Regulation of sheep α-TTP by dietary vitamin E and preparation of monoclonal antibody for sheep α-TTP

α-Tocopherol transfer protein (α-TTP) is a cytosolic protein that plays an important role in regulating concentrations of plasma α-tocopherol (the most bio-active form of vitamin E). Despite the central roles that α-TTP plays in maintaining vitamin E adequacy, we have only recently proved the existence of the α-TTP gene in sheep and, for the first time, cloned its full-length cDNA. However, the study of sheep α-TTP is still in its infancy. In the present study, thirty-five local male lambs of Tan sheep with similar initial body weight were randomly divided into five groups and fed with diets supplemented with 0 (control group), 20, 100, 200, 2000 IU·sheep^− 1·d^− 1 vitamin E for 120 days. At the end of the experiment, the plasma and liver vitamin E contents were analyzed first and then α-TTP mRNA and protein expression levels were determined by quantitative real-time PCR (qRT-PCR) and Western-blot analysis, respectively. In addition, as no sheep α-TTP antibody was available, a specific monoclonal antibody (McAb) against the ovine α-TTP protein was prepared. The effect of vitamin E supplementation was confirmed by the significant changes in the concentrations of vitamin E in the plasma and liver. As shown by qRT-PCR and Western-blot analysis, dietary vitamin E does not affect sheep α-TTP gene expression, except for high levels of vitamin E supplementation, which significantly increased expression at the protein level. Importantly, the specific sheep anti-α-TTP McAb we generated could provide optimal recognition in ELISA, Western-blot and immunohistochemistry assays, which will be a powerful tool in future studies of the biological functions of sheep α-TTP.     

Effects of supplementation with vitamin E on the performance and the tissue peroxidation of broiler chicks and the stability of thigh meat against oxidative deterioration

Two experiments were conducted: Expt 1 determined the optimal allowance of vitamin E in the diet for broiler chicks aged 0–3 weeks; Expt 2 investigated the effects of different dietary levels of vitamin E (α-tocopherol) on the performance and the oxidative stability of thigh meat of broiler chicks during storage. In Expt 1, 1-day-old 900 broiler chicks were allocated to five treatments, each with six replicates (cages) of 22 as-hatched chicks for performance evaluation, and another cage of 45 male chicks for determining plasma and hepatic α-tocopherol and thiobarbituric acid reactive substances (TBARS) concentration in blood and liver. The basal dietary α-tocopherol concentration was 13 mg/kg, and the five α-tocopherol acetate supplementation levels were 0, 5, 10, 50 and 100 mg/kg. For 0–3-week-old broiler chicks fed with maize–soya bean meal–soya oil type diet, supplementation of vitamin E did not influence the feed intake, but tended to improve growth and feed utilization, however there was no significant correlation between performance and vitamin E supplementation level. Significant positive correlations existed between dietary supplemental vitamin E level and plasma or hepatic α-tocopherol concentrations (P<0.05), and a negative correlation with hepatic TBARS levels no matter at what age (11, 16 and 21 days). In Expt 2, 2200 broiler chicks were randomly allocated to five treatments with four replicates (pens) in each. Chicks were fed ad libitum five pellet diets supplemented with vitamin E at 5, 10, 20, 50 and 100 mg/kg of diet, respectively. The basal dietary α-tocopherol level of grower and finisher diets were 7 and 6 mg/kg, respectively. Supplementation of vitamin E tended to improve growth and feed utilization of birds during 0–3 weeks of age, but the performance from 0 to 6 weeks of age were not influenced. The hepatic α-tocopherol concentrations of 6-week-old chicks linearly increased with the dietary vitamin E levels (R²=0.98, P<0.001). The content of TBARS in the thigh meat over 4 days of storage under 4°C was significantly decreased by increasing dietary vitamin E level (P<0.05). There was a significant inverse relationship between TBARS value in the thigh meat and the dietary vitamin E level (R²=0.93, P<0.01). Supplementation of vitamin E significantly improved the meat quality stability substantially against oxidative deterioration. Comparing the hepatic α-tocopherol levels of chicks in Expts 1 and 2, total allowance of dietary α-tocopherol of 20–30 mg/kg could sustain relatively constant hepatic α-tocopherol level at round about 2–2.5 μg/kg.     

