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© 2015 R. Haddad, R. H. Japelaghi
Department of Agricultural Biotechnology, Faculty of Engineering and Technology, Imam Khomeini International University, Qazvin, Islamic Republic of Iran
Received November 22, 2014
Peroxiredoxins (Prxs) are peroxidases that reduce hydrogen peroxide (H2O2) and various alkyl hydroperoxides and act as reductants. A full-length cDNA encoding for a Prx polypeptide was isolated and cloned from grape (Vitis vinifera L. cv. Askari) berries. The cDNA was 773 nucleotides in length with a deduced amino acid of 162 residues, possessing one conserved cysteine, which belongs to the type II Prx C. The calculated molecular mass and the predicted isoelectric point of the deduced polypeptide are 17.26 kD and 5.15, respectively. The deduced protein sequence showed a high similarity to PrxII C from other plants, in particular from cotton (Gossypium hirsutum), poplar (Populus trichocarpa), and Citrus sP. The in silico analysis of the promoter region of grape Prx demonstrated the presence of a number of potential cis-acting elements to respond to environmental signals, suggesting that VvPrxII C may respond to a variety of environmental signals, including dehydration, heat, heavy metals, light, pathogens, wounding, and plant hormones. The grape Prx gene was also analyzed for its expressional response to abiotic stress, oxidative stress, and antioxidants application. The results revealed a highly induced response to abiotic stress conditions due to the presence of different putative regulatory elements in its promoter.
1 This text was submitted by the authors in English.
Abbreviations: APX ascorbate peroxidase; CAT catalase; CTAB cetyltrimethylamonium bromide; DHAR dehydroascorbate reductase; GST glutathione-S-transferase; HSE heat stress responsive element; MBS MYB binding site; MRE metal responsive element; MYB myeloblastosis; ORF open reading frame; POX peroxidase; Prx peroxiredoxin; RWC relative water content; SA salicylic acid; SOD superoxide dismutase; Trx thioredoxin.
Corresponding author: Raheem Haddad. Department of Agricultural Biotechnology, Faculty of Engineering and Technology, Imam Khomeini International University, Qazvin, Islamic Republic of Iran; fax: +98281 33901175, e-mail: email@example.com
Keywords: Vitis vinifera abiotic stress antioxidants oxidative stress peroxiredoxin
Peroxiredoxins (Prxs) catalyze the reduction of hydrogen peroxide (H2O2) and various alkyl hydroperoxides via catalytic cysteine and thiol-containing proteins that act as reductants [1]. The importance of these enzymes is underlined by their presence in all groups of organisms, high abundance and involvement in multiple cellular processes such as antioxidant defense, H2O2-mediated cellular signaling [1] and molecular chaperones [2]. In plants, based on amino acid sequence similarities and specific structural features, mainly the number and position of conserved Cys residues, the Prxs can be divided into four different classes: (1) 2-Cys Prx; (2) Prx Q; (3) Prx II, which all contain two catalytic Cys residues in distinct sequence environment, and (4) 1-Cys Prx with one conserved Cys residue only [3]. A phylogenetic distance analysis suggests that 2-Cys Prx, Prx Q, and 1-Cys Prx are related proteins, whereas the group of Prx II is likely to have evolved independently [4].
The first Prxs were identified from bacteria such as Salmonella typhimurium and Escherichia coli [5]. Since then, they have been isolated from many organisms. For example, there are at least five Prx isoforms in yeast (Saccharomyces cerevisiae) and six isoforms characterized in mammalian cells [6]. The Prx family has been also intensively studied in different plants. In Arabidopsis thaliana genome there are 10 ORFs (open reading frames) with sequence similarity to Prxs that can be assigned to the four well-known groups of Prxs: two ORFs code for 2-Cys Prx, one for 1-Cys Prx, one for Prx Q, and six ORFs for Prx II [3, 4]. One of the reasons explaining this multiplicity is the multiple subcellular localization of these proteins in different organisms including the chloroplasts, cytosol, peroxisomes, mitochondria, and possibly the nucleus [3].
The Prxs have been also reported to be involved in response to abiotic stress conditions, such as divergent light, drought, heat, salinity, heavy metals, and plant hormones [711]. Kim et al. [2] reported that dual functions of Chinese cabbage 2-Cys Prx acting as a peroxidase and as a molecular chaperone are alternatively switched by heat shock and oxidative stresses, accompanying by its structural changes. In addition, overexpression of Prx genes in tall fescue (Festuca arundinacea) [12] and yeast (S. cerevisiae) [11] increased tolerance to abiotic stress conditions compared with the control plants. Moreover, studies indicated that expression levels of Prx genes are differentially modified under different stress conditions [4, 10], suggesting unique roles for Prxs in plants during various aspects of the plant life cycle.
We described here the isolation and characterization of a Prx of the type C from grape berry and showed that this gene differentially expressed in different organs. Using in silico analysis, we indicated that the promoter region of PrxII C gene contained a large number of putative regulatory elements and respond to a wide variety of environmental signals. Moreover, we revealed that PrxII C gene is differentially induced upon abiotic stress conditions.
Plant materials and extraction of total RNA. One-year-old cuttings with different organs of grape (Vitis vinifera L. cv. Askari) including berries, leaves, clusters, petioles, stems, tendrils, buds, roots, and seeds, were prepared from plants in the field collection of the Grape Research Station, Takistan-Qazvin, Iran, during the 2010 field season. The cuttings were grown in pots containing soil culture with a 16-h light period at 21°C, 50% humidity, and a photon fluence rate of 120 µmol quanta/(m2 s) in a greenhouse, and different grape organs were immediately frozen in liquid nitrogen at the time of collection and then stored at –80°C until extraction. Total RNA was also extracted from different grape organs by cetyltrimethylamonium bromide (CTAB) method [13].
3′- and 5′-RACE reactions. For 3′-RACE reaction, first strand cDNA was synthesized using 3′-RACE primer as the initiation primer and amplifications were performed using prxF (5′-ataggatccATGGCTCCGATTGCAGTTGG-3′), 3′-RACE (5′-tatggatccgagctcctcgagT18-3′), and adaptor primer (5′-TATGGATCCGAGCTCCTCGAG-3′) primers [14]. For 5′-RACE reaction, the reverse transcription reaction was also carried out using oligo (dT)18 primer as the initiation primer and RNA was degraded by RNase H. A homopolymeric C-tail was added to the 5′ end of the purified cDNA by Terminal Deoxynucleotidyl Transferase ("Fermentas", Germany). Amplifications were performed using prxR (5′-actctcgagTCAGATAGCTTTGAGGATGTC-3′), 5′-RACE (5′-tatggatccgagctcctcgagG15-3′), and adaptor primer primers [14]. The oligonucleotides primers were designed based on the available expressed sequence tag (EST FQ437352), identified with the BLAST program (http://www.ncbi.nlm.nih.gov).
Cloning and sequencing. The PCR products were purified by AccuPrep Gel Purification kit ("Bioneer", South Korea) and subcloned into the pTG19-T vector ("Vivantis", Malaysia) according to the manufacturer's instructions. The nucleotide sequence of the inserts was also determined in both directions by dideoxynucleotide sequencing ("Bioneer").
Abiotic stress treatments. To investigate the response of the different grape Trx h genes to various stresses, the cuttings were treated with abiotic stimuli in three independent replicates. Progressive water deficit was applied by withholding watering for 10–12 days and leaf RWC (relative water content) was determined as described by Pruvot et al. [15]. For salt stress, the cuttings were treated with different concentrations of NaCl, including 50, 100, 150, 200, 250, and 300 mM. The cuttings were gradually exposed salt stress during 57 days and then the youngest fully expanded leaves were harvested after a period of approximately 7 days. Control and abiotic treated cuttings were held under a 16-h light period at 21°C, 50% humidity, and a photon fluence rate of 120 µM quanta/(m2 s) in a greenhouse. Heat stress was also applied by exposing the cuttings to a temperature of 40°C for 6, 12, 18, 24, 48, and 72 h under a 16-h light period, 50% humidity, and a photon fluence rate of 120 µM quanta/(m2 s) in a chamber growth ("Grouc", Iran). For recovery, the cuttings were held in under a 16-h light period at 21°C, 50% humidity, and a photon fluence rate of 120 µmol quanta/(m2 s) in a greenhouse for 7 days.
