Effects of negative dietary cation-anion difference and calcidiol supplementation in transition diets fed to sows on piglet survival, piglet weight, and sow metabolism.

Diets that provide a negative dietary anion cation difference (DCAD) and supplement with a vitamin D metabolite 25-OH-D3 (calcidiol) may increase calcium availability at parturition, and enhance piglet survival and performance. This factorial study assessed the effects of DCAD, calcidiol (50 µg/kg), and parity (parity 1 or >1) and their interactions. Large White and Landrace sows (n = 328), parity 1 to 8 were randomly allocated in blocks to treatment diets from day 103 of gestation until day 3 postfarrow: 1) negative DCAD without calcidiol (negative DCAD + no CA), n = 84, 2) negative DCAD with calcidiol (negative DCAD + CA) n = 84, 3) positive DCAD without calcidiol (negative DCAD + no CA), n = 81, and 4) positive DCAD with calcidiol (positive DCAD + CA), n = 79. Negative DCAD diets were acidified with an anionic feed (2 kg/t) and magnesium sulfate (2 kg/t). All treatment diets contained cholecalciferol at 1,000 IU/kg. Dry sow diets contained 14.8% crude protein (CP), 5.4% crude fiber (CF), 0.8% Ca, and 83 mEq/kg DCAD. Treatment diets 1 and 2 contained 17.5% CP, 7.3% CF, 0.8% Ca, and -2 mEq/kg DCAD. Treatment diets 3 and 4 contained 17.4% CP, 7.4% CF, 0.8% Ca, and 68 mEq/kg DCAD. Before farrowing, all negative DCAD sows had lower urine pH than all sows fed a positive DCAD (5.66 ± 0.05 and 6.29 ± 0.05, respectively; P < 0.01); urinary pH was acidified for both DCAD treatments indicating metabolic acidification. The percentage of sows with stillborn piglets was not affected by DCAD, calcidiol, or parity alone but sows fed the negative DCAD + CA diet had a 28% reduction in odds of stillbirth compared to the negative DCAD + no CA diet and even lesser odds to the positive DCAD + CA diet. At day 1 after farrowing, blood gas, and mineral and metabolite concentrations were consistent with feeding a negative DCAD diet and that negative DCAD diets influence energy metabolism, as indicated by increased glucose, cholesterol, and osteocalcin concentrations and reduced nonesterified free fatty acids and 3-hydroxybutyrate concentrations. In the subsequent litter, total piglets born and born alive (14.7 ± 0.3 and 13.8 ± 0.3 piglets, respectively; P = 0.029) was greater for positive DCAD diets compared to negative DCAD diets; and there was an interaction between DCAD, calcidiol, and parity (P = 0.002). Feeding a negative DCAD diet influenced stillbirth, subsequent litter size, and metabolic responses at farrowing. More studies are needed to define optimal diets prefarrowing for sows.

The transition period between late gestation and lactation is critical to farrowing and successful lactation; sows with higher blood calcium have less risk of dystocia. We evaluated transition diets that provided a negative dietary cation­anion difference (DCAD) and supplemented with calcidiol (CA), both of which influence calcium metabolism. Purebred Landrace or Large White sows (n = 328) were enrolled in the experiment and selected sows that were either primiparous (n = 99) or multiparous (n = 229; average parity = 2.59 ± 1.51; parity range = 1 to 8) were fed a dry sow ration until day 103 of gestation and were then fed transition diets until day 3 postfarrowing in a factorial study. The diets were formulated to include 1) negative DCAD + no CA, 2) negative DCAD + CA, 3) positive DCAD + no CA, or 4) positive DCAD + CA. All diets induced a metabolic acidosis as indicated by urinary pH. Sows fed the negative DCAD with added calcidiol had a >28% reduction in odds of stillbirth over negative DCAD + no CA and positive DCAD + CA diets. Following weaning and re-mating, there were 0.9 more piglets born in the subsequent litter for both positive DCAD diets compared to negative DCAD diets. Blood gas, and mineral and metabolite concentrations provided evidence that negative DCAD diets positively influenced energy metabolism.