Dietary vitamin E affects α-TTP mRNA levels in different tissues of the Tan sheep

The α-tocopherol transfer protein (α-TTP) is a ~ 32 kDa cytosolic protein that plays an important role in the efficient circulation of plasma α-tocopherol in the body, a factor with great relevance in reproduction. The α-TTP gene has been studied in a number of tissues; however, its expression and function in some ovine tissues remain unclear. A previous study from our laboratory has demonstrated α-TTP expression in sheep liver. In the present study we determined whether α-TTP is expressed in non-liver tissues and investigated the effects of dietary vitamin E on the α-TTP mRNA levels. Thirty-five male Tan sheep with similar body weight were randomly allocated into five groups and supplemented 0, 20, 100, 200 and 2000 IU sheep^− 1 day^− 1 vitamin E, for four months, respectively. At the end of the study, the animals were slaughtered and tissue samples from the heart, spleen, lung, kidney, longissimus dorsi muscle and gluteus muscle were immediately collected. We found that the α-TTP gene is expressed in sheep tissues other than the liver. Moreover, dietary vitamin E levels had influenced the expression levels of α-TTP gene in these tissues in a tissue-specific way. The technique of immunohistochemistry was used to detect α-TTP in tissues of the heart, spleen, lung, and kidney and we found that α-TTP was mainly located in the cytoplasm while no α-TTP immunoreactivity was detected in the cytoplasm of longissimus dorsi and gluteus muscle samples. Importantly, our findings lay the foundation for additional experiments focusing on the absorption and metabolism of vitamin E in tissues other than the liver.     

Effects of dietary selenium supplementation on parasitemia,anemia and serum proteins of Trypanosoma brucei brucei infected rats

Trypanosomosis has been associated with immunosuppression, anemia and oxidative damage while selenium possesses both immunostimulatory and antioxidative effects. This study was designed to assess the effect of dietary selenium supplementation on parasitemia, anemia, survival pattern and serum protein profiles of trypanosome-infected rats. Twenty five rats, divided into five groups (A–E) of 5 each, were treated as follows: 4, 8 and 16 ppm (ppm) of selenium in their feed, respectively throughout the experimental period and were infected with Trypanosoma brucei brucei on day 14 post supplementation, infected not supplemented and the negative control. Supplementation at 4 and 8 ppm increased the packed cell volume (PCV) and hemoglobin (Hb) concentration on day 7 of supplementation (PS) when compared with the unsupplemented groups. Following infection on day 14 PS, the PCV, Hb of 16 ppm and infected not supplemented groups were significantly (P < 0.05) lower than other groups on days 28 and 35 PS. Supplementation did not lead to significant (P > 0.05) changes on the total protein, albumin and globulin by day 14 PS. Infection, however, caused significant (P > 0.05) decrease in the total protein and albumin from day 28. The supplementation did not significantly (P > 0.05) increase the pre-patent period but caused a significant reduction in the parasitemia levels and increased survival intervals. Dietary selenium supplementation, from the results, may show promise in the management of African trypanosomosis as the supplementation was able to: reduce anemia and parasitemia and increase survival intervals of trypanosome infected rats.     

Effect of sodium selenite, Se-yeast and nano-elemental selenium on growth performance, Se concentration and antioxidant status in growing male goats

The objective of this experiment was to study the effect of inorganic, organic and elemental nano-selenium on growth performance, Se concentration and antioxidant status in growing male goats. A total of 40 weaned Taihang black goats were randomly divided into four equal groups, given the basal diet either unsupplemented (CTRL only received 0.03 mg/kg Se background) or supplemented with 0.3 mg/kg Se as sodium selenite (SS), Se-yeast (SY) or elemental nano-selenium (NS) for a 90 days experiment (from weaning to maturity). Average initial and finial body weight (BW) and average daily gain (ADG) were recorded. Serum and whole blood were collected for serum glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) activity and Se content analysis. At the end of the feeding trail five bucks in each group were killed and samples of heart, liver, spleen, lung, kidney, muscle and testis were collected for Se determination. The result showed that the final BW was increased (P < 0.05) in bucks supplemented with Se compared to the controls, and ADG in NS and SY were greater (P < 0.05) than SS or CTRL bucks. Whole blood, serum and tissue Se concentration, serum antioxidant enzymes activity were also affected by dietary Se supplementation. Serum GSH-Px, SOD and CAT in NS were higher (P < 0.05) than those in SS and SY, and Se retention of whole blood, serum and some organs in NS were also higher than SS or SY (P < 0.05). It could be concluded that supplementation of Se can improve growth performance, serum oxidant status and Se concentration in blood and tissues in growing male goat. The dietary supplementation of elemental nano-Se could be utilized more effectively when compared to inorganic or organic Se.     

