Autotetraploid Emergence via Somatic Embryogenesis in Vitis vinifera Induces Marked Morphological Changes in Shoots, Mature Leaves, and Stomata.

Polyploidy plays an important role in plant adaptation to biotic and abiotic stresses. Alterations of the ploidy in grapevine plants regenerated via somatic embryogenesis (SE) may provide a source of genetic variability useful for the improvement of agronomic characteristics of crops. In the grapevine, the SE induction process may cause ploidy changes without alterations in DNA profile. In the present research, tetraploid plants were observed for 9.3% of 'Frappato' grapevine somatic embryos regenerated in medium supplemented with the growth regulators ß-naphthoxyacetic acid (10 µM) and N6-benzylaminopurine (4.4 µM). Autotetraploid plants regenerated via SE without detectable changes in the DNA profiles were transferred in field conditions to analyze the effect of polyploidization. Different ploidy levels induced several anatomical and morphological changes of the shoots and mature leaves. Alterations have been also observed in stomata. The length and width of stomata of tetraploid leaves were 39.9 and 18.6% higher than diploids, respectively. The chloroplast number per guard cell pair was higher (5.2%) in tetraploid leaves. On the contrary, the stomatal index was markedly decreased (12%) in tetraploid leaves. The observed morphological alterations might be useful traits for breeding of grapevine varieties in a changing environment.

Evaluating the radioprotective effect of single dose and daily oral consumption of green tea, grape seed, and coffee bean extracts against gamma irradiation.

INTRODUCTION: The aim of this study was to investigate and compare the radio-protective effect of green tea, grape seed, and coffee bean extracts in different oral consumption methods in mice. MATERIALS AND METHODS: In this experimental-quantitative study 150 mice in 15 equally sized groups were used. For each extract, two groups received 200 mg/kg of herbal extracts' combination for 7 and 30 consecutive days before irradiation, and one group received 800 mg/kg of the extract 2 h before irradiation (3 Gy gamma-rays of Co-60). The similar groups were classified to receive a combination of the plant extracts (green tea, grape seed, and coffee bean). Irradiation without consuming plant extract (irradiated group), and a control group were also devised. Alkaline comet and micronucleus assays were used to investigate the radioprotective effect on mice blood and bone marrow cells, respectively. RESULTS: Consumption of all plant extracts significantly decreased the radiation damage to blood and bone marrow cells, compared to the irradiated group (p < 0.01), with grape seed extract showing higher protective effect. Continuous daily oral consumption (one week/month) showed a significant higher radioprotective effect compared to single consumption (p < 0.05). Continuous consumption of the combination of the extracts showed a higher radio-protection in comparison to each of the plant extracts (p < 0.03). CONCLUSIONS: The radioprotective effect of continuous consumption (for one week/month) of the plant extracts was greater than single dose. In continuous consumption protocols, we found the synergetic property and higher radioprotective effect of the plant extract combination compared to each one.

Valorisation of black mulberry and grape seeds: Chemical characterization and bioactive potential.

Grape (Vitis vinifera L. var. Albariño) and mulberry (Morus nigra L.) seeds pomace were characterized in terms of tocopherols, organic acids, phenolic compounds and bioactive properties. Higher contents of tocopherols (28 ± 1 mg/100 g fw) were obtained in mulberry, whilst grape seeds were richer in organic acids (79 ± 4 mg/100 g fw). The phenolic analysis of hydroethanolic extracts characterised grape seeds by catechin oligomers (36.0 ± 0.3 mg/g) and mulberry seeds by ellagic acid derivatives (3.14 ± 0.02 mg/g). Both exhibited high antimicrobial activity against multiresistant Staphylococcus aureus MIC = 5 mg/mL) and no cytotoxicity against carcinogenic and non-tumour primary liver (PLP) cells. Mulberry seeds revealed the strongest inhibition (p < 0.05) against thiobarbituric reactive substances (IC50 = 23 ± 2 µg/mL) and oxidative haemolysis (IC50 at 60 min = 46.0 ± 0.8 µg/mL). Both seed by-products could be exploited for the developing of antioxidant-rich ingredients with health benefits for industrial application.

Enzyme Inhibition and Antimicrobial Activities of Fractionated Wild Grape (Ampelocissus martinii Planch.) Seed Extracts.