Molecular basis for selective uptake and elimination of organic anions in the kidney by OAT1.

In mammals, the kidney plays an essential role in maintaining blood homeostasis through the selective uptake, retention or elimination of toxins, drugs and metabolites. Organic anion transporters (OATs) are responsible for the recognition of metabolites and toxins in the nephron and their eventual urinary excretion. Inhibition of OATs is used therapeutically to improve drug efficacy and reduce nephrotoxicity. The founding member of the renal organic anion transporter family, OAT1 (also known as SLC22A6), uses the export of α-ketoglutarate (α-KG), a key intermediate in the Krebs cycle, to drive selective transport and is allosterically regulated by intracellular chloride. However, the mechanisms linking metabolite cycling, drug transport and intracellular chloride remain obscure. Here, we present cryogenic-electron microscopy structures of OAT1 bound to α-KG, the antiviral tenofovir and clinical inhibitor probenecid, used in the treatment of Gout. Complementary in vivo cellular assays explain the molecular basis for α-KG driven drug elimination and the allosteric regulation of organic anion transport in the kidney by chloride.

Negative dietary cation-anion difference in prepartum dairy cow diets: a pragmatic study in two commercial dairy farms.

Pragmatic studies, evaluating the effectiveness of an intervention under its usual conditions, are less commonly reported than the explanatory trials. For instance, the effectiveness of prepartum negative dietary cation-anion difference (DCAD) diets on inducing a compensated metabolic acidosis that promotes a higher blood Ca concentration at calving has not been frequently described under commercial farm management conditions without researchers' interference. Thus, the objectives were to study cows under commercial farm management conditions to (1) describe the daily close-up dairy cows' urine pH and fed DCAD, and (2) evaluate the association between urine pH and fed DCAD, and preceding urine pH and blood Ca at calving. A total of 129 close-up Jersey cows about to commence their ≥2nd lactation were enrolled in the study after 7 days of exposure to DCAD diets in two commercial dairy herds. Urine pH was determined daily from mid-stream urine samples from enrollment to calving. Fed DCAD was determined from feed bunk samples obtained during 29 (Herd 1) and 23 (Herd 2) consecutive days. Plasma Ca concentration was determined within 12 h after calving. Descriptive statistics were generated at the herd- and cow-level. Multiple linear regression was used to evaluate the associations between urine pH and fed DCAD for each herd, and preceding urine pH and plasma Ca concentration at calving for both herds. At herd-level, the average urine pH and CV during the study period were 6.1 and 12.0% (Herd 1) and 5.9 and 10.9% (Herd 2), respectively. At the cow-level, the average urine pH and CV during the study period were 6.1 and 10.3% (Herd 1) and 6.1 and 12.3% (Herd 2), respectively. During the study period, fed DCAD averages were -121.3 and -165.7 mEq/kg of DM and CV 22.8 and 60.6% for Herd 1 and Herd 2, respectively. No evidence of association between cows' urine pH and fed DCAD was observed in Herd 1, whereas a quadratic association was observed in Herd 2. When both herds were combined, a quadratic association was observed between the urine pH intercept (at calving) and plasma Ca concentration. Although average urine pH and fed DCAD were within recommended ranges, the high variability observed indicates that acidification and fed DCAD are not constant, and often outside the recommended ranges in commercial settings. Monitoring of DCAD programs is warranted to ensure their effectiveness under commercial settings.

Review: Dietary cation-anion difference to prevent hypocalcemia with emphasis on over-acidification in prepartum dairy cows.

Hypocalcemia remains a common metabolic disorder of dairy cattle; therefore, an efficient prevention is still challenging. Among the various prevention strategies for hypocalcemia is the use of anionic compounds to induce a mild metabolic acidosis during the prepartum period. Acid-base status can be readily assessed through urine pH. Accordingly, a target urine pH during the prepartum period between 6.0 and 6.8 has been recommended for Holstein cows; however, in several countries, including the US, certain nutritional strategies are still focused on benchmarking the urine pH to below 6.0. Unfortunately, over-acidification can have no advantages and/or detrimental effects on both the dam and her offspring. In this review, updated information regarding the use of anionic diets on prepartum dairy cows and the potential negative impact of such diets on both cow and calf performance are discussed. There is an urgent need for studies that will elucidate the pathophysiological mechanisms by which very acidotic diets may impact the well-being and productive efficiency of dairy cows, and the transgenerational effects of such diets on offspring performance and survival.