Vitamin D status and vitamin D receptor gene polymorphisms and susceptibility to type 1 diabetes in Egyptian children

Background

Type 1 diabetes mellitus (T1DM) is recognized as a T-cell-mediated autoimmune disease. Vitamin D compounds are known to suppress T-cell activation by binding to vitamin D receptor (VDR); and thus, VDR gene polymorphisms may be related to T-cell-mediated autoimmune diseases. The aim of this study was to investigate the association between vitamin D status and VDR gene polymorphisms and T1DM.

Materials and methods

One hundred and twenty patients with T1DM and one hundred and twenty controls were enrolled in the study. VDR gene BsmI, FokI, ApaI and TaqI polymorphisms were determined using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). Serum 25-hydroxyvitamin D (25(OH)D) was determined using ELISA.

Result

Serum 25(OH)D levels revealed a vitamin D deficiency or insufficiency in 75% of the patients. The mean levels of vitamin D were significantly lower in patients as compared to their controls (P = < 0.001). VDR BsmI Bb and bb genotypes and VDR FokI Ff and ff genotypes were associated with increased risk of T1DM (OR = 2.3, 95% CI = 1.3–4.2, P = 0.005; OR = 2.2, 95% CI = 1.1–4.7, P = 0.04; OR = 1.8, 95% CI = 1.03–3.04, P = 0.04; OR = 4.03, 95% CI = 1.2–13.1, P = 0.01 respectively), while the VDR ApaI and TaqI polymorphisms were not.

Conclusion

Our study indicated that vitamin D deficiency and VDR BsmI and FokI polymorphisms were associated with T1DM in Egyptian children.     

Zinc supplementation does not alter plasma homocysteine,vitamin B12 and red blood cell folate concentrations in French elderly subjects

Background/aimsIn ageing, low folates and vitamin B12 status are frequent and can explain the increase of plasma homocysteine level. Zinc is involved in the folates and vitamin B12 metabolism with opposite actions. The aim of this study was to investigate the effects of zinc supplementation on homocysteine and vitamin B12 plasma levels as well as red blood cell folate level in French ageing subjects participating in the ZENITH study.MethodsApparently healthy middle-aged (55–70 years) and free-living older (70–85 years) subjects were enrolled. They were randomly allocated to three groups: 0, 15 or 30 mg Zn per day for 6 months as zinc gluconate in addition to their usual dietary intake.ResultsAt baseline, plasma homocysteine levels (15.2±3.5 μmol/L) in older people were higher than in the middle-aged subjects (12.7±2.7 μmol/L) and was negatively correlated with vitamin B12 values (p=0.0036, r=?0.215) and with RBC folate levels (p<0.0001, r=?0.30). These results are in agreement with previous data. However, we found no correlation between the biomarkers of zinc status and homocysteine, vitamin B12 or folate levels at baseline. Moreover, 6-month zinc supplementation did not modify homocysteine, vitamin B12 and RBC folate values in either of the groups.ConclusionsZinc supplementation at moderate doses do not lead to deleterious effect on folate or vitamin B12 status in ageing healthy free-living people, but does not have any beneficial effects on homocysteine metabolism either.     

