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Comparative proteomic and metabolomic profiling of citrus fruit with enhancement of disease resistance by postharvest heat treatment
- Ze Yun†1,
- Huijun Gao†1,
- Ping Liu1,
- Shuzhen Liu1,
- Tao Luo1,
- Shuai Jin1,
- Qiang Xu1,
- Juan Xu1,
- Yunjiang Cheng1Email author and
- Xiuxin Deng1
© Yun et al.; licensee BioMed Central Ltd. 2013
Received: 4 September 2012
Accepted: 5 March 2013
Published: 16 March 2013
From field harvest to the consumer's table, fresh citrus fruit spends a considerable amount of time in shipment and storage. During these processes, physiological disorders and pathological diseases are the main causes of fruit loss. Heat treatment (HT) has been widely used to maintain fruit quality during postharvest storage; however, limited molecular information related to this treatment is currently available at a systemic biological level.
Mature 'Kamei' Satsuma mandarin (Citrus unshiu Marc.) fruits were selected for exploring the disease resistance mechanisms induced by HT during postharvest storage. Proteomic analyses based on two-dimensional gel electrophoresis (2-DE), and metabolomic research based on gas chromatography coupled to mass spectrometry (GC-MS), and liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS) were conducted. The results show resistance associated proteins were up-regulated in heat treated pericarp, such as beta-1, 3-glucanase, Class III chitinase, 17.7 kDa heat shock protein and low molecular weight heat-shock protein. Also, redox metabolism enzymes were down-regulated in heat treated pericarp, including isoflavone reductase, oxidoreductase and superoxide dismutase. Primary metabolic profiling revealed organic acids and amino acids were down-regulated in heat treated pericarp; but significant accumulation of metabolites, including tetradecanoic acid, oleic acid, ornithine, 2-keto-d-gluconic acid, succinic acid, turanose, sucrose, galactose, myo-inositol, glucose and fructose were detected. Noticeably, H2O2 content decreased, while, lignin content increased in heat treated pericarp compared to the control, which might increase fruit resistibility in response to external stress. Also, flavonoids, substances which are well-known to be effective in reducing external stress, were up-regulated in heat treated pericarp.
This study provides a broad picture of differential accumulation of proteins and metabolites in postharvest citrus fruit, and gives new insights into HT improved fruit disease resistance during subsequent storage of 'Kamei' Satsuma mandarin. Interpretation of the data for the proteins and metabolites revealed reactive oxygen species (ROS) and lignin play important roles in heat treatment induced fruit resistance to pathogens and physiological disorders.
Disease resistance H2O2 Heat treatment Lignin postharvest storage Metabolomics ProteomicsBackground
Fresh citrus fruits, from harvest to human consumption, require a lengthy period for shipping, storing and marketing. During that time, the main damage to fruit comes from biological diseases and physical damage. Storage pre-treatments, such as biotic or abiotic typological treatments, can be used to inhibit disease and extend the shelf-life. Among the various types of treatment available, heat treatment has previously been reported to induce disease resistance in apples, citrus, grapes, peaches, pears and mangos against a multitude of pathogens [1–3].
In the fruit industry, hot water treatment is an alternative physical treatment, which has been used in citrus fruit storage since 1922 [4]. More recently, hot air and hot water systems have been widely adopted in several countries to inhibit postharvest decay of fruits and to alleviate chilling injuries during storage. Heat treatment (HT) has the advantage of minimizing surface infection in pericarp without leaving any chemical residues [5]. HT can prevent the development of plant pathogens and disease by heat-killing disease organisms or by inhibiting pathogen mycelial growth, by removing surface insects and by structurally altering epicuticular wax [6]. HT can also enhance fruit resistance to chilling injury in cold-sensitive cultivars, and help the fruit retain quality during cold storage and help maintain shelf life [7]. Aside from controlling pathogenic organisms, HT may also reduce disease by changing the physical characteristics of the interior pericarp. For example, HT not only delayed fruit ripening and coloration, inhibited fruit softening, and slowed ethylene production [3], it also increased fruit hardness, as well as increasing the content of polyphenolics, flavonoids and dopamine [6–8]. However, less is understood about the regulatory genes which are active in the response to microbial attack, and there is no exhaustive metabolomics profile detailing the changes in heat treated pericarp.
In the past few decades, researchers have given increasing attention to the study of gene expression in heat treated fruits. Sapinitskaya et al.[9] reported HT activates stress genes and lipid modification genes in response to chilling stress in grapefruit, such as glutathione-dependent formaldehyde dehydrogenase, dehydrin, heat shock protein (HSP), ascorbate peroxidase, superoxide dismutase, fatty acid desaturase and lipid transfer protein. Some scientists discovered HT induces phenylalanine ammonia lyase and accumulates phenylpropanoid compounds, which play a vital role in heat-induced fruit resistibility [2]. Dixon et al.[10] studied the functions of phenylpropanoid compounds in plants involved in local and systemic signalling for defence-gene induction. However, little information is available related to which particular class of phenylpropanoid compounds plays a core role in defensive functions, and even less is known about the regulatory genes which orchestrate the rapid induction of phenylpropanoid defences in response to HT. Proteome analysis reveals the induction of HSPs, allergen proteins, dehydrin, and other proteins involved in the stress response. Repression of polyphenol oxidase caused by HT may constitute the molecular basis for the protection against chilling stress in peach fruit [3]. However, few proteins involved in HT were reported to have induced fruit resistance to deterioration, especially in citrus fruit.
Also, proteomics analysis and metabolic profiling approaches make possible comparative parallel analyses of global changes at the proteome and metabolome levels, facilitating an understanding of the relationships between changes in specific proteins and subsequent alterations in metabolites in response to HT. In the present study, fruits of 'Kamei' Satsuma mandarin (Citrus unshiu Marc.) received HT. The comparative proteomics analysis and metabolic profiling (the primary metabolome and the secondary metabolome) analysis were performed based on two-dimensional electrophoresis (2-DE), gas chromatography and mass spectrometry (GC-MS) and high-performance liquid chromatography/electrospray ionization-time of flight-mass spectrometry (HPLC-QTOF-MS). Comprehensive physiological and biochemical analysis were completed. Results will provide additional theoretical evidence to improvement of disease resistance in heat treated pericarp.
Satsuma mandarin is one of the main citrus crops in East Asia, including China, South Korea and Japan. However, this cultivar has poor storability mainly because it is susceptible to biotic stress and physical damage. HT improved fruit resistance to stress and damage effectively and inhibited postharvest decay of fruits. In present study, HT fruits were dipped in a 52°C warm water bath for 2 min, and control fruits were dipped in a 25°C water bath for 2 min. After treatments, fruits were stored in a ventilated warehouse (storage temperature: 12–16°C; relative humidity: 90–95%). Samples were collected at 2 h, and 1, 2, 3, 4, 6, 9, 12, 16, 20, 24, 28, 32, 38, 44, 50 and 60 days after treatments (DAT) for further analysis.