Oxidative stress treatments. For the different oxidative stress treatments, 3 to 4 youngest fully expanded leaves from one-year-old cuttings were pooled and cut into 1 cm diameter leaf slices. After vacuum infiltration with distilled water, the leaf slices were suspended in effector solutions (pH 56) including 10 mM H2O2, 1 mM diamide, 100 µM CuSO4, 100 µM CoCl2, 100 µM CdCl2, 100 µM AlCl3, 100 µM ABA, and 100 µM SA (salicylic acid). The leaf slices were incubated at 21°C and a photon fluence rate of 120 µM quanta/(m2 s) for 4 h.
Semi-quantitative RT-PCR. Semi-quantitative RT-PCR was performed using 5 µg of DNase I-treated total RNA for first-strand synthesis of cDNA and about 1/20 of the reverse transcription reaction was used for RT-PCR with specific primers for distinct VvPrx cDNA. PCR amplifications were performed in a thermal cycler programmed with the following temperature parameters: 3 min at 94°C, followed by 35 cycles of 30 s at 94°C, 1 min at 58°C, 30 s at 72°C, and the final extension of 5 min at 72°C. The grape actin gene (VvAct2) was used as an internal control to normalize each sample for variations in the amounts of RNA used. To control for possible genomic DNA contamination, parallel reactions were carried out where reverse transcriptase activity was inactivated by incubation at 95°C. A negative control lacking a template was included for each set of RT-PCR reactions. Reactions were performed in triplicates. Amplification products were separated by agarose gel electrophoresis and quantified using ImageJ software (W.S. Rasband; 1997–2007; National Institutes of Health; http://rsb.info.nih.gov/ij). Signal intensities were normalized with respect to VvAct2 from the same sample.
Sequence analysis and molecular modeling. The properties of deduced amino acid sequence were estimated using ProtParam (http://www.expasy.ch/tools/protparam.html) program and its subcellular localization prediction was performed by using a combination of three programs, TargetP (http://www.cbs.dtu.dk/services/TargetP/), iPSORT (http://ipsort.hgc.jp/), and YLOC (http://www-bs.informatik.uni-tuebingen.de/Services/YLoc/). Promoter region of the grape Prx gene was obtained from the Phytozome website (http://www.phytozome.net) and searched to find regulatory elements controlling several types of plant stress responses using PlantCare software (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The three-dimensional (3D) structure of deduced amino acid sequence was determined by I-TASSER (http://zhang.bioinformatics.ku.edu/I-TASSER) and superimposition analysis of the 3D models of grape Prx and its templates, such as human (Homo sapiens) Prx V protein (PDB ID code 3mngA), poplar (Populus tremula) PrxII C (PDB ID code 1tp9A), and PsPrxII F (PDB ID code 2pwjA) from pea (Pisum sativum) was done using 3-dimensional Structural Superposition (3d-SS) service (http://cluster.physics.iisc.ernet.in/3dss/severalinput.html).
Phylogenetic analysis. Protein sequences were aligned using the ClustalW2 at European Bioinformatics Institute (http://www.abi.ac.uk) using default parameters. Neighbor Joining tree was constructed with MEGA4.1 Beta 2 software [16] and bootstrap analysis with 1000 replicates was also conducted in order to obtain confidence levels for the branches. The NCBI and SwissProt accession numbers for the sequences described and mentioned in this study are as follows: Arabidopsis (Arabidopsis thaliana): At-2CPrx A (At3g11630), At-2CPrx B (At5g06290), At-1CPrx (At1g48130), AtPrx Q (At3g26060), AtPrxII A (At1g65990), AtPrxII B (At1g65980), AtPrxII C (At1g65970), AtPrxII D (At1g60740), AtPrxII E (At3g52960); (A. lyrata): AlPrxII A (EFH64732); castor oil (Ricinus communis): Rc-2CPrx A (EEF32207), RcPrx Q (EEF46226), RcPrxII C (EEF48227), RcPrxII E (EEF33787), RcPrxII F (EEF45351); Chlamydomonas reinhardtii (EDP07991); Citrus sP.: CitrusPrxII C (ABL67649); cotton (Gossypium hirsutum): GhPrxII C (ACJ11720); Escherichia coli (EDV66373); human (H. sapiens): Prx V (P30044); maize (Zea mays): Zm-1CPrx (DAA63833); pea (P. sativum): PsPrxII F (CAG30523); poplar (Populus trichocarpa): Pt-2CPrx A (EEF05155), PtPrx Q (EEE71975), PtPrxII C (EEF03570), PtPrxII E (ABK92641), PtPrxII F (EEE99739); (P. tremula): PtrPrxII C (AAL90751); soybean (Glycine max): Gm-2CPrx A (XP_003535806), Gm-1CPrx (XP_003531110), GmPrxII F (ACU14896).
Cloning and sequence analysis of grape Prx gene
One EST sequence (NCBI GenBank accession no. FQ437352) corresponding to a putative full-length Prx cDNA was identified using the BLASTn program. In order to characterize the full-length cDNA, 3′ and 5′ RACE reactions were performed and the PCR products were cloned into pTG19-T plasmid vector to generate the pTG19-Prx plasmid. The grape Prx cDNA (submitted at NCBI GenBank under accession no. KC195963) was 773 nucleotides long and contained a single ORF of 489 bp coding for a polypeptide of 162 amino acid residues. The calculated molecular mass and the predicated isoelectric point of the deduced polypeptide sequence were 17.26 kD and 5.15, respectively.
A phylogenetic tree was constructed using the grape Prx and Prx sequences from other plants (fig. 1a). The tree is divided into two major clusters. One cluster contains type II Prxs including A, B, C, D, E, and F that show different subcellular localizations. The PrxsII E and F possess an N-terminus extension that presumably codes for a chloroplastic or mitochondrial transit peptide [3]. Nevertheless, expression activity has not been observed for PrxII A and D, indicating that the two Prxs might be pseudogenes [4, 7]. In general, PrxII B and C do not possess N-terminus extensions and are thus probably not routed to mitochondria, chloroplasts, or to vacuolar and extracellular compartments [1]. The second cluster has three main branches, including the nuclear 1-Cys Prxs, the chloroplastic Prxs Q, and the chloroplastic 2-Cys Prxs [3], which are further subdivided into A and B types. The grape Prx displays strict identity with type II Prx C sequences from other plants, such as cotton (G. hirsutum; GhPrxII C, 89%), poplar (P. trichocarpa; PtPrxII C, 89%), Citrus sP. (CiPrxII C, 88%), and castor oil (R. communis; RcPrxII C, 86%). In contrast, the grape Prx shares lower degree of identity to Prx of human, C. reinhardtii, and E. coli with 40, 51, and 30%, respectively. The absence of an N-terminal extension and of identified signals as Ser-Lys-Leu for the peroxisomes [1] suggest that the subcellular localization of the grape Prx might be the cytosol and the interrogation of the three programs, TargetP, iPSORT, and YLOC, support this proposal. The amino acid sequences comparison of PrxII C from plants reveals the presence of one Cys residue (Cys51 in the grape Prx) that is strictly conserved with the surrounding consensus sequence: P44GAFTPTCS52 (fig. 1b). Site-directed mutagenesis has shown that this residue is the catalytic one, whereas the second Cys residue (Cys76) is not essential for PrxII C activity [17]. Nevertheless, an important difference is the presence of an additional Cys in human and C. reinhardtii Prxs in positions 152 and 151, respectively, and two atypical Cys residues (51 and 100 positions) in E. coli Prx. The grape and all other plant sequences do not possess these additional Cys residues. The grape sequence isolated in this study shows only one Cys (Cys51) and it is strongly associated with the type II Prx C from plants. A predicted 3D structure was determined for VvPrxII C by applying I-TASSER simulation. VvPrxII C and all Prxs are organized in a fold similar to Trx (thioredoxin) with a central pleated β-sheet surrounded by α-helices [18]. VvPrxII C contains five α-helices and seven β-sheets with secondary structural elements: β1β2α1β3α2β4α3β5α4β6β7α5 (fig. 2a). VvPrxII C 3D structure matches almost perfectly with Prx crystal structures from human (H. sapiens), poplar (P. tremula), and pea (P. sativum) (fig. 2b).