BACKGROUND AND OBJECTIVE: Study on medicinal plant extract is gradually interested and distributed, especially their biological activities. The present study aimed to determine the enzyme inhibition and antimicrobial activities of the fractionated extracts of wild grape (Ampelocissus martinii Planch.) seeds. MATERIALS AND METHODS: Wild grape seeds in different growth stages were extracted with methanol before fractionation by silica gel chromatography. The anti-glucosidase and anti-tyrosinase enzyme activities of the extracts were then tested by using UV-Vis spectrophotometry and antimicrobial activities were observed from MIC, MBC values and time killing assay. RESULTS: The sub-fraction of immature stage eluted by ethyl acetate/methanol at 75/25 (%v/v) has the highest enzyme inhibition activity and the most potent efficiency for time kills profiles. The MIC values of the potent immature, mature and ripe fractioned extracts were ranging from 1.25-50.00, 1.25-50.00 and 1.56-25.00 mg mL-1, respectively, while the MBC values ranged from 3.12-6.25, 3.12-25.00 and 3.12-25.00 mg mL-1, respectively. CONCLUSION: The wild grape seed composed of α-glucosidase and tyrosinase inhibition and antibacterial activities compounds. The wild grape seed extracts may be used as active ingredients sources of health-supporting products or cosmetics.

Genomic Organization of the B3-Domain Transcription Factor Family in Grapevine (Vitis vinifera L.) and Expression during Seed Development in Seedless and Seeded Cultivars.

Members of the plant-specific B3-domain transcription factor family have important and varied functions, especially with respect to vegetative and reproductive growth. Although B3 genes have been studied in many other plants, there is limited information on the genomic organization and expression of B3 genes in grapevine (Vitis vinifera L.). In this study, we identified 50 B3 genes in the grapevine genome and analyzed these genes in terms of chromosomal location and syntenic relationships, intron-exon organization, and promoter cis-element content. We also analyzed the presumed proteins in terms of domain structure and phylogenetic relationships. Based on the results, we classified these genes into five subfamilies. The syntenic relationships suggest that approximately half of the genes resulted from genome duplication, contributing to the expansion of the B3 family in grapevine. The analysis of cis-element composition suggested that most of these genes may function in response to hormones, light, and stress. We also analyzed expression of members of the B3 family in various structures of grapevine plants, including the seed during seed development. Many B3 genes were expressed preferentially in one or more structures of the developed plant, suggesting specific roles in growth and development. Furthermore, several of the genes were expressed differentially in early developing seeds from representative seeded and seedless cultivars, suggesting a role in seed development or abortion. The results of this study provide a foundation for functional analysis of B3 genes and new resources for future molecular breeding of grapevine.

Non-Destructive and Rapid Variety Discrimination and Visualization of Single Grape Seed Using Near-Infrared Hyperspectral Imaging Technique and Multivariate Analysis.

Hyperspectral images in the spectral range of 874⁻1734 nm were collected for 14,015, 14,300 and 15,042 grape seeds of three varieties, respectively. Pixel-wise spectra were preprocessed by wavelet transform, and then, spectra of each single grape seed were extracted. Principal component analysis (PCA) was conducted on the hyperspectral images. Scores for images of the first six principal components (PCs) were used to qualitatively recognize the patterns among different varieties. Loadings of the first six PCs were used to identify the effective wavelengths (EWs). Support vector machine (SVM) was used to build the discriminant model using the spectra based on the EWs. The results indicated that the variety of each single grape seed was accurately identified with a calibration accuracy of 94.3% and a prediction accuracy of 88.7%. An external validation image of each variety was used to evaluate the proposed model and to form the classification maps where each single grape seed was explicitly identified as belonging to a distinct variety. The overall results indicated that a hyperspectral imaging (HSI) technique combined with multivariate analysis could be used as an effective tool for non-destructive and rapid variety discrimination and visualization of grape seeds. The proposed method showed great potential for developing a multi-spectral imaging system for practical application in the future.

Somatic embryogenesis from seeds in a broad range of Vitis vinifera L. varieties: rescue of true-to-type virus-free plants.

