Detection and molecular characterization of Slovak tomato isolates belonging to two recombinant strains of potato virus Y.

Total RNAs from a symptomless tomato plant were subjected to next-generation sequencing (NGS) analysis, revealing the presence of a single viral agent - potato virus Y (PVY). The analysis of determined full-length genome sequence assigned the PVY SL16 isolate to the recombinant PVY-N-Wi strain group. A series of primers targeting the four main recombinant junction (RJ) sites were used for characterization of additional 5 tomato PVY isolates recovered in Western Slovakia. Based on the partial sequences, the isolates could be classified as belonging to PVY-N-Wi and PVY-NTNa strain groups. Interestingly, both these distinct recombinant PVY types were identified in mixed infection in one tomato sample (SL31). Our results further reinforce the data on the complexity of PVY infection and confirm the recombination as a significant evolutionary process shaping the PVY diversity.

Cloning of the complete infectious cDNA of the plum pox virus strain PPV-Rec.

UNLABELLED: Plum pox virus (PPV) is the causal agent of Sharka, considered to be the most detrimental viral disease of Prunus spp. worldwide. So far, several PPV strains have been recognized, three of them (PPV-D, PPV-M, and PPV-Rec) having shown serious economic impact in the European area. Infectious cDNA clones of plant RNA viruses are excellent tools for functional studies of viral genomes. Preparation and use of PPV-D and PPV-M infectious clones have been previously reported. Here we describe the construction of an infectious cDNA clone of the strain PPV-Rec (isolate BOR-3) by the strategy involving the subsequent exchanges of homologous BOR-3 genome parts in the backbone of the previously prepared PPV-D infectious construct. The infectivity of each intermediate chimeric cDNA as well as that of the final construct (pIC-PPV-Rec) was confirmed by biolistic transfection of Nicotiana benthamiana plants. Complete sequence of the cloned viral BOR-3 cDNA revealed 0.14% of difference at the nucleotide level compared to original BOR-3 sequence, resulting in four amino acid changes. This slight inequality was related to the population heterogeneity of the initial BOR-3 isolate; no difference in the amino acid sequence resulted from the cloning steps performed. KEYWORDS: inter-strain chimera; biolistics; genome sequence.

Simple and efficient biolistic procedure for the plant transfection with cDNA clones of RNA viruses.

Simple, fast, low-cost, and efficient procedure for DNA delivery to the cell nuclei of whole plants was developed. The procedure was optimized for the Plum pox virus (PPV) and its host Nicotiana benthamiana. It is based on the leaf bombardment with tungsten microparticles with bound DNA using common air gun. The procedure did not require special equipment and source of driving gas. The transfection efficiency obtained by the newly developed procedure was close to 100%, but this level dropped with the age of bombarded plants.

A single amino acid mutation alters the capsid protein electrophoretic double-band phenotype of the Plum pox virus strain PPV-Rec.

Plum pox virus (PPV) isolates differ by their capsid protein (CP) mobility in SDS-PAGE. These electrophoretic phenotypes are likely to result from post-translational modifications of the CP. We demonstrated that the CP mobility was solely determined by the CP N-terminal region. Sequence comparison pinpointed a possible role of mutations at position 66 in determining the CP phenotype of PPV-Rec isolates. Site-directed mutagenesis of a chimeric clone demonstrated that Gly(66) in the CP resulted in the double-band phenotype, while Arg(66) led to a single-band CP pattern, possibly by preventing the phosphorylation of a nearby Ser residue by steric hindrance.

Infectious full-length clones of plant viruses and their use for construction of viral vectors.

In the last two decades, preparation of infectious RNA or DNA clones of plant viruses has transformed to a standard laboratory technique, providing an excellent tool for the research of viral gene functions and virus-host interactions. A full-length clone can be transformed in vitro to a viral vector expressing foreign poly/oligopeptides in soluble form or fused to a viral capsid protein. The use of the plants as producers of antigens, allergens, and other pharmaceuticals is cheaper than the use other eukaryotic bioreactors. Transient expression of proteins using the vector technique has several advantages in comparison with a transgenic approach. Virus-induced gene silencing vectors have been widely adapted for research of plant genes functions and screening of plant genomes for resistance genes. The potential instability of the constructs remains the main problem of virus vector-based techniques leading to a wild-type virus recovery by recombination. A novel approach including "a deconstructed virus-strategy" may overcome these difficulties. A preparative-scale production of numerous biologically active compounds in the various plants using viral vectors are expected in the near future.

Lipid peroxidation in men after dietary supplementation with a mixture of antioxidant nutrients.