Tea plant roots respond to aluminum-induced mineral nutrient imbalances by transcriptional regulation of multiple cation and anion transporters.

BACKGROUND: Tea is one of the most popular non-alcoholic beverages in the world for its flavors and numerous health benefits. The tea tree (Camellia sinensis L.) is a well-known aluminum (Al) hyperaccumulator. However, it is not fully understood how tea plants have adapted to tolerate high concentrations of Al, which causes an imbalance of mineral nutrition in the roots. RESULTS: Here, we combined ionomic and transcriptomic profiling alongside biochemical characterization, to probe the changes of metal nutrients and Al responsive genes in tea roots grown under increasing concentrations of Al. It was found that a low level of Al (~ 0.4 mM) maintains proper nutrient balance, whereas a higher Al concentration (2.5 mM) compromised tea plants by altering micro- and macro-nutrient accumulation into roots, including a decrease in calcium (Ca), manganese (Mn), and magnesium (Mg) and an increase in iron (Fe), which corresponded with oxidative stress, cellular damage, and retarded root growth. Transcriptome analysis revealed more than 1000 transporter genes that were significantly changed in expression upon Al exposure compared to control (no Al) treatments. These included transporters related to Ca and Fe uptake and translocation, while genes required for N, P, and S nutrition in roots did not significantly alter. Transporters related to organic acid secretion, together with other putative Al-tolerance genes also significantly changed in response to Al. Two of these transporters, CsALMT1 and CsALS8, were functionally tested by yeast heterologous expression and confirmed to provide Al tolerance. CONCLUSION: This study shows that tea plant roots respond to high Al-induced mineral nutrient imbalances by transcriptional regulation of both cation and anion transporters, and therefore provides new insights into Al tolerance mechanism of tea plants. The altered transporter gene expression profiles partly explain the imbalanced metal ion accumulation that occurred in the Al-stressed roots, while increases to organic acid and Al tolerance gene expression partly explains the ability of tea plants to be able to grow in high Al containing soils. The improved transcriptomic understanding of Al exposure gained here has highlighted potential gene targets for breeding or genetic engineering approaches to develop safer tea products.

Another building block in the plant cell wall: Barley xyloglucan xyloglucosyl transferases link covalently xyloglucan and anionic oligosaccharides derived from pectin.

We report on the homo- and hetero-transglycosylation activities of the HvXET3 and HvXET4 xyloglucan xyloglucosyl transferases (XET; EC 2.4.1.207) from barley (Hordeum vulgare L.), and the visualisation of these activities in young barley roots using Alexa Fluor 488-labelled oligosaccharides. We discover that these isozymes catalyse the transglycosylation reactions with the chemically defined donor and acceptor substrates, specifically with the xyloglucan donor and the penta-galacturonide [α(1-4)GalAp]5 acceptor - the homogalacturonan (pectin) fragment. This activity is supported by 3D molecular models of HvXET3 and HvXET4 with the docked XXXG donor and [α(1-4)GalAp]5 acceptor substrates at the -4 to +5 subsites in the active sites. Comparative sequence analyses of barley isoforms and seed-localised TmXET6.3 from nasturtium (Tropaeolum majus L.) permitted the engineering of mutants of TmXET6.3 that could catalyse the hetero-transglycosylation reaction with the xyloglucan/[α(1-4)GalAp]5 substrate pair, while wild-type TmXET6.3 lacked this activity. Expression data obtained by real-time quantitative polymerase chain reaction of HvXET transcripts and a clustered heatmap of expression profiles of the gene family revealed that HvXET3 and HvXET6 co-expressed but did not share the monophyletic origin. Conversely, HvXET3 and HvXET4 shared this relationship, when we examined the evolutionary history of 419 glycoside hydrolase 16 family members, spanning monocots, eudicots and a basal Angiosperm. The discovered hetero-transglycosylation activity in HvXET3 and HvXET4 with the xyloglucan/[α(1-4)GalAp]5 substrate pair is discussed against the background of roles of xyloglucan-pectin heteropolymers and how they may participate in spatial patterns of cell wall formation and re-modelling, and affect the structural features of walls.