The grape Prx promoter contains regulatory elements
in response to environmental signals
To obtain insight into the regulation of grape Prx gene by abiotic stresses, its promoter region was obtained from the Phytozome website and looked for relevant cis-acting elements by PlantCare software. The analysis of the promoter region of grape Prx showed that a number of potential cis-acting elements to respond to environmental signals were present (table). A heat stress responsive element (HSE) was present at position 1274. However, a functional heat shock element contains at least three basic repeats that must be arranged in alternating orientations [19]. Thus, the HSE motif in VvPrxII C promoter no appears to be functional. Four putative basic motifs (CAACTG) of MYB binding site (MBS) involved in drought inducibility were located at positions 169, 810, 925, and 1209. The MBS element is essential for water stress-induced gene expression but is not under the regulation of ABA [20]. Therefore, it seems that VvPrxII C gene may be induced by the ABA-independent pathway. An metal responsive element (MRE)-like sequence (TGCAGAC), similar to the conserved core MRE sequence involved in heavy metal-induced expression of metallothionein genes in animals [21], was located at position 233. A putative TCA-element (382) thought to be involved in SA response was also found [22]. Lastly, a W-box was located in promoter region of the grape Prx (799), indicating its possible implication in response to pathogens. Nevertheless, mutational experiments were exhibited that at least two copies of the W-box are necessary to perform the WRKY6-dependent gene activation [23]. The WRKY proteins comprise a family of plant-specific zinc-finger-type factors implicated in the regulation of genes associated with pathogen defense [23]. These putative regulatory elements suggest that the promoter region of the grape Prx may respond to a variety of environmental signals, including dehydration, heat, heavy metals, light, pathogens, wounding, and plant hormones. The sequence also contains a putative TATA box at 44 relative to the transcriptional start site and a putative CAAT box at 219. The other potential cis-acting elements to respond to environmental signals in the promoter of grape Prx are indicated in the table.
Expression analysis of VvPrxII C in different grape organs
The pattern of expression of the VvPrxII C gene was analyzed in different organs of grape by semi-quantitative RT-PCR. The expression level of VvPrxII C gene was also compared with the expression of VvAct2 gene that is used as an internal control in the PCR reaction. With the exception petiole, the grape Prx gene was expressed in all organs (fig. 3). Studying of the number of ESTs coding for VvPrxII C in the NCBI GenBank database also revealed around 113 ESTs at most grape organs, such as berry, leaf, cluster, stem, root, seed, bud, and flower, suggesting a high level of expression for this gene in grape. However, no EST coding for VvPrxII C was found in petiole and tendril organs in the NCBI EST sequence database. Based on the number of identified ESTs in different organs, VvPrxII C thus appears to be transcribed at high levels in berries (41 hits) and leaves (19 hits) tissues, and at low levels in stems (4 hits) and clusters (7 hits).
The highest levels of expression were detected in berry, leaf, and tendril organs, whereas the lowest amount of grape Prx transcripts is related to clusters, stems, and buds. Similarly, the expression profiles of four Prx genes were analyzed in Tamarix hispida roots, stems, and leaves under normal growing conditions. The results indicated that these genes were expressed in all organs but with different expression patterns than each other [10]. In poplar (P. trichocarpa), Rouhier et al. [1] reported that the type C Prx gene is efficiently transcribed and translated in plant. The northern-blot experiment revealed that transcription level of this gene was high in leaves, lower in roots, and weak in stems and the western-blot experiment confirmed the results of the northern-blot experiment.
Expression of Prx gene under abiotic stress conditions
To analyze the response of grape Prx gene to abiotic stress, the grape cuttings were treated under drought, salt, and heat stresses in three independent replicates and expression analysis was performed by semi-quantitative RT-PCR. Under the abiotic stress, the expression of grape Prx gene was clearly induced (fig. 4). In response to drought stress (fig. 4a), the transcript amounts of VvPrxII C were detected at a low level in leaves from 90 to 70% RWC, but at much higher level in plants subjected to severe deficit (60% RWC). The transcript abundances considerably increased at RWC around 55% and followed to a relatively dramatic decrease upon very severe stress conditions (50% RWC). When wilted plants (50% RWC) were rewatered for one week, plants recovered a leaf RWC close to 95%, and level of transcripts decreased to control level. The VvPrxII C was also up-regulated under salt stress (fig. 4b). The transcript levels of VvPrxII C gene remarkably increased after incubation with 150 mM NaCl and then there was a severe decreased at 300 mM NaCl. The presence of MBS in promoter region of VvPrxII C gene is demonstrated that induction of its expression under salinity or drought may transcriptionally not be controlled by ABA, and it may be induced by the ABA-independent pathway [20]. The up-regulation of Prx genes during drought and high salt stress was also observed in wheat (Triticum aestivum) [8] and T. hispida [10].
Similar to the drought and salt stresses, the grape Prx was also induced upon heat treatment (fig. 4c). The transcripts of VvPrxII C gradually increased to 48 h and then slightly decreased to 72 h of heat treatment. It has been indicated that hydrogen peroxide is rapidly accumulated upon high temperature, among other stresses, and may act as an important signaling molecule [24]. The up-regulation of several enzymes involved in redox homeostasis in response to oxidative stress generated by heat, including SOD (superoxide dismutase), CAT (catalase), DHAR (dehydroascorbate reductase), POX (peroxidase), and GST (glutathione-S-transferase) was reported [25]. Kim et al. [12] reported that overexpression an Arabidopsis 2-Cys Prx in transgenic tall fescue (F. arundinacea) plants confers tolerance against heat stress and protects leaves from oxidative damage probably due to chaperon activity. Kim et al. [11] also showed that heterologous expression of salt-induced 2-Cys Prx from rice (Oryza sativa) increased heat stress tolerance and fermentation capacity in the transgenic yeast (S. cerevisiae) cells. Therefore, under heat stress, it seems that induction of VvPrxII C expression may be exposed by ROS.
Expression of Prx gene under oxidative stress
To test whether the steady-state transcript amounts respond to oxidative stress, various treatments were used including chemical inducers, heavy metals, and hormonal treatments to trigger production of ROS. The effectors were applied to leaf slices by incubation in effector solution after infiltration to ensure fast and homogenous application (fig. 5). VvPrxII C showed a strong increase in the transcript amount with H2O2, heavy metals, and hormonal treatments, whereas its transcript level was almost unaffected by the diamide (fig. 5). H2O2 is a compound with directly oxidizing properties, whereas diamide acts indirectly as oxidative stressor by depletion of the cellular thiol pool [7]. Reactive oxygen species and peroxides are known to be inducers which via Keap1/Nrf2 signaling pathway up-regulate the transcription of antioxidant and associated enzymes including SOD, CAT, Prx, glutathione peroxidase, thioredoxin reductase, and glutathione reductase [26]. The high up-regulation of grape Prx under H2O2 treatment thus suggests that VvPrxII C may be implicated in response to oxidative stress and be involved in antioxidant defense. Similarly, in Arabidopsis, PrxII C gene also showed a general strong increase in the transcript amount with H2O2 than other Prxs [7].
