starved cells, as demonstrated by FACS analysis. 5'-Rapid amplification of cDNA ends analysis demonstrated that the increase of hST6Gal I transcript by SS is mediated by P1 promoter. To elucidate transcriptional regulation of hST6Gal I in SS-induced MG-63 cells, we functionally characterized the P1 promoter region of the hST6Gal I gene. The 5'-deletion analysis of P1 promoter region revealed that the 189 bp upstream region of transcription start site is critical for transcriptional activity of hST6Gal I gene in SS-induced MG-63 cells. This region contains the predicted binding sites for several transcription factors, including AREB6, FOXP1, SIX3, HNF1, YY2, and MOK2. The mutagenesis analysis for these sites and chromatin immunoprecipitation assay demonstrated that the YY2 binding site at -98 to -77 was essential for the SS-induced hST6Gal I gene expression during differentiation of MG-63 cells.

Characterization of a Type-2 Diacylglycerol Acyltransferase from Haematococcus pluvialis Reveals Possible Allostery of the Recombinant Enzyme.

Haematococcus pluvialis is a green microalga used in the algal biotechnology industry that can accumulate considerable amounts of storage triacylglycerol (TAG) and astaxanthin, which is a high-value carotenoid with strong antioxidant activity, under stress conditions. Diacylglycerol acyltransferase (DGAT) catalyzes the last step of the acyl-CoA-dependent TAG biosynthesis and appears to represent a bottleneck in algal TAG formation. In this study, putative H. pluvialis DGAT2 cDNA (HpDGAT2A, B, D and E) were identified from a transcriptome database and were subjected to sequence-based in silico analyses. The coding sequences of HpDGAT2B, D, and E were then isolated and characterized through heterologous expression in a TAG-deficient Saccharomyces cerevisiae strain H1246. The expression of HpDGAT2D allowed the recovery of TAG biosynthesis in this yeast mutant, and further in vitro enzymatic assays confirmed that the recombinant HpDGAT2D possessed strong DGAT activity. Interestingly, the recombinant HpDGAT2D displayed sigmoidal kinetics in response to increasing acyl-CoA concentrations, which has not been reported in plant or algal DGAT2 in previous studies.

Ascorbic acid-induced TET activation mitigates adverse hydroxymethylcytosine loss in renal cell carcinoma.

Although clear cell renal cell carcinoma (ccRCC) has been shown to result in widespread aberrant cytosine methylation and loss of 5-hydroxymethylcytosine (5hmC), the prognostic impact and therapeutic targeting of this epigenetic aberrancy has not been fully explored. Analysis of 576 primary ccRCC samples demonstrated that loss of 5hmC was strongly associated with aggressive clinicopathologic features and was an independent adverse prognostic factor. Loss of 5hmC also predicted reduced progression-free survival after resection of nonmetastatic disease. The loss of 5hmC in ccRCC was not due to mutational or transcriptional inactivation of ten eleven translocation (TET) enzymes, but to their functional inactivation by l-2-hydroxyglutarate (L2HG), which was overexpressed due to the deletion and underexpression of L2HG dehydrogenase (L2HGDH). Ascorbic acid (AA) reduced methylation and restored genome-wide 5hmC levels via TET activation. Fluorescence quenching of the recombinant TET-2 protein was unaffected by L2HG in the presence of AA. Pharmacologic AA treatment led to reduced growth of ccRCC in vitro and reduced tumor growth in vivo, with increased intratumoral 5hmC. These data demonstrate that reduced 5hmC is associated with reduced survival in ccRCC and provide a preclinical rationale for exploring the therapeutic potential of high-dose AA in ccRCC.

Heterologous expression of phaC2 gene and poly-3-hydroxyalkanoate production by recombinant Cupriavidus necator strains using canola oil as carbon source.