Effect of anion type on enzymatic hydrolysis of starch-(thermostable α-amylase)-calcium system in a low-moisture solid microenvironment of bioextrusion.

The effect of six anions (Cl-, OH-, NO3-, SO42-, C6H10O62- and PO43-) on a starch (St)-enzyme (thermostable α-amylase, TαA)-calcium (Ca) system was investigated in a low-moisture solid state. Two levels of Ca salts (1 and 10 mmol/100 g St) added to potato starch with and without TαA were analyzed by FT-IR, DSC and SEM. The surface morphologies of the St-Ca complexes were different in the presence of various anions, and the residual Ca salts around the St granules might decrease the enzymatic action. For bioextrusion, TαA (0.5‰ and 1.5‰) were introduced for a relatively low Ca content (1 mmol/100 g). Significant differences in enzyme activity were observed, increasing the activity of TαA by SO42- (146.54 %) > C6H10O62- > Cl- > control > NO3- > OH- ≈ PO43- and C6H10O62- (123.20 %) ≈ Cl- ≈ SO42- > control > PO43 > OH- > NO3- for the low and high enzyme levels, respectively.

Negative dietary cation-anion difference and amount of calcium in prepartum diets: Effects on milk production, blood calcium, and health.

Acidogenic prepartum diets with negative dietary cation-anion difference (DCAD) induce compensated metabolic acidosis, which stimulates calcium (Ca) mobilization before calving and decreases clinical and subclinical hypocalcemia postpartum. This strategy is often combined with limiting dietary Ca concentrations, which historically has been used to mobilize Ca prepartum to prepare cows for lactation. Supplemental dietary Ca in combination with a negative DCAD formulation that does not reverse the effect of compensated metabolic acidosis may be beneficial. Our objective was to determine the effects of prepartum dietary strategies on dry matter intake (DMI), milk production, peripartal Ca status, and health during the transition period in multiparous Holstein cows (n = 81). Treatments during the last 28 d before calving were: (1) positive DCAD diet, +6 mEq/100 g of DM, target urine pH >7.5, low dietary Ca (0.40% DM; CON); (2) negative DCAD diet, -24 mEq/100 g of DM, target urine pH 5.5 to 6.0, low dietary Ca (0.40% DM; ND); or (3) negative DCAD diet, -24 mEq/100 g of DM, target urine pH 5.5 to 6.0, , high dietary Ca (2.0% DM; NDCA). Preplanned treatment contrasts were: (1) CON versus (ND and NDCA), and (2) ND versus NDCA. Individual DMI were recorded daily. Cows were milked 3 times daily, with individual DMI and milk yield summarized by week. Whole blood sampled at calving and 24 h, 48 h, and 4 d after calving was analyzed for ionized Ca concentration, and serum was analyzed for total Ca. Prepartum urine pH for cows fed ND or NDCA averaged 5.7, whereas cows fed CON remained >7.5. During the 3 wk before calving, cows fed CON had greater DMI than cows fed ND or NDCA, with NDCA greater than ND. Postpartum DMI (% of body weight) tended to be less for cows fed CON than for those fed ND or NDCA prepartum. Thresholds for subclinical hypocalcemia were ionized Ca <1.0 mM at 24 h, and total Ca ≤2.125 mM at 48 h after calving. On average, blood Ca for cows fed CON indicated subclinical hypocalcemia, whereas blood Ca for cows fed ND or NDCA was greater than subclinical hypocalcemia thresholds for both ionized Ca and total Ca. No milk production differences were detected. Cows fed CON had an elevated adverse health score (calculated by assigning numerical values to recorded health events) and tended to have an elevated somatic cell count during the fresh period compared with cows fed ND or NDCA. Overall, an acidogenic diet prepartum without or with high Ca improved postpartum Ca status and health. Supplementation of additional Ca to the acidogenic diet had little effect.