The accumulating evidence suggests that heavy metals and plant hormones including ABA and SA induce ROS production and cause oxidative stress in plants [9]. Contreras-Porcia et al. [9] have reported that the activities of antioxidant enzymes APX (ascorbate peroxidase), Prx, Trx, and GST increased in response to copper excess, indicating that the marine alga (Ulva compressa) was under oxidative stress. Studies also proposed that the expression of Prx genes increased under heavy metal treatments [10, 27]. In Arabidopsis, Cd application increased the mitochondrial PrxII F content and demonstrated a principal role for this gene in antioxidant defense and potential redox signaling in plant cells [27]. The ABA-induced expression of Prx genes was also reported in T. hispida [10]. It seems that the induction of VvPrxII C expression by heavy metals and SA was probably a direct response to them due to the presence of cis-acting elements (MRE and TCA-element) in the its promoter region no by ROS. In contrast, the activation of VvPrxII C by ABA as well as the absence of ABA-responsive element (ABRE) in the VvPrxII C promoter are likely in favor of an activation of expression mediated by ROS.
Antioxidants effects on Prx gene expression
Ascorbate is the most abundant, powerful and water soluble antioxidant acts to prevent or minimize the damage caused by ROS in plants. It occurs in all plant tissues, usually being highest in mature leaves with fully developed chloroplast and highest chlorophyll. Proline is also a potent antioxidant, a potential inhibitor of programmed cell death, and a ROS scavenger [28]. To test antioxidants effects on VvPrxII C gene expression, leaf slices were suspended in media supplemented with ascorbate or proline. VvPrxII C was largely affected by antioxidants and its transcript amounts increased upon external application of antioxidants (fig. 6). In Arabidopsis, after addition of ascorbate, the transcript levels of chloroplastic Prx genes declined, whereas PrxII C gene showed high level of mRNAs [9]. A growing body of evidence displays that abiotic stress conditions significantly increased the ascorbate and proline contents in plants [28]. It strongly proposes that abiotic stress causes increased generation of ROS and induced the effect of an oxidative stress condition. Ascorbate and proline are considered as ROS scavengers because of their ability to donate electrons in a number of enzymatic and non-enzymatic reactions. Ascorbate is directly involved in the Apx-mediated detoxification of H2O2 and indirectly of lipid peroxides [29]. Proline has been also proposed to act as an osmoprotectant, a protein stabilizer, a metal chelator, an inhibitor of lipid peroxidation, and OH and 1O2 scavenger [30]. Furthermore, Prx proteins are known as alternative enzymes in H2O2 and lipid peroxide reduction. These data indicate that antioxidants can regulate expression of Prx genes and may communicate with each other in the cell redox regulation. However, further studies are needed to understand the potential role of antioxidants in regulation of Prx genes expression.
In conclusion, Prx gene, designated VvPrxII C, was isolated from grape berry organs of Iranian cultivar, called Askari. The constructed phylogenetic tree revealed that the grape Prx belongs to type II Prx C and contains a high degree of identity with type II Prx C form other plants. A modeling analysis indicated that VvPrxII C shares a common structure with Trxs in which the central β-sheets surrounded by flanking α-helices. The grape Prx gene was also found to be highly induced under abiotic stress conditions, oxidative stress, and antioxidants application. Collectively, the specific expression in almost all tissues, responses to environmental stress conditions, and specially antioxidants-induced regulation of expression suggest that VvPrxII C could play a critical role in the cell redox regulation.
We thank M. Sc. A. Alborzian (Imam Khomeini International University, Qazvin, Iran) for technical assistance.
This work was supported by research grant from the Iranian Imam Khomeini International University.
The potential cis-acting regulatory elements identified in the promoter region of VvPrxII C gene using PlantCare software
Fig. 1. Phylogenetic tree and multiple alignment of VvPrxII C and Prxs from different sources.
a – phylogenetic tree of VvPrxII C and Prxs from other plants using MEGA4.1 Beta 2 software. The phylogenetic tree is divided into two major clusters, including type II Prx and 2Cys Prx with 1Cys Prx and Prx Q; b – multiple sequence alignment of VvPrxII C and Prx sequences from different organisms. The protein sequence deduced from the VvPrxII C gene was aligned with its homologs and human Prx V, C. reinhardtii Prx, and Prx protein from E. coli using ClustalW2. The typical Cys residues in Prx sequences and atypical Cys residues in Prxs of human, Chlamydomonas, and E. coli are shown by black triangles and squares, respectively. The consensus sequence of PGAFTPTCS is also indicated in black box. Accession numbers are given in materials and methods.
Fig. 2. Three-dimensional models and conserved residue prediction for VvPrxII C.
a – cartoon display of the three-dimensional structure of VvPrxII C. The α-helices, β-sheets, and coiled coil regions are marked as α, β, and cc, respectively. The cysteine, initiation methionine, and end isoleucine residues are indicated as C51, M1, and I162 spheres, respectively. The consensus sequence of P44GAFTPT50 is also showed by sticks; b – superimposition of 3D model of VvPrxII C (1) and the top three templates of: human Prx V protein (2; PDB ID code 3mngA), poplar PrxII C (3; PDB ID code 1tp9A), and PsPrxII F from pea (4; PDB ID code 2pwjA) using 3d-SS (3 Dimensional Structural Superposition) service.
Fig. 3. Expression of the VvPrxII C gene in different grape organs by semi-quantitative RT-PCR.
RT-PCR analysis was performed using gene specific (VvPrxII C) and reference gene (VvAct2) primers in berry, leaf, cluster, petiole, stem, tendril, bud, root, and seed organs. One representative gel is shown from three independent replicates. Relative band intensities were normalized to the VvAct2 band intensity (100%). Each histogram represents the mean ± SD obtained from three independent RT-PCR reactions.
Fig. 4. Expression analysis of the VvPrxII C gene in youngest fully expanded leaves in response to drought, salinity, and heat by semi-quantitative RT-PCR.
a – expression analysis of VvPrxII C gene upon water deficit and after rewatering. Progressive water deficit was applied by withholding watering for 10–12 days and leaf RWC was determined ranged from 90 to 50%; b – expression analysis of VvPrxII C gene under salt stress. For salt stress, different concentrations of NaCl were used, including 50, 100, 150, 200, 250, and 300 mM; c – expression analysis of VvPrxII C gene upon high temperature and after recovery. The heat stress was applied by exposing the cuttings to a temperature of 40°C for 6, 12, 18, 24, 48, and 72 h in a chamber growth. The grape cuttings were treated under heat stress in three independent replicates and the semi-quantitative RT-PCR was performed using gene specific (VvPrxII C) and reference gene (VvAct2) primers in leaf tissue. One representative gel is shown from three independent replicates. Relative band intensities were normalized to the VvAct2 band intensity (100%). Each histogram represents the mean ± SD obtained from three independent RT-PCR reactions.
Fig. 5. Effect of oxidative stressors on the expression of VvPrxII C gene in grape leaf slices.
Leaf slices were incubated in the presence of H2O2 (10 mM), diamide (1 mM), CuSO4 (100 µM), CoCl2 (100 µM), CdCl2 (100 µM), AlCl3 (100 µM), ABA (100 µM), and SA (100 µM) as mediators of oxidative stress for 4 h before total RNA extraction. The experiments were carried out in three independent replicates and one representative gel is shown from three independent replicates. Relative band intensities were normalized to the VvAct2 band intensity (100%). Each histogram represents the mean ± SD obtained from three independent RT-PCR reactions.
Fig. 6. Effect of exogenous application of antioxidants on VvPrxII C gene expression of grape leaf slices.
Leaf slices were incubated in the presence of 50 mM ascorbate and proline for 2, 4, 6, and 8 h before total RNA extraction. The experiments were carried out in three independent replicates and one representative gel is shown from three independent replicates. Relative band intensities were normalized to the VvAct2 band intensity (100%). Each histogram represents the mean ± SD obtained from three independent RT-PCR reactions.