Effectiveness of vitamin D supplementation in Swedish children may be negatively impacted by BMI and serum fructose

In regions where sunlight exposure is limited, dietary vitamin D intake becomes important for maintaining status. However, Swedish children have been shown to have deficient or marginal status during the winter months even if the recommended dietary intake is met. Since low vitamin D status has been associated with several disease states, this study investigated the metabolic changes associated with improved vitamin D status due to supplementation.During the 3 winter months, 5–7-year-old children (n=170) in northern (Umeå, 63° N) and southern (Malmö, 55° N) Sweden were supplemented daily with 2 (placebo), 10 or 25 μg of vitamin D. BMI-for-age z-scores (BAZ), S-25(OH)D concentrations, insulin concentrations and the serum metabolome were assessed at baseline and follow-up.S-25(OH)D concentrations increased significantly in both supplementation groups (P<.001). Only arginine and isopropanol concentrations exhibited significant associations with improvements in S-25(OH)D. Furthermore, the extent to which S-25(OH)D increased was correlated with a combination of baseline BAZ and the change in serum fructose concentrations from baseline to follow up (P=.012). In particular, the change in S-25(OH)D concentrations was negatively correlated (P=.030) with the change in fructose concentrations for subjects with BAZ ≥0 and consuming at least 20 μg vitamin D daily. These results suggest that although the metabolic changes associated with improved vitamin D status are small, the effectiveness of dietary supplementation may be influenced by serum fructose concentrations.     

Association of the rs6235 variant in the proprotein convertase subtilisin/kexin type 1 (PCSK1) gene with obesity and related traits in a Taiwanese population

One particularly interesting single nucleotide polymorphism (SNP), rs6235 (encoding an S690T substitution), in the proprotein convertase subtilisin/kexin type 1 (PCSK1) gene has been widely associated with obesity in several European cohorts. The present study was intended to investigate the association between the PCSK1 rs6235 SNP and the prevalence of overweight or obesity, or obesity-related metabolic traits in a Taiwanese population. A total of 964 Taiwanese subjects with general health examinations were analyzed. Our data revealed no association of PCSK1 rs6235 with the risk of obesity or overweight in the complete subjects. However, the PCSK1 rs6235 SNP exhibited a significant association with overweight among the male subjects (P = 0.03), but not among the female subjects. Furthermore, the carriers of GG variant had a significantly higher waist circumference than those with the CC variant (82.5 ± 11.5 vs. 81.2 ± 10.2 cm; P = 0.01) and those with the CG variant (82.5 ± 11.5 vs. 81.4 ± 10.4 cm; P = 0.021). In addition, the carriers of GG variant had a higher diastolic blood pressure than those with the CC variant (81.9 ± 14.2 vs. 80.3 ± 12.9 mm Hg; P = 0.023). Our study indicates that the PCSK1 rs6235 SNP may contribute to the risk of overweight in men and predict obesity-related metabolic traits such as waist circumference and diastolic blood pressure in Taiwanese subjects.     

Vanadium and 1, 25 (OH)2 vitamin D3 combination in inhibitions of 1,2, dimethylhydrazine-induced rat colon carcinogenesis

The current investigation demonstrates the antitumor effects of combined supplementations of vanadium (V) (4.27 µmol/L drinking water ad libitum) and1α, 25-dihydroxy vitamin D₃ (Vitamin D₃) (0.3 μg/100 μL propylene glycol per os twice a week) on 1, 2 dimethylhydrazine (DMH) (20 mg/kg body weight) induced rat colon carcinogenesis. There was a significant reduction in incidence (70%), multiplicity (P < 0.0001) and volume (P < 0.01) of colon tumors. HPLC-fluorescence assay detected the combinatorial actions of V and Vitamin D₃ against DMH-induced colonic O⁶-methylguanine DNA adducts formation (at four sequential time points; ANOVA, F = 13.56, P < 0.01). Simultaneous inhibition of DNA single strand breaks (P < 0.001) indicates the potency of the combination regimen in limiting the initiation event of colon carcinogenesis. Immunohistochemical analysis revealed that the effect of V and vitamin D₃ occurred through suppression of cell proliferation (BrdU-LI: P < 0.001) along with an induction of apoptosis (TUNEL-LI: P < 0.01). The immunoexpression of tumor suppressor p53 and downregulation of antiapoptotic protein BCl-2 in subsequent immunofluorescence assay further provide strong evidence for the combinatorial inhibitory actions of vanadium and vitamin D₃ against DMH-induced rat colon carcinogenesis.     

