The Use of Kosher Phenotyping for Mapping QTL Affecting Susceptibility to Bovine Respiratory Disease

Bovine respiratory disease (BRD) is the leading cause of morbidity and mortality in feedlot cattle, caused by multiple pathogens that become more virulent in response to stress. As clinical signs often go undetected and various preventive strategies failed, identification of genes affecting BRD is essential for selection for resistance. Selective DNA pooling (SDP) was applied in a genome wide association study (GWAS) to map BRD QTLs in Israeli Holstein male calves. Kosher scoring of lung adhesions was used to allocate 122 and 62 animals to High (Glatt Kosher) and Low (Non-Kosher) resistant groups, respectively. Genotyping was performed using the Illumina BovineHD BeadChip according to the Infinium protocol. Moving average of -logP was used to map QTLs and Log drop was used to define their boundaries (QTLRs). The combined procedure was efficient for high resolution mapping. Nineteen QTLRs distributed over 13 autosomes were found, some overlapping previous studies. The QTLRs contain polymorphic functional and expression candidate genes to affect kosher status, with putative immunological and wound healing activities. Kosher phenotyping was shown to be a reliable means to map QTLs affecting BRD morbidity.     

Oxidative modification of human low-density lipoprotein by horseradish peroxidase in the absence of hydrogen peroxide

Heme-peroxidases, such as horseradish peroxidase (HRP), are among the most popular catalysts of low density lipoprotein (LDL) peroxidation. In this model system, a suitable oxidant such as H₂O₂ is required to generate the hypervalent iron species able to initiate the peroxidative chain. However, we observed that traces of hydroperoxides present in a fresh solution of linoleic acid can promote lipid peroxidation and apo B oxidation, substituting H₂O₂.

Spectral analysis of HRP showed that an hypervalent iron is generated in the presence of H₂O₂ and peroxidizing linoleic acid. Accordingly, careful reduction of the traces of linoleic acid lipid hydroperoxide prevented formation of the ferryl species in HRP and lipid peroxidation. However, when LDL was oxidized in the presence of HRP, the ferryl form of HRP was not detectable, suggesting a Fenton-like reaction as an alternative mechanism. This was supported by the observation that carbon monoxide, a ligand for the ferrous HRP, completely inhibited peroxidation of LDL.

These results are in agreement with previous studies showing that myoglobin ferryl species is not produced in the presence of phospholipid hydroperoxides, and emphasize the relevance of a Fenton-like chemistry in peroxidation of LDL and indirectly, the role of pre-existing lipid hydroperoxides.     

Red wine mitigates the postprandial increase of LDL susceptibility to oxidation

The aim of the present study was to verify the extent of oxidative stress induced by a meal at plasma and LDL level, and to investigate the capacity of red wine to counteract this action. In two different sessions, six healthy men ate the same test meal consisting of "Milanese" meat and fried potatoes. The meal was taken either with 400 ml red wine or with an isocaloric hydroalcoholic solution. Oxidative stress at plasma level was estimated through the measure of ascorbic acid, alpha-tocopherol, protein SH groups, uric acid, and antioxidant capacity, measured before and 1 and 3 h after the meal. The change in the resistance of LDL to oxidative modification was taken as an index of exposure to pro-oxidants. The susceptibility to Cu(II)-catalyzed oxidation of baseline and postprandial LDL was measured as conjugated dienes formation, tryptophan residues, and relative electrophoretic mobility. The experimental meal taken with wine provoked a significant increase in the total plasma antioxidant capacity and in the plasma concentration of alpha-tocopherol and SH groups. Postprandial LDL was more susceptible to metal-catalyzed oxidation than the homologous baseline LDL after the ethanol meal. On the contrary, postprandial LDL obtained after the wine meal was as resistant or more resistant to lipid peroxidation than fasting LDL.     