Effects of anionic supplement source in prepartum negative dietary cation-anion difference diets on serum calcium, feed intake, and lactational performance of multiparous dairy cows.

Incidence of subclinical hypocalcemia in early postpartum dairy cows continues to be an animal welfare concern and an economic burden for producers. Feeding prepartum negative dietary cation-anion difference (DCAD) diets produces metabolic acidosis, which supports mobilization of bone calcium and reduces the incidence of hypocalcemia. Achieving a sufficient degree of metabolic acidosis without reducing dry matter intake (DMI) can be difficult. This study compared the ability of MegAnion (MA; Origination O2D Inc., Maplewood, MN), a new DCAD supplement designed to be more palatable than typical anionic salt sources, and another palatable commercial DCAD product, SoyChlor (SC; Landus Cooperative, Ralston, IA), to reduce urine pH (a surrogate for metabolic acidosis) without reducing prepartum DMI. A secondary objective was to assess the effect of these anionic supplements on postpartum serum calcium concentrations and DMI. Prepartum multiparous Holstein (HO) and crossbred (XX) cows were blocked by breed and expected calving date and randomly assigned within breed to total mixed rations (TMR) with MA or SC and DCAD values of -215 mEq/kg of DM. Cows (n = 56; 15 MA-HO, 12 SC-HO, 15 MA-XX, 14 SC-XX) consumed the treatment TMR for at least 19 d and completed the 28 d in milk (DIM) phase of the study. Urine and blood samples were collected weekly and at 1, 2, and 3 DIM. Data were analyzed as a randomized block design by repeated measures with week or DIM as the repeated effect. Prepartum urine pH decreased from 8.15 ± 0.27 before treatment to 6.12 ± 0.14 during treatment, was not affected by anionic supplement, and increased immediately after calving when all cows consumed the same early-lactation TMR. Prepartum serum calcium concentrations were not affected (2.34 vs. 2.33 ± 0.02 mmol/L) by treatment, whereas nonesterified fatty acids were lower (86 vs. 120 ± 10 mmol/L) and insulin was greater (215 vs. 174 ± 10 pmol/L) in cows fed MA than in cows fed SC. These differences are supported by the numerically greater prepartum DMI (1.2 kg/d) and energy balance (1.8 Mcal/d) of cows fed MA. However, pre- and postpartum DMI and other production variables, including body weight, body condition score, milk yield, and energy balance, were not affected by treatment. This lack of difference indicates that MA provides another effective source of anionic salts for diets designed to reduce urine pH and induce metabolic acidosis in prepartum dairy cows.

Scutellarin inhibition of the rosuvastatin uptake in rat hepatocytes and the competition for organic anion transporting polypeptide 1B1 in HEK293T cells.

In this report, we investigated the hepatocytic uptake of rosuvastatin when administered with scutellarin (a Chinese herbal medicine) in rats and the role of organic anion transporting polypeptide 1B1 (OATP1B1) plays in the uptake. Forty-eight rats were randomly divided into two groups according to the medicine administered: rosuvastatin alone and rosuvastatin in combination with a series concentration of scutellarin. Rosuvastatin concentrations in blood and liver were measured using the liquid chromatography-tandem mass spectrometry (LC-MS) method. The uptake was also measured in rat primary hepatocytes and OATP1B1 transfected human embryonic kidney 293 T (HEK293T) cells. The uptake was investigated under the optimal intake conditions. The rosuvastatin Cmax and AUC0-∞ in rat plasma increased 55% and 61%, respectively in the combination treatment group; and the liver scutellarin concentrations decreased 32%, 34%, and 33% at 1 h, 2 h, and 6 h, respectively. All scutellarin dosages (20, 50, and 100 µM) inhibited the uptake of rosuvastatin in rat primary hepatocytes (4.71%, 22.73%, and 45.89%). Scutellarin of 10 µM significantly inhibited the in vitro uptake of rosuvastatin in OATP1B1-HEK293T cells (P < 0.05), with an IC50 of 60.53 ± 5.74 µM. Scutellarin increases the plasma concentration of rosuvastatin and inhibits the uptake in rat primary hepatocytes and OATP1B1-HEK293T cells, suggesting a drug interaction between scutellarin and rosuvastatin and OATP1B1 as a potential mechanism.