Many heterologous transformation studies have been carried out using the Cupriavidus necator PHB-4 strain to investigate the expression characteristics of various polyhydroxyalkanoate (PHA) synthase enzymes. In this study, we generated a recombinant C. necator PHB-4 strain by transforming a plasmid (pMRC03) harbouring the synthetic phaC2 gene of Pseudomonas putida CA-3. Under conditions favourable for expression of the phaC2 P.putCA-3 gene, canola oil was used as carbon source for the synthesis of PHAs. The expressed synthase polymerised monomers of 3-hydroxybutyrate (3-HB), 3-hydroxyvalerate (3-HV) and 3-hydroxyhexanoate (3-HHx) in the recombinant C. necator PHB-4 (pMRC03) strain. We then co-expressed the phaC2P.putCA-3 gene with the native phaC1C.ne gene in wild type Cupriavidus necator H16 (C. necator H16 (pMRC03)). This co-expression produced a PHA blend of 3-HB, 3-HV, 3-HHx and 3-hydroxyoctanoate (3-HO) monomers in the presence of canola oil. Gas chromatography analysis revealed the presence of 94mol% 3-HB, 1mol% 3-HV, 4mol% 3-HHx and 1mol% 3-HO in a tetra-polymer. Thus, we confirmed that a synthetic phaC2 gene encoding the synthase enzyme is functionally active with substrates ranging from short to medium chain length PHAs.

Lannea coromandelica (Houtt.) Merr. Induces Heme Oxygenase 1 (HO-1) Expression and Reduces Oxidative Stress via the p38/c-Jun N-Terminal Kinase-Nuclear Factor Erythroid 2-Related Factor 2 (p38/JNK-NRF2)-Mediated Antioxidant Pathway.

The leaves of Lannea coromandelica (Houtt.) Merr. are used in the Garo, Pahan, and Teli tribal communities of Bangladesh as a traditional medicinal plant to treat hepatitis, diabetes, ulcers, heart disease, and dysentery. However, there have been limited phytochemical and biological studies on the bark of L. coromandelica. This study aimed to investigate the antioxidant activities of L. coromandelica bark extract (LCBE) and the underlying mechanism using RAW 264.7 cells. The LCBE was analysed by high-pressure liquid chromatography (HPLC) to detect its key polyphenolic compounds. Various in vitro antioxidant assays were performed using RAW 264.7 cells to assess the antioxidant effects of the LCBE and to understand the underlying molecular mechanism. HPLC revealed the presence of gallic acid, (-)-epigallocatechin-3-gallate, catechin, chlorogenic acid, and caffeic acid in the LCBE. The extract showed a very potent capacity to scavenge numerous free radicals through hydrogen atom transfer and/or electron donation and also quenched cellular reactive oxygen species (ROS) generation without showing any toxicity. The LCBE was found to combat the oxidative stress by enhancing the expression, at both transcriptional and translational levels, of primary antioxidant enzymes as well as phase II detoxifying enzymes, especially heme oxygenase 1, through the upregulation of the nuclear factor erythroid 2-related factor 2 (NRF2)-mediated pathway in RAW 264.7 cells via the phosphorylation of p38 kinase and c-Jun N-terminal kinase (JNK). The LCBE exhibited strong antioxidant activities and mitigated the cellular ROS production. These results provide scientific evidence of its potential as an ideal applicant for a cost-effective, readily available, and natural phytochemical, as well as a strategy for preventing diseases associated with oxidative stress and attenuating disease progress.

Clinacanthus nutans Extracts Modulate Epigenetic Link to Cytosolic Phospholipase A2 Expression in SH-SY5Y Cells and Primary Cortical Neurons.