HT increased fruit weight loss slightly, but did not affect fruit quality
HT suppression of Penicilliumgermination on pericarp
Differentially accumulated proteins in HT citrus pericarp
Identities of HT induced/decreased proteins in the peels of Satsuma mandarin fruit
Glycolysis and TCA cycle: 9 | ||||||||
No. | Protein name | Protein accumulation | Source organism | GI no. | TheoMr/pI | Expt Mr/pI | PC | MS |
H1 | Enolase | | Glycine max | 42521309 | 47.97/5.31 | 50.84/5.23 | 8 | 443 |
H2 | Phosphoglycerate kinase, putative | | Arabidopsis thaliana | 15223484 | 50.02/8.27 | 41.88/4.90 | 9 | 108 |
H3 | Putative 2-oxoglutarate dehydrogenase E2 subunit | | Oryza sativa | 48716382 | 49.46/6.78 | 43.64/4.62 | 4 | 105 |
H4 | Malate dehydrogenase, cytoplasmic | | Beta vulgaris | 11133601 | 35.81/5.89 | 37.61/4.61 | 7 | 347 |
H5 | Triosephosphate isomerase, cytosolic (TIM) | | Zea mays | 136063 | 27.23/5.52 | 24.97/4.47 | 9 | 310 |
H6 | Malate dehydrogenase, mitochondrial precursor | | Citrullus lanatus | 126896 | 36.40/8.88 | 36.36/4.71 | 8 | 376 |
H7 | Pyruvate dehydrogenase E1 beta subunit isoform 2 | | Zea mays | 162458637 | 40.23/5.56 | 35.91/5.72 | 5 | 302 |
H8 | NAD-malate dehydrogenase | | Nicotiana tabacum | 5123836 | 43.67/8.03 | 34.10/5.38 | 11 | 436 |
H9 | Putative2,3-bisphosphoglycerate- independent phosphoglycerate mutase | | Arabidopsis thaliana | 15982735 | 60.67/5.27 | 61.18/5.12 | 9 | 207 |
Redox regulation: 9 | ||||||||
No. | Protein name | Protein accumulation | Source organism | GI no. | TheoMr/pI | Expt Mr/pI | PC | MS |
H10 | Isoflavone reductase related protein | | Pyrus communis | 3243234 | 33.80/6.02 | 33.12/4.53 | 5 | 92 |
H11 | Oxidoreductase, zinc-binding dehydrogenase family protein | | Arabidopsis thaliana | 15220854 | 41.13/8.46 | 37.58/5.41 | 6 | 93 |
H12 | Putative NAD(P)H oxidoreductase, isoflavone reductase | | Arabidopsis thaliana | 19310585 | 34.24/6.61 | 34.43/4.71 | 4 | 67 |
H13 | Aldo/keto reductase family protein | | Arabidopsis thaliana | 42571931 | 27.76/6.33 | 38.36/5.16 | 6 | 164 |
H14 | 2-oxoacid dehydrogenase family protein | | Arabidopsis thaliana | 15240454 | 50.27/9.19 | 47.44/4.92 | 7 | 168 |
H15 | Mitochondrial processing peptidase | | Solanum tuberosum | 587562 | 54.62/5.99 | 49.29/5.42 | 4 | 65 |
H16 | Copper/zinc superoxide dismutase | | Citrus limon | 33340236 | 15.20/5.46 | 17.02/5.27 | 4 | 263 |
H17 | Putative NAD(P)H oxidoreductase, isoflavone reductase | | Arabidopsis thaliana | 19310585 | 34.24/6.61 | 34.47/4.92 | 3 | 85 |
H18 | Chloroplast stromal ascorbate peroxidase | | Vigna unguiculata | 45268439 | 39.95/7.06 | 31.00/5.06 | 9 | 191 |
Stress response: 7 | ||||||||
No. | Protein name | Protein accumulation | Source organism | GI no. | TheoMr/pI | Expt Mr/pI | PC | MS |
H19 | Abscisic stress ripening-like protein | | Prunus persica | 16588758 | 20.74/5.68 | 29.89/5.25 | 4 | 316 |
H20 | Class III chitinase | | Benincasa hispida | 5919201 | 33.03/9.8 | 26.84/6.32 | 3 | 65 |
H21 | Heat shock protein 60 | | Prunus dulcis | 24637539 | 58.06/5.26 | 88.79/5.66 | 10 | 205 |
H22 | 17.7 kDa heat shock protein | | Carica papaya | 37933812 | 17.77/6.39 | 19.12/5.95 | 2 | 98 |
H23 | Putative heat shock 70 KD protein, mitochondrial precursor | | Oryza sativa | 27476086 | 70.68/5.45 | 92.99/5.65 | 17 | 358 |
H24 | Low molecular weight heat-shock protein | | Pseudotsuga menziesii | 1213116 | 18.18/5.82 | 17.78/6.39 | 4 | 122 |
H25 | Beta-1, 3-glucanase | | Citrus sinensis | 2274915 | 37.32/9.19 | 34.38/6.18 | 7 | 98 |
Other metabolism: 6 | ||||||||
No. | Protein name | Protein accumulation | Source organism | GI no. | TheoMr/pI | Expt Mr/pI | PC | MS |
H26 | Fiber annexin | | Gossypium hirsutum | 3493172 | 36.20/6.34 | 36.23/5.53 | 5 | 70 |
H27 | Alpha-amylase | | Citrus reticulata | 20336385 | 17.05/4.91 | 44.16/5.64 | 8 | 177 |
H28 | Glutamine synthetase | | Elaeagnus umbellata | 47933888 | 39.33/6.10 | 39.38/5.16 | 5 | 134 |
H29 | Putative inorganic pyrophosphatase | | Oryza sativa | 46805453 | 20.45/4.61 | 28.58/6.31 | 4 | 74 |
H30 | Thiazole biosynthetic enzyme, chloroplast precursor | | Citrus sinensis | 6094476 | 37.74/5.40 | 31.35/6.04 | 12 | 447 |
H31 | Lipocalin protein | | Capsicum annuum | 50236424 | 21.41/7.66 | 19.0/4.33 | 3 | 143 |
Protein folding: 6 | ||||||||
No. | Protein name | Protein accumulation | Source organism | GI no. | TheoMr/pI | Expt Mr/pI | PC | MS |
H32 | Catalytic/coenzyme binding | | Arabidopsis thaliana | 18404496 | 34.97/8.37 | 25.54/4.50 | 6 | 252 |
H33 | Chaperonin-60 beta subunit | | Solanum tuberosum | 1762130 | 63.26/5.72 | 57.6/5.77 | 12 | 283 |
H34 | Protein disulfide isomerase | | Zea mays] | 162461925 | 40.43/6.29 | 37.7/5.26 | 9 | 165 |
H35 | Transcription factor APFI | | Arabidopsis thaliana | 13507025 | 30.14/6.24 | 28.15/5.14 | 3 | 89 |
H36 | Putative ATP synthase beta subunit | | Oryza sativa | 56784991 | 45.93/5.33 | 51.13/5.66 | 20 | 695 |
H37 | Putative ATP synthase beta subunit | | Oryza sativa | 56784991 | 45.93/5.33 | 50.0/5.6 | 20 | 808 |
Differentially accumulated metabolites in HT citrus pericarp
Differentially accumulated metabolites were studied to investigate the changes in metabolic composition between HT and control pericarp. The biochemical changes were determined by GC-MS and HPLC-QTOF-MS. A parallel analysis of non-polar and polar extracts using methanol extraction enabled the detection of a large number of compounds of different classes.
Differential accumulated primary metabolites in heat treated pericarp compared to control pericarp
Sugars | 2 h | 1 d | 6 d | 32 d |
Turanose | 1.19 * | 1.13 | 1.36 * | 1.25 * |
Galactose | 1.44 * | 1.59 * | 1.09 | 1.17 |
Fructose | 1.29 * | 1.36 * | 1.02 | 1.08 |
Glucose | 1.4 * | 1.51 * | 1.06 | 1.14 |
Sucrose | 1.21 * | 1.09 | 1.07 | 1.19 |
4-Keto-glucose | 1.59 * | 1.09 | 1.43 * | 0.84 |
Arabinose | 1.36 * | 0.71 * | 1.23 * | 0.18 * |
Mannose | 1.06 | 1.11 | 0.88 | 0.73 * |
Xylose | 0.82 * | 1.37 * | 0.82 * | 0.98 |
Glucopyranose | 0.81 * | 2.09 * | 0.84 | 1.41 * |
Organic acids | 2 h | 1 d | 6 d | 32 d |
Pentonic acid | 1.77 * | 0.78 * | 1.04 | 1.46 * |
Succinic acid | 1.13 | 1.2 * | 1.26 * | 1.54 * |
Hydroxypyruvic acid | 1.16 | 1.07 | 1.28 * | 0.71 * |
2-Ketoglutaric acid | 1.06 | 1.1 | 0.65 * | 1.31 * |
2-Keto-d-gluconic acid | 1.05 | 1.56 * | 1.13 | 2.52 * |
Gluconic acid | 1 | 1.81 * | 0.92 | 1.04 |
Isocitric acid | 0.85 | 2.1 * | 1.11 | 1.15 |
2,3,4-Trihydroxybutyric acid | 0.84 | 1.67 * | 0.61 * | 1.2 * |
Ethanedioic acid | 0.83 * | 1.26 * | 1.11 | 1.55 * |
Citric acid | 0.83 * | 1.53 * | 1.22 * | 1.92 * |
Malic acid | 0.63 * | 1 | 0.77 * | 1.41 * |
4-N-Trimethylsilylmethylaminobutyric acid | 0.56 * | 1.03 | 0.76 * | 1.33 * |
Amino acids | 2 h | 1 d | 6 d | 32 d |
Ornithine | 2.63 * | 3.69 * | 2.34 * | 2.87 * |
Valine | 0.48 * | 0.99 | 0.46 * | 0.7 * |
5-oxo-L-proline | 0.45 * | 1.08 | 0.73 * | 1.14 |
Glycine | 0.37 * | 1.42 * | 0.65 * | 1.44 * |
Alanine | 0.31 * | 2.22 * | 0.69 * | 1.25 * |
Threonine | 0.29 * | 1.12 | 0.47 * | 0.92 |
Aspartic acid | 0.14 * | 0.99 | 0.35 * | 0.8 * |
L-proline | 0.13 * | 0.99 | 0.19 * | 0.65 * |
Serine | 0.13 * | 1.27 * | 0.2 * | 0.64 * |
Glutamine | 0.11 * | 0.86 | 0.3 * | 0.73 * |
Asparagine | 0.03 * | 0.34 * | 0.25 * | 0.89 |
Alcohols | 2 h | 1 d | 6 d | 32 d |
Glycerol | 1.61 * | 1.2 * | 0.91 | 2.23 * |
Arabitol | 1.05 | 1.04 | 0.39 * | 0.92 |
Rhamnitol | 0.98 | 0.97 | 0.76 * | 1.73 * |
Sorbitol | 0.94 | 0.68 * | 1.87 * | 1.8 * |
Fatty acids | 2 h | 1 d | 6 d | 32 d |
Oleic acid | UP * | UP * | UP * | UP * |
Tetradecanoic acid | UP * | UP * | UP * | UP * |
Octadecanoic acid | 0.65 * | 1.11 | 0.94 | 1.66 * |
9,12-Octadecadienoic acid | 0.57 * | 0.84 | 1.05 | 0.93 |
Hexadecanoic acid | 0.62 * | 1.06 | 0.99 | 1.38 * |
Hexadecanoic acid,2,3-bisoxypropylester | 0.68 * | 0.39 * | 0.81 * | 2.18 * |
Others | 2 h | 1 d | 6 d | 32 d |
Phosphate | 0.89 | 1.48 * | 1.33 * | 1.48 * |
Secondary metabolites specifically accumulated in heat treated pericarp
Secondary metabolites | 2 h | 1 d | 6 d | 32 d |
Quercetin-dihexose-deoxyhexose | 16.71 | 14.81 | 15.42 | n.s. |
Hesperetin | 12.32 | 2.08 | n.s. | n.s. |
Naringenin chalcone-hexose | 15.04 | n.s. | 4.77 | n.s. |
Vanillic acid | 14.73 | n.s. | n.s. | 14.37 |
Hydroxybenzoic acid-hexose | 14.99 | n.s. | n.s. | n.s. |
Diosmin | 4.06 | n.s. | n.s. | n.s. |
Rutin | 3.69 | n.s. | n.s. | n.s. |
Phloretin-C-diglycoside | 14.97 | n.s. | 0.06 | n.s. |
Chlorogenic acid | 15.96 | 0.06 | 0.12 | n.s. |
Isosakuranetin | n.s. | 16.42 | n.s. | n.s. |
Qucercetin | n.s. | 16.03 | 0.14 | 0.12 |
Neohesperidin | n.s. | 1.31 | 0.14 | n.s. |
Protocatechuic acid | n.s. | 0.09 | n.s. | n.s. |
Caffeic acid hexose | n.s. | 0.06 | 0.20 | 15.90 |
Naringenin | n.s. | n.s. | 15.31 | n.s. |
Caffeic acid | n.s. | n.s. | 0.09 | n.s. |
Hydroxylated naringenin-hexose | n.s. | n.s. | 0.10 | n.s. |
Sinensetin | n.s. | n.s. | 0.08 | n.s. |
p-Coumaroyl quinic acid | n.s. | n.s. | 0.07 | n.s. |
Feruloylquinic acid | n.s. | n.s. | 0.06 | n.s. |
Pantothenic acid -hexose | n.s. | n.s. | 0.06 | n.s. |
Quercetin-O-dihexoside | 0.08 | n.s. | 1.13 | 0.46 |
Cinnamic acid | 0.11 | n.s. | n.s. | n.s. |
Eriodictyol-O-dihexoside | 0.06 | n.s. | n.s. | 0.06 |
Ferulic acid | 0.42 | n.s. | 0.09 | n.s. |
Sinapic acid | 0.05 | n.s. | 0.07 | n.s. |
Jasmonic acid | 0.07 | n.s. | 0.06 | n.s. |
HT up-regulated stress response proteins in citrus pericarp
In disease response, HT induced Beta-1, 3-glucanase (H25) and Class III chitinase (H20) accumulated in pericarp (Table 1), where these proteins are involved in reducing stress resistance in plants. In the stress response, HT up-regulated 17.7 kDa heat shock protein (H22) and low molecular weight heat-shock protein (H24) accumulated in pericarp, especially at early storage stage (Table 1). Also, other proteins accumulated in heat treated pericarp in higher amounts when compared to control pericarp during the same period of storage, which might be indirectly involved in the fruit's response to stress, including the pyruvate dehydrogenase E1 beta subunit isoform 2 (H7), chloroplast stromal ascorbate peroxidase (H18), putative ATP synthase beta subunit (H36), putative inorganic pyrophosphatase, isoflavone reductase related protein (H10), and cytoplasmic malate dehydrogenase (H4, Table 1).