Antioxidants and antioxidant enzymes protect living organisms against the attack of reactive oxygen species. An adequate daily intake of the individual antioxidants is therefore important to prevent the cells against oxidative damage. We investigated the effect of a modest dietary supplementation with a mixture of antioxidant nutrients (100 mg vitamin E, 100 mg vitamin C,6 mg beta-carotene and 50 microg of selenium per day) for 3 months on the plasma antioxidant capacity and indices of oxidative stress. Two groups of middle-age men were selected: group 1 with survivors of myocardial infarction (MI), and group 2 with clinically normal controls. The values of total antioxidant capacity of plasma (FRAP) significantly increased after supplementation with antioxidants in the both groups. Markers of in vivo lipid peroxidation, plasma malondialdehyde (MDA) and conjugated diene (CD) levels significantly decreased in the both supplemented groups. MDA and CD values were significantly higher at baseline in the group of survivors of myocardial infarction when compared with the group of healthy men. The results demonstrate that short-term and modest supplementation with a mixture of antioxidant nutrients improves antioxidative capacity and reduces products of lipid peroxidation in plasma. Since a more pronounced effect was observed within the group of survivors of myocardial infarction, a recommendation of antioxidant supplements seems appropriate for patients with a history of cardiovascular disease. (Fig. 1, Ref. 19.)

Effects of dietary extra virgin olive oil on serum lipid resistance to oxidation and fatty acid composition in elderly lipidemic patients.

An inverse relation between high consumption of olive oil and low incidence of coronary heart disease among the people living in Mediterranean countries has been proposed. It has been shown, that an oleic acid-rich diet could increase the resistance of human LDL to in vitro oxidation which is postulated to play an important role in the development of atherosclerotic lesions. The aim of this study was to assess the effect of extra virgin olive oil consumptiom on the resistance of serum lipids to in vitro oxidation and on fatty acid composition in the serum of elderly lipidemic patients. A total of 26 patients (mean age 69 years) with combined hyperlipidemia consumed daily 2 table spoons (approx. 20 g) of extra virgin olive oil for 6 weeks. Plasma lipids, total antioxidant capacity, indices of serum lipid oxidizability (lag time and maximal rate of oxidation) and the content of fatty acids in serum phospholipids were determined before and after dietary supplementation with olive oil. Plasma total cholesterol and LDL cholesterol decreased significantly after 6 weeks of dietary intervention. A significant increase in the lag time of conjugated diene formation (p = 0.026) and the decrease in the rate of lipid oxidation (p = 0.030) were observed after olive oil consumption. The changes in the fatty acid profile were characterized by an increase in oleic acid content (p = 0.005) as well as by a decline in the content of linoleic acid (p = 0.020) and arachidonic acid (p = 0.022). Linear regression analysis revealed some interesting and significant correlations between indices of serum lipid resistance to oxidation and individual fatty acids, suggesting a protective effects of olive oil in lipoprotein oxidation. In conclusion, the daily consumption of extra virgin olive oil in elderly lipidemic patients favourably affected serum lipoprotein spectrum and fatty acid composition that probably contributed to the increased resistance of serum lipids to oxidation. (Tab. 2, Ref. 18).

Serum ex vivo lipoprotein oxidizability in patients with ischemic heart disease supplemented with vitamin E.

The decreased oxidizability of plasma lipoproteins is related to the increased vitamin E intake and its association with a relatively lower incidence of coronary heart disease has been proposed. We investigated the effect of the in vivo vitamin E supplementation on the oxidizability of serum lipids in patients with ischemic heart disease and a moderate hypercholesterolemia. Thirty-two patients (16 males and 16 postmenopausal women) participated in this placebo-controlled, randomized trial. They were treated with 400 mg vitamin E/day for 6 weeks. The copper-induced serum lipid oxidizability ex vivo was assessed by measuring conjugated diene formation at 245 nm. We also measured vitamin E, malondialdehyde (MDA) and uric acid concentrations in the plasma. Because of observed significant differences in parameters of serum lipid oxidizability (lag time and maximal rate of oxidation), plasma alpha-tocopherol and MDA levels between male patients and postmenopausal women supplemented with vitamin E, the results were compared between both genders. Six weeks of vitamin E supplementation significantly increased plasma vitamin E levels (by 87 %) in male patients but in postmenopausal women only by 34 %. Concomitantly with increased plasma levels of vitamin E the decrease in plasma MDA levels was observed in male patients (decrease by 20 %; p=0.008), but in postmenopausal women the decrease did not attain statistical significance. Plasma uric acid levels were not apparently changed in placebo or vitamin E supplemented groups of patients. The changes in ex vivo serum lipid oxidizability after vitamin E, supplementation have shown a significantly prolonged lag time (by 11 %; p=0.048) and lowered rate of lipid oxidation (by 21 %; p=0.004) in male patients in comparison with postmenopausal women. Linear regression analysis revealed a significant correlation between plasma vitamin E levels and the lag time (r=0.77; p=0.03) and the maximal rate of serum lipid oxidation (r=-0.70; p=0.05) in male patients. However, in postmenopausal women the correlations were not significant. We conclude that 400 mg vitamin E/day supplementation in patients with ischemic heart disease and a moderate hypercholesterolemia influenced favorably ex vivo serum lipid oxidation of male patients when compared with postmenopausal women. The observed differences between both genders could be useful in the selection of the effective vitamin E doses in the prevention of coronary heart disease.