BACKGROUND: Somatic embryogenesis is the preferred method for cell to plant regeneration in Vitis vinifera L. However, low frequencies of plant embryo conversion are commonly found. In a previous work we obtained from cut-seeds of a grapevine infected with the Grapevine leafroll associated viruses 1 and 3 (GLRaV-1 and GLRaV-3), high rates of direct regeneration, embryo plant conversion and sanitation. The aim of this study is to evaluate the usefulness of this procedure for regeneration of other grapevine varieties which include some infected with one to three common grapevine viruses (GLRaV-3, Grapevine fanleaf virus (GFLV) and Grapevine fleck virus (GFkV)). As grapevine is highly heterozygous, it was necessary to select from among the virus-free plants those that regenerated from mother tissues around the embryo, (true-to-type). RESULTS: Somatic embryogenesis and plant regeneration were achieved in a first experiment, using cut-seeds from the 14 grapevine varieties Airén, Cabernet Franc, Cabernet Sauvignon, Mencía, Merlot, Monastrell, Petit Verdot, Pinot Blanc (infected by GFLV and GFkV), Pinot Gris, Pinot Meunier, Pinot Noir, Syrah, Tempranillo (infected by GFLV), and Verdil. All regenerated plants were confirmed to be free of GFkV whereas at least 68% sanitation was obtained for GFLV. The SSR profiles of the virus-free plants showed, in both varieties, around 10% regeneration from mother tissue (the same genetic make-up as the mother plant). In a second experiment, this procedure was used to sanitize the varieties Cabernet Franc, Godello, Merlot and Valencí Blanc infected by GLRaV-3, GFkV and/or GFLV. CONCLUSIONS: Cut-seeds can be used as explants for embryogenesis induction and plant conversion in a broad range of grapevine varieties. The high regeneration rates obtained with this procedure facilitate the posterior selection of true-to-type virus-free plants. A sanitation rate of 100% was obtained for GFkV as this virus is not seed-transmitted. However, the presence of GLRaV-3 and GFLV in some of the regenerated plants showed that both viruses are seed-transmitted. The regeneration of true-to-type virus-free plants from all infected varieties indicates that this methodology may represent an alternative procedure for virus cleaning in grapevine.

Ameliorative Effects of Grape Seed Proanthocyanidin Extract on Growth Performance, Immune Function, Antioxidant Capacity, Biochemical Constituents, Liver Histopathology and Aflatoxin Residues in Broilers Exposed to Aflatoxin B1.

Aflatoxicosis is a grave threat to the poultry industry. Dietary supplementation with antioxidants showed a great potential in enhancing the immune system; hence, protecting animals against aflatoxin B1-induced toxicity. Grape seed proanthocyanidin extract (GSPE) one of the most well-known and powerful antioxidants. Therefore, the purpose of this research was to investigate the effectiveness of GSPE in the detoxification of AFB1 in broilers. A total of 300 one-day-old Cobb chicks were randomly allocated into five treatments of six replicates (10 birds per replicate), fed ad libitum for four weeks with the following dietary treatments: 1. Basal diet (control); 2. Basal diet + 1 mg/kg AFB1 contaminated corn (AFB1); 3. Basal diet + GSPE 250 mg/kg; (GSPE 250 mg/kg) 4. Basal diet + AFB1 (1 mg/kg) + GSPE 250 mg/kg; (AFB1 + GSPE 250 mg/kg) 5. Basal diet + AFB1 (1mg/kg) + GSPE 500 mg/kg, (AFB1 + GSPE 500 mg/kg). When compared with the control group, feeding broilers with AFB1 alone significantly reduced growth performance, serum immunoglobulin contents, negatively altered serum biochemical contents, and enzyme activities, and induced histopathological lesion in the liver. In addition, AFB1 significantly increased malondialdehyde content and decreased total superoxide dismutase, catalase, glutathione peroxide, glutathione-S transferase, glutathione reductase activities, and glutathione concentration within the liver and serum. The supplementation of GSPE (250 and 500 mg/kg) to AFB1 contaminated diet reduced AFB1 residue in the liver and significantly mitigated AFB1 negative effects. From these results, it can be concluded that dietary supplementation of GSPE has protective effects against aflatoxicosis caused by AFB1 in broiler chickens.

Cultivar-specific gene modulation in Vitis vinifera: analysis of the promoters regulating the expression of WOX transcription factors.