HT down-regulated reactive oxygen species metabolism-related proteins in citrus pericarp
HT down-regulated reactive oxygen species metabolism-related proteins, especially during later periods of storage (Table 1), including oxidoreductase, zinc-binding dehydrogenase family protein (H11), putative NAD(P)H oxidoreductase, isoflavone reductase (H12), aldo/keto reductase family protein (H13), copper/zinc superoxide dismutase (H16), and putative NAD(P)H oxidoreductase, isoflavone reductase (H17). Also, other proteins were down-regulated in heat treated pericarp (relative to control), such as triosephosphate isomerase, cytosolic (H5), abscisic stress ripening-like protein (H19), and thiazole biosynthetic enzyme, chloroplast precursor (H30). In addition, some proteins were up-regulated in heat treated pericarp (relative to control) only in the middle of the experiment, including, phosphoglycerate kinase, putative (H2), pyruvate dehydrogenase E1 beta subunit isoform 2 (H7), aldo/keto reductase family protein (H13), mitochondrial processing peptidase (H15), putative inorganic pyrophosphatase (H29). Putative ATP synthase beta subunit (H36) was up-regulated at the end but down-regulated at the beginning in heat treated pericarps (relative to control).
HT increased contents of sugars and flavonoids in citrus pericarp
Primary metabolite profiling of the HT and control pericarp revealed prominent changes in a group of metabolites during the entire storage phases (Table 2), mainly including sugars and fatty acids. Interestingly, the contents of many kinds of sugars were increased in heat treated pericarp (relative to control) at 2 h after HT, but decreased to the same level with control at later storage phases, such as galactose, fructose, glucose, sucrose and 4-keto-glucose. Some primary metabolites contents were increased in heat treated pericarp (relative to control) during whole storage, including oleic acid, tetradecanoic acid and ornithine (Table 2).
Secondary metabolite profiling of the HT and control pericarp revealed that a group of metabolites were increased in heat treated pericarp (relative to control) during some periods of storage, including quercetin-dihexose-deoxyhexose, hesperetin, hydroxybenzoic acid-hexose, naringenin, naringenin chalcone-hexose, vanillic acid, diosmin, rutin and isosakuranetin, which all might play a vital role in the response to stress.
HT decreased contents of organic acids, amino acids and phenylpropanoids
Primary metabolic profiling revealed organic acids, fatty acids and amino acids were down-regulated in heat treated pericarp when compared to control pericarp, especially at 2 h after heat treatment. Levels of five types of organic acids (citric acid, malic acid, 4-N-Trimethylsilylmethylaminobutyric acid, oxalic acid and GABA) had decreased in heat treated pericarp compared to the control at 2 h after HT (Table 2), but levels of ten types of organic acid increased in heat treated pericarp (relative to the control) at 32 DAT (Table 2). The content of ten types of amino acids were lower in heat treated pericarp than in the control at 2 h after HT, while only five of them were lower in heat treated pericarp than in the control at 32 DAT, although two of them were higher in heat treated pericarp than in the control at 32 DAT (Table 2). In a similar way, compared to organic acids, levels of four types of fatty acids decreased in heat treated pericarp compared to the control at 2 h after heat treatment; levels of three of them increased in heat treated pericarp compared to the control at 32 DAT (Table 2). There is common occurrence here; levels of many types of organic acids, amino acids and fatty acids decreased in heat treated pericarp compared to the control at 2 h after HT, but levels of many of them increased in heat treated pericarp compared to the control at 32 DAT. Compared to increased sugar concentrations, it appears there is a sudden transformation of organic, amino and fatty acids into sugars which is driven by HT; also, levels of many types of organic, amino and fatty acids increased in heat treated pericarp compared to the control during the later stages of storage.
Further metabolic characterization of chemicals which induced resistance in heat treated pericarp was performed using an HPLC-QTOF-MS. Contents of ferulic acid, sinapic acid, cinnamic acid and caffeic acid, the precursors of lignin synthesis, decreased in heat treated pericarp when compared to the control pericarp, especially at 2 h and 12 DAT. Moreover, the contents of other secondary metabolites decreased in heat treated pericarp, especially at 2 h or 12 DAT, such as hydroxylated naringenin-hexose, sinensetin, p-coumaroyl quinic acid, feruloylquinic acid, pantothenic acid-hexose, quercetin-O-dihexoside, eriodictyol-O-dihexoside, and jasmonic acid.
HT decreased H2O2content in citrus pericarp
Superoxide dismutase (SOD) and peroxidase (POD) play important roles in deoxidization of H2O2. In this study, the activity of POD showed no significant change between HT and the control (Figure 4C). The activity of SOD was higher in heat treated pericarp relative to the control at 6 DAT, but was lower in heat treated pericarp relative to the control at 12 and 32 DAT (Figure 4D). So, the activity of SOD fluctuated during the entire storage period in heat treated pericarp compared to the control (Figure 4D).
Concerning redox regulation, most of the proteins were down-regulated in heat treated pericarp when compared to control pericarp, especially in the later stages of storage (Table 1). This suggests the redox reaction was weaker in heat treated pericarp during storage.
HT increased fruit firmness and the content of lignin in citrus pericarp
HT improved fruit pathogen resistance without negatively impacting fruit quality
Physical treatments are used frequently to keep fruit fresh, including microporous membranes, pre-storage treatment (sweating), ultraviolet illumination, radiofrequency treatment, heat treatments (heat therapy), and other storage techniques. Postharvest heat treatments such as hot water treatment and hot air treatment provide a type of quarantine, inhibit the development of pathogens, enhance fruit resistance to chilling injury in cold-sensitive cultivars, and help preserve fruit quality during storage and extend its shelf life [7]. In the present study, HT effectively inhibited the development of pathogens. Also, HT (52°C, 2 min) caused a statistically significant reduction the incidence of chilling injury [9, 11].Also, postharvest HT had no obvious negative effects on fruit flesh quality during subsequent storage. This suggests HT increased the ability of fruit to tolerate storage stress without affecting their commercial qualities, and shows strong potential application in the citrus industry.
HT induced stress response proteins
HT can effectively inhibit fruit chilling injury during cold storage [12]. After HT, some proteins were up-regulated, while others were down-regulated. The up-regulated proteins might be responsible for heat induced fruit resistance. Interesting, some proteins were up-regulated at 1 d after HT, and decreased to the previous level of control during the storage period. Those kinds of proteins might be just involved in response to HT, but not play a vital role in fruit disease resistance. The proteins, which are up-regulated during whole storage period might be directly involved in fruit disease resistance.
In the present study, heat shock protein (HSP) 17.7, putative HSP 70 and low molecular weight HSP were up-regulated in heat treated pericarp compared to the control during entire storage period (Table 1). HSPs are encoded by a multigene family and are located in different subcellular compartments [13]. These molecular chaperones play a crucial role in protecting plants against stress by re-establishing normal protein configuration and thus helping to maintain cellular homeostasis [14]. Abiotic stresses usually causes protein dysfunction; so maintaining proteins in their functional configurations and preventing the aggregation of non-native proteins is particularly important for cell survival under stress [14]. HSPs are responsible for protein folding, assembly, translocation and degradation during cellular processes. HSPs also stabilize proteins and membranes, and can assist in protein refolding under stressful conditions [15]. Our results suggest HSPs play a crucial role in reducing the susceptibility of HT fruit to biotic and abiotic stress.