Glutathione S-transferase polymorphisms influence the level of oxidative DNA damage and antioxidant protection in humans.

Glutathione S-transferase genotypes GSTT1, GSTM1, GSTP1 were characterised in 155 middle-aged men and compared with parameters of oxidative stress at the level of DNA and lipids, with antioxidant enzymes, and with plasma antioxidants in smokers and non-smokers. Smokers had on average significantly lower levels of Vitamin C, beta-carotene and beta-cryptoxanthin and higher amounts of oxidised purines and pyrimidines in lymphocyte DNA. The GSTM1 null genotype was associated with elevated glutathione as well as with higher Vitamin C concentration in plasma. Vitamin C was higher in GSTT1+ compared with GSTT1 null--as was glucose-6-phosphate dehydrogenase activity. The homozygous GSTP1 a/a genotype was associated with significantly higher levels of GST activity measured in lymphocytes, in comparison with the b/b genotype. Using multifactorial statistical analysis we found significant associations between smoking, GSTP1 genotype, plasma Vitamin C, and purine base damage in lymphocyte DNA. The difference in Vitamin C plasma levels between smokers and non-smokers was seen only with the GSTP1 b/b genotype. This group accounted also for most of the increase in purine oxidation in smokers. In contrast, the link between smoking and oxidised pyrimidines in DNA was seen only in the GSTT1 null group. It seems that polymorphisms in the phase II metabolising enzyme glutathione S-transferase may be important determinants of commonly measured biomarkers.

LDL and HDL oxidation and fatty acid composition in vegetarians.

The oxidation of low-density lipoproteins (LDL) and high-density lipoproteins (HDL) and fatty acid composition, which may affect the resistance of lipoproteins to oxidation, were determined in 24 lacto-ovo vegetarians. Vegetarian diets contain more essential polyunsaturated fatty acids (PUFAs). Therefore, the relationship between LDL and HDL oxidation resistance measured by the kinetics of the formation of fatty acid conjugated dienes (lag time, maximal rate of oxidation and maximal amount of conjugated dienes), LDL and HDL fatty acid composition and vitamin E content were evaluated. All parameters of in vitro oxidation were significantly lower in HDL when compared with LDL. The relative values of arachidonic, dihomo-gamma-linoleic and docosahexaenoic acid were significantly higher in HDL, alpha-linolenic acid content significantly lower, when compared with LDL; the peroxidizability index was significantly higher in HDL. The content of vitamin E was found more than 2-fold lower in HDL particles. The results show the importance of fatty acid composition in the resistance of LDL and HDL to oxidation and from this aspect, the composition of LDL isolated from vegetarians seems to be more favourable compared to HDL.

Serum lipid resistance to oxidation and uric acid levels in subjects with Down's syndrome.

In subjects with Down's syndrome (DS) increased oxidative stress and consequent oxidative cell damage have been reported. The aim of this study was to assess whether the excessive production of free oxygen radicals in these subjects can affect the copper-induced lipid oxidation resistance measured in fresh whole serum. Since a significant elevation of serum uric acid levels, which is an efficient hydrophilic antioxidant, has been repeatedly reported in subjects with DS, we studied the association between increased serum uric acid levels and lipid resistance to oxidation measured directly in serum samples by monitoring the change in absorbance at 234 nm. The group of subjects with Down's syndrome consisted of 25 individuals (aged 18+/-5 years). Control group included brothers and sisters of subjects with DS (n = 25, aged 17+/-7 years). In subjects with DS, the serum lipid resistance to oxidation (lag time) was significantly higher than in controls (p<0.05) and a concomitant increase in serum uric acid levels was observed (p<0.001). A significant positive correlation between lag time and serum uric acid concentration was found in subjects with DS (r = 0.48, p<0.05), while the positive correlation in the control group was not significant. The results suggest that increased serum uric acid levels repeatedly observed in subjects with DS may be associated with an enhanced resistance of serum lipids to oxidation which is thought to play an important role in the atherogenic process.