Clinacanthus nutans Lindau (C. nutans), commonly known as Sabah Snake Grass in southeast Asia, is widely used in folk medicine due to its analgesic, antiviral, and anti-inflammatory properties. Our recent study provided evidence for the regulation of cytosolic phospholipase A2 (cPLA2) mRNA expression by epigenetic factors (Tan et al. in Mol Neurobiol. doi: 10.1007/s12035-015-9314-z , 2015). This enzyme catalyzes the release of arachidonic acid from glycerophospholipids, and formation of pro-inflammatory eicosanoids or toxic lipid peroxidation products such as 4-hydroxynonenal. In this study, we examined the effects of C. nutans ethanol leaf extracts on epigenetic regulation of cPLA2 mRNA expression in SH-SY5Y human neuroblastoma cells and mouse primary cortical neurons. C. nutans modulated induction of cPLA2 expression in SH-SY5Y cells by histone deacetylase (HDAC) inhibitors, MS-275, MC-1568, and TSA. C. nutans extracts also inhibited histone acetylase (HAT) activity. Levels of cPLA2 mRNA expression were increased in primary cortical neurons subjected to 0.5-h oxygen-glucose deprivation injury (OGD). This increase was significantly inhibited by C. nutans treatment. Treatment of primary neurons with the HDAC inhibitor MS-275 augmented OGD-induced cPLA2 mRNA expression, and this increase was modulated by C. nutans extracts. OGD-stimulated increase in cPLA2 mRNA expression was also reduced by a Tip60 HAT inhibitor, NU9056. In view of a key role of cPLA2 in the production of pro-inflammatory eicosanoids and free radical damage, and the fact that epigenetic effects on genes are often long-lasting, results suggest a role for C. nutans and phytochemicals to inhibit the production of arachidonic acid-derived pro-inflammatory eicosanoids and chronic inflammation, through epigenetic regulation of cPLA2 expression.

Expression of ecto-nucleoside triphosphate diphosphohydrolase3 (NTPDase3) in the female rat brain during postnatal development.

Nucleoside triphosphate diphosphohydrolase3 (NTPDase3) is membrane-bound ecto-enzyme which hydrolyzes extracellular ATP, thus modulating the function of purinergic receptors and the pattern of purinergic signaling. Here we analyzed the developmental expression of NTPDase3 in female hypothalamus, cerebral cortex and hippocampal formation at different postnatal ages (PD7-PD90) by qRT-PCR and immunohistochemistry. In hypothalamus and hippocampus, a similar developmental profile was seen: NTPDase3 gene expression was stable during postnatal development and increased in adults. In the cortex, upregulation of NTPDase3 mRNA expression was seen at PD15 and further increase was evidenced in adults. Immunohistochemical analysis at PD7 revealed faint neuronal NTPDase3 localization in a dorsal hypothalamus. The immunoreactivity (ir) gradually increased in PD15 and PD20, in clusters of cells in the lateral, ventral and dorsomedial hypothalamus. Furthermore, in PD20 animals, NTPDase3-ir was detected on short fibers in the posterior hypothalamic area, while in PD30 the fibers appeared progressively longer and markedly varicose. In adults, the strongest NTPDase3-ir was observed in collections of cells in dorsomedial hypothalamic nucleus, dorsal and lateral hypothalamus and in several thalamic areas, whereas the varicose fibers traversed entire diencephalon, particularly paraventricular thalamic nucleus, ventromedial and dorsomedial hypothalamic nuclei, the arcuate nucleus and the prefornical part of the lateral hypothalamus. The presumably ascending NTPDase3-ir fibers were first observed in PD20; their density and the varicose appearance increased until the adulthood. Prominent enhancement of NTPDase3-ir in the hypothalamus coincides with age when animals acquire diurnal rhythms of sleeping and feeding, supporting the hypothesis that this enzyme may be involved in regulation of homeostatic functions.

Effects of Maternal Choline Supplementation on the Septohippocampal Cholinergic System in the Ts65Dn Mouse Model of Down Syndrome.

Down syndrome (DS), caused by trisomy of chromosome 21, is marked by intellectual disability (ID) and early onset of Alzheimer's disease (AD) neuropathology including hippocampal cholinergic projection system degeneration. Here we determined the effects of age and maternal choline supplementation (MCS) on hippocampal cholinergic deficits in Ts65Dn mice compared to 2N mice sacrificed at 6-8 and 14-18 months of age. Ts65Dn mice and disomic (2N) littermates sacrificed at ages 6-8 and 14-18 mos were used for an aging study and Ts65Dn and 2N mice derived from Ts65Dn dams were maintained on either a choline-supplemented or a choline-controlled diet (conception to weaning) and examined at 14-18 mos for MCS studies. In the latter, mice were behaviorally tested on the radial arm Morris water maze (RAWM) and hippocampal tissue was examined for intensity of choline acetyltransferase (ChAT) immunoreactivity. Hippocampal ChAT activity was evaluated in a separate cohort. ChAT-positive fiber innervation was significantly higher in the hippocampus and dentate gyrus in Ts65Dn mice compared with 2N mice, independent of age or maternal diet. Similarly, hippocampal ChAT activity was significantly elevated in Ts65Dn mice compared to 2N mice, independent of maternal diet. A significant increase with age was seen in hippocampal cholinergic innervation of 2N mice, but not Ts65Dn mice. Degree of ChAT intensity correlated negatively with spatial memory ability in unsupplemented 2N and Ts65Dn mice, but positively in MCS 2N mice. The increased innervation produced by MCS appears to improve hippocampal function, making this a therapy that may be exploited for future translational approaches in human DS.