PDCD10 Gene Mutations in Multiple Cerebral Cavernous Malformations

Cerebral cavernous malformations (CCMs) are vascular abnormalities that may cause seizures, intracerebral haemorrhages, and focal neurological deficits. Familial form shows an autosomal dominant pattern of inheritance with incomplete penetrance and variable clinical expression. Three genes have been identified causing familial CCM: KRIT1/CCM1, MGC4607/CCM2, and PDCD10/CCM3. Aim of this study is to report additional PDCD10/CCM3 families poorly described so far which account for 10-15% of hereditary cerebral cavernous malformations. Our group investigated 87 consecutive Italian affected individuals (i.e. positive Magnetic Resonance Imaging) with multiple/familial CCM through direct sequencing and Multiplex Ligation-Dependent Probe Amplification (MLPA) analysis. We identified mutations in over 97.7% of cases, and PDCD10/CCM3 accounts for 13.1%. PDCD10/CCM3 molecular screening revealed four already known mutations and four novel ones. The mutated patients show an earlier onset of clinical manifestations as compared to CCM1/CCM2 mutated patients. The study of further families carrying mutations in PDCD10/CCM3 may help define a possible correlation between genotype and phenotype; an accurate clinical follow up of the subjects would help define more precisely whether mutations in PDCD10/CCM3 lead to a characteristic phenotype.     

Lipid Peroxidation and Antioxidant Systems in Rat Brain: Effect of Chronic Alcohol Consumption

The effect of chronic ethanol exposure, in a liquid diet, on lipid peroxidation and some antioxidant systems of rat brain was investigated. Chronic ethanol administration induced a greater susceptibility to iron/ascorbate-induced lipid peroxidation, estimated as thiobarbituric reactive substances (TBARS) production, in the microsomal fraction, but a lower lipid peroxidation in the total homogenate. Glutathione (GSH) levels as well as GSH peroxidase and GSH reductase were unaffected, while the activity of Cu-Zn superoxide dismutase was decreased and that of catalase increased. Lipid peroxidation experiments performed in the presence of some hydroxyl radical scavengers suggested that a greater OH^· generation may be responsible of the greater TBARS production in the microsomal fraction of ethanol treated rats; differently, in total homogenate of control and ethanol rats a relationship was found between the redox state of iron and TBARS production, suggesting that the lower lipid peroxidation in treated rats may depend on a different modulation of the iron redox state.     

Phenolic acids from beer are absorbed and extensively metabolized in humans

In spite of the wide literature describing the biological effects of phenolic compounds, scarce data are available on their absorption from diet. In the present work, we studied the absorption in humans of phenolic acids from beer, a common beverage rich in different phenolic acids with related chemical structures. Beer was analyzed for free and total (free+bound) phenolic acids. Ferulic, caffeic and sinapic acids were present in beer mainly as bound forms, while 4-hydroxyphenylacetic acid and p-coumaric acid were present mainly as free forms. Vanillic acid was present equally in the free and bound forms. Plasma samples were collected before and 30 and 60 min after beer administration and analyzed for free and conjugated phenolic acid content. A significant two- to fourfold increase in plasma levels of phenolic acids was detected with peak concentrations at 30 min after beer ingestion. 4-Hydroxyphenylacetic acid was present in plasma mainly as nonconjugated forms while p-coumaric acid was present equally as nonconjugated and conjugated forms. Ferulic, vanillic and caffeic acids were present in plasma predominantly as conjugated forms, with a slight prevalence of sulfates with respect to glucuronates. Our results indicate that phenolic acids from beer are absorbed from the gastrointestinal tract and are present in blood after being largely metabolized to the form of glucuronide and sulfate conjugates. The extent of conjugation is related to the chemical structure of phenolic acids: the monohydroxy derivatives showing the lowest conjugation degree and the dihydroxy derivatives showing the highest one.     