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Orlando's NBA franchise since 1989, the Magic's mission is to be world champions on and off the court, delivering legendary moments every step of the way. On the court, Orlando has won five division championships (1995, 1996, 2008, 2009, 2010), had seven 50-plus win seasons, and won the Eastern Conference title in 1995 and 2009.
Off the court, on an annual basis, the Orlando Magic gives more than $2 million to the local community by way of sponsorships of events, donated tickets, autographed merchandise, scholarships and grants. Orlando Magic community relations programs impact an estimated 75,000 kids each year, while a Magic staff-wide initiative provides more than 6,000 volunteer hours annually.
In addition, over the last 22 years more than $17 million has been distributed to local non-profit community organizations via the Orlando Magic Youth Fund (OMYF-MFF), a McCormick Foundation Fund since 1994, which serves at-risk youth.
Ticket highlights for the 2012-13 season in the Amway Center include: 2,500 seats priced $20 or less, 8,000 seats priced $40 or less and 10,000 seats priced $50 or under.
Mailing Address:
Orlando Magic
8701 Maitland Summit Boulevard
Orlando, FL 32810
Telephone Number: (407) 916-2400
Email: firstname.lastname@example.com
RDV Sportsplex
The RDV Sportsplex, a joint venture between RDV Sports, Inc. and Florida Hospital, is a trend-setting destination for sports and wellness. It is a multi-faceted, public/private facility unmatched in the world. With activities and services available to both club members and non-club members, the facility includes an athletic club, tennis center, ice rinks, cafe and concessions, pro shops, salon & spa, Kids' Stuff member daycare facility, and a medical complex. Situated on 21 acres in Orange County, Florida, the 365,000-square-foot RDV Sportsplex opened in February 1998.
RDV Sportsplex provides comprehensive learning and training for all ages and skill levels. The goal is to serve all levels of performance and ability from novice to the professional athlete - all with access to the same skilled staff. RDV Sportsplex's commitment extends beyond physical fitness to health and wellness. Reinforcing the athletic club are medical facilities that include pediatrics, corporate and international health, orthopaedics, and a dentist.
Providing total care for families and individuals is the most important service.
The DeVos Family
The DeVos Family
The DeVos family purchased the Orlando Magic in September of 1991. At that time, family head Rich DeVos stated that the DeVos family is simply the team's current caretaker, making it clear that the real owners of the NBA franchise would continue to be the Central Florida community and the loyal Magic fans. Since purchasing the team, the franchise has won five division championships (Atlantic Division: 1994-95, 1995-96; Southeast Division: 2007-08, 2008-09, 2009-10), two Eastern Conference titles (1994-95, 2008-09) and had seven 50-win seasons, including a franchise-record 60 victories in 1995-96. The Magic have advanced to the playoffs 13 times and finished with a .500 record or better in 15 of the last 19 seasons.
The acquisition and operation of the Magic is a family affair involving Rich, his wife, Helen, and their four children and spouses: Dick and Betsy DeVos, Dan and Pamella DeVos, Cheri DeVos Vander Weide, and Doug and Maria DeVos.
Although the senior DeVoses have an estab- lished residence in southern Florida, the family was introduced to the Central Florida community during Orlando's drive to bring major league baseball to "The City Beautiful." An ensuing relationship was formed between the DeVos family and the city of Orlando.
The baseball venture left them with a height- ened interest in professional sports ownership. The 85-year-old co-founder of Amway – today a global leader in direct selling – envisioned incorporating the philosophies that made him a successful business- man with that of a solid, young NBA franchise. He understands the impact professional sports can have on the upcoming generation, and he is committed to ensuring the team provides a positive, encouraging example.
The DeVos family played a key role in helping to secure the new state-of-the-art Amway Center in Orlando. The Amway Center is part of a public community venues program that also includes a new Performing Arts Center and a renovation of the Citrus Bowl Stadium. The new Amway Center opened in October 2010. In addition, the Magic also contributed $12.5 million toward construction of five community recreation centers throughout Orange County, Florida, as part of its commitment to build the new Amway Center.
From 1995-2001, RDV Sports owned and oper- ated the Orlando Solar Bears of the International Hockey League. During their six seasons of existence, the Solar Bears captured one division title (Central Division, 1995-96), three Eastern Conference crowns (1996, 1999, 2001) and the IHL's Turner Cup championship in 2000-01. Orlando compiled an all-time regular season record of 286-162-44 (.626), the best in the league over that time frame.
From 1998-2002, the Orlando Miracle of the WNBA was part of the RDV Sports portfolio. The Miracle played in their inaugural season during the summer of 1999 and made the playoffs in 2000.
RDV Sports, named for Rich DeVos and the general partner of the Magic, has been and continues to be an active partner in the Central Florida community. In February of 1998, RDV Sports, in partnership with Florida Hospital, opened the $50 million, 365,000-square-foot RDV Sportsplex. The RDV Sportsplex is a state-of-the-art health and wellness facility, which includes a world-class health club, medical offices, restaurant, retail shops, and front office facilities for the Magic.
The DeVos family remains involved in numerous charities and community-enhancement endeavors, both in the Central Florida area and their hometown region of Grand Rapids, Mich. Pamella DeVos serves as Chairman of the Orlando Magic Youth Fund, a fund of the McCormick Foundation. The Orlando Magic Youth Fund has positively impacted more than one million children. Through private do- nations, fund-raising events and contributions, more than $16 million has been raised and distributed to non-profit organizations that support the OMYF mission statement.

subset: commoncrawl


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Rodney King won this round against cops unanimously.
The 44-year old whose beating at the hands of white LAPD officers who were later acquitted sparked deadly race riots in Los Angeles nearly two decades ago was the unanimous winner in a celebrity boxing match outside of Philadelphia.
"This is a new chapter in my life," King said after the fight, according to a statement. "I am sober, fit and now a winner."
King took on ex-cop Simon "The Renegade" Aouad, 31, at the Philadelphia Airport Ramada Inn in three rounds and drew a crowd of about 500 fans, according to the New York Post.
The 6-foot, 215-pound tattooed Aouad, who worked as an officer outside of Philly, came to the fight with a 36-0 record and 34 knock outs. The 6-foot-4, 250-pound King trained for the fight by sparring, hitting bags and jumping rope, according to the Post.
"I know some people will see the irony here. ... But I would have fought anyone who was worthy," King said before the fight. "I take this very seriously. Never felt better, physically and in the mind."
The three-round match was sponsored by Celebrity Boxing.


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Man Sentenced For Abducting Montana Girl
29 year- old Edgard Enrique Le from Escondido, California plead guilty last February to a charge of transportation with intent to engage in criminal sexual activity. He was sentenced yesterday to 10 years in prison and 5 years of supervised release.
Le drove to Kalispell to pick up a 12 year old girl he met online and take the girl back to California to get married. They were spotted by police as they traveled through Las Vegas and were arrested at the California-Nevada border.


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The center will offer emerging Chinese software companies the ability to tap into a virtual computing environment to support their development activities. It will be established through an agreement signed today between IBM and Wuxi Tai Lake Industry Investment and Development Company Limited.
IBM will work with Wuxi Tai Lake Industry Investment and Development Company Limited; the Wuxi municipal government; and its business partners to build the China Cloud Computing Center, which will be a shared facility providing each software company in the park with its own virtualized computing resource. For example, a company will be able to use the allocated resource for designing, developing and testing its software products. Such virtual environments can replace the traditional data center model, in which each company owns and manages its own hardware and software.
Companies in the park will be able to access these common services provided by the center at any time -- just as they use utilities and other shared services. The technologies being offered to the community include IBM Rational software development tools, WebSphere Application Server software and DB2 database software running on IBM System x, System p and BladeCenter servers. IBM Tivoli systems management software will manage the cloud computing environment.