Quantitative Polymerase Chain Reaction Analysis of the Mouse Cyp2j Subfamily: Tissue Distribution and Regulation.

Members of the cytochrome P450 CYP2J subfamily are expressed in multiple tissues in mice and humans. These enzymes are active in the metabolism of fatty acids to generate bioactive compounds. Herein we report new methods and results for quantitative polymerase chain reaction (qPCR) analysis for the seven genes (Cyp2j5, Cyp2j6, Cyp2j8, Cyp2j9, Cyp2j11, Cyp2j12, and Cyp2j13) of the mouse Cyp2j subfamily. SYBR Green primer sets were developed and compared with commercially available TaqMan primer/probe assays for specificity toward mouse Cyp2j cDNA, and analysis of tissue distribution and regulation of Cyp2j genes. Each TaqMan primer/probe set and SYBR Green primer set were shown to be specific for their intended mouse Cyp2j cDNA. Tissue distribution of the mouse Cyp2j isoforms confirmed similar patterns of expression between the two qPCR methods. Cyp2j5 and Cyp2j13 were highly expressed in male kidneys, and Cyp2j11 was highly expressed in both male and female kidneys. Cyp2j6 was expressed in multiple tissues, with the highest expression in the small intestine and duodenum. Cyp2j8 was detected in various tissues, with highest expression found in the skin. Cyp2j9 was highly expressed in the brain, liver, and lung. Cyp2j12 was predominately expressed in the brain. We also determined the Cyp2j isoform expression in Cyp2j5 knockout mice to determine whether there was compensatory regulation of other Cyp2j isoforms, and we assessed Cyp2j isoform regulation during various inflammatory models, including influenza A, bacterial lipopolysaccharide, house dust mite allergen, and corn pollen. Both qPCR methods detected similar suppression of Cyp2j6 and Cyp2j9 during inflammation in the lung.

Genotype-Associated Arginase 1 Expression in Rat Peritoneal Macrophages Induced by Toxoplasma gondii.

Toxoplasma gondii induces polarization of mouse macrophages, including both classically activated macrophages (M1) and alternatively activated macrophages (M2) in a genotype-related manner. Here we present a novel result that the Wh6 strain with type Chinese 1, which is predominantly prevalent in China, induces Arg1 expression in a STAT6-dependent manner in primary rat peritoneal macrophages as compared to the PRU stain with type II, which elicited a high expression of Arg1 in a C/EBPß-dependent manner. In addition, dexamethasone inhibited Arg1 expression in rat macrophages in both treatments. Our data suggest that Arg1 expression, which is abundant in polarized M2 cells, is associated with strain/genotype differences from different pathways.

Molecular Cloning, Characterization, and Expression Analysis of a Prolyl 4-Hydroxylase from the Marine Sponge Chondrosia reniformis.