Neutrophil ß-defensin gene expression of postpartum dairy cows is altered by prepartum dietary cation-anion difference.

The objective of this study was to evaluate expression of a cluster of genes encoding ß-defensin antimicrobial peptides in neutrophils of postpartum cows in relation to prepartum dietary cation-anion difference (DCAD), vitamin D, and postpartum disease. Pregnant dry Holstein cows (28 nulliparous and 51 parous) at 255 d gestation were blocked by parity and randomly assigned to 4 prepartum diets of positive (+130 mEq/kg) or negative (-130 mEq/kg) DCAD and either 3 mg vitamin D3 or 3 mg of 25-hydroxyvitamin D3 per 11 kg of dry matter/d. Treatment diets were fed from 255 d of gestation until calving. Peripheral blood neutrophils of 35 parous cows were collected at 0 and 3 d after calving and stimulated with 0 or 100 ng/mL of lipopolysaccharide (LPS). Furthermore, serum Ca and incidences of postpartum diseases were recorded for all cows. The mRNA transcripts of ß-defensin genes were quantified by real-time PCR, and data were analyzed with a general linear mixed model to test for fixed effects and interactions of day, level of DCAD, source of vitamin D, and incidence of disease. Effects of DCAD and vitamin D on neutrophil oxidative burst and phagocytosis were previously reported but were analyzed for effects of disease in the present study. Transcripts for DEFB1, DEFB3, DEFB4, DEFB5, DEFB7, DEFB10, and lingual antimicrobial peptide (LAP) in neutrophils were upregulated by LPS at 0 d but not at 3 d. Transcripts for DEFB4 and DEFB7 in LPS-stimulated neutrophils were greater in cows fed negative DCAD diets compared with positive DCAD. Source of vitamin D (vitamin D3 vs. 25-hydroxyvitamin D3) did not affect expression of ß-defensins in neutrophils. Cows with postpartum subclinical hypocalcemia (serum Ca <2.0 mM) had decreased DEFB3, DEFB4, DEFB6, DEFB7, DEFB10, and LAP expression in LPS-stimulated neutrophils compared with cows that did not experience subclinical hypocalcemia. Likewise, DEFB4, DEFB6, DEFB7, DEFB10, and LAP in LPS-stimulated neutrophils at 3 d postpartum were positively associated with serum Ca at 0 d postpartum. Transcripts for DEFB7, DEFB10 and LAP also were less abundant in neutrophils from cows with metritis compared with healthy cows. In conclusion, feeding a prepartum negative DCAD to improve postpartum serum Ca resulted in greater neutrophil ß-defensin expression, and greater neutrophil ß-defensin expression was positively associated with postpartum health.

Bfd, a New Class of [2Fe-2S] Protein That Functions in Bacterial Iron Homeostasis, Requires a Structural Anion Binding Site.

Mobilization of iron from bacterioferritin (BfrB) requires specific interactions with a [2Fe-2S] ferredoxin (Bfd). Blocking the BfrB:Bfd interaction results in irreversible iron accumulation in BfrB and iron deficiency in the cytosol [Eshelman, K., et al. (2017) Metallomics 9, 646-659]. The only known Bfd structure, which was obtained in complex with BfrB (Protein Data Bank entry 4E6K ), indicated a new fold and suggested that the stability of Bfd is aided by an anion binding site consisting of R26, R29, and K46. We investigated the Bfd fold using site-directed mutagenesis, X-ray crystallography, and biochemistry in solution. The X-ray structure, which is nearly identical to that of Bfd in the BfrB:Bfd complex, shows that the [2Fe-2S] cluster preorganizes residues at the BfrB:Bfd interface into a structure complementary to the Bfd binding site on BfrB. Studies in solution showed rapid loss of the [2Fe-2S] cluster at a low ionic strength but higher stability with an increasing ionic strength, thus supporting a structural anion binding site. Structures of the R26E and R26E/K46Y mutants are nearly identical to that of Bfd, except for a new network of hydrogen bonds stabilizing the region encompassing the former anion binding site. The stability of the R26E and R26E/K46Y mutants, which is weakly and completely independent of solution ionic strength, respectively, corroborates that Bfd requires an anion binding site. The mutations, which caused only small changes to the strength of the BfrB:Bfd interaction and mobilization of iron from BfrB, indicate that the anion binding site in Bfd serves primarily a structural role.