The family of Wuschel-related Homeobox (WOX) genes is a class of transcription factors involved in the early stages of embryogenesis and organ development in plants. Some of these genes have shown different transcription levels in embryogenic tissues and mature organs in two different cultivars of Vitis vinifera: 'Chardonnay' (CH) and 'Cabernet Sauvignon' (CS). Therefore, we investigated the genetic basis responsible for these differences by cloning and sequencing in both the cultivars the promoter regions (~2000 bp) proximal to the transcription start site of five VvWOX genes. We then introduced these promoters into Arabidopsis thaliana for expression pattern characterisation using the GUS reporter gene. In the transgenic Arabidopsis, two promoters isolated from CS (pVvWOX13C_CS and pVvWOX6_CS) induced increased expression compared to the sequence isolated in CH, confirming the data obtained in grapevine tissues. These results were corroborated by transient expression assays using the agroinfiltration approach in grapevine somatic embryos. Truncated versions of pVvWOX13C demonstrated that few nucleotide differences between the sequences isolated from CH and CS are pivotal for the transcriptional regulation of VvWOX13C. Analysis of promoters using heterologous and homologous systems appear to be effective for exploring gene modulation linked with intervarietal sequence variation in grapevine.

Molecularly Imprinted Dispersive Solid-Phase Extraction for the Determination of Triazine Herbicides in Grape Seeds by High-Performance Liquid Chromatography.

Molecular imprinting technique, regarded as one of the current state-of-the-art researches, was incorporated with the simple dispersive solid-phase extraction (MI-DSPE) in this work for the extraction of triazine herbicides in grape seeds. The atrazine molecularly imprinted polymers (MIPs) were successfully prepared and characterized by scanning electron microscopy and Fourier transform infrared spectroscopy. The imprinting particles were used as the adsorbent in DSPE. Thus, a simple, rapid and selective method based on MIPs coupled with DSPE was established for the simultaneous cleaning-up and quantitative extraction of four triazine herbicides in grape seeds. The experiment parameters, including type of washing solvents, washing time and type of eluting solvents, were investigated and optimized. The performance of the present method was validated by high-performance liquid chromatography. Good linear responses were obtained in concentration range of 0.010-5.0 µg g(-1)with correlation coefficients (r(2)) higher than 0.9993. The recoveries at two spiked levels (1.0 and 2.0 µg g(-1)) were between 81.2 and 113.0% with relative deviations ranging from 1.2 to 10.7%. The limits of detection were ranged between 0.006 and 0.013 µg g(-1), which were lower than the values required by European regulations.

Formulation and evaluation on human skin of a water-in-oil emulsion containing Muscat hamburg black grape seed extract.

BACKGROUND: Vitis vinifera 'muscat hamburg' (Vitaceae) is a blue-black grape variety commonly found in Pakistan. It has been consumed and used in traditional medicine for centuries. Compared to other grapes, M. hamburg records one of the greatest amount of polyphenols and displays potent antioxidant activities, which make it a great candidate for its exploitation in the development of stable cream emulsions destined to improve the skin appearance. OBJECTIVE: Evaluate the effects of stable water-in-oil (W/O) emulsion containing 2% M. hamburg grape seed extract ('formulation') on human cheek skin in comparison with the placebo ('base'). METHODS: An occlusive patch test, containing either the formulation or the base, was topically tested for 8 weeks during a winter period in young adult and healthy Pakistani male volunteers. The subjects were instructed to use twice a day the base and the formulation on their right and left cheek skin, respectively. Non-invasive measurements on these skin areas were carried out every week to assess any effects produced on melanin, elasticity and sebum. Skin compatibility assay (Burchard test) was used to report any potential skin reactivity. ANOVA, paired sample t-test and LSD test were applied to determine the statistical data significance. RESULTS: Significant differences (P ≤ 0.05) were found between the placebo and the formulation in terms of their respective skin effects elicited on melanin, elasticity and sebum content. Nevertheless, placebo and formulation exerted similar effects on skin erythema and moisture contents. Importantly, no skin hypersensitivity cases were reported during the whole course of the study. CONCLUSION: The developed grape-based cream could be efficiently and safely applied to improve a number of skin conditions (e.g. hyper-pigmentation, premature ageing, acne).

Determination of abscisic acid and its glucosyl ester in embryogenic callus cultures of Vitis vinifera in relation to the maturation of somatic embryos using a new liquid chromatography-ELISA analysis method.