HT significantly inhibited pathogen development and fruit decay during storage [16]. Chitinase and β-1, 3-glucanase were up-regulated in heat treated fruit in this study; these enzymes have long been thought to be active in fruit antifungal defences. Pathogenesis-related (PR) proteins have been classified into five groups and two of these groups contain proteins with β-1, 3-glucanase and chitinase [17]. Chitinase catalyses the hydrolysis of chitin, a β-1, 4-linked polymer of N-acetyl D-glucosamine and is a major component of cell walls of most phytopathogenic fungi [18]. Crude protein extracts of chitinase and β-1, 3-glucanase were observed to effectively inhibit fungal activity [19]. Also, β-1, 3-glucanase and chitinase are thought to contribute to the level of resistance provided and are known to inhibit mycelial growth of a wide range of fungal pathogens [20]. This suggests β-1, 3-glucanase and chitinase were directly involved in HT induced pathogen resistance in fruits.
HT increased the content of stress response metabolites
Primary metabolites are directly involved in normal growth, development, and reproduction. In the present study, the content of ornithine, oleic acid and tetradecanoic acid increased in heat treated pericarp compared to the control during storage, and levels of seven types of sugars increased in heat treated pericarp compared to the control at 2 h after HT (Table 2). In plants, ornithine is required for the synthesis of polyamines and alkaloids, which contribute to oxidative stress tolerance in plants subjected to severe water stress [21].
Flavonoids are important defensive compounds which protect fruit against the development of pathogens, acting either as phytoanticipins or phytoalexins [22]. Flavonoids were observed to be up-regulated in heat treated pericarp (compared to the control), including quercetin-dihexose-deoxyhexose, hesperetin, naringenin, naringenin chalcone-hexose, hydroxybenzoic acid-hexose, isosakuranetin, diosmin, and rutin, which may play a vital role in response to stress [23]. Rutin is a bioflavonoid with strong antioxidant activity, which had an antimicrobial effect on pathogens, as evidenced by reduced conidial germination and appressorium formation of the pathogen [22]. Quercetin protected cells from oxidative stress-induced cell death by blocking both the cyclooxygenase and lipooxygenase pathways [24]. Quercetin has a protective function against oxidative stress by reducing oxidative injury to the cells during H2O2 treatment [25]. Also, plant extracts rich in quercetin have been applied as an interesting resource for functional food products and other foods used to reduce the risks of age-related macular degeneration [25]. Hesperidin acts as an antioxidant, based on in vitro studies, and hesperidin also provides strong cellular antioxidant protection against the damaging effects induced by stress [26]. Diosmin, hesperidin and naringin are flavonoid glycosides that occur naturally in citrus fruits; they exert a variety of properties such as antioxidant and free radical scavenging [27]. Interestingly, naringenin and rutin are not only involved in response to stress, but also were assumed to be signalling molecules [23].
HT induced fruit pathogen resistance and increased lignin content
The functions of phenylpropanoid compounds in plant defence range from preformed or inducible physical and chemical barriers against infection [10]. Defensive functions are not restricted to a particular class of phenylpropanoid compounds, but are found in the lignification of monolignols to lignin [28]. In the present study, HT decreased the level of precursors needed in lignin synthesis (ferulic acid, sinapic acid, cinnamic acid and caffeic acid), but increased lignin content (Table 3, Figure 5). Lignin is one of the major components of secondary cell walls, providing cells with mechanical support and isolation from the environment. In plants, cells can sense cell wall polymer structure and this activates trans-plasma membrane sensor proteins which might trigger a cascade of signal transduction events interwoven with other pathways that might modulate cellular functions by the activation or inhibition of specific transcription factors as well as by affecting posttranslational control of gene expression and protein function [29]. If a cell wall is damaged, cells activate a series of responses to the invading disease organisms, which might negatively impact fruit quality. The accumulation of lignin induced by HT creates a thickening of cell walls which forms an effective physical barrier to pathogens, delaying the invasion of disease organisms. Also, HT increased fruit firmness; this might be a result of HT induced lignin thickened fruit cell walls.
HT reduced the content of H2O2
HT is an effective method for improving citrus storability. In storage, fruits suffer a variety of biotic and abiotic environmental stresses. All these stresses could lead to the generation of reactive oxygen species (ROS). To avoid the accumulation of ROS, which may lead to cell death, plants have up-regulated enzymatic and non-enzymatic antioxidants to scavenge ROS and alleviate their negative effects [30]. In this study, HT down-regulated H2O2 content in pericarp, which was consistent with the effect of HT on yeast cells. HT yeast cells accumulated less ROS than untreated cells in response to stresses [31].
Also, isoflavone reductase (IFR) might have an important role as an antioxidant [32]. IFR is an enzyme involved in the production of isoflavone phytoalexins and it accumulates in response to pathogenic attack, fungal elicitor and biotic stress [33]. Isoflavonoids are important secondary metabolites for essential physiological processes. Kim et al.[32] reported the isoflavone reductase-like gene may act as a down-regulator by producing antioxidant chemicals to prevent the elevation of ROS. In this study, isoflavone reductase, a related protein (H10), was up-regulated in heat treated pericarp when compared to control pericarp (Table 1), which might be another factor leading to lower H2O2 levels and stronger resistance to pathogens in heat treated pericarp when compared to control pericarp.
Ascorbate peroxidase scavenges H2O2 in specific organelles of the cell [34]. Also, Cu/Zn-SOD is the main cellular isoform of SODs which play a key role in the antioxidant defence system through the disproportionation of H2O2[34]. Both of these can prevent ROS caused damages to the cellular membrane and act as a primary defence mechanism when organisms were exposed to oxidative stress. These proteins are considered the main enzymatic and non-enzymatic systems for protecting cells against oxidative damage, and are responsible for disease resistance. In response to biotic stress, these proteins also act as the primary defence mechanism against oxidative stresses caused by pathogens, therefore preventing damage of ROS to cellular membranes. However, the activities of POD and SOD were not elevated in Heat treated pericarp when compared to control pericarp, which might be caused by the lower H2O2 content in heat treated pericarp. Also, other proteins involved in scavenging ROS were down-regulated in heat treated pericarp when compared to control pericarp, including oxidoreductase (H11), oxidoreductase (H12, 17), aldo/keto reductase (H13), 2-oxoacid dehydrogenase (H14), superoxide dismutase (H16). This suggests HT induced a level of resistance to pathogens attacking fruits by causing a decline in fruit H2O2 content and by down-regulated scavenging ROS enzymes.
Role of sugars, organic acids and amino acids in improving storability
In this study, the profiling of primary metabolics showed HT induced the accumulation of sugars at early stages of storage (Table 2). Soluble sugars, especially sucrose, glucose, and fructose, accumulated when fruits were subjected to stress, and accumulated soluble sugars play an obviously vital role in the response of fruit to a number of stressors [35]. Soluble sugars do not only act as nutrients and metabolites, but also act as signal substance in the modification of redox homeostasis [36]. However, less information was available on how other sugars are involved in the stress response, especially arabinose, mannose, galactose, and ribose.
Moreover, primary metabolic data shows HT reduced organic acids and amino acids content especially at 2 h after treatment in this study. Although organic acids and amino acids play an obvious central role in energy and metabolism at the cellular and whole-organism levels, there seems to be a rapid conversion from organic acids and amino acids to sugars in heat treated pericarp. However, little is known about the decreased levels of organic acids and amino acids seen during a stress response, and further study will be required to validate this response.
This study provides a broad picture of differential accumulation of proteins and metabolites, and gives a new insight into how HT increases fruit stress resistance during subsequent storage of citrus fruit. In metabolic substrates, the up-accumulation of secondary metabolites played vital roles in increasing the ability of fruit to deal with stress. Flavonoids are directly involved in the response to external stress. The increased lignin and decreased H2O2 contents involved in HT probably increased fruit resistibility in response to external stress.
Sample collection
'Kamei' Satsuma mandarin (Citrus unshiu Marc.) fruits were harvested at commercial maturity (fruit colour has turned completely orange and the level of soluble solids approaches 9%) from an orchard in the city of Yichang, Hubei Province, China, in October 2009. Fruits of a uniform size and colour, free of visible injury or blemishes, were selected for the experiments. Three hundred kilograms of fruit were divided into two equal parts, 150 kg were used for the HT and the other 150 kg used for a control. After treatments, fruits were stored in a ventilated warehouse (storage temperature: 12–16°C; relative humidity: 90–95%). Samples were collected at 2 h, and 1, 2, 3, 4, 6, 9, 12, 16, 20, 24, 28, 32, 38, 44, 50 and 60 DAT. The pericarp was sampled along the equatorial plane of each fruit; 30 fruits were selected as one sample and ground to powder in liquid nitrogen, then stored at –80°C for further analysis.
Heat treatment (HT)
Heat treated fruits were dipped in a 52°C warm water bath for 2 min, and control fruits were dipped in a 25°C water bath for 2 min. After treatments, fruits were air dried and stored in a ventilated warehouse prior to sampling.
Fruit quality determination
Fruits were checked for weight loss (%), respiration rate (mg kg–1 h–1) and total soluble solids (TSS,%) at 1, 2, 3, 4, 6, 9, 12, 16, 20, 24, 28, 32, 38, 44, 50 and 60 DAT. The weight was measured using an electronic hydrostatic balance (Model: MP31001, Shanghai Selon Scientific Instrument, Co., Ltd, Shanghai, China) with an accuracy of ± 0.01 g. The respiration rate of fruit was measured with an infrared gas analyser (Model: GXH-305H, Junfang Science & Technology Institute of Physical and Chemical Research, Beijing, China). TSS were determined with a refractometer (Model: Pocket PAL-1, Atago Inc., Toyko, Japan) according to the manufacturer's instructions.
