Body weight loss in beef cows: II. Increased antioxidant messenger ribonucleic acid levels in skeletal muscle but not erythrocyte antioxidant activity.

Twenty-six Angus-cross cows were used to examine the effect of BW loss (WL) on skeletal muscle and erythrocyte markers of oxidative stress. Serum NEFA concentrations, erythrocyte superoxide dismutase, and glutathione peroxidase activities were measured during WL and BW maintenance. Real-time reverse-transcription-PCR was used to determine mRNA levels of antioxidant genes during both periods to assess skeletal muscle response to WL. Body weight loss resulted in elevated serum NEFA concentrations but no change in erythrocyte superoxide dismutase and glutathione peroxidase activities. During WL, mRNA levels of the antioxidant genes glutathione peroxidase 4, mitochondrial superoxide dismutase, thioredoxin reductase 1, and selenoprotein W increased. Abundance of mRNA of genes involved in antioxidant signaling, specifically, PPARgamma coactivator-1 alpha, nuclear respiratory factor 1, estrogen-related receptor alpha, and tumor protein 53, was also increased. In summary, during WL cows had no change in peripheral antioxidant enzyme activity, but mRNA abundance of proteins involved in protecting the body from oxidative stress increased in skeletal muscle. During times when NEFA are used as a fuel source, signals such as mild reactive oxygen species production or increased concentration of lipid by-products activate the transcription of nuclear signaling molecules such as PPARgamma gamma coactivator-1 alpha, nuclear respiratory factor 1, estrogen-related receptor alpha, and tumor protein 53. These genes work to activate antioxidant genes such as mitochondrial superoxide dismutase, glutathione peroxidase 4, and thioredoxin reductase 1 to aid in the detoxification of reactive oxygen species. These data suggest an important role for antioxidant genes to protect cattle that are mobilizing body fat.

Effect of cobalt supplementation during late gestation and early lactation on milk and serum measures.

Thirty-six multiparous cows were assigned to a study to determine the effects of dietary Co supplementation during late gestation and early lactation on concentrations of Co in serum and liver, vitamin B12 concentrations in serum and milk, and milk yield. Nonlactating cows received diets containing 0.15, 0.89, or 1.71 mg/ kg of Co (dry matter basis) from 55 d before parturition, and lactating cows received diets containing 0.19, 0.57, or 0.93 mg/kg of Co (dry matter basis) from parturition through 120 d postpartum. Serum vitamin B12 concentrations declined sharply in all cows between 55 and 20 d prepartum. Dietary Co supplementation tended to cause an increase in the concentration of vitamin B12 in colostrum and milk. Cobalt intake did not affect concentrations of Co in liver or serum, but increased the Co concentration of milk (0.089, 0.120, and 0.130 microg of Co/mL) at 120 days in milk. There was no effect of Co supplementation on dry matter intake or yield of milk and milk components. In conclusion, serum concentrations of vitamin B12 are reduced in the early dry period, and added dietary Co may increase ruminal synthesis of vitamin B12 as indicated by a tendency for increased vitamin B12 concentrations in colostrum and milk of cows supplemented with dietary Co.

Effect of selenium intake and fetal age on mRNA levels of two selenoproteins in porcine fetal and maternal liver.

The objective of this study was to determine if levels of mRNA encoding cytosolic glutathione peroxidase (cGPx) and thioredoxin reductase (TrxR-1) change during fetal development, and if maternal Se intake during gestation affects the mRNA levels of these proteins. Prepubertal gilts (n = 24) were randomly assigned to either Se-adequate (0.39 ppm of Se; n = 12) or Se-deficient (0.05 ppm of Se; n = 12) diets, 6 wk before breeding. Maternal liver was collected at d 10, 45, 70, and 114 of pregnancy, and fetal liver samples were collected at the same times except d 10. Complementary DNA sequences encoding cGPx and TrxR-1 were cloned and sequenced. Quantitative real-time PCR analysis indicated that levels of mRNA for cGPx in fetal liver decreased more than 3-fold between d 45 and 114 of gestation. Although the gilts were only marginally deficient in Se, and maternal Se intake did not affect cGPx mRNA levels in fetal liver, the low-Se diet tended (P = 0.1) to reduce fetal TrxR-1 mRNA levels. In the liver of the dams, the low Se intake did not affect mRNA levels for either cGPx or TrxR-1. Compared with the liver of the dams, mRNA levels for cGPx were about 3.5 times lower in fetal liver. Results of this study support the hypothesis that neonatal pigs are born with reduced cGPx corresponding to reduced cGPx mRNA levels during late gestation.