The levels of abscisic acid (ABA), its conjugate ABA-GE, and IAA were determined in embryogenic calli of Vitis vinifera L. (cv. Mencía) cultured in DM1 differentiation medium, to relate them to the maturation process of somatic embryos. To achieve this goal, we developed an analytical method that included two steps of solid-phase extraction, chromatographic separation by HPLC, ABA-GE hydrolysis, and sensitive ELISA quantification. Because the ABA immunoassay was based on new polyclonal antibodies raised against a C4'-ABA conjugate, the assay was characterized (detection limit, midrange, measure range, and cross-reaction) and validated by a comparison of the ABA data obtained with this ELISA procedure and with a physicochemical method (LC-ESI-MS/MS). Radioactive-labeled internal standards were initially added to callus extracts to correct the losses of plant hormones, and thus assure the accuracy of the measurements. The endogenous concentration of ABA in the embryogenic callus cultured in DM1 medium was doubled at the fifth week of culture, concurring with the maturation process of somatic embryos, as indicated by the accumulation of carbohydrates observed through histological analysis. The ABA-GE content was higher than ABA, decreasing at 21 days of culture in DM1 medium but increasing thereafter. The data suggest the involvement of the synthesis and conjugation of ABA in the final stages of development in grapevine somatic embryos from embryogenic callus. IAA levels were low, suggesting that auxin plays no significant role during the maturation of somatic embryos. In addition, the lower ABA levels in calli cultured in DM differentiation medium with PGRs, a medium presenting high precocious germination and deficiencies in somatic embryo development indicate that an increase in ABA content during the development of somatic embryos in grapevine is necessary for their correct maturation.

Cryopreservation of embryogenic cell suspensions by encapsulation-vitrification and encapsulation-dehydration.

Encapsulation-vitrification and encapsulation-dehydration are two newly developed techniques for cryopreservation of embryogenic cell suspensions. Here, we describe the two protocols using grapevine (Vitis) as a model plant. Cell suspensions at the exponential growth stage cultured in a cell suspension maintenance medium are encapsulated to form beads, each being about 4 mm in diameter and containing 25% cells. In the encapsulation-vitrification procedure, the beads are stepwise precultured in increasing concentrations of sucrose medium up to 0.75 M, with 1 day for each concentration. The precultured beads are treated with a loading solution for 60 min and then dehydrated with plant vitrification solution 2 at 0°C for 270 min before a direct immersion in liquid nitrogen. Following cryostorage, the beads are rapidly rewarmed at 40°C for 3 min and then unloaded with 1 M sucrose solution for 30 min. In the encapsulation-dehydration procedure, the beads are precultured in increasing concentrations of sucrose medium up to 1 M, with 1 day for each concentration, and then maintained on 1 M sucrose medium for 3 days. The precultured beads are dehydrated for 6 h under a sterile air flow, prior to rapid freezing in liquid nitrogen. The freezing and rewarming procedures are the same as used in the encapsulation-vitrification technique. The unloaded beads from encapsulation-vitrification and rewarmed beads from encapsulation-dehydration are postcultured on a recovery medium for 3 days at 25°C in the dark for survival. Surviving cells are transferred to a regrowth medium to induce cell proliferation. Embryogenic cell suspensions are reestablished by suspending the cells in a cell suspension maintenance medium maintained on a gyratory shaker at 25°C in the dark. For plant regeneration, surviving cells are transferred from the recovery medium to an embryo maturation medium and maintained at 25°C under light conditions. Embryos at the torpedo stage are cultured on a rooting medium until whole plantlet regenerates.

Transient expression of artificial microRNAs targeting Grapevine fanleaf virus and evidence for RNA silencing in grapevine somatic embryos.