"Being one of the model zones to offer outsourcing services in China, the city of Wuxi is committed to providing companies in our park with the environment and tools that are needed for rapid growth," said Mr. Zhu Weiping, Party Secretary of Bin Hu District, Wuxi Municipal Government. "We are proud to be the first in the world to offer the power of cloud computing to companies in Wuxi Tai Hu New Town Science and Education Industrial Park."
Wuxi is classified as an investment zone by the Chinese government and has been named a "National Model City of Science and Technology Advancement" for five consecutive years.
"Cloud computing is helping to foster the growth of new software companies in China," said Steve Mills, Senior Vice President and Group Executive, IBM Software Group. "Like many new software companies seeking growth opportunities both locally and abroad, these Chinese software companies will rely on technical infrastructures built on open standards and delivered as a service. This open approach to computing will help them deliver innovation and pursue global market opportunities."
The Wuxi Tai Hu New Town Science and Education Industrial Park will be home to dozens of new software companies and represents a multimillion dollar investment. iSoftStone, one of the new software companies in the park, plans to develop account settlement software for the financial services industry.
"The China Cloud Computing Center represents a milestone in service-oriented computing," said T. W. Liu, the chairman and CEO of iSoftStone. "It will allow companies in the Wuxi Software Park to leapfrog to the newest computing models and will provide an efficient IT platform for software development."
Cloud computing is an approach to shared information technology (IT) infrastructure in which large pools of systems are linked together to provide IT services. Cloud computing allows corporate data centers to operate more like the Internet by enabling computing resources to be accessed and shared as virtual resources in a secure and scalable manner. The center will be built using IBM's "Blue Cloud" technologies, a series of cloud computing offerings based on open standards and open source software which link together computers to deliver Web 2.0 capabilities such as mashups, open collaboration, social networking and mobile commerce.
IBM has established a significant research and development presence in China. There are currently more than 3,200 IBM engineers and scientists employed by IBM labs in Beijing and Shanghai. Engineers from the High Performance on Demand Solutions (HiPODS) team in IBM China will work with their colleagues in the IBM Silicon Valley Laboratory to establish the center.
For more information about IBM's High Performance On Demand Solutions Lab, please visit http://www-128.ibm.com/developerworks/websphere/zones/hipods/
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Chemistry, computer science, electrical engineering, materials and mathematical sciences, physics and services science
Information Management, Lotus, Tivoli, Rational, WebSphere, Open standards, open source


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McCutchen stays hot, Pirates top Cubs 4-0
PITTSBURGH (AP) — Pittsburgh Pirates manager Clint Hurdle has coined a phrase for where star center fielder Andrew McCutchen is at the moment.
"(He's) McHot," Hurdle said.
For once, it might be an understatement.
The reigning National League MVP had two doubles and two RBIs to lift the Pirates to a 4-0 victory over the Chicago Cubs on Thursday night. McCutchen is batting .428 (18 of 42) this month, with 15 of his hits going for extra bases.
"I'm seeing the ball and I get a pitch to hit and I hit it and it's finding the gaps and that's basically what's happening right now for me," said McCutchen, a career .324 hitter in June. "Things are just working out right now and I want to continue to stay where I'm at."
Russell Martin also doubled twice for the Pirates and rookie Gregory Polanco got a hit in the third straight game since his call-up. Edinson Volquez (4-5) pitched seven effective innings to win for the third time in his last five starts.
Volquez struck out five and walked two while outpitching Chicago ace Jeff Samardzija (2-6). It helped the Cubs could do little when given the chance. Chicago went 0 for 8 with runners in scoring position.
"He was commanding the zone like he does when he is in a good place and we were on the other side of it," Cubs manager Rick Renteria said of Volquez.
McCutchen broke it open with a two-run double in the fifth. The Pirates had seven doubles in all, their highest total since a win over Houston in July 2010.
"The next team that comes in here is probably better off pitching around him and letting him go to first base," Samardzija said.
Luis Valbuena had two doubles for the Cubs, who went 0 for 8 with runners in scoring position to fall for the fourth time in five games. The Cubs have scored two runs or fewer in nine of Samardzija's 14 starts.
Chicago's troubles weren't just on offense on a night the worst road team in baseball fell to 0-9-3 in series played away from Wrigley Field this year. Samardzija struggled against a team that seems to be rounding into form behind its star center fielder.
McCutchen extended his hitting streak to eight straight by doubling with one out in the fourth before coming around to score when Martin smacked a ball off the wall in right field. Ike Davis scored two batters later on a sacrifice fly by Jordy Mercer to give the Pirates a 2-0 lead.
Pittsburgh made it 4-0 in the fifth when Polanco — batting leadoff for the first time — and Starling Marte hit consecutive singles followed by drive by McCutchen to the notch in left-center. McCutchen slid into the third on the play before making a rare mental miscue when he stepped off the base, allowing him to get tagged out in the process.
"I guess that's why they say stay humble in this game, because crazy stuff like that will happen," McCutchen said. "Popping up, getting all excited and next thing you know I'm floating all over the bag and his glove is right there so that's why you stay humble."
It hardly mattered. The Cubs were too busy wasting chances to get to Volquez, who has suddenly emerged as a constant on Pittsburgh's injury muddled pitching staff. After a bumpy patch in early May, Volquez is 3-1 with a 2.48 ERA in his last five starts.
NOTES: The Pirates called up INF Michael Martinez from Triple-A Indianapolis before the game and optioned RHP Casey Sandler back to Indianapolis. ... Chicago CF Emilio Bonifacio left in the first inning with a rib injury and did not return. ... Chicago signed free agent C Yorvit Torrealba to a minor league deal Thursday. Torrealba, who played for Colorado last season, will report to Chicago's training complex in Mesa, Arizona. ... The Pirates begin a three-game set in Miami on Friday night against the Marlins. Jeff Locke (0-1) starts for Pittsburgh against Nathan Eovaldi (4-2, 3.27). ... The Cubs continue a 10-game road trip on Friday in Philadelphia. Jake Arrieta (1-1, 2.50) starts for the Cubs against Roberto Hernandez (2-4, 4.35).
Follow Will Graves at www.twitter.com/WillGravesAP


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|A cloth pack left by the villagers at Bey
We were already aware from the fact that the village called Bey in north Sikkim has been abandoned by the villagers and they were now living at a Relief Camp at Char Mile. Even though, we decided to go there to see the enormity of the huge shake which has thrilled the entire state of Sikkim. In the evening of 7th October, a native of Lingzya had come to our camp at the Panchayat Bhavan of the village. He told us about the death of a 16 years old girl at Bey due to the landslide after the earthquake. She was Lakit Lepcha, a student of Class VII of Lingzya Jr. High School. She was a smiling and a beautiful child and was staying at Lingzya with her friends. On 18th September which will be remembered as a "Black Day" by every Sikkimese in the coming days, she asked her friends to visit her house at Bey to have Momo (a traditional Tibetan dish mostly popular in the entire Himalayan region). Her friends refused to go with her and they stay back at Lingzya. After their refusal she took a small boy from Lingzya to her house at Bey. Her brother, a saw puller (I have forgotten his Name) who was working at lower Dzongu, had also come to his house. When the earthquake occurred, the brother-sister duo was preparing Momo for the evening. After a few seconds of the earthquake the mountain above their village fall down with a huge spark (possibly occurred due to rubbing of the rocks) and swept the house of Lakit leading her to bury inside the debris of her house. Her smiles, her happiness, her containment and cheerfulness were also buried along with her in the wreckage of her house. The small boy, who accompanied Lakit on her way to Bey, fortunately was at another house where a Puja was going on. The second house remains untouched and many other villagers of Bey survived.
|Relief Team of Namchi Govt. College on the way to Bey
After hearing this account about Lakit, I decided to meet the surviving members of her family. They had come to Bey to lift Dhaja in the name of those who lost their lives in the catastrophe of September 18th. Her surviving family members (I did not have the courage to ask their relation with Lakit) cried in front of us while telling about her. The family has lost almost all of its members. Five people including Lakit lost their lives at Bey. Three bodies had been recovered, but they could not find the bodies of another two people.