Prediction and evaluation of urine and urinary nitrogen and mineral excretion from dairy cattle.

Urine excretion is a substantial factor in the amount of manure that needs to be managed, and urinary N can contribute to ammonia volatilization. Development and validation of prediction equations focusing on dietary factors to decrease urine and urinary nutrient excretion will provide information for managing urine and feces separately or for other future technologies. The objective of this study was to develop equations for prediction of urine excretion and excretion of urinary N, Na, and K and to evaluate both new and previously published prediction equations for estimation of urine and urinary nutrient excretion from lactating dairy cows. Data sets from metabolism studies conducted at Washington State University were compiled and evaluated for excretion of minerals. Urine excretion averaged 24.1 kg/d and urinary nitrogen excretion ranged from 63 to 499 g/d in the calibration data set. Regression equations were developed to predict urine excretion, urinary N excretion, and urinary Na and K excretion. Predictors used in the regression equations included milk yield, body weight, dietary crude protein percentage, milk urea nitrogen, and nutrient intakes. Previously published prediction equations were evaluated using data sets from Washington State University and the University of Wisconsin. Mean and linear biases were evaluated by determining the regression of residuals on predicted values. Evaluation and validation of prediction equations are important to develop equations that will more accurately estimate urine and urinary nitrogen excretion from lactating dairy cows.

Prediction of manure and nutrient excretion from dairy cattle.

Accurate estimates of manure excretion are needed for planning manure storage facilities and for nutrient management. Data sets from metabolism studies conducted at several universities were compiled and evaluated for excretion of total manure, N, P, and K. Animal groups included calves weighing up to 204 kg, heifers weighing between 274 and 613 kg, nonlactating cows, and lactating cows. Regression equations were developed to predict excretion of total manure, total dry matter, N, P, and K. Predictors used in the regression equations for lactating cows included milk yield, percentages of protein and fat in milk, dietary concentrations of crude protein and neutral detergent fiber, and intakes of nutrients. The regression equations provide improved predictions of excretion and enable more accurate planning of manure storage and nutrients to be managed at the farm level.

Effect of grain source and exogenous phytase on phosphorus digestibility in dairy cows.

Two experiments were conducted to determine P digestibility in lactating dairy cows fed corn or barley as grain sources. The first experiment utilized a replicated incomplete 5 x 4 Latin square design with 8 lactating Holstein cows fed diets containing either corn alone or corn in combination with one of 4 barley varieties that differed in chemical composition. Total tract digestibility of P ranged from 11 to 29% for diets containing the barley varieties and was approximately 35% for the corn diet. A second experiment compared P digestibility in cows fed diets containing corn or barley when exogenous phytase was added to the diets. Lactating Holstein cows (n = 16) were arranged in 4 replications of a Latin square with 2 grains (barley or corn), fed separately or with added exogenous phytase (427 phytase units/kg of total mixed ration and 4 periods of 21 d. Phytate P comprised about 50% of the total P (0.46% P) in the total mixed ration. The concentration of serum inorganic P was higher in cows fed diets with exogenous phytase (5.8 vs. 6.5 mg/dL in cows fed barley diets and 5.5 vs 6.0 mg/dL in cows fed corn diets). Using acid detergent lignin as an internal marker, hydrolysis of phytate P was increased by the exogenous phytase, and total P digestibility tended to be increased. In contrast to Experiment 1, in Experiment 2 there was no effect of grain source on P digestibility and total fecal P. Dry matter intake and efficiency of milk production were not affected by exogenous phytase or grain type. Although phytase activity occurs in the rumen, physical properties of the diet and ruminal passage rates may prevent total hydrolysis of phytate in the rumen of lactating cows. Thus, exogenous dietary phytase might improve P digestibility in dairy cows in some dietary situations.