Grapevines are affected worldwide by viruses that compromise fruit yield and quality. Grapevine fanleaf virus (GFLV) causes fanleaf degeneration disease, a major threat to grapevine production. Transgenic approaches exploiting the RNA silencing machinery have proven suitable for engineering viral resistance in several crop species. However, the artificial microRNA (amiRNA)-based strategy has not yet been reported in grapevine. We developed two amiRNA precursors (pre-amiRNAs) targeting the coat protein (CP) gene of GFLV and characterised their functionality in grapevine somatic embryos. To create these pre-amiRNAs, natural pre-miR319a of Arabidopsis thaliana was modified by overlapping PCR in order to replace miR319a with two amiRNAs targeting different regions of the CP gene: amiR(CP)-1 or amiR(CP)-2. Transient expression of these two pre-amiRNA constructs was tested in grapevine somatic embryos after co-cultivation with Agrobacterium tumefaciens. Expression of amiR(CP)-1 and amiR(CP)-2 was detected in plant tissues by an endpoint stem-loop RT-PCR as early as 1 day after a 48-h co-cultivation, indicating active processing of pre-amiRNAs by the plant machinery. In parallel, GUS-sensor constructs (G(CP)-1 and G(CP)-2) were obtained by fusing the target sequence of amiR(CP)-1 or amiR(CP)-2 to the 3' terminus of the GUS gene. Co-transformation assays with GUS-sensors and the pre-amiRNA constructs provided evidence for in vivo recognition and cleavage of the 21-nt target sequence of GUS-sensors by the corresponding amiRNA. This is the first report of amiRNA ectopic expression in grapevine. The constructs we developed could be useful for engineering GFLV-resistant grapes in the future.

Co-transformation of grapevine somatic embryos to produce transgenic plants free of marker genes.

A cotransformation system using somatic embryos was developed to produce grapevines free of selectable marker genes. This was achieved by transforming Vitis vinifera L. "Thompson Seedless" somatic embryos with a mixture of two Agrobacterium strains. The first strain contained a binary plasmid with an egfp gene of interest between the T-DNA borders. The second strain harbored the neomycin phosphotransferase (nptII) gene for positive selection and the cytosine deaminase (codA) gene for negative selection, linked together by a bidirectional dual promoter complex. Our technique included a short positive selection phase of cotransformed somatic embryos on liquid medium containing 100 mg/L kanamycin before subjecting cultures to prolonged negative selection on medium containing 250 mg/L 5-fluorocytosine.

Initiation and transformation of grapevine embryogenic cultures.

Protocols for the production and transformation of grapevine embryogenic cultures are described. Embryogenic cultures are initiated from leaves or stamens and pistils and transformed with Agrobacterium containing an enhanced green fluorescent protein/neomycin phosphotransferase II (egfp/nptII) fusion gene. Cultures are transferred to induction medium in the dark for callus formation and proliferation. Resulting cultures are transferred to somatic embryo development medium to induce secondary embryogenesis and formation of transgenic somatic embryos. Transgenic embryos identified on the basis on GFP fluorescence and kanamycin resistance are transferred to germination medium to regenerate transgenic plants. The presence of transgenes in independent plant lines is confirmed by PCR.

Determination of vitamin E isomers of grape seeds by high-performance liquid chromatography-UV detection.

A simple analytical method for the determination of vitamin E isomers in grape seeds by reversed-phase high-performance liquid chromatography with UV detection is described. The method is based on a solid-liquid extraction separation on an ODS column, and the analytes are monitored at 295 nm with a UV detector. Tocopherols are extracted in n-hexane and directly injected onto the column without using any purification step, such as saponification, prior to the separation and determination. The chromatographic separation of tocopherols is achieved in 12 min with a mobile phase that consists of n-hexane and isopropyl alcohol (99.99:0.01, v/v). The method is reproducible and accurate, with respect to demonstrating a relative standard deviation between 2.57% and 3.30% (n = 10, for 500 ng/mL) and a relative error between 0.84% and 6.54% (n = 10, for 500 ng/mL), respectively. The theoretical limits are estimated as 25 ng/mL for α-tocopherol, 43 ng/mL for γ-tocopherol, and 83 ng/mL for δ-tocopherols. The method is then applied for the determination of tocopherols in grape seeds grown in Turkey. The amounts of tocopherols are calculated by using the standard addition method.

Generation of reactive oxygen species by grape seed extract causes irreparable DNA damage leading to G2/M arrest and apoptosis selectively in head and neck squamous cell carcinoma cells.