After hearing about the untimed and a most unfortunate demise of Lakit, every person in our team got tearful. The account about the smiling daughter of Bey is not an overstatement as we have been hearing; it is undeniably a heartbreaking fact of the September 18th. I wanted to acquire more information about her from Lingzya. After our arrival from Bey, I met a student of Lingzya Jr. High School and asked her about Lakit. She told me that Lakit was her senior and was a pleasant and a kindly girl. On 8th of October, we left Lingzya and proceeded towards Singtam for Namchi. Everybody in our team was silently praying for the soul of the Smiling Daughter of Bey to rest in peace.


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Maine lawmaker seeks to make pot more accessible to patients
Rep. Mark Dion proposing new bill
A bill, proposed by Rep. Mark Dion, D-Portland, would allow any physician to write a prescription for the plant and eliminate the lists of ailments and diseases for which medical marijuana is prescribed.
Dion announced the bill and its details at a news conference Monday afternoon.
"The principal is simple. I believe in patient-centered care. Behind that is the idea that the best people positioned are in fact the patient and his or her doctor," said Dion.
Veteran Ryan Begin has had more than 50 surgeries after his arm was serverely injured by a bomb during a tour in Iraq. He also suffers from post-traumatic stress disorder.
Because doctors in Maine currently can't prescribe medical marijuana for mental illness, Begin said the only reason he is prescribed medical marijuana is because of his arm.
"I have to be thankful for my arm getting blown off cause had my arm not got wounded, I would not have qualified for medical marijuana. My PTSD would still be uncontrolled," said Begin.
Gordon Smith, executive vice president for Maine Medical Association, said the association has opposed another similar bill in a previous session and expects to oppose Dion's bill in January.
He said medical marijuana is unlike any other medication and still considered a Schedule I substance under federal law.
Smith said schedule I substances have a high potential for abuse, have no currently accepted medical use in treatment in the United States, and have a lack of accepted safety for use under medical supervision.
"I think this sets a dangerous precedent somewhere down the road that a doctor could prescribe medical marijuana for anything from anxiety to depression," Smith said.
Begin's doctor said medical marijuana can help with conditions like anxiety and depression.
"I know if cannabis is able to replace such a wide variety of medications, including those in the mental health realm, there must be some mental health benefits for these patients," said Dr. Dustin Sulak, DO.


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|Experts in car diagnostics since 2001
Direct-fit & Universal Oxygen Sensor Application List
Also known as O2 sensors, HEGO sensors, Lambda sensors
Please select your car specification from the list below, or return to the model list, or return to the Oxygen Sensor category page.
PLEASE NOTE: All sensors listed on these page are front/pre-cat/upstream sensors unless they have "(Post Catalytic Convertor)" in their description. ( Unsure what front/pre-cat/upstream means? Click here for information )
If your model is not listed here, please complete our online form for more information. We can normally get hold of most sensors within a few days.
Pre-cat / Post-cat? What does that mean?All petrol cars sold within the EU from 2001 onwards (and some before then) will have 2 or more oxygen sensors:
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"The Impossible" is based on the true story of one family's experience during the Indian Ocean tsunami of 2004.
It was one of the deadliest natural disasters in recorded history. Over 230,000 people lost their lives.
This film will place you squarely in the center of that event.
"The Impossible" briefly but successfully establishes the loving, happy family on vacation by showing a bit of their flight in to Thailand and their arrival at the luxurious ocean-side resort they're staying at. A husband (Ewan McGregor), wife (Naomi Watts) and three boys (the eldest played by Tom Holland) are on a vacation for Christmas. They spend a joyful Christmas Day opening presents and playing, and that night they enjoy a beautiful beachside celebration, releasing candle-kites into the night sky.
Their idyllic peace is not long-lived, however. The very next day, while the family is poolside, the tsunami strikes.
The actual tsunami scene is as terrifying and realistic a disaster re-creation as you could imagine. The family has a few seconds to see the massive wall of water coming at them before it smashes through everything in sight and washes some of them miles inland. As the wave hits, the family is separated and the story focuses in on the eldest son and the mother, who are swept along not far apart from each other. Aside from the very real danger of drowning, or being washed out to sea, the two risk being slammed into objects or protrusions and being fatally gouged or knocked unconscious. A second wave tosses them about. During it all, they frantically try to stay together, each of them giving up the possibility of safety alone at some point, in order to pursue the other, still in danger.
Back at the resort, unbeknownst to them, the husband and other two boys have also survived. Once the immediate danger has passed, the story shifts back to them, and to his efforts to find his wife and eldest son. He's left with nothing, and has no idea where they might be, but he doggedly refuses to give up hope.
Aside from the personal struggle of this individual family, this movie imparts a feeling of the scope of the tragedy. Obviously, the real event was of such a massive scale that no film could adequately impart it, but "The Impossible" makes a valiant effort. Wreckage and desolation stretch as far as the eye can see. The hospitals are teeming with people and stretched beyond their resources. Every time one of the family members mentions they're searching for a loved one, multiple people plead with them to be on the look-out for those that they've lost as well. Humanity is reduced to the basics. Life becomes about simply surviving and finding the people you love.
"The Impossible" is one of the most emotionally draining films that I can recall. Most "tear-jerkers" (not to call "The Impossible" a tear-jerker, mind you) will set you up for one or two big moments of sorrow and then try to hit you with the big cry. "The Impossible" exists in a continuous zone of shock and fear, with sudden bursts of sorrow and equally sudden bursts of great, joyous elation and/or relief. You'll feel the family's pain as they get bettered by the event, and then the fear that they experience afterwards not knowing if everyone survived (or will still survive). When you're not experiencing their plight, the film is showing the scope of the event, and you're witnessing the plight of others. There are easily 30-40 moments here that could justifiably cause people to weep, and the audience I watched the film with was indeed sniffling throughout.
Watts and McGregor both were astonishing. McGregor's character is doing his damndest to be resolute and courageous, but he's faced with insurmountable odds and extremely difficult decisions. It's too much for him at points, and he occasionally loses it. Watts takes the worst of the damage from the wave, and while she starts out a protective, driven mother… her strength fades along the course of the movie and her battle for life continues once she survives the water. Her performance is utterly genuine, you can feel her fear and love for her child, and then feel for her as her strength slowly slips away. Together, the two of them perfectly communicate the emotional experience that one might undergo in such a traumatic event.
"The Impossible" is one of the most powerful, moving, intense films of the year. It wowed me with its horrifying special effects, stunned me with its scope, and had me on the verge of tears from beginning to end. I highly recommend that you see it in theatres to get the maximum impact, and I'd also recommend seeing it with family. You will undeniably feel lucky to have them safe and sound with you after viewing this incredible film.


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<bt>When Kate Ziegler's parents first put her into a swimming pool, there was no way they could have known what they were getting themselves into.
At 17, Ziegler has become one of the top swimmers in the world, recently winning first place in the 800-meter and 1500-meter races at the World Swimming Championships in Montreal.
"I just got back from Montreal, so I'm taking a little break," Ziegler said from her home in Great Falls. "I get about one break a year so I'm taking it easy right now."
She began swimming in a summer league at age 7, and it became a year-round hobby three years later. "When I was little, I just loved being in the water. My parents would take me to the pool … my mom says I used to do laps in the baby pool," she said with a laugh.
After joining the FISH swim team in McLean, she was placed in their top group after about six months and began swimming competitively.
"In the past two years, I've been to Montreal, Australia, New York and California," she said, in addition to some regional competitions. She narrowly missed out on being a member of the 2004 Olympic swim team, something she hopes to correct in time for the 2008 trials.