Studies into using manure in a biorefinery concept.

Animal manure is an underutilized biomass resource containing a large amount of organic carbon that is often wasted with the existing manure disposal practices. A research project funded by the US Department of Energy explored the feasibility of using manure via the sugar platform in a biorefinery, converting the carbon from fiber to biochemicals. The results showed that (1) fiber was the major component of manure dry material making up approx 50%, 40%, and 36% of the dry dairy, swine, and poultry manure material, respectively; within dairy manure, more than 56% of the dry matter was in particles larger than 1.680 mm; (2) in addition to being a carbon source, manure could provide a variety of nutrient for fungi T. reesei and A. phoenicis to produce cellulase; (3) the hemicellulose component in the manure fiber could be readily converted to sugar through acid hydrolysis; while concentrated acid decrystallization treatment was most effective in manure cellulose hydrolysis; (4) purification and separation was necessary for further chemical conversion of the manure hydrolysate to polyols through hydrogenation; and (5) the manure utilization strategy studied in this work is currently not profitable.

Maternal selenium deficiency increases hydrogen peroxide and total lipid peroxides in porcine fetal liver.

To investigate the role of selenium (Se) in the developing porcine fetus, prepubertal gilts (n = 42) were randomly assigned to either Se-adequate (0.39 ppm Se) or Se-deficient (0.05 ppm Se) gestation diets 6 wk prior to breeding. Maternal and fetal liver was collected at d 30, 45, 70, 90, and 114 of pregnancy. Concentrations of Se in maternal liver decreased during gestation in gilts fed the low-Se diet. The activity of cellular glutathione peroxidase (GPx) was decreased at d 30 and 45 of gestation in liver of gilts fed the low-Se diet. Concentrations of malondialdehyde (MDA) and hydrogen peroxide (H2O2) were greater in liver homogenates from gilts fed the low-Se diet. Within the fetuses, liver Se decreased in those fetuses of gilts fed the low-Se diet. Although the activity of GPx in fetal liver was not affected by the maternal diet, concentrations of H2O2 and MDA in fetal liver were greater in fetuses from gilts fed the low-Se diet. Maternal liver GPx activity was approx 12-fold greater than fetal liver GPx activity regardless of dietary treatment. These results indicate that maternal dietary Se intake affects fetal liver Se concentration and feeding a low-Se diet during gestation increases oxidative stress to the fetus, as measured by fetal liver H2O2 and MDA.

Gestational changes in concentrations of selenium and zinc in the porcine fetus and the effects of maternal intake of selenium.

The effect of maternal dietary selenium (Se) and gestation on the concentrations of Se and zinc (Zn) in the porcine fetus were determined. Mature gilts were randomly assigned to treatments of either adequate (0.39 ppm Se) or low (0.05 ppm Se) dietary Se. Gilts were bred and fetuses were collected throughout gestation. Concentrations of Se in maternal whole blood and liver decreased during gestation in sows fed the low-Se diet compared to sows fed the Se-supplemented diet. Maternal intake of Se did not affect the concentration of Se in the whole fetus; however, the concentration of Se in fetal liver was decreased in fetuses of sows fed the low-Se diet. Although fetal liver Se decreased in both treatments as gestation progressed, the decrease was greater in liver of fetuses from sows fed the low- Se diet. Dietary Se did not affect concentrations of Zn in maternal whole blood or liver or in the whole fetus and fetal liver. The concentration of Se in fetal liver was lower but the concentration of Zn was greater than in maternal liver when sows were fed the adequate Se diet. These results indicate that maternal intake of Se affects fetal liver Se and newborn piglets have lower liver Se concentrations compared to their dams, regardless of the Se intake of sows during gestation. Thus, the piglet is more susceptible Se deficiency than the sow.