Effect of ciprofibrate on lipoprotien metabolism and oxidative stress parameters in patients with type 2 diabetes mellitus and atherogenic lipoprotein phenotype.

The effect of ciprofibrate therapy on plasma lipids and lipoproteins, HDL and LDL subfraction profile, fractional esterification rate of HDL cholesterol (FER(HDL)) and the resistance of LDL and serum lipids to oxidation was studied in 24 males with type 2 diabetes and atherogenic lipoprotein phenotype (ALP). We also examined the effect of ciprofibrate therapy on oxidative DNA damage in peripheral lymphocytes. No differences in glucose, HbA1C and BMI levels were found after three months of ciprofibrate therapy. Ciprofibrate significantly decreased total cholesterol and triglyceride levels by 5.5% and 50% (p = 0.05; 0.001, respectively) and increased HDL-cholesterol levels by 8.5% (p = 0.05). FER(HDL) and LDL subfraction profile were also favorably affected. However, no effect on HDL subclasses was found. There were no statistically significant differences in lipid resistance to oxidation measured in serum and in LDL (lag time and Vmax) before and after therapy. No significant effect of ciprofibrate was found on oxidative DNA damage. The evaluation of the relationship between oxidative damage of purines with lag time in LDL and maximal rate of serum lipid oxidation showed significant correlations after therapy (r = -0.58; 0.47, p = 0.01; 0.05, respectively), but only trends before starting ciprofibrate treatment. Type 2 diabetes mellitus represents a complex metabolic disorder expressed in glucose and lipoprotein disturbances and increased oxidative stress. Ciprofibrate therapy favorably affected major features of lipid abnormalities of diabetic patients, but the level of oxidative stress assessed by in vitro and in vivo methods was not changed. The evaluation of expected logical correlations between the parameters of lipoprotein metabolism, lipid resistance in serum and LDL, and oxidative DNA damage showed that those correlations were more relevant and significant after ciprofibrate treatment and were not related with glucose homeostasis.

Oxidation resistance of LDL in hypertriglyceridaemic patients treated with ciprofibrate.

The oxidative modification of low density lipoprotein (LDL) plays an important role in the pathogenesis of atherosclerosis. LDL of subjects with atherogenic lipoprotein phenotype (ALP) is known to be more susceptible to oxidation. We studied the effect of the hypolipidaemic drug ciprofibrate on the susceptibility of LDL to in vitro oxidation. Nine patients with primary hypertriglyceridaemia and hypoalphalipoproteinaemia (mean plasma triglycerides 3.76 mmol.l-1 and HDL-cholesterol 0.74 mmol.l-1) were treated with ciprofibrate for 12 weeks. The susceptibility of LDL to in vitro Cu(2+)-mediated oxidation was assessed by measuring conjugated diene formation at 234 nm. Ciprofibrate therapy significantly prolonged the lag time (93 +/- 7 min vs. 102 +/- 11 min, P = 0.02). The maximal rate of diene production was 11% lower, but the decrease was not significant. A significant positive correlation was observed between maximal rate and maximal amount of dienes formed. Thiobarbituric acid reacting substances (TBARS) and lipid hydroperoxides (LPO) in oxidatively-modified LDL, isolated from the plasma of patients before and after drug treatment, were unchanged. The results suggest that ciprofibrate therapy has a favourable effect by increasing the in vitro resistance of LDL against oxidation.

Ciprofibrate treatment in patients with atherogenic lipoprotein phenotype: effects on HDL quality, LDL susceptibility to oxidation and DNA damage.

OBJECTIVES: This study was conducted to examine a complex effect of ciprofibrate therapy in patients with atherogenic lipoprotein phenotype. METHODS: Effects of ciprofibrate were studied on HDL subpopulations, HDL ability to esterify cholesterol (FER(HDL)), susceptibility of LDL to oxidation as well as on in vivo oxidative DNA damage in peripheral lymphocytes, measured as strand breaks (SBs) by the comet assay. RESULTS: Ciprofibrate treatment significantly decreased total cholesterol, and triglycerides, and increased HDL-cholesterol. The FER(HDL) showed a significant reduction (29.5+/-7.4 to 23+/-7.5% x h(-1), P=0.0001). The relative concentrations of HDL subclasses did not differ between baseline and after treatment. Ciprofibrate induced a significant increase in LDL oxidation lag time (93+/-7 to 102=11 min, P=0.02) and a decrease in DNA strand breaks (34.0+/-16.2 to 17.8+/-7.5, P=0.02). A significant correlation between maximal rate of diene production and strand breaks was found (r=0.55, P=0.01). These findings may be explained by an improvement of LDL resistance to oxidation, resulting in a decrease in oxidatively modified LDL's cytotoxic effect. CONCLUSION: Ciprofibrate treatment favourably affected the quality of plasma HDL, probably by the improvement of triglyceride rich lipoprotein metabolism and/or LDL subpopulation profile, increased LDL resistance to oxidation, and decreased the level of DNA damage in peripheral lymphocytes.