Antifungal assay of HT in wounded citrus fruits
Two-dimensional gel electrophoresis and MAILDI-TOF/TOF
Samples at 1, 6, 12 and 32 DAT were used for total protein extraction based on the method described by Isaacson et al.[37]. The precipitate was then nitrogen gas-dried and solubilized in the lysis buffer containing 8 M urea, 0.2% (w/v) Bio-Lyte, 4% CHAPS, 65 mM DTT. The protein concentration was determined with a Bio-Rad protein assay kit (Bio-Rad Laboratories Inc., Hercules, CA, USA) based on the Bradford method using BSA as the standard. Two-dimensional gel electrophoresis and gel staining was carried out based on the method of Yun et al.[38]. The stained gels were imaged with a UVP imager (Bio-Rad) in the 'trans white' mode using PDQuest 2-D analysis software version 7.4 (Bio-Rad). Protein spots matching and differential protein spots analysis were completed based on the method of Yun et al.[38]. Isoelectric points and molecular weights of the proteins were determined by comparison with the markers.
In-gel digestion and protein identification were performed using a 4800 Proteomics Analyzer MALDI TOF/TOF (Applied Biosystems, Foster City, CA) as described by Yun et al.[38]. MS/MS data were then submitted to the MASCOT program for protein identification against the green plants database. The following are the search parameters: trypsin enzyme, one missed cleavage, fixed modifications of carbamidomethyl (C), fragment mass tolerance of ± 0.5 Da, variable modifications of oxidation (Met), peptide tolerance of 100 ppm, and peptide charge of 1+. Only results of peptides with MS/MS P < 0.05 confidence were accepted.
The primary metabolic profiling
Samples collected at 2 h and 1, 6 and 32 DAT were used for the differential primary metabolic profiling analysis. A 300 mg sample was extracted in 2,700 μl of methanol as described by Roessner-Tunali et al.[39]. 300 μl of 0.2 mg ml–1 ribitol in water was added as a quantification internal standard. A derivatization reaction was performed based on the protocol of Zhang et al.[40] with little modification. Extracts were incubated in 50 μl of 20 mg ml–1 methoxyamine hydrochloride in pyridine for 30 min at 50°C, followed by a 40 min treatment at 60°C using 50 μl BSTFA (containing 1% TMCS). Each sample (1 μl) was injected into the gas chromatograph system through a fused-silica capillary column (30 m × 0.25 mm i.d., 0.25 μm) DB-5 MS stationary phase. The injector temperature was 250°C, with a carrier gas flow rate of 1.0 ml min–1. The column temperature was held at 100°C for 1 min; increased to 184°C at a rate of 3°C min–1, increased to 190°C at a rate of 0.5°C min–1, increased to 280°C at 15°C min–1. The flow rate of the carrier helium (99.999%) gas was 1 ml min–1. The following were MS operating parameters: ionization voltage, 70 eV (electron impact ionization); ion source temperature, 200°C; interface temperature, 250°C. TIC (total ioncurrent) spectra were recorded in the mass range of 45–600 atomic mass units in scanning mode.
The secondary metabolic profiling
Samples at 2 h and 1, 6 and 32 DAT were used for differential secondary metabolic profiling analysis using HPLC-MS. 300 mg dried powder was extracted with methanol, the mixture was filtered through a 0.22–mm polytetrafluoroethylene membrane filter.
Metabolite profiling was performed using a QTOF 6520 mass spectrometer (Agilent Technologies, Palo Alto, CA, USA) coupled to a 1200 series Rapid Resolution HPLC system as described by Page et al.[41]. 2 μl of sample extract was loaded onto a Zorbax Eclipse Plus C18 1.8 μm, 2.1 × 100 mm reverse-phase analytical column (Agilent Technologies). Mobile phase A comprised 0.1% formic acid in water and mobile phase B was acetonitrile. The following gradient was used: 0 min–10% B; 20 min–95% B; 22 min–95% B; 22.1 min–10% B; 30 min–10% B. The flow rate was 0.3 ml min–1 and the column temperature was held at 35°C for the duration. The source conditions for electrospray ionization were as follows: gas temperature was 350°C with a drying gas flow rate of 10 l min–1 and a nebulizer pressure of 40 psig. The capillary voltage was 3.5 kV in both positive and negative ion mode. The fragmentor voltage was 135 V and the skimmer was 65 V.
Assay of phenylalanine ammonia-lyase (PAL), superoxide dismutase (SOD) and peroxidase (POD) activities
Samples collected at 1, 6, 12 and 32 DAT were used for enzyme activity determination. A 5 g sample was homogenized in 25 ml of 0.05 M sodium borate buffer (pH 8.8 for PAL, containing 5 mM β-mercaptoethanol) and 25 ml of ice-cold 100 mM sodium phosphate (pH 7.8 for POD and SOD, containing 0.5 g polyvinyl polypyrrolidone). PAL activity was determined by measuring the absorbance of cinnamic acid at 290 nm over a period of 30 min at 30°C [42]. POD activity was analysed using guaiacol as the substrate [43]. SOD activity was determined using the xanthine-xanthine oxidase method, and nitro blue tetrazolium was used as the indicator of superoxide radical production [44].
Determination the content of H2O2in the peel
Samples collected at 2 h and 1, 6 and 32 DAT were used to measure hydrogen peroxide levels based on the methods of Patrick and Wagner [45] with some modifications. Briefly, peel tissues (0.5 g) were ground in liquid nitrogen and homogenized in 5 ml of physiological saline. Samples were then subjected to an ultrasonic bath for 15 min and centrifuged at 6,000 g for 20 min. The supernatants (50 μl) were subsequently added into 0.5 ml of solution I and warmed in a 37°C water bath for 10 min before 0.5 ml of solution II was added. The absorbance of the mixture was measured at 405 nm and the content of H2O2 in each sample was calculated by comparison with a standard calibration curve.
Determination of fruit firmness
Samples collected at 2 h and 1, 6 and 32 DAT were used for the determination of fruit firmness. Eighteen fruits were measured and every fruit was measured on three different spots around the equatorial plane. Firmness was measured with a fruit sclerometer (GY-B, Jilin, China) with 4 mm diameter and expressed as N cm–2 and the average value per fruit was used for further analysis, after removing three largest and three smallest measurements; a total twelve replicates were comparative statistically analysed per sample using Student's t-test (P < 0.05).
Determination of lignin in pericarp
Samples collected at 2 h and 1, 6 and 32 DAT were used to measure fruit lignin content. Lignin levels were determined using the method described by Lei et al.[46].
Statistical analysis
The experimental design was completely randomized with more than three replications. Four individual replicates were completed in 2-DE, GC-MS, HPLC-MS, H2O2 content, lignin content and enzyme activity determination. Twelve replicates were completed to measure weight loss, respiration rate, total soluble solids and fruit firmness. Data for each sample was statistically analysed using Student's t-test (P < 0.05).
Two-dimensional gel electrophoresis
Gas chromatography coupled to mass spectrometry
Heat shock protein
Heat treatment
Isoflavone reductase
Liquid chromatography quadrupole time-of-flight mass spectrometry
Matrix-assisted laser desorption/ionization-time-of-flight tandem mass spectrometry
National center for biotechnology information
Phenylalanine ammonia-lyase
Reactive oxygen species
Superoxide dismutase
Total soluble solids.
We thank Prof. Hanhui Kuang of Huazhong Agricultural University for help in revising the manuscript. This work was supported by Huazhong Agricultural University Scientific & Technological Self-innovation Foundation, the Natural Science Foundation of China (NSFC No. 31271968), The Important Project of Ministry of Education (311029), and National Modern Agriculture (Citrus) Technology Systems of China (No. CARS-27).
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Bella has delivered 7 healthy puppies on 22nd February
2 solid liver boys
2 liver and white boys
and 3 liver and white girls.
Sired Keigame Nasrullah,it is a first litter for both.They are both hip,heart and eye tested and have delightful natures.
Both have been showed with success at champ show level,tho only lightly.Both work on the shoot as beating,picking up dogs.
We are expecting both liver and white and solid livers.
Puppies will be reared in house,well socalised,docked and dewed,regularly wormed,and will be endorsed not .... (click to read more) -
Redmires High Society x Sh Ch Cushatlaw Illustrious JW
Tyne & Wear
30th January 2017
Parents Health TestedDue to unforeseen circumstances we now have a Dog puppy for sale to the right home, enquiries welcome...... Boudi's puppies arrived safely on 28th January, 4 Dogs and 4 Bitches. Mum and pups are all doing very well.
We are pleased to announce that Boudi ( SH CH Cushatlaw Illustrious JW) is in whelp to Denver(Redmires High Society).The litter is due on 30th January, and has been carefully planned. Puppies will be KC Registered, wormed regularly, and microchipped.Both the Sire and the Dam have been very successful in the show ring, and Denver is worked on a regular basis. Both dogs are .... (click to read more) -
Katelands Caisteal A'Torr x Fern'S Folly
Nr Rugby
6th March 2017
Parents Health TestedUPDATED 20th MARCH
Skye's litter arrived a few days earlier than expected.
Many thanks for all the expressions of interest.
We have six wonderful puppies (2 bitches, 4 dogs) and we are suspending our list of potential new owners at this point because we are already going to have to disappoint some excellent potential homes.
We are working through the list of interested parties, therefore if we already have your details we will be contacting you shortly.