Prolyl 4-hydroxylase (P4H) catalyzes the hydroxylation of proline residues in collagen. P4H has two functional subunits, α and ß. Here, we report the cDNA cloning, characterization, and expression analysis of the α and ß subunits of the P4H derived from the marine sponge Chondrosia reniformis. The amino acid sequence of the α subunit is 533 residues long with an M r of 59.14 kDa, while the ß subunit counts 526 residues with an M r of 58.75 kDa. Phylogenetic analyses showed that αP4H and ßP4H are more related to the mammalian sequences than to known invertebrate P4Hs. Western blot analysis of sponge lysate protein cross-linking revealed a band of 240 kDa corresponding to an α2ß2 tetramer structure. This result suggests that P4H from marine sponges shares the same quaternary structure with vertebrate homologous enzymes. Gene expression analyses showed that αP4H transcript is higher in the choanosome than in the ectosome, while the study of factors affecting its expression in sponge fragmorphs revealed that soluble silicates had no effect on the αP4H levels, whereas ascorbic acid strongly upregulated the αP4H mRNA. Finally, treatment with two different tumor necrosis factor (TNF)-alpha inhibitors determined a significant downregulation of αP4H gene expression in fragmorphs demonstrating, for the first time in Porifera, a positive involvement of TNF in sponge matrix biosynthesis. The molecular characterization of P4H genes involved in collagen hydroxylation, including the mechanisms that regulate their expression, is a key step for future recombinant sponge collagen production and may be pivotal to understand pathological mechanisms related to extracellular matrix deposition in higher organisms.

Methotrexate-related response on human peripheral blood mononuclear cells may be modulated by the Ala16Val-SOD2 gene polymorphism.

Methotrexate (MTX) is a folic acid antagonist used in high doses as an anti-cancer treatment and in low doses for the treatment of some autoimmune diseases. MTX use has been linked to oxidative imbalance, which may cause multi-organ toxicities that can be attenuated by antioxidant supplementation. Despite the oxidative effect of MTX, the influence of antioxidant gene polymorphisms on MTX toxicity is not well studied. Therefore, we analyzed here whether a genetic imbalance of the manganese-dependent superoxide dismutase (SOD2) gene could have some impact on the MTX cytotoxic response. An in vitro study using human peripheral blood mononuclear cells (PBMCs) obtained from carriers with different Ala16Val-SOD2 genotypes (AA, VV and AV) was carried out, and the effect on cell viability and proliferation was analyzed, as well as the effect on oxidative, inflammatory and apoptotic markers. AA-PBMCs that present higher SOD2 efficiencies were more resistance to high MTX doses (10 and 100 µM) than were the VV and AV genotypes. Both lipoperoxidation and ROS levels increased significantly in PBMCs exposed to MTX independent of Ala16Val-SOD2 genotypes, whereas increased protein carbonylation was observed only in PBMCs from V allele carriers. The AA-PBMCs exposed to MTX showed decreasing SOD2 activity, but a concomitant up regulation of the SOD2 gene was observed. A significant increase in glutathione peroxidase (GPX) levels was observed in all PBMCs exposed to MTX. However, this effect was more intense in AA-PBMCs. Caspase-8 and -3 levels were increased in cells exposed to MTX, but the modulation of these genes, as well as that of the Bax and Bcl-2 genes involved in the apoptosis pathway, presented a modulation that was dependent on the SOD2 genotype. MTX at a concentration of 10 µM also increased inflammatory cytokines (IL-1ß, IL-6, TNFα and Igγ) and decreased the level of IL-10 anti-inflammatory cytokine, independent of SOD2 genetic background. The results suggest that potential pharmacogenetic effect on the cytotoxic response to MTX due differential redox status of cells carriers different SOD2 genotypes.

Cystathionine γ-lyase deficiency mediates neurodegeneration in Huntington's disease.

Huntington's disease is an autosomal dominant disease associated with a mutation in the gene encoding huntingtin (Htt) leading to expanded polyglutamine repeats of mutant Htt (mHtt) that elicit oxidative stress, neurotoxicity, and motor and behavioural changes. Huntington's disease is characterized by highly selective and profound damage to the corpus striatum, which regulates motor function. Striatal selectivity of Huntington's disease may reflect the striatally selective small G protein Rhes binding to mHtt and enhancing its neurotoxicity. Specific molecular mechanisms by which mHtt elicits neurodegeneration have been hard to determine. Here we show a major depletion of cystathionine γ-lyase (CSE), the biosynthetic enzyme for cysteine, in Huntington's disease tissues, which may mediate Huntington's disease pathophysiology. The defect occurs at the transcriptional level and seems to reflect influences of mHtt on specificity protein 1, a transcriptional activator for CSE. Consistent with the notion of loss of CSE as a pathogenic mechanism, supplementation with cysteine reverses abnormalities in cultures of Huntington's disease tissues and in intact mouse models of Huntington's disease, suggesting therapeutic potential.