Quality and lipid composition of spermatozoa in rabbits fed DHA and vitamin E rich diets

The effects of fish oil (FO) and vitamin E (vE) dietary supplementation on semen quality, sperm susceptibility to lipid peroxidation, tocopherols content and fatty acid profiles were studied in rabbits. Fifty-two rabbit bucks randomly divided in four groups received a control diet and enriched diets containing either FO (1.5%, w/w), vE (200 mg/kg) or both. Semen volume, concentration, motility and viability were analysed at various time-points and the lipid composition was assessed on sperm cells. The phospholipid fatty acid profile was determined: n-6 PUFA were the major fatty acids found, with a proportion of 42%, whereas the n-3 PUFA accounted for nearly 1%, mainly represented by C22:6n-3 (docosahexaenoic acid, DHA). FO supplementation produced a seven-fold increase in the content of DHA in sperm phospholipids and a comprehensive rearrangement of the phospholipid fatty acid composition, while an unexpected negative effect of feeding high level of vE on the proportion of total PUFA was found. Despite the remarkable changes observed in sperm lipid composition, semen quality parameters were not affected by the dietary treatments and the interaction between the two dietary supplements had a significant effect only on sperm concentration. An increase in semen production by ageing and a concomitant rise in sperm susceptibility to in vitro peroxidation was found. α- and δ-tocopherol, present in rabbit sperm in similar amount, were not affected by dietary treatment. δ-tocopherol content had a significant linear negative regression with age and showed a significant negative correlation with the susceptibility to peroxidation values.     

Physiological changes in Labeo rohita during nitrite exposure: Detoxification through dietary vitamin E

This study investigated the effect of sub-lethal nitrite–nitrogen exposure on Labeo rohita. Fishes fed with different levels of vitamin E (VE) for 60 days were exposed to nitrite for another 45 days with same feeding regime. There were four treatment groups, viz., VE₁₀₀− N, VE₁₀₀ + N, VE₁₅₀ + N and VE₃₀₀ + N. After 45 days of exposure, lowest specific growth rate was observed in VE₁₀₀ + N and highest in VE₁₀₀− N. Reno-somatic index and methaemoglobin reductase activity were significantly increased by nitrite exposure. Highest Hb and Hct were observed in VE₁₀₀− N and significantly decreased upon nitrite exposure. Significant differences were observed in the activities of catalase and SOD as well as serum potassium and chloride levels among different treatments. However, serum calcium and osmolality was not significantly varied. Nitrite exposure caused marked increase in nitrite concentration in gill, liver and muscle. In liver and muscle dietary supplementation of higher amounts of VE found to reduce nitrite accumulation. It was noticed that nitrite exposure has adversely affected growth, haematological variables, ionic balance and dietary supplementation with additional amounts of VE found to overcome the adverse effects of nitrite–nitrogen. Detoxification of nitrite by methaemoglobin reductase system was enhanced by dietary supplementation of additional amounts of VE.     

Effect of finishing period length with α-tocopherol supplementation on the expression of vitamin E-related genes in the muscle and subcutaneous fat of light lambs

The aim of this study was to investigate how different finishing period lengths with α-tocopherol supplementation or alfalfa grazing affect mRNA expression levels of genes related to vitamin E metabolism in L. thoracis (LT) muscle and subcutaneous fat (SF) from lambs of the Rasa Aragonesa breed. Indoors, concentrate-fed light lambs (n = 48) were supplemented with 500 dl-α-tocopheryl acetate/kg concentrate for an average finishing period length of 0 (C), 10.7 (VE10d), 21.2 (VE20d) and, 32.3 (VE30d) days before slaughtering. Simultaneously, 8 lambs with their dams were alfalfa-grazed. The α-tocopherol affected in a short-term the expression of genes in LT muscle (ABCA1, LPL, APOE, and SREBP1) and SF (ABCA1, SCARB1, LPL, and PPARG). On the contrary, PPARA gene expression showed a long-term α-tocopherol effect because the highest levels of PPARA mRNA were found in the VE30d.     