'SKYE' (Fern's Folly) our elegant three year old black & white GSP has been successfully mated to OLLIE (Katelands .... (click to read more) -
Winterwell Mr Hobbs x Ladyhawke California Star JW
North Yorkshire
10th February 2017
Parents Health TestedSTOP PRESS: Due to unforeseen circumstances we have a beautiful dog puppy available.
Ruby's Puppies Arrived safely on 8th February, 5 dogs and 6 bitches. Thank you to everyone who expressed an interest in the litter. We have some fantastic homes for them and we were so sorry to disappoint others who would have also provided super homes for a GSP.
Ladyhawke California Star JW [Ruby] has been confirmed in whelp to Winterwell Mr Hobbs [Hobbs]. Puppies are due mid February 2016.
This is a repeat mating and brings together some of the best English and American lines. .... (click to read more) -
Sh Ch Indijazz Originale at Soellis x Indijazz Vivacious
10th April 2017
Parents Health TestedDue to the huge interest in this wonderful mating I am suspending the list until after puppies are born.
We are pleased to announce the mating of Indijazz Vivacious (Tilly)with Indijazz Originale at Soellis (Rudi),litter due around 10th April. The sire is both a working dog and show dog, made up to Champ show level. The dam is a huge part of our family and comes from both show and working lines. Both dogs have excellent temperaments.
The puppies will be KC registered, micro chipped, have their dew claws removed but their tails will not be docked. Puppies will leave with four weeks pet .... (click to read more) -
Pruso Blue My Cover x Laganglen Jasmine
Haywards Heath
West Sussex
5th April 2017
Parents Health TestedPLEASE READ UPDATE BELOW
Suzi and David are thrilled to announce that Maisie (Laganglen Jasmine) is soon to have her first litter of puppies thanks to the planned attention of the fabulous Morse.
Maisie's puppies will be socialised, regularly wormed, legally docked and have dew-claws removed. Of course, they will also be microchipped.
The puppies, due end of March/early April, will be brought up inside. Following their sale (no earlier than 8 weeks after birth) some breeding restrictions/conditions will be placed on them. The puppies are not for export.
Three years .... (click to read more) -
Katelands Caisteal A'Torr x Avafields Clareleyview Smart Rosa
16th April 2017
Parents Health TestedWe are expecting a litter of puppies on Easter weekend, Beau is our loving family pet with excellent lines and looks to match,
Father of the pups is Ollie from Katelands Gundogs who's pedigree and hip score are second to none
All puppies will come KC Registered,
Health checked and micro chipped,
All pups will have Dew claws removed but NOT docked,
Please feel free to call anytime regarding our puppies -
Sh Ch Isara Kurzhaar Wieser JW ShCM x Zarozinia Cleopatra Star
10th December 2016
Parents Health TestedWe are really pleased to announce Esse's pups arrived safely and mum and pups are all well. All the pups are reserved.
Our pups are brought up in a busy family home with lots of love and social interaction with adults and children. They will be ready to go to their new families around the middle of Feb, when they are 8 weeks of age, KC Reg, 1st Vaccination, Micro chipped, 4 weeks insurance, along with a comprehensive puppy pack and on going help and support.
Esse is a wonderful family dog that we bred ourselves with a great temperament and boundless energy and great instinct. She .... (click to read more) -
Puddledub Pyjama x Waltzing Tilly
19th January 2017
Parents Health TestedTilly has had 11 healthy adorable puppies due to a carefully planned litter. The puppies will be kc registered with all relevant documentation including 5 weeks insurance, wormed at 2/4/6/&8 weeks,microchipped & vet checked. They have excellent breeding lines for work & showing.Tilly is a beautiful girl with a fantastic temperament she has been health checked & has a hip score of 4/5. Her 5 generation pedigree boasts 14 FT champions. The sire is a stunning liver& white ticked dog,he's intelligent,obedient & very eager to please,he has a hip score of 2/5,his 5 generation pedigree boasts 17 .... (click to read more)
Sh Ch Magregor Chocolossus x Sh Ch Barleyarch Polkadot JW
27th February 2017
Parents Health Tested**WE MAY HAVE 1 PUPPY AVAILABLE TO A SPECIAL HOME IN 2 WEEKS TIME FOLLOWING OUR CHOICE OF PUPPY***
We are delighted to announce Dotty has had her puppies and mum and pups are thriving! The pedigree is star studded as you can see but more importantly both parents are house pets and have wonderful temperaments, steady natures and are health tested.
Both are proven to produce happy healthy dogs that can work, show or simply make active pets. As you would expect from us, no puppy goes without our help and support throughout its lifetime and in return we ask they are given early .... (click to read more) -
Sh Ch Isara Kurzhaar Wieser JW ShCM x Eairycor Tigerlily at Orchidstar
Thornton Cleveleys
14th February 2017
Parents Health TestedDue to the amount of interest in my forthcoming litter I am suspending the waiting list till I know how many puppies are available.
Our beautiful Frazzell and the stunning Flymm are confirmed to be having their second litter together.Both Frazzell and Flymm have faultless temperaments, good with kids and other dogs.
First and foremost my GSP's are family pets but are also very successful in the show ring. Showing is a hobby enjoyed by myself,son and the dogs.
Pups have been reared in our home with the other dogs, children and all day to day life noises. They have had their dew .... (click to read more) -
Youlthorpe Granite x Milebushfarm Belinda
E Yorks
7th February 2017
Sire Health TestedA fabulous litter of beautifully marked gsp's. Both parents are working dogs and loving family pets. Quality puppies with tails legally docked and dew-claws removed. All vet checked , microchipped, wormed up to date. I am looking for lifelong, knowledgeable, loving & active homes for Indies puppies. I am a gundog trainer with many years experience & all new owners will be offered a one to one training session for the new puppy , along with lifetime support & advice . A contract of sale with each puppy plus a care sheet and a puppy pack from Eukanuba.
Stewtonian Dude x Sunbeam Seeker
23rd April 2017
Parents Health TestedZiggy (Sunbeam Seeker) has been mated to Tommy Brechney's young dog Stewtonian Dude (Kane). Tommy is well known in the Field Trial world and thinks highly of this dog.
Ziggy is an excellent working dog. She is very biddable and is a fast and stylish hunter. She is also soft mouthed as is Kane.
Litter is due 23rd April and puppies should be suitable for Field Trials, working or even as a very good friend.
They will be K.C. Reg. Microchipped, vet checked and have their tails docked and dew claws removed. -
Sh Ch Isara Kurzhaar Wieser JW ShCM x Alayglow Hasina
17th February 2017
Parents Health TestedLuca's litter have arrived she has given us 10 beautiful puppies " all pups are reserved " thank you everyone for the massive interest in them and so sorry we had to disappoint so many ! We are delighted that Luca is in whelp to Flymm, this is a carefully planned litter, due about the 17th February 2017. The pups will be KC reg'd, micro chipped, 4 weeks insurance, a comprehensive puppy pack, and a life time support from us.
Luca is a beautiful girl and a joy to live with, with a fantastic temperament, she has been heart tested clear and hip scored. Flymm is a stunning boy, with a equally .... (click to read more)

subset: commoncrawl


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Pessoas | 5 |
Quartos | 1 |
Banheiros | 2 |
Área | 74 m² |
Tipo de imóvel | apartamento |
Estadia Mínima | noites |
Wonderful apartment. 2 bathrooms allowed 2 women space to get ready for the day or evening. Quiet location with eatery next door for the tired. Balcony with late afternoon sun for chatting over sangria. Short walk to the handbag shop and all the eateries you could ever want. The fish dishes are fabulous, as are the steaks and the pastas. There is a walkway along the side of the harbour which takes you to the beach and a choice of bars in which to quench your thirst. The weather was extra warm and sunny all week, so we sampled the cafes along the harbour prom cos the views of the harbour changed every day. Fantastic place for a little r and r. You can do as much or as little you want as there boat trips available as well buses to other towns. We loved the place. I bought two handbags this time, so we need to go back to buy matching shoes. Returning to this apartment in Alvor will give us the greatest of pleasure. Ah, just loved it.
We have just returned from a three week stay in Alvor at this apartment and it really is superb! Spotlessly clean with an abundance of clean towels and bed linen. it's the first apartment we've stayed in where everything actually works! The kitchen has everything you could possibly need (apart from a chef!) it really is lovely. The air con was so good to have, it was reallyyyyyyyyy hot whilst we were there and it was nice to be able to escape from it for a bit of a cooler. I think you definately need air con if going in the hotter months. I think if anything, the photos really dont do this apartment justice, and the owner was so lovely to deal with, always prompt with any reply to queries etc.
We used a man recommended by the owner for airport transfer, it was lovely not to have to sit on a coach and to be taken straight to your holiday destination and cheaper also !
The location is excellent, although there are plenty of bars and restaurants close by, it isn't noisy attall and you're only a five min stroll from the centre of Alvor if you fancy a good night out.Numerous eating places to chose from , but our favourite was Vagabondos, great food reasonably priced and great service.
The beach is just a stroll away and definately worth a visit, really beautiful
All in all a fantastic holiday and we certainly hope to return next year............. hopefully will have paid for this one by then! lol
Many many thanks
The appartment is well kept, spacious,good location.There is a lift to the appartment block which could be usefull if needed. Although we didnt use it, the Sofa bed is of very good quality and the appartment could accomodate 4.It was nice to have the air conditioning during our stay.The restaurant next door gets busy ,so get in early but its not noisey.The Por do Sol restuarant is our favorite go by car or taxi.The Grand Muralha is best for Chinese food near the Taxi rank,there's pleanty of bars & restuarants to choose from up in the old town of Alvor a short walk away.The beach is long and pleanty of space for sunbathing.We hired a car and toured around there's pleanty of beaches and some nice countryside inland.Something for everyone to enjoy,Lidle is the best place to get your food stuffs but you need a car.Would'nt hesitate to recomend this appartment.