Effect of dietary cobalt supplementation on cobalt metabolism and performance of dairy cattle.

Three studies were conducted with dairy cattle fed diets with added Co. The first study examined cow age and added dietary Co on Co in liver and blood. Nonpregnant, nonlactating Holstein cows were blocked by age (2.5 or 6.5 yr) and assigned to either a control diet or a diet supplemented with 9 mg Co per day. The Co concentration of liver, taken on d 60, was not affected by dietary Co but was higher in the younger cows. The cytosolic fraction of liver contained the most Co, and the subcellular distribution of Co was not affected by total Co in liver. In a second study, Holstein cows were assigned to one of three treatments of dietary Co from 21 d prepartum until 120 d postpartum. There was an interaction of time x treatment x parity such that milk yield response to Co supplementation differed between multiparous cows and primiparous cows. Supplemental Co did not increase Co in serum, colostrum, milk, or liver. Primiparous cows secreted colostrum and milk with higher Co concentrations than did multiparous cows. Likewise, serum B12 levels were higher in primiparous than multiparous cows and declined with increasing days in milk (DIM). Serum Co also decreased from 7 to 120 DIM. In a final study, a Co supplement in the starter diet did not affect Co in serum or liver of young calves. In conclusion, supplemental dietary Co did not affect secretion of Co in milk, tissue retention, or subcellular distribution of Co within the liver. Primiparous and multiparous cows differed in their milk yield response to dietary Co supplementation.

The effect of oilseeds in diets of lactating cows on milk production and methane emissions.

Thirty-six lactating multiparous Holstein cows were assigned to diets that contained 2.3, 4.0, and 5.6% fat for an entire lactation to determine the effect of oilseeds on milk composition, production, and methane emissions. The diets were formulated so that whole cottonseeds and canola oilseeds provided equal amounts of added fat. Methane emissions were measured every 3 mo from two replicates of four cows per treatment using a room tracer approach. Dry matter intakes and yields of milk and FCM were greater for cows fed the diets containing oilseeds. Although the concentration of protein in milk was reduced, yields of both protein and fat tended to be increased by the addition of fat. Within the milk fat, the concentrations of C10, C12, C14:0, and C16:0 were reduced and concentrations of C18, C18:1, and trans-C18:1 were increased in response to dietary oilseeds. In serum, urea-N was increased by the dietary oilseeds. Supplementation of diets with oilseeds did not affect methane emissions but tended to increase the efficiency of milk produced per unit of methane emitted. A 1.7% addition of fat to the control diet from a combination of oilseed types increased yields of milk without reducing methane emission rates. The strategy of using unsaturated fats from oilseeds to substantially reduce methane emissions was ineffective, although yield of milk was increased.

A modified risk assessment to establish molybdenum standards for land application of biosolids.

The USEPA standards (40 CFR Part 503) for the use or disposal of sewage sludge (biosolids) derived risk-based numerical values for Mo for the biosolids --> land --> plant --> animal pathway (Pathway 6). Following legal challenge, most Mo numerical standards were withdrawn, pending additional field-generated data using modern biosolids (Mo concentrations <75 mg kg(-1) and a reassessment of this pathway. This paper presents a reevaluation of biosolids Mo data, refinement of the risk assessment algorithms, and a reassessment of Mo-induced hypocuprosis from land application of biosolids. Forage Mo uptake coefficients (UC) are derived from field studies, many of which used modern biosolids applied to numerous soil types, with varying soil pH values, and supporting various crops. Typical cattle diet scenarios are used to calculate a diet-weighted UC value that realistically represents forage Mo exposure to cattle. Recent biosolids use data are employed to estimate the fraction of animal forage (FC) likely to be affected by biosolids applications nationally. Field data are used to estimate long-term Mo leaching and a leaching correction factor (LC) is used to adjust cumulative biosolids application limits. The modified UC and new FC and LC factors are used in a new algorithm to calculate biosolids Mo Pathway 6 risk. The resulting numerical standards for Mo are cumulative limit (RPc)=40 kg Mo ha(-1), and alternate pollutant limit (APL) = 40 mg Mo kg(-1) We regard the modifications to algorithms and parameters and calculations as conservative, and believe that the risk of Mo-induced hypocuprosis from biosolids Mo is small. Providing adequate Cu mineral supplements, standard procedure in proper herd management, would augment the conservatism of the new risk assessment.