Photoperiod history-dependent responses to intermediate day lengths engage hypothalamic iodothyronine deiodinase type III mRNA expression.

Perihypothalamic thyroid hormone signaling features prominently in the seasonal control of reproductive physiology. Triiodothyronine (T(3)) signaling stimulates gonadal development, and decrements in T(3) signaling are associated with gonadal regression. Type 3 iodothyronine deiodinase (DIO3) converts the prohormone thyroxine (T(4)) into biologically inactive 3,3',5'-triiodothyronine, and in long-day breeding Siberian hamsters exposure to long (LD) and short (SD) photoperiods, respectively, inhibit and stimulate hypothalamic dio3 mRNA expression. Reproductive responses to intermediate-duration photoperiods (IntD) occur in a history-dependent manner; IntDs are interpreted as inhibitory only when preceded by longer photoperiods. Because dio3 expression has only been evaluated under LD or SD photoperiods, it is not known whether hypothalamic dio3 encodes absolute photoperiod duration or the reproductive interpretation of photoperiod. Male Siberian hamsters with and without a prior history of LD were exposed to IntD photoperiods, and hypothalamic dio3 mRNA expression was measured 6 wk later. Hamsters with a LD photoperiod history exhibited gonadal regression in IntD and a marked upregulation of hypothalamic dio3 expression, whereas in hamsters without prior exposure to LD, gonadal responses to IntD were absent, and dio3 expression remained low. Patterns of deiodinase expression in hamsters maintained in chronic IntD photoperiods did not appear to reflect feedback effects of gonadal status. Hypothalamic expression of dio3 does not exclusively reflect ambient photoperiod, but rather the context-dependent reproductive interpretation of photoperiod. Neuroendocrine mechanisms that compare current and prior photoperiods, which permit detection of directional changes in day length, occur either upstream, or at the level, of hypothalamic dio3 expression.

SirT1 mediates hyperbaric oxygen preconditioning-induced ischemic tolerance in rat brain.

Our previous studies have shown that hyperbaric oxygen preconditioning (HBO-PC) induces tolerance to cerebral ischemia/reperfusion (I/R). This study aimed to investigate whether SirT1, a class III histone deacetylase, is involved in neuroprotection elicited by HBO-PC in animal and cell culture models of ischemia. Rats were subjected to middle cerebral artery occlusion for 120 minutes after HBO-PC (once a day for 5 days). Primary cultured cortical neurons were exposed to 2 hours of HBO-PC after 2 hours of oxygen-glucose deprivation (OGD). We showed that HBO-PC increased SirT1 protein and mRNA expression, promoted neurobehavioral score, reduced infarct volume, and improved morphology at 24 hours and 7 days after cerebral I/R. Neuroprotection of HBO-PC was attenuated by SirT1 inhibitor EX527 and SirT1 knockdown by short interfering RNA (siRNA), whereas it was mimicked by SirT1 activator resveratrol. Furthermore, HBO-PC enhanced SirT1 expression and cell viability and reduced lactate dehydrogenase release 24 hours after OGD/re-oxygenation. The neuroprotective effect of HBO-PC was emulated through upregulating SirT1 and, reversely, attenuated through downregulating SirT1. The modulation of SirT1 was made by adenovirus infection carrying SirT1 or SirT1 siRNA. Besides, SirT1 increased B-cell lymphoma 2 (Bcl-2) expression and decrease cleaved caspase 3. These results indicate that SirT1 mediates HBO-PC-induced tolerance to cerebral I/R through inhibition of apoptosis.

The novel Solanum tuberosum calcium dependent protein kinase, StCDPK3, is expressed in actively growing organs.