LDL oxidizability and antioxidative status of plasma in vegetarians.

Vegetarian food with a higher content of natural antioxidants may decrease the risk of developing cardiovascular diseases. The oxidative modification of low-density lipoprotein (LDL) has been implicated in the pathogenesis of atherosclerosis. The objectives of this study were to investigate in vitro resistance of LDL against oxidation and antioxidative status of plasma in 19 lactoovovegetarians and nonvegetarians (n = 19). The kinetics of conjugated diene formation (lag time and maximal rate of oxidation, Vmax) and thiobarbituric acid reacting substances (TBARS) were determined as indices of LDL oxidation. No significant differences in lag time and Vmax were observed between vegetarians and nonvegetarians, while TBARS in native and oxidatively modified LDL were significantly lower in vegetarians. Plasma vitamin (C, E, beta-carotene) levels were increased in vegetarians, but only the vitamin C increase was statistically significant. Vegetarians also had a significantly increased plasma total antioxidant status (TAS) and molar ratio vitamin E/cholesterol which indicates a more effective protection of lipoproteins against oxidation. A significant positive correlation was observed between lag time and plasma TAS in both vegetarians and nonvegetarians. We conclude that although the resistance of LDL to in vitro oxidation, as measured by the kinetics of conjugated diene formation was not altered in vegetarians, the effect of vegetarian nutritional habits favorably affected TBARS levels in LDL and total plasma antioxidant capacity.

Effects of blockade of Kupffer cells by gadolinium chloride on hepatobiliary function in cold ischemia-reperfusion injury of rat liver.

The mechanisms of liver injury from cold storage and reperfusion are not completely understood. The aim of the present study was to investigate: 1) whether the inactivation of Kupffer cells (KCs) by gadolinium chloride (GadCl) modulates cold ischemia-reperfusion injury of rat liver; and 2) whether cold storage of rat liver involves injury to biliary epithelial cells (BECs). Hepatobiliary function was assessed using an isolated perfused rat liver model. Compared with control livers, in livers subjected to cold storage at 4 degrees C in Euro-Collins solution (EC) for 18 hours or in University of Wisconsin solution (UW) for 48 hours, portal flow was lower and resistance significantly higher, taurocholate (TC) and bromosulfophthalein (BSP) elimination were markedly impaired, bile flow was reduced, and lactate dehydrogenase (LDH) leakage into the perfusate was increased. Pretreatment of rats with GadCl, a selective KC toxicant, abrogated disturbances of the microcirculation in both models, but it did not influence viability and functional parameters of the liver. Most of the parameters studied in livers stored in UW solution for 18 hours were not significantly different from those found in control livers. As to biliary activity of gamma-glutamyl transferase (GGT), as an index of BEC integrity, it was increased with increasing time of cold storage. The reabsorption of glucose from the bile decreased with longer storage time. The results suggest the following: 1) that cold ischemia-reperfusion injury of rat liver is mediated by KC-dependent (hepatic microcirculation) and -independent (parenchymal cell function) mechanisms; and 2) that cold storage of rat liver induces functional impairment of BECs.

The influence of ascorbic acid on the hepatic cytochrome P-450, and glutathione in guinea-pigs exposed to 2,4-dichlorophenol.

Chlorophenols, mainly used as biocides, are compounds with a wide spectrum of toxic effects including teratogenic and carcinogenic actions. In this study, the effects of 2,4-dichlorophenol (2,4-DCP) on hepatic microsomal cytochrome P-450, NADPH-cytochrome c reductase activity, liver ascorbic acid (AA) and glutathione (GSH) content were studied in guinea-pigs with a low (2 mg/day/animal) or a high (50 mg/day/animal) ascorbic acid intake. The high AA intake significantly increased liver AA and GSH levels. There was a clear-cut correlation between liver AA and GSH levels. Administration of 2,4-DCP significantly decreased cytochrome P-450 and NADPH-cytochrome c reductase activity in hepatic microsomes isolated from guinea-pigs with the low AA intake. Such a reduction was not observed in intoxicated guinea-pigs with the high AA intake. The results suggest that AA can play a protective role in 2,4-DCP toxicity.