Effects of prenatal source and level of dietary selenium on passive immunity and thermometabolism of newborn lambs.

The objective of this study was to determine the effects of the amount and chemical form of Se fed to pregnant ewes on concentrations of thyroid hormones, Se in tissues, immunoglobulins (Ig) in serum, and measures of thermometabolism in ewes and their newborn lambs. Pregnant ewes (n=21) were randomly assigned to receive a diet deficient of Se (<0.02ppm) or supplemented to provide 0.3ppm Se from either sodium selenite or selenized yeast (SeY). Pregnant ewes which were fed additional Se had increased (P<0.05) concentrations of Se in whole blood and serum, greater activity of glutathione peroxidase (GSHpx), and higher concentrations of tri-iodothyronine (T(3)) and thyroxine (T(4)). At 12h of age, lambs of ewes given Se had higher (P<0.05) concentrations of Se in blood and liver, greater activities of GSHpx, and tended (P<0.1) to have higher T(3) levels. When the two Se supplements were compared, lambs of ewes which were fed with SeY had higher (P<0.001) concentrations of Se and activities of GSHpx (P<0.05) in blood than lambs of ewes fed with selenite. Concentrations of Se in colostrum were increased (P<0.05) with Se supplementation and the ewes fed with SeY tended (P<0.1) to have higher Se in colostrum than ewes fed with selenite. Although IgG in serum and colostrum of ewes was not affected by supplemental Se, IgM in serum of ewes was increased (P<0.05). Lambs of ewes which were given Se had increased (P<0.05) absorption of IgG, but not IgM. No treatment effects on measures of thermometabolism were detected. In conclusion, Se supplementation of pregnant ewes affected measures of thyroxine metabolism and immunity. Compared to selenite, Se from SeY was more readily transferred to the fetus and colostrum.

Phosphorylation of Syk activation loop tyrosines is essential for Syk function. An in vivo study using a specific anti-Syk activation loop phosphotyrosine antibody.

Syk is an important protein-tyrosine kinase in immunoreceptor signaling. FcepsilonRI aggregation in mast cells induces tyrosine phosphorylation and increased enzymatic activity of Syk. The two adjacent tyrosines in the Syk activation loop are thought to be important for the propagation of FcepsilonRI signaling. To evaluate the phosphorylation of these tyrosines in vivo and further understand the relationship of Syk tyrosine phosphorylation with its function, an antibody was developed specific for phosphorylated tyrosines in the activation loop of Syk. FcepsilonRI aggregation on mast cells induced the phosphorylation of both tyrosine residues of the activation loop. The kinase activity of Syk played the major role in phosphorylating its activation loop tyrosines both in vivo and in vitro. In FcepsilonRI-stimulated mast cells, the total Syk tyrosine phosphorylation paralleled the phosphorylation of its activation loop tyrosines and downstream propagation of signals for histamine release. In contrast, the cell surface binding of anti-ganglioside monoclonal antibody AA4 induced only strong general tyrosine phosphorylation of Syk and minimal histamine release and weak phosphorylation of activation loop tyrosines. These results demonstrate that phosphorylation of the activation loop tyrosines is important for mediating receptor signaling and is a better marker of Syk function than is total Syk tyrosine phosphorylation.