Effects of dietary amino acids and repeated handling on stress response and brain monoaminergic neurotransmitters in Senegalese sole (Solea senegalensis) juveniles

The present study aimed to assess the effects of increased availability of dietary amino acids (AA) on brain monoamine neurotransmitters and the metabolic processes resulting from stressful situations in fish. Senegalese sole (Solea senegalensis) juveniles (24.2 ± 0.4 g wet mass) were weekly subjected to an acute handling stressor (HDLG) or remained undisturbed (CTL). Additionally, both treatments were fed a control or a high protein (HP) diet (CTL, CTL HP, HDLG and HDLG HP). The HP diet slightly increased the levels of digestible indispensable AA, together with tyrosine and cysteine. Repeated handling induced a stress response after 14 and 28 days in fish held at both HDLG and HDLG HP treatments. While dietary treatment and handling stress activated the serotonergic system at 14 days, these effects were not observed after 28 days. In addition, the HP diet minimized the decrease in plasma indispensable AA due to repeated handling stress after 28 days. It was concluded that HP diet decreased post-stress plasma glucose and lactate levels in HDLG HP specimens only at 14 days of treatment. Moreover, dietary treatment was also effective in stimulating DA synthesis and release, thus dietary phenylalanine supplementation can increase DA biosynthesis in fish.     

Primary hemocyte culture of Penaeus monodon as an in vitro model for white spot syndrome virus titration, viral and immune related gene expression and cytotoxicity assays

Immortal cell lines have not yet been reported from Penaeus monodon, which delimits the prospects of investigating the associated viral pathogens especially white spot syndrome virus (WSSV). In this context, a method of developing primary hemocyte culture from this crustacean has been standardized by employing modified double strength Leibovitz-15 (L-15) growth medium supplemented with 2% glucose, MEM vitamins (1×), tryptose phosphate broth (2.95 g l⁻¹), 20% FBS, N-phenylthiourea (0.2 mM), 0.06 μg ml⁻¹ chloramphenicol, 100 μg ml⁻¹ streptomycin and 100 IU ml⁻¹ penicillin and hemolymph drawn from shrimp grown under a bio-secured recirculating aquaculture system (RAS). In this medium the hemocytes remained viable up to 8 days. 5-Bromo-2′-deoxyuridine (BrdU) labeling assay revealed its incorporation in 22 ± 7% of cells at 24 h. Susceptibility of the cells to WSSV was confirmed by immunofluoresence assay using a monoclonal antibody against 28 kDa envelope protein of WSSV. A convenient method for determining virus titer as MTT₅₀/ml was standardized employing the primary hemocyte culture. Expression of viral genes and cellular immune genes were also investigated. The cell culture could be demonstrated for determining toxicity of a management chemical (benzalkonium chloride) by determining its IC₅₀. The primary hemocyte culture could serve as a model for WSSV titration and viral and cellular immune related gene expression and also for investigations on cytotoxicity of aquaculture drugs and chemicals.     

Dietary supplementation of pyrroloquinoline quinone disodium protects against oxidative stress and liver damage in laying hens fed an oxidized sunflower oil-added diet