Calcium-dependent protein kinases (CDPKs) are key components of calcium regulated signaling cascades in plants. In this work, isoform StCDPK3 from Solanum tuberosum was studied and fully described. StCDPK3 encodes a 63 kDa protein with an N-terminal variable domain (NTV), rich in prolines and glutamines, which presents myristoylation and palmitoylation consensus sites and a PEST sequence indicative of rapid protein degradation. StCDPK3 gene (circa 11 kb) is localized in chromosome 3, shares the eight exons and seven introns structure with other isoforms from subgroup IIa and contains an additional intron in the 5'UTR region. StCDPK3 expression is ubiquitous being transcripts more abundant in early elongating stolons (ES), leaves and roots, however isoform specific antibodies only detected the protein in leaf particulate extracts. The recombinant 6xHis-StCDPK3 is an active kinase that differs in its kinetic parameters and calcium requirements from StCDPK1 and 2 isoforms. In vitro, StCDPK3 undergoes autophosphorylation regardless of the addition of calcium. The StCDPK3 promoter region (circa 1,800 bp) was subcloned by genome walking and fused to GUS. Light and ABRE responsive elements were identified in the promoter region as well as elements associated to expression in roots. StCDPK3 expression was enhanced by ABA while GA decreased it. Potato transgenic lines harboring StCDPK3 promoter∷GUS construct were generated by Agrobacterium tumefaciens mediated plant transformation. Promoter activity was detected in leaves, root tips and branching points, early ES, tuber eyes and developing sprouts indicating that StCDPK3 is expressed in actively growing organs.

Characterization and expression analysis of phospholipid hydroperoxide glutathione peroxidase cDNA from Chironomus riparius on exposure to cadmium.

Phospholipid-hydroperoxide glutathione peroxidase (PHGPx) is an antioxidant enzyme in the glutathione peroxidases (GPx) family that reduces hydroperoxides of phospholipids and maintains the integrity of biomembranes. Here, we report the identification and characterization of a full length cDNA of PHGPx from the ecotoxicologically important aquatic midge Chironomus riparius (CrPHGPx1) from the Expressed Sequence Tags (ESTs) database generated through pyrosequencing. The 837 base pair (bp) cDNA contained an open reading frame of 597 bp, and a 75 bp 5' and a 159 bp 3'untranslated region. The theoretical molecular mass of the deduced amino acid (aa) sequence (197 aa) was 22.40 kDa with an estimated pI of 8.77. The Cys-codon was present at residue 74 and also the active site residues Gln(91) and Trp(164). The active-site motifs and GPx family signature motifs LAFPCNQF(101-108) and WNFTK(163-168) were also found. Phylogenetic analysis showed that CrPHGPx1 is grouped with PHGPx1 from other species and is more closely related to insects belonging to the dipteran order. The mRNA of CrPHGPx1 was detected in larvae, pupae and adults. The expression of CrPHGPx1 is induced by cadmium exposure indicating that the mRNA expression of CrPHGPx1 is differently regulated in response to oxidative stress caused by environmental stressors.

Seasonal influence on gene expression of monoterpene synthases in Salvia officinalis (Lamiaceae).

Garden sage (Salvia officinalis L., Lamiaceae) is one of the most important medicinal and aromatic plants and possesses antioxidant, antimicrobial, spasmolytic, astringent, antihidrotic and specific sensorial properties. The essential oil of the plant, formed mainly in very young leaves, is in part responsible for these activities. It is mainly composed of the monoterpenes 1,8-cineole, α- and ß-thujone and camphor synthesized by the 1,8-cineole synthase, the (+)-sabinene synthase and the (+)-bornyl diphosphate synthase, respectively, and is produced and stored in epidermal glands. In this study, the seasonal influence on the formation of the main monoterpenes in young, still expanding leaves of field-grown sage plants was studied in two cultivars at the level of mRNA expression, analyzed by qRT-PCR, and at the level of end-products, analyzed by gas chromatography. All monoterpene synthases and monoterpenes were significantly influenced by cultivar and season. 1,8-Cineole synthase and its end product 1,8-cineole remained constant until August and then decreased slightly. The thujones increased steadily during the vegetative period. The transcript level of their corresponding terpene synthase, however, showed its maximum in the middle of the vegetative period and declined afterwards. Camphor remained constant until August and then declined, exactly correlated with the mRNA level of the corresponding terpene synthase. In summary, terpene synthase mRNA expression and respective end product levels were concordant in the case of 1,8-cineole (r=0.51 and 0.67 for the two cultivars, respectively; p<0.05) and camphor (r=0.75 and 0.82; p<0.05) indicating basically transcriptional control, but discordant for α-/ß-thujone (r=-0.05 and 0.42; p=0.87 and 0.13, respectively).