Metabolic and yield responses of multiparous Holstein cows to prepartum rumen-undegradable protein.

The effects of concentration of rumen-undegradable protein (RUP) in prepartum diets on performance and metabolic measures were determined. Pregnant, nonlactating cows were assigned to one of three dietary treatments that differed in concentrations of crude protein and RUP. The crude protein and RUP percentages (dry matter basis) in the diets were 11.7 and 3.1%, 15.6 and 6.8%, and 20.6 and 10.6%, which were identified as low, medium, and high protein diets, respectively. Dietary treatments were initiated 6 wk prior to expected calving date and were fed until parturition. All cows were fed similar postpartum diets through wk 10 of lactation. Prepartum intakes of dry matter and rumen-degradable protein were similar among treatments. Yields of milk, protein, and fat were not affected by prepartum RUP. Concentrations of plasma urea N in cows fed the medium and high protein diets were elevated during the prepartum period even though intakes of rumen-degradable protein were similar for cows on all treatments. Cows fed the medium and high protein diets had greater prepartum concentrations of Val, Ile, and Leu but not other essential amino acids in plasma. Dietary treatments did not affect prepartum or postpartum contents of triglycerides in liver or plasma nonesterified fatty acid concentrations. Thus, supplemental RUP fed to cows for 6 wk prepartum affected protein metabolism but not measures of energy metabolism.

Reduction of calcineurin activity in brain by antisense oligonucleotides leads to persistent phosphorylation of tau protein at Thr181 and Thr231.

Phosphorylation of tau protein promotes stability of the axonal cytoskeleton; aberrant tau phosphorylation is implicated in the biogenesis of paired helical filaments (PHF) seen in Alzheimer's disease. Protein kinases and phosphatases that modulate tau phosphorylation have been identified using in vitro techniques; however, the role of these enzymes in vivo has not been determined. We used intraventricular infusions of antisense oligodeoxynucleotides (ODNs) directed against the major brain isoforms of the Ca2+/calmodulin-dependent phosphatase calcineurin to determine how reduced activity of this enzyme would affect tau dephosphorylation. Five-day infusions of antisense ODNs (5 and 10 nmol/day) in rats decreased immunoreactive levels and activity of calcineurin throughout the brain; sense ODNs, scrambled ODNs, and infusion vehicle alone had no effect. When neocortical slices were prepared from antisense ODN-treated rats and incubated for 1 to 2 h in vitro, tau protein remained phosphorylated as determined by using the phosphorylation-sensitive monoclonal antibodies AT-180 (Thr231) and AT-270 (Thr181). In contrast, AT-180 and AT-270 sites were completely dephosphorylated during incubation of neocortical slices from vehicle-infused controls and sense ODN-treated rats. Neocortical slices from antisense-treated rats were incubated with the phosphatase inhibitors okadaic acid (100 nM; 10 microM) and FK-520 (5 microM); these preparations showed enhanced tau phosphorylation, consistent with a significant loss of calcineurin activity. Thus, we conclude that phosphorylation of at least two sites on tau protein, namely, Thr181 and Thr231, is regulated by calcineurin.

Mineral deficiencies in grazing dairy cattle in Kordofan and Darfur regions in western Sudan.