Transcriptome and metabolome analyses provide insights into root and root-released organic anion responses to phosphorus deficiency in oat.

Roots and root-released organic anions play important roles in uptake of phosphorus (P), an essential macronutrient for food production. Oat, ranking sixth in the world's cereal production, contains valuable nutritional compounds and can withstand poor soil conditions. Our aim was to investigate root transcriptional and metabolic responses of oat grown under P-deficient and P-sufficient conditions. We conducted a hydroponic experiment and measured root morphology and organic anion exudation, and analysed changes in the transcriptome and metabolome. Oat roots showed enhanced citrate and malate exudation after 4 weeks of P deficiency. After 10 d of P deficiency, we identified 9371 differentially expressed transcripts with a 2-fold or greater change (P<0.05): 48 sequences predicted to be involved in organic anion biosynthesis and efflux were consistently up-regulated; 24 up-regulated transcripts in oat were also found to be up-regulated upon P starvation in rice and wheat under similar conditions. Phosphorylated metabolites (i.e. glucose-6-phosphate, myo-inositol phosphate) were reduced dramatically, while citrate and malate, some sugars and amino acids increased slightly in P-deficient oat roots. Our data are consistent with a strategy of increased organic anion efflux and a shift in primary metabolism in response to P deficiency in oat.

Comparison of 0.46% calcium diets with and without added anions with a 0.7% calcium anionic diet as a means to reduce periparturient hypocalcemia.

Most studies demonstrating that diets with low dietary cation-anion difference (DCAD) reduce hypocalcemia in cows add enough anions to the diet to reduce urine pH below 7.0. One objective of these experiments was to determine whether there is any benefit to periparturient plasma Ca concentration if diet anion addition results in a lesser degree of acidification of the cow and urine pH does not go below 7.0. Another method for reducing hypocalcemia involves feeding a prepartal diet that is Ca deficient. This places the cow in negative Ca balance before calving, stimulating parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D secretion before calving and thus promoting Ca homeostasis at calving. As practiced in the field, low-Ca diets are often about 0.5% Ca. Our second objective was to determine whether a 0.46% Ca diet would be sufficiently low in Ca to stimulate PTH secretion before calving. A meta-analysis of the literature suggests that a 0.5% Ca, low-DCAD diet will reduce hypocalcemia better than a 0.7% Ca diet. A third objective was to compare periparturient plasma Ca in cows fed 0.46 or 0.72% Ca diets with similar DCAD. In experiment 1, anions (primarily chloride) or anions plus Ca were added to a 1.4% K basal diet to create the following diets: 0.46% Ca and +167 mEq/kg of DCAD, 0.46% Ca and -13 mEq/kg of DCAD, and 0.72% Ca and -17 mEq/kg of DCAD. In experiment 2, the same amounts of anion were added to a 2.05% K basal diet to create the following diets: 0.46% Ca and +327 mEq/kg of DCAD, 0.46% Ca and +146 mEq/kg of DCAD, and 0.72% Ca and +140 mEq/kg of DCAD. In experiment 1, cows fed the diet with 0.46% Ca and +167 mEq/kg of DCAD had significantly lower plasma Ca concentration after calving than cows fed the 0.46 or 0.72% Ca diets with anions. Periparturient plasma Ca concentrations did not differ in cows fed the low-DCAD diets with 0.46 or 0.72% Ca. Urine pH was reduced from 8.27 in the diet with 0.46% Ca and +167 mEq/kg of DCAD to 7.07 and 7.41 in the 0.46 and 0.72% Ca anion diets, respectively. Precalving plasma PTH and 1,25-dihydroxyvitamin D concentrations were similar in cows fed the 0.46% Ca diets and the 0.72% Ca diets, suggesting that the 0.46% Ca diets were not low enough in Ca to place the cow in negative Ca balance before calving. In experiment 2, adding the anion supplements to a 2.05% K diet did not reduce urine pH below 8.0. Periparturient plasma Ca concentrations did not differ in cows in any group in experiment 2. Precalving diets that are 0.46% Ca fed ad libitum are too high in Ca to stimulate Ca homeostasis before calving. Adding anions to a diet can benefit periparturient cow plasma Ca concentration, but only if it alters acid-base status enough to reduce urine pH below 7.5.