Head and neck squamous cell carcinoma (HNSCC) accounts for 6% of all malignancies in USA and unfortunately the recurrence of secondary primary tumors and resistance against conventional treatments decrease the overall 5 year survival rate in HNSCC patients. Thus, additional approaches are needed to control HNSCC. Here, for the first time, employing human HNSCC Detroit 562 and FaDu cells as well as normal human epidermal keratinocytes, we investigate grape seed extract (GSE) efficacy and associated mechanism in both cell culture and nude mice xenografts. GSE selectively inhibited the growth and caused cell cycle arrest and apoptotic death in both Detroit 562 and FaDu cells by activating DNA damage checkpoint cascade, including ataxia telangiectasia mutated/ataxia telangiectasia-Rad3-related-checkpoint kinase 1/2-cell division cycle 25C as well as caspases 8, 9 and 3. Consistent with these results, GSE treatment resulted in a strong DNA damage and a decrease in the levels of DNA repair molecules breast cancer gene 1 and Rad51 and DNA repair foci. GSE-caused accumulation of intracellular reactive oxygen species was identified as a major mechanism of its effect for growth inhibition, DNA damage and apoptosis, which was remarkably reversed by antioxidant N-acetylcysteine. GSE feeding to nude mice decreased Detroit 562 and FaDu xenograft tumor growth by 67 and 65% (P < 0.001), respectively. In immunohistochemical analysis, xenografts from GSE-fed groups showed decreased proliferation but increased DNA damage and apoptosis. Together, these findings show that GSE targets both DNA damage and repair and provide mechanistic insights for its efficacy selectively against HNSCC both in cell culture and mouse xenograft, supporting its translational potential against HNSCC.

Effects of grape seed proanthocyanidin extract on oxidative stress induced by diabetes in rat kidney.

BACKGROUND: This study examined the effect of grape seed proanthocyanidin extract (GSPE) on lipid peroxidation content and activity of tissue antioxidant enzymes, including catalase, superoxide dismutase and glutathione peroxidase in diabetic rats. METHODS: Thirty male rats were divided into three groups of 10 rats each: control, diabetic and diabetic groups that received 500 mg/kg GSPE for 6 weeks. Diabetes was induced by a single intraperitoneal injection of streptozotocin (50 mg/kg body weight). Rats with fasting blood glucose levels above 250 mg/dl were used as diabetic animals. The first 24-hour urinary albumin excretion (UAE) was measured two weeks after diabetes induction and then each week until the end of the experimental period in all groups. Lipid peroxidation content and activities of catalase, superoxide dismutase and glutathione peroxidase were measured in kidney homogenate supernatants. Statistical significance of differences was assessed with one-way ANOVA by SPSS followed by Tukey's t-test. P < 0.05 was assumed statistically significant. RESULTS: UAE in diabetic nephropathy rats were significantly higher than in control. In addition, an increase in lipid peroxidation content and decrease in catalase, superoxide dismutase and glutathione peroxidase activities in kidney of diabetic nephropathy rats were observed. The GSPE administration did not affect on body weight, but significantly decreased lipid peroxidation and augmented the activities of antioxidant enzymes studied in kidney of diabetic nephropathy rats as well as reduced UAE and decreased kidney weight. CONCLUSION: The results suggested that GSPE could ameliorate diabetic nephropathy rats through reduction of oxidative stress and increase in renal antioxidant enzyme activity.

Proteomic changes in grape embryogenic callus in response to Agrobacterium tumefaciens-mediated transformation.

Agrobacterium tumefaciens-mediated transformation is highly required for studies of grapevine gene function and of huge potential for tailored variety improvements. However, grape is recalcitrant to transformation, and the underlying mechanism is largely unknown. To better understand the overall response of grapevine to A. tumefaciens-mediated transformation, the proteomic profile of cv. Prime embryogenic callus (EC) after co-cultivation with A. tumefaciens was investigated by two-dimensional electrophoresis and MALDI-TOF-MS analysis. Over 1100 protein spots were detected in both inoculated and control EC, 69 of which showed significantly differential expression; 38 of these were successfully identified. The proteins significantly up-regulated 3 d after inoculation were PR10, resistance protein Pto, secretory peroxidase, cinnamoyl-CoA reductase and different expression regulators; down-regulated proteins were ascorbate peroxidase, tocopherol cyclase, Hsp 70 and proteins involved in the ubiquitin-associated protein-degradation pathway. A. tumefaciens transformation-induced oxidative burst and modified protein-degradation pathways were further validated with biochemical measurements. Our results reveal that agrobacterial transformation markedly inhibits the cellular ROS-removal system, mitochondrial energy metabolism and the protein-degradation machinery for misfolded proteins, while the apoptosis signaling pathway and hypersensitive response are strengthened, which might partially explain the low efficiency and severe EC necrosis in grape transformation.