The seasonal changes in mineral profiles in serum of grazing dairy cattle and the concentrations of nutrients available from forages were determined in western Sudan. Blood samples were collected seasonally from dairy cows, Kenana and Botana breeds, in 6 locations in Kordofan and Darfur. Data were analysed as a split-plot design with repeated measures. The results indicated there were significant seasonal changes in concentrations of P, Cu and K, Ca, Mg, Na, Co, and Zn in blood serum of grazing cows. Concentrations of P, Ca and Na in serum were lowest during the late dry season (4.5 mg/dl, 8.21 mg/dl and 129 mEq/L respectively), while concentrations of Cu, deficient throughout the year, were lowest during the rainy season (0.35 microgram/ml). The interaction of location x season was significant for serum P, Ca, Cu, Mg, Na, Co and K. Kenana cattle had lower Zn in serum (0.96 microgram/ml) than Botana cattle (1.13 microgram/ml). During the dry season, forages had very low CP (3.5 +/- 0.5%), high neutral detergent fibre (NDF) (67.5 +/- 1.8%) and high acid detergent (ADF) (38.5 +/- 1.2%) content. Calcium, P and Na levels in forages were deficient during the dry season (0.38 +/- 0.03%, 0.08 +/- 0.03% and 0.047 +/- 0.01% respectively). Copper also was deficient in forages during the dry season, but adequate during the mid-rainy season (3.0 +/- 2.4 and 26.1 +/- 1.5 mg/kg respectively). In conclusion, the low Cu concentration in sera of cattle indicated a possible nutritional deficiency throughout the year, while P, Ca, and Na were low in sera during the late dry season. Thus, seasonal deficiencies of minerals may affect productivity of cattle in the Kordofan and Darfur regions of Sudan.

Effect of anionic salts on selenium metabolism in nonlactating, pregnant dairy cows.

The objective of this trial was to determine whether anionic salts in the diets of nonlactating, pregnant cows for 14 to 21 d prepartum affected measures of Se status. One of three dietary treatments (control, anionic salts, and anionic salts plus Se) was administered to 34 nonlactating, pregnant cows using a completely randomized design with repeated measures. The anionic salts were delivered via gelatin capsules that were administered orally in two equal amounts per day, and the Se (3 mg/d) was administered via an intraruminal bolus. The incidence of milk fever among cows was not significantly different across dietary treatments. The severity of hypocalcemia as indicated by concentrations of ionized Ca in serum collected < 2 h postpartum was significantly lessened by dietary anionic salts. Supplementation of anionic salts to the diet did not significantly affect serum Ca concentrations at either 7 d prepartum or 7 d postpartum. Anionic salts did not affect concentrations of Se in blood; however, Se supplementation of the diets of cows significantly increased postpartum concentrations of Se in serum. No treatment effects were detected for concentrations of Se in either serum or whole blood of newborn calves. In conclusion, these data indicate that diets supplemented with anionic salts for 14 to 21 d prepartum and the supplementation of diets with Se had independent effects on concentrations of minerals in blood. Thus, anionic salts administered to prevent milk fever without danger of significantly reducing the transfer of Se from the dam to the calf and without compromising the Se status of the cow when the anionic salts are limited to administration for 14 to 21 d before calving.

Direct evidence that FK506 inhibition of FcepsilonRI-mediated exocytosis from RBL mast cells involves calcineurin.

FcepsilonRI-mediated exocytosis of preformed mediators from mast cells and basophils (e.g. histamine, serotonin, beta-hexosaminidase) is sensitive to the immunosuppressants cyclosporin A and FK506 (IC50 200 and 4 nM, respectively) but not rapamycin. The mechanism of inhibition does not appear to involve tyrosine phosphorylation, hydrolysis of inositol phosphates or calcium flux. Here we report experiments using a molecular approach to assess the role of calcineurin, a serine/threonine phosphatase thought to be the primary pharmacological target of these drugs. Calcineurin's activity requires association of its catalytic (A) subunit with an intrinsic regulatory (B) subunit. We hypothesized that calcineurin-sensitive signalling events should be affected by the depletion of calcineurin B subunits, thereby reducing the number of active A:B complexes. We therefore transfected rat basophilic leukemia (RBL) cells with an inhibitory (dominant negative) form of the calcineurin A subunit, which binds the calcineurin B subunit with high affinity but does not possess catalytic activity (B subunit knock-out, BKO). In these transfected cells, the dose-response curve for the inhibition of FcepsilonRI-mediated exocytosis by FK506 was shifted to the left, indicating an increased drug sensitivity of BKO-transfected cells. We conclude that FK506 inhibition of FcepsilonRI-mediated exocytosis in mast cells specifically targets calcineurin activity.