"Going to the Olympics is definitely a goal of mine," she said. "It's my three-year goal, but you never know what's going to happen. I love being in the water. My best friends are all on my team."
It is a goal and a dream shared by her parents, Don and Cathy Ziegler.
"I lasted one day on my high school swimming team myself," Cathy Ziegler said. "I can't count how many meets I've been to now, between her meets at school and summer meets and competitions."
She remembers when Kate started taking swimming lessons and the realization that she would have to get up early during the summer to drive her to the pool. "As much as I hated getting up early, I realized that there was some value teaching her structure in the summer," she said.
From there, they encouraged her to try out for the FISH team. "She was reluctant at first because she'd never done it before, but after she met the coach she agreed," Ziegler said.
Countless laps, practices and meets later, the Zieglers have become friends with many of the other swim parents they see regularly.
"The meets are long and they get a little boring, but I enjoy sitting in the stands and talking with the other parents," she said. "We all sit and do our needlework together. I enjoy watching her do well."
"Kate is the athlete of the family," said her father, Don Ziegler. "Parents want their kids to try things and have a broad experience in life. This is just one aspect of that for her and it's a fabulous experience."
Even though they have watched her swim and win many times before, he said, his daughter can still surprise him from time to time.
"I'm amazed by how professional she is when she's being interviewed," he said. "I thought that when they raised the American flag and played the national anthem at Worlds she'd start crying but she was very mature. I was getting choked up when they raised the flag. It's one thing to represent your school or yourself at meets, but it's an entirely different thing to represent your country."
AS SHE PREPARES to graduate from Bishop O'Connell High School next June, Don Ziegler hopes she continues to live a good life.
"I told her she might be a good swimmer for 10 years but I hope she'll continue to be a good person for 70 years. That's more important," he said. "The life lessons she can learn from this are numerous. I hope she can continue to be happy in swimming or whatever else she chooses to do. She sacrifices a lot to do this."
"Kate's been given an incredible gift," Cathy Ziegler said. "You can look at swimming and ask what value is it to the world and you'd be right, but when you have a talent like this, it gives you the ability to reach people," she said, adding, "I feel God has given her this talent for a reason, and I hope we'll be sensitive to that purpose."
Ray Benicki has been Kate's coach for the past four years and has been able to accompany her to many of her competitions.
"When she first came to the team, she didn't have a strong background in being a fast swimmer, so it took her a while to move into the top team," he said.
She was a fast swimmer, but he wanted to see if she could make it as a distance swimmer. "At that age, she was too young to specialize in the 50-meter freestyle race, but she's been able to take that speed and spread it out over 800 and 1500 meters, which is the exact opposite of what most distance swimmers do," he said, adding that most swimmers lose some of their speed over long distances and would prefer to stick with shorter races. "It's a lot of extra laps, a lot of longer swims with shorter rests and endurance training."
There have been some complications along the way, he said, including realizing she had asthma two summers ago.
"She got into the Olympic trials after a good meet at George Mason University, and the next week she had difficulty breathing after a meet," he said. He talked with some other coaches and encouraged her to go to a specialist who was able to give her an inhaler specifically for athletes. "Being a swimmer, her lung capacity is probably a littler larger that most people. It has to be adjusted from time to time, but I can tell when it needs to be tweaked, usually because of the change in seasons," Benicki said.
THE TIME COMMITMENT required to be a competitive swimmer creates a family-like bond among teammates, he said, all of whom wish Kate well when she competes and console her if a meet does not go well.
"The more mature swimmers look at what she's doing and think it'll work for them too. We all support each other, but obviously we know there can only be one world champion. It might be different if she didn't earn the respect of her teammates, but she's so sweet and nice everyone wishes her the best," Benicki said.
Although the Olympic trials are still a few years off, they discuss the trials often, he said.
"She dropped 18 seconds from January to July before the trails last year. A lot of people at the Olympic trials said that if she just had another year she'd be on the team," he said. "She's got three years to go. A lot of experts at USA Swimming said that if she was able to gain one inch per stroke, she'd have the world record. You can't rush things," he said.
Kate and her coach spend a lot of time together, training and practicing. "I have eight practices each week in the winter, two morning swims, four afternoon swims and then one on Saturday and Sunday. In the spring we add a morning practice," she said. "My major goal is to keep improving. I know if I keep going it'll get me to where I want to be."
Now that she has a little time off before starting her final year at Bishop O'Connoll, acting as a captain for her school swim team and deciding which college she will attend, she and her coach took the opportunity for a quick reward: This past weekend, they flew to Florida to swim with dolphins, something she has always wanted to do.


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You will need this permit if you wish to use your vehicle for commercial purposes within the council area - for example, delivery drivers, couriers. This permit will enable you to stand in regulated traffic areas while conducting business.
A licence defines the need to obtain recognition / certification and registration to undertake a certain business activity.
Douglas Shire Council - Local Law 5 (Parking) 2011 - PDF
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Douglas Shire Council - Subordinate Local Law 5 (Parking) 2020 - PDF
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Contact council for more information on the current application process and relevant form.
Douglas Shire Council
Contact Email, Phone and Address Details for this service in simple two column table format, header then data.
Douglas Shire Council


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Global Information Assurance Certification
GIAC Certified Incident Handler
Oral or Written Exam Required? Yes Renew through Re-Examination? YesDoes applicant have choice of at least two options from above for renewal (CEU, CPD, or exam)? No This certification is In Demand.Read more about In Demand.
This certification is related to military occupational specialties. Read more about certifications related to military occupations. This certification is accredited by ANSI. Read more about ANSI.


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Before I became a mother my life was impossibly crazy - working, going to school full-time (for 2 degrees at once), interning, writing books, maintaining two blogs and trying to preserve my sanity with zero time left for myself at the end of the day. Cooking has always been a way for me to release stress at the end of a busy day so making quick and easy meals became my obsession - and helped me create 3 phenomenal cookbooks , if I do say so myself J. After I had my daughter all the things that made my life crazy were still in place but now there is a tiny person who my whole world revolves around. When she started eating solids it was an easy transition, she just ate whatever I ate – never any pureed, smashed or mushy foods just full, bold flavors and lots of variety. In the last 3 months she went from dabbling in food to eating portions that rival my own and suddenly I found myself stressed out about dinner time. When it was just me I could grab some Taco Bell on the way home and call it dinner, or graze on snacks all night long while finishing up homework or writing a new chapter. But now, I have to have a wholesome, nutritious, home cooked meal on the table every night and the pressure of the "have to" can make life crazy sometimes.
Thankfully, I'm starting to find a balance. Cooking double portions of everything and freezing half for the times when I just have no time to cook has been a life saver. My other secret to stress free cooking is a good store-bought simmer sauce. I have a growing list of favorites which I will share over the coming year – all low sodium, no crazy scientific ingredients and full of flavor. With a good simmer sauce you simply throw whatever protein and/or vegetables you have hanging out in your freezer or fridge in a pan, sauté them with a little oil, add the sauce and voila! You have a home cooked meal.
Last night's simmer sauce of choice was Seeds of Change Organic Madras Simmer Sauce. It was surprisingly difficult to find a vegan Indian simmer sauce. Many of them have some form of dairy in them but this one is a pure simple sauce. Just tomatoes, onions, and a laundry list of spices. And that's it, no other crazy ingredients or preservatives. The label says it is a "red hot curry" so I was a little worried it might be too spicy for my little one but it actually doesn't have much heat at all. So if you're looking for a spicy sauce you might want to add a pinch of red pepper flakes to it. I sautéed a bag of Garden chik'n strips, frozen broccoli and frozen peas together in a large sauce pan with canola oil, poured the sauce over everything and stirred until it was warmed through and then spooned it over a bed of brown basmati rice. Pure heaven! And my little one gobbled it up! In fact, she ate more of it than I did. You can find the Seeds of Change simmer sauce down the "ethnic" (I hate that word) or international food aisle.