4-Methylcoumarins as antioxidants: Scavenging of peroxyl radicals and inhibition of human low-density lipoprotein oxidation

The antioxidant activity of eight synthetic 4-methylcoumarins was systematically studied. The antioxidant capacity was measured using: (i) a competition kinetic test, to measure the relative capacity to quench peroxyl radical; (ii) the in vitro oxidative modification of human low-density lipoprotein, initiated by AAPH or catalyzed by copper. In both models, the ortho-OH substitutes were found to be better antioxidant than the meta one. The most efficient antioxidant was the 7,8-dihydroxy-4-methylcoumarin and the corresponding diacetoxy-substituted was unexpectedly a good antioxidant. Finally, the presence of an ethoxycarbonylethyl substituent at the C-3 position increased the antioxidant capacity of both 7,8-dihydroxy-4-methylcoumarin and 7,8-diacetoxy-4-methylcoumarin.     

Effect of caffeic acid on tert-butyl hydroperoxide-induced oxidative stress in U937

Nonvitamin phenolic compounds are ubiquitous in food plants and therefore potentially present in human plasma in a diet-dependent concentration. The aim of this study was to evaluate the ability of caffeic acid, a phenolic acid with antioxidant activity, to affect cellular response in U937 human monocytic cells to t-butyl hydroperoxide-induced oxidative stress. In our experimental conditions caffeic acid was incorporated into cells without any cytotoxic effect. Caffeic acid-treated cells showed an increased resistance to oxidative challenge, as revealed by an higher percent of survival and the maintenance of an higher proliferative capacity in respect to control cells. This effect seems to be due to the ability of caffeic acid to reduce glutathione depletion and to inhibit lipid peroxidation during tBOOH treatment. It can be concluded that caffeic acid exerts an antioxidant action inside the cell, responsible for the observed modulation of the cellular response to oxidative challenge. Due to its presence in the diet, therefore, caffeic acid may play a role in the modulation of oxidative processes in vivo.     

Biphasic effect of iron on human intestinal Caco-2 cells: early effect on tight junction permeability with delayed onset of oxidative cytotoxic damage.

Treatment of differentiated human intestinal Caco-2 cells with Fe(II) ascorbate altered tight junction permeability in a dose and time-dependent way for up to 3 hr of treatment Upon iron removal and transfer to complete culture medium, the effect was reversible up to 10 microM Fe(II), while at higher concentrations a late phase toxic effect was observed. Reduction of intracellular energy abolished the short term effect of iron on tight junction permeability without affecting its cellular uptake, suggesting that active processes, other than transport, were involved. The short term effect of iron the permeability of tight junctions did not appear to result from the generation of reactive oxygen species, as it was not prevented by antioxidant treatment under normal energy conditions. Conversely, the late phase effect leading to both apoptosis and necrosis during the 24 hr following iron removal could be reduced by antioxidant treatment and was exacebated by GSH depletion. Iron induced oxidative stress may therefore be responsible for membrane damage and cellular death occurring in the late phase. The reported effects of iron on intestinal tight junction permeability followed by more widespread cytotoxicity from oxidative events should be considered in light of the extensive use of iron supplementation in different phases of human life.     

Regulation of human erythrocyte glyceraldehyde-3-phosphate dehydrogenase by ferriprotoporphyrin IX

Erythrocyte glyceraldehyde-3-phosphate dehydrogenase (G3PD) is a glycolytic enzyme containing critical thiol groups and whose activity is reversibly inhibited by binding to the cell membrane. Here, we demonstrate that the insertion of ferriprotoporphyrin IX (FP) into the red cell membranes exerts two opposite effects on membrane bound G3PD. First, the enzyme is partially inactivated through oxidation of critical thiols. Dithiothreitol restores part of the activity, but some critical thiols are irreversibly oxidized or crosslinked to products of FP-induced lipid peroxidation. Second, G3PD binding to the membrane is modified and the enzyme is activated through displacement into the cytosol and/or release from its binding site.