The protective effects of dietary pyrroloquinoline quinone disodium (PQQ.Na₂) supplementation against oxidized sunflower oil-induced oxidative stress and liver injury in laying hens were examined. Three hundred and sixty 53-week-old Hy-Line Gray laying hens were randomly allocated into one of the five dietary treatments. The treatments included: (1) a diet containing 2% fresh sunflower oil; (2) a diet containing 2% thermally oxidized sunflower oil; (3) an oxidized sunflower oil diet with 100 mg/kg of added vitamin E; (4) an oxidized sunflower oil diet with 0.08 mg/kg of PQQ.Na₂; and (5) an oxidized sunflower oil diet with 0.12 mg/kg of PQQ.Na₂. Birds fed the oxidized sunflower oil diet showed a lower feed intake compared to birds fed the fresh oil diet or oxidized oil diet supplemented with vitamin E (P=0.009). Exposure to oxidized sunflower oil increased plasma malondialdehyde (P<0.001), hepatic reactive oxygen species (P<0.05) and carbonyl group levels (P<0.001), but decreased plasma glutathione levels (P=0.006) in laying hens. These unfavorable changes induced by the oxidized sunflower oil diet were modulated by dietary vitamin E or PQQ.Na₂ supplementation to levels comparable to the fresh oil group. Dietary supplementation with PQQ.Na₂ or vitamin E increased the activities of total superoxide dismutase and glutathione peroxidase in plasma and the liver, when compared with the oxidized sunflower oil group (P<0.05). PQQ.Na₂ or vitamin E diminished the oxidized sunflower oil diet induced elevation of liver weight (P=0.026), liver to BW ratio (P=0.001) and plasma activities of alanine aminotransferase (P=0.001) and aspartate aminotransferase (P<0.001) and maintained these indices at the similar levels to the fresh oil diet. Furthermore, oxidized sunflower oil increased hepatic DNA tail length (P<0.05) and tail moment (P<0.05) compared with the fresh oil group. Dietary supplementation of PQQ.Na₂ or vitamin E decreased the oxidized oil diet induced DNA tail length and tail moment to the basal levels in fresh oil diet. These results indicate that PQQ.Na₂ is a potential antioxidant and is as effective against oxidized oil-related liver injury in laying hens as vitamin E. The protective effects of PQQ.Na₂ against liver damage induced by oxidized oil may be partially due to its role in the scavenging of free radicals, inhibiting of lipid peroxidation and enhancing of antioxidant defense systems.     

Enhanced binding of calmodulin to RyR2 corrects arrhythmogenic channel disorder in CPVT-associated myocytes

Aims

Calmodulin (CaM) plays a key role in modulating channel gating in ryanodine receptor (RyR2). Here, we investigated (a) the pathogenic role of CaM in the channel disorder in CPVT and (b) the possibility of correcting the CPVT-linked channel disorder, using knock-in (KI) mouse model with CPVT-associated RyR2 mutation (R2474S).

Methods and results

Transmembrane potentials were recorded in whole cell current mode before and after pacing (1–5 Hz) in isolated ventricular myocytes. CaM binding was assessed by incorporation of exogenous CaM fluorescently labeled with HiLyte Fluor® in saponin-permeabilized myocytes. In the presence of cAMP (1 μM) the apparent affinity of CaM binding to the RyR decreased in KI cells (Kd: 140–400 nM), but not in WT cells (Kd: 110–120 nM). Gly-Ser-His-CaM (GSH-CaM that has much higher RyR-binding than CaM) restored normal binding to the RyR of cAMP-treated KI cells (140 nM). Neither delayed afterdepolarization (DAD) nor triggered activity (TA) were observed in WT cells even at 5 Hz pacing, whereas both DAD and TA were observed in 20% and 12% of KI cells, respectively. In response to 10 nM isoproterenol, only DAD (but not TA) was observed in 11% of WT cells, whereas in KI cells the incidence of DAD and TA further increased to 60% and 38% of cells, respectively. Addition of GSH-CaM (100 nM) to KI cells decreased both DADs and TA (DAD: 38% of cells; TA: 10% of cells), whereas CaM (100 nM) had no appreciable effect. Addition of GSH-CaM to saponin-permeabilized KI cells decreased Ca²⁺ spark frequency (+33% of WT cells), which otherwise markedly increased without GSH-CaM (+100% of WT cells), whereas CaM revealed much less effect on the Ca²⁺ spark frequency (+76% of WT cells). Then, by incorporating CaM or GSH-CaM to intact cells (with protein delivery kit), we assessed the in situ effect of GSH-CaM (cytosolic [CaM] = ∼240 nM, cytosolic [GSH-CaM] = ∼230 nM) on the frequency of spontaneous Ca²⁺ transient (sCaT, % of total cells). Addition of 10 nM isoproterenol to KI cells increased sCaT after transient 5 Hz pacing (37%), whereas it was much more attenuated by GSH-CaM (9%) than by CaM (26%) (P < 0.01 vs CaM).

Conclusions

Several disorders in the RyR channel function characteristic of the CPVT-mutant cells (increased spontaneous Ca²⁺ leak, delayed afterdepolarization, triggered activity, Ca²⁺ spark frequency, spontaneous Ca²⁺ transients) can be corrected to a normal function by increasing the affinity of CaM binding to the RyR.