Expression of antioxidant genes in renal cortex of PTU-induced hypothyroid rats: effect of vitamin E and curcumin.

The present study was undertaken to investigate the effect of vitamin E and curcumin on the expression of antioxidant genes in 6-propyl-2-thiouracil (PTU)-induced hypothyroid rat renal cortex. The levels of lipid peroxidation and protein carbonylation were increased in hypothyroid rat kidney. Co-administration of vitamin E and curcumin to hypothyroid rats resulted in amelioration of lipid peroxidation level, whereas curcumin alone alleviated the protein carbonylation level. The mRNA levels of SOD1 and SOD2 were decreased in hypothyroid rats. Decreased level of SOD1 transcripts was observed in hypothyroid rats supplemented with curcumin alone or co-administrated with vitamin E. Translated products of SOD1 and SOD2 in hypothyroid rats was elevated in response to supplementation of both the antioxidants. Decreased SOD1 and SOD2 activities in hypothyroid rats compared to control were either unaltered or further decreased in response to the antioxidants. Expressions of CAT at transcript and translate level along with its activity were down regulated in hypothyroid rats. Administration of vitamin E to hypothyroid rats resulted in elevated CAT mRNA level. In contrast, expression of CAT protein was elevated in response to both the antioxidants. However, CAT activity was unaltered in response to vitamin E and curcumin. GPx1 and GR mRNA level and the activity of glutathione peroxidase (GPx) were not affected in response to induced hypothyroidism. The activity of GPx was increased in response to vitamin E treatment, whereas decreased GR activity in hypothyroid rats was further declined by the administration of antioxidants. The over all results suggest that vitamin E and curcumin differentially modulate the altered antioxidant defence mechanism of rat kidney cortex under experimental hypothyroidism.

Isolation and characterization of a novel peroxisomal choline monooxygenase in barley.

Glycine betaine (GB) is a compatible solute accumulated by many plants under various abiotic stresses. GB is synthesized in two steps, choline â†’ betaine aldehyde â†’ GB, where a functional choline-oxidizing enzyme has only been reported in Amaranthaceae (a chloroplastic ferredoxin-dependent choline monooxygenase) thus far. Here, we have cloned a cDNA encoding a choline monooxygenase (CMO) from barley (Hordeum vulgare) plants, HvCMO. In barley plants under non-stress condition, GB had accumulated in all the determined organs (leaves, internodes, awn and floret proper), mostly in the leaves. The expression of HvCMO protein was abundant in the leaves, whereas the expression of betaine aldehyde dehydrogenase (BADH) protein was abundant in the awn, floret proper and the youngest internode than in the leaves. The accumulation of HvCMO mRNA was increased by high osmotic and low-temperature environments. Also, the expression of HvCMO protein was increased by the presence of high NaCl. Immunofluorescent labeling of HvCMO protein and subcellular fractionation analysis showed that HvCMO protein was localized to peroxisomes. [(14)C]choline was oxidized to betaine aldehyde and GB in spinach (Spinacia oleracea) chloroplasts but not in barley, which indicates that the subcellular localization of choline-oxidizing enzyme is different between two plant species. We investigated the choline-oxidizing reaction using recombinant HvCMO protein expressed in yeast (Saccharomyces cerevisiae). The crude extract of HvCMO-expressing yeast coupled with recombinant BBD2 protein converted [(14)C]choline to GB when NADPH was added as a cofactor. These results suggest that choline oxidation in GB synthesis is mediated by a peroxisomal NADPH-dependent choline monooxygenase in barley plants.