The apartment we rented at Quinta Da Praia in Alvor was first class.. It was very spacious, clean, comfortable and well furnished. The kitchen was really well equipped and there was loads of drawer and cupboard space. The location was perfect and although quite close to a bar we were never bothered with noise. The Al Dente restaurant is next door and I would certainly recommend trying it. The food is wonderful! I would certainly try and rent it again for our next visit to Alvor.


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Oyo State Governor Abiola Ajimobi and his wife, Florence, will on Saturday be conferred with the chieftaincy titles of Aare Atunluse and Yeye Aare Atunluse of Ibadanland (the beautifier and restorer of the dignity of Ibadan) by the Olubadan, Oba Samuel Odulana Odugade I.
The ceremony will hold at the Olubadan's palace in Monatan, Ibadan, the state capital.
In a statement yesterday, the governor's media aide, Dr. Festus Adedayo, said the chieftaincy titles were in recognition of Ajimobi's achievements in two years.
He said the Ajimobi administration's urban renewal programme and road projects have transformed Ibadan, which used to be the dirtiest city in Nigeria.
Eminent personalities from all walks of life are expected at the ceremony.


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Description and amenities
Main amenities
Apartment is located in the city center. 5 minutes walk to the pomp room. Total area 54 sq.m. The apartment is located on the 3 floor, the elevator is open 24 hours a day. The apartment has a large double bed and a folding sofa, TV, a big kitchen equipped with cooking facilities, underfloor heating, Internet access, washing machine. Cold and hot water round the clock. Near the House there is a parking. Autonomous heating system.
From the railway station (bus station) free shuttle service.
Maximum occupancy is 4 people.
Availability and rates
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8 Krushelnytskoyi Street, Truskavets
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Friday, January 30 – Leduc, Alberta
Day 1
Despite snowy conditions, the U.S. National Sled Hockey Team arrived safely in Leduc, Alberta, today for the World Sled Hockey Challenge that is set to begin Sunday, Feb. 1. Reunited a week after spending training camp in North Carolina, the team will get to work quickly. After checking into the hotel, the team will have lunch and then head to the Leduc Recreation Center for an 80-minute practice.
The 2015 World Sled Hockey Challenge is being staged at the 309,000 square foot Leduc Recreation Centre. The one-stop multi-use facility includes three hockey rinks, an aquatic centre, an eight-sheet BMO curling rink, a 9,000 square foot fitness center and track, and a field house that can accommodate badminton, basketball, floor hockey, pickle ball, volleyball and other sports.
Big Show, a WWE professional wrestler, was on the same plane to Edmonton as several members of the U.S. National Sled Hockey Team. The 7'0", 425 pound wrestler nicknamed the World's Largest Athlete spoke and took photos with some of the players, wishing them the best of luck for this year's World Sled Hockey Challenge. The seven-time world champion is in Edmonton for the WWE LIVE Road to WrestleMania event on Jan. 31.
No news currently found.
Tag(s): World Sled Challenge


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One of the latest ideas that has been floating around the the health and fitness-sphere these days is the concept of Intermittent Fasting, often referred to as IF. While not exactly the same as traditional dieting, IF is a method of controlled intake of food throughout the day, which claims to offer a host of health benefits and optimization of body composition.
The primary concept of IF is a longer than normal period of daily fasting, followed by more regular eating. For example, perhaps the easiest form of IF to follow is one where the individual eats their last meal at 8pm, skips any sort of breakfast, and doesn't eat again until noon, which is effectively a 16 hour daily fast.
Others subscribe to a more extreme version, whereby they don't eat for 24 hours or so. In other words, a day of normal eating, followed by an entire day of fasting. For many, it's much easier to stick to the much more moderate form of 16 hour fasts.
In fact, for someone like me and for many others, the 16-hour fast is something you don't even think about. Personally, I rarely eat breakfast anyways, and so this form of IF already falls right in line with my normal schedule, plus I receive the purported benefits of intermittent fasting:
- Increased fat loss: to begin with, employing intermittent fasting will allow someone's body to enter into the fasted state, which typically occurs about 12 hours after the most recent meal. Since most people don't go this long before eating their next meal, the body doesn't typically go into its natural thermogenesis (fat-burning) mode on a regular basis. However, by following intermittent fasting, your body goes into this state by happenstance, based just on the timing.
- Enhanced insulin sensitivity: now this is where things get really interesting. The working theory is that by following intermittent fasting, one is able to essentially reset/recalibrate their body's sensitivity to insulin. For those that don't know, insulin is the primary regulatory hormone in the human body. Without delving too far into the geeky rabbit hole, all you really need to know is that increased insulin sensitivity helps to lose fast and gain muscle from the food you eat. Essentially, it makes your body more efficient when it comes to processing what you put into it. The reason this works is because insulin sensitivity is higher when glycogen levels are depleted; and since glycogen levels are lower during a fasted state…well, I think you know where I'm going with this.
- Increased health benefits: beyond just the aesthetic benefits of IF, there are also reports that conclude intermittent fasting can actually increase the health of an individual and amazing, perhaps contribute to one's longevity. Now, I'm sure nothing is yet conclusive, but it is encouraging to hear that there is some correlation to IF and increased lifespan, at least in a research environment. I wouldn't go so far as to say this is definite just yet, but personally, that's just icing on the cake.
Pour Conclude…
All in all, intermittent fasting just fits in with my lifestyle. I tend to be on the run from the moment I wake up, and subsequently I rarely eat breakfast. Now, with all the information about IF out there that indicates it's actually helping me to become a sexy beast (yeah baby), consider me all-in.
Of course, like anything else, it's not a silver bullet. It's not a replacement for proper nutrition, regular exercise and restful sleep. That will always remain true. But, it's just one more bullet in my chamber. If you're interested in learning more, there's plenty of resources out there on the topic. I think I've learned the most from the site Leangains, which is run by Martin Berkhan who is one of the guys who really popularized IF. Check it out!


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Home to all things epic & awesome.
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Replace your scratched Oakley Elevate Goggle Lens with the original Plutonite lens to get your old gogs working like new. Plutonite offers zero-distortion, a high level of clarity, 100% UV and unsurpassed impact protection. - $45.00
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Listing Format: | Auction |
Current price : | $79.99 |
Starting bid: | $79.99 |
Shipping Cost: | |
# of bids: | 1 |
Closes: | Auction is closed |
Location: | California, United States |
Started: | 1/28/2014 4:40:26 PM |
Ended: | 1/31/2014 4:40:26 PM |
Seller: |
Ginger Lynn
View this user's other listings |
High Bidder(s): | Tater (409) |
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Mail this listing to a friend! Watch this item! |
Item Description
Well this is a VERY SPECIAL auction guys. I've been in Playboy, I've been in Penthouse, I've even shot numerous Penthouse Pets just for you. But this shoot and this girl are TRULY special. www.GingerLynnAuctions.com is pleased to offer to you here, and ONLY here, the most intimate and personal items of the
I've had trouble keeping my hands off of girls I've shot before, but Jenna is just special. Her personality is incredible, and her body is just to DIE FOR! Take a look at those breasts, her perfectly bald little pussy, those legs, and that face. Jenna is the definition of perfection… Imagine those legs wrapped around your head, those perfect titties in your mouth, her clit between your teeth as you finger her wet pussy. Well, that was actually me, but you can smell and taste the aftermath she left behind in each of these special items. And imagine it was you…
Because let me tell you, she is the real deal. The best that Penthouse has to offer. She just may be one of the best that women have to offer. She is that good!!! You can even catch her in some hard core action. Take a look guys, and then watch it with a pair of her lingerie wrapped around your cock.
It's the next best thing to being there…
A CD of ALL the photos from ALL the sets is also available for $79 (www.GingerLynnAuctions.com retains all rights to the photos). Check out auction #2349
This listing is for:
ONE (1) Black Bra and Black and White Zebra Striped Panties with Black Lace worn by JENNA ROSE
ONE (1) Autographed Polaroid of JENNA ROSE wearing the Bra and Panties
ONE (1) 8 X 10 color photo of JENNA ROSE wearing the Bra and Panties
ONE (1) Certificate of Authenticity
Postage and Handling
United States: $15.00 US Postal Service Priority Mail (2-3 business days)
International: $30.00 US Postal Service Priority International Mail
Handling time 5-10 business days
I will send an invoice at auctions end.
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Major sections: Historical Background and Locke's Life - The Limits of Human Understanding - Locke's Major Works on Education - The Two Treatises Of Government - Locke and Religious Toleration - Bibliography - Other Internet Resources
John Locke (29 August 1632 - 28 October 1704), was an English philosopher and physician regarded as one of the most influential of Enlightenment thinkers and known as the "Father of Classical Liberalism". Considered one of the first of the British empiricists, following the tradition of Francis Bacon, he is equally important to social contract theory. His work greatly affected the development of epistemology and political philosophy. His writings influenced Voltaire and Rousseau, many Scottish Enlightenment thinkers, as well as the American revolutionaries. His contributions to classical republicanism and liberal theory are reflected in the United States Declaration of Independence.
Major sections: Historical Background and Locke's Life - The Limits of Human Understanding - Locke's Major Works on Education - The Two Treatises Of Government - Locke and Religious Toleration - Bibliography - Other Internet Resources
Includes list of selected works
Major sections: Life - Method - Human Nature - Religion - Moral Philosophy - Political Philosophy - Assessment - References and Further Reading
Oregon State University, Philosophy Department: includes short biography, detailed timeline, links to his principal works (three at the same site) and list of selected texts about Locke
Web Pages
Includes short profile, picture and link to related articles by James Bovard, Roderick Long, Wendy McElroy, Jim Powell, Joseph Stromberg and others
Includes portrait, short biography, links to several related articles, to various versions of Locke's works and to selected quotations
Extensive biographical essay, including summaries of his major works
Short biographical essay including details on how the Essay Concerning Human Understanding came about
John Locke Foundation: explores Locke's principle of property rights and government's role in protecting them
Explains the absurdity of California Senator Barbara Boxer's statement that public lands are "owned ... by the American people"
Chapter 9; originally written in 1973 for the Enciclopedia del Novicento; covers both the history of both strands of liberalism as well as a systematic description of the "classical" or "evolutionary" type
Examines the arguments made by Rothbard from the premise that slavery is wrong, self-ownership, private property rights and a free market without government interventions follows
Volume II, Part II "Intercolonial Developments", Chapter 33: Starts by considering the influence of English writers Sidney and Locke and then considers Trenchard and Gordon's Cato's Letters
Examines Hillary Clinton's assertion about "wanting" a universal health care plan
Originally "Epistolia de Tolerentia", translated by William Popple
Books Authored
Electronic text available at Wikisource; contents: Book I: Neither Principles nor Ideas Are Innate - Book II: Of Ideas - Book III: Of Words - Book IV: Of Knowledge and Probability
- ISBN 9562910911: Audio CD, bnpublishing.com, 2006
- ISBN 0844624780: Hardcover, Peter Smith Publisher, 1959
- ISBN 0872202178: Hardcover, Hackett Pub Co, Abridged edition, 1996
- ISBN 0879759178: Paperback, Prometheus Books, 1994
Electronic text available at The University of Adelaide; partial contents: Of the State of Nature - Of the State of War - Of Slavery - Of Property - Of Paternal Power - Of Political or Civil Society - Of the Beginning of Political Societies
- ISBN 0879753374: Paperback, Prometheus Books, 1986
The introductory paragraph uses material from a Wikipedia article, which is released under the Creative Commons Attribution-Share-Alike License 3.0.


text:
First Look podcast: Wal-Mart, 10th SBAs Wal-Mart goes big ServiceMaster joins MiLB sponsor roster Holograms produce real results in sports Logano driving off-track studio concept Lefton Report: Pepsi challenge Toyota first in-ice sponsor of Knights Etihad renews MLS deal, plans content More NBA teams sign jersey sponsors Emirates to sponsor USA Rugby series
Upcoming Conferences and Events
Apr 19-20
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SBJ/December 10 - 16, 2001/Marketingsponsorship
UPS to gear up Jarrett campaign for all Winston Cup races
Published December 10, 2001
UPS said it will expand its popular campaign in which Dale Jarrett repeatedly refuses to drive the company's "big brown truck" during a NASCAR race.
While many companies are cutting back on advertising, the company expects to run television commercials next year during each of the 36 NASCAR Winston Cup races, up from 32 races with commercials this year.
UPS public relations manager Susan Rosenberg said shooting will start in January and the first ad will run during the Daytona 500 on Feb. 17 on NBC. The Martin Agency in Richmond, Va., is producing the ads.
The company also is increasing its print campaign, expanding into auto-supply publications. Rosenberg wouldn't comment on the cost of the entire ad campaign.
"The campaign has had a lot of legs," she said. "We've had a lot of fun with it."
UPS was named marketing company of the year during the recent NASCAR banquet in New York, in part because of its 12 Jarrett ads, said Robert Conner, vice president of corporate sponsorships and events for UPS.
Daily traffic at the company's racing Web site, racing.ups.com, jumped threefold in July when it was announced that Jarrett's UPS-sponsored car would be painted like a UPS truck, and that the changes could be viewed on the site.
"One weekend when I was flying to a race, I'm wearing my UPS racing shirt and an elderly lady asked, 'Are you going to try to get Dale to drive that truck?' " Conner said.
Will Jarrett drive it in 2002, when the ads appear on NBC, TNT, Fox and FX? Rosenberg was coy. "You're going to have to wait and see," she said.


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Holy Bible: New Revised Standard Version (NRSV) Anglicised Cross-Reference Edition Hardback
For the first time the Anglicized Edition of the New Revised Standard Version is available from Collins, in a new centre column cross-reference setting, to create the best-ever edition of the translation most widely-used across the churches.
The New Revised Standard Version of the Bible is steadily increasing in popularity.
It is highly respected among academics, including evangelicals, and used in theological colleges across the board as the most accurate translation available.
It has been widely adopted across the Church of England and is in the process of being adopted for use in the Catholic liturgy.
The NRSV is an accurate translation in the tradition of the King James Version, with special sensitivity to gender inclusiveness, without any loss of clarity, and retaining the traditional style of referring to God as 'He' or 'Him.' Completely reset in the highly-respected Anglicized Edition, along with cross references in all the standard text Bibles, this is the ideal Bible for personal reading, academic study, and for church use.
- Format: Hardback
- Pages: 1152 pages
- Publisher: HarperCollins Publishers
- Publication Date: 10/02/2007


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Let us know when your receive great advice so we can thank our lawyers and point others with similar questions in the right direction.
Got it!I have been on felony probation for the sale of marijuana for almost 2 years now. I was supposed to petition for early probation violation in a few months, but I was recently arrested in the state of Nevada for an alleged DUI.
I have contacted my probation officer, and they said they will want to see what happens with the DUI court case. I didn't want to bother my p.o. that much so I didn't ask any further questions.
Does this mean that if my DUI court case goes well (which I think I can get it dismissed or dropped to a lesser crime), and my p.o. decides not to file a violation of probation, that nothing will happen? Or will the court eventually find out and attempt to violate my probation?
This is a general answer...but generally, a DUI arrest while on Probation will raise a probation violation issue. Even if the court case for the new charge goes well, usually there would still be a probation violation hearing that can take place separate and apart from the new criminal charge. That being said, the better the result of the new charge, the better off you will be for the probation violation, either getting it dismissed, or minimizing penalty if the probation violation is found. I would highly recommend that you speak to a good DUI attorney in NV where the new charge is, and your attorney, or another criminal defense attorney in San Jose regarding the probation violation issue.
Legal disclaimer: This message does not constitute legal advice and is for informational purposes only. This message does not establish an attorney-client relationship, which can only be established once a retainer agreement has been fully executed between you and this firm.
If my PO didn't violate me, does that mean the court will not know about it? Or will they find out eventually?
Just because your P.O. did not immediately take action, does not mean that it will go away. Did you mention the issue to the attorney who handled the underlying matter? That, or contacting another Attorney in your area to discuss your situation in more detail is important. I am assuming your probation is out of San Jose since that is the city that you listed to coincide with this post. I do not practice in San Jose, so although I can give a general answer, an attorney familiar with the Courts in San Jose would be in a better position to advise once they knew more information about your situation. Many Attorneys give free initial consultations, so making a call at minimum is a good idea. There are many qualified attorneys in your area that are on Avvo. In the event you would like a referral, let me know.
It is impossible to predict the outcome, as my colleague suggests.
The fact that your PO did not violate you for the arrest is a good sign. Continue to comply in all other respects with your Probation, and make sure you have a capable attorney to represent you on your pending DUI/DWI. Many such attorney post here on Avvo, and this is an excellent source to find capable representation.
Good luck to you,
If my PO didn't violate me, does that mean the court will not know about it? Or will they find out eventually?
I assume they will find out about it - a court may request a PO file a UCR (Uniform Court Report) or direct the PO to file a violation. There are a number of different scenarios all of which are best discussed in person with an attorney who can assist you and not in a public forum.
If you had an attorney on the felony case, I suggest giving them a call to make them aware of the current situation. I also suggest hiring a locally experienced DUI attorney for the Vegas case. Having an attorney will generally increase your chances of a positive outcome. Best of luck.
Jasen Nielsen
If my PO didn't violate me, does that mean the court will not know about it? Or will they find out eventually?
It depends on the outcome of the Vegas case.
Everyone's answer is accurate, except that you need to recall that it is not the PO but the court who violates your probation. Unless aggravated, an out of state misd. DUI is not likely to result in any serious probation issues here.
Get an attorney local to you that can handle any potential violation of probation and hire an attorney in nv to fight your DUI. Good luck
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Across the ocean in Canada, folks are getting ready for the implementation of a major new anti-spam law, which comes into effect from 1 July this year.
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Under the terms of the law, Canadian companies who have already got your consent to email you have a 36 month grace period in which continued consent to email is implied.
But if they can get you to consent to receiving commercial emails before 1 July, the 36 month grace period no longer applies, and you will receive email until you choose to opt out.
To this end, businesses that happen to operate in Canada have begun sending a tide of emails requesting reconfirmation that it is okay to email, and it is starting to look, well, a bit like spam.
I really don't think the Canadians have thought this one through. But typically, they are being very polite about the whole thing.


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Hi, I'm not new on mac, in fact I've been using mac since '85, 21 years (wow time goes fast), I post here because I'm having trouble with my 12" 867Mhz Superdrive PowerBook G4, The battery is pissing me off, It says it's full and when it's on 95% it goes to sleep, I connect the power adaptor and it says battery is 0% and starts charging, it never says XX time to go, is always calculatieng and when it's in 5% it turns the green led on and says Battery Fully Charged. I don't know what's happening, I did the ctrl+option+shift+power 5 seconds, and it resets the date( so it works) but no change in battery. Does anyone know how to fix it. Thanks Sorry about my english
You might be entitled to a new batter under the recent battery recall. I have the exact same machine and got a replacement battery.


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