Effects of prepartum dietary cation-anion difference and source of vitamin D in dairy cows: Lactation performance and energy metabolism.

The objectives of this experiment were to evaluate the effects of feeding diets with 2 dietary cation-anion difference (DCAD) levels and supplemented with either cholecalciferol (CH) or calcidiol (CA) during late gestation on lactation performance and energetic metabolism in dairy cows. The hypothesis was that combining a prepartum acidogenic diet with calcidiol supplementation would benefit peripartum Ca metabolism and, thus, improve energy metabolism and lactation performance compared with cows fed an alkalogenic diet or cholecalciferol. Holstein cows at 252 d of gestation were blocked by parity (28 nulliparous and 51 parous cows) and milk yield within parous cows, and randomly assigned to 1 of 4 treatments arranged as a 2 × 2 factorial, with 2 levels of DCAD (positive, +130, and negative, -130 mEq/kg) and 2 sources of vitamin D, CH or CA, fed at 3 mg per 11 kg of diet dry matter (DM). The resulting treatment combinations were positive DCAD with CH (PCH), positive DCAD with CA (PCA), negative DCAD with CH (NCH), or negative DCAD with CA (NCA), which were fed for the last 21 d of gestation. After calving, cows were fed the same lactation diet. Body weight and body condition were evaluated prepartum and for the first 49 d postpartum. Blood was sampled thrice weekly prepartum, and on d 0, 1, 2, 3, and every 3 d thereafter until 30 d postpartum for quantification of hormones and metabolites. Lactation performance was evaluated for the first 49 d postpartum. Feeding a diet with negative DCAD reduced DM intake in parous cows by 2.1 kg/d, but no effect was observed in nulliparous cows. The negative DCAD reduced concentrations of glucose (positive = 4.05 vs. negative = 3.95 mM), insulin (positive = 0.57 vs. negative = 0.45 ng/mL), and insulin-like growth factor-1 (positive = 110 vs. negative = 95 ng/mL) prepartum. Treatments did not affect DM intake postpartum, but CA-supplemented cows tended to produce more colostrum (PCH = 5.86, PCA = 7.68 NCH = 6.21, NCA = 7.96 ± 1.06 kg) and produced more fat-corrected milk (PCH = 37.0, PCA = 40.1 NCH = 37.5, NCA = 41.9 ± 1.8 kg) and milk components compared with CH-supplemented cows. Feeding the negative DCAD numerically increased yield of fat-corrected milk by 1.0 kg/d in both nulliparous and 1.4 kg/d in parous cows. Minor differences were observed in postpartum concentrations of hormones and metabolites linked to energy metabolism among treatments. Results from this experiment indicate that replacing CH with CA supplemented at 3 mg/d during the prepartum period improved postpartum lactation performance in dairy cows.

Effects of prepartum dietary cation-anion difference and source of vitamin D in dairy cows: Vitamin D, mineral, and bone metabolism.

Pregnant Holstein cows, 28 nulliparous and 51 parous, were blocked by parity and milk yield and randomly allocated to receive diets that differed in dietary cation-anion difference (DCAD), +130 or -130 mEq/kg, and supplemented with either calcidiol or cholecalciferol at 3 mg/11 kg of dry matter from 255 d of gestation until parturition. Blood was sampled thrice weekly prepartum, and on d 0, 1, 2, 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30 postpartum to evaluate effects of the diets on vitamin D, mineral and bone metabolism, and acid-base status. Blood pH and concentrations of minerals, vitamin D metabolites, and bone-related hormones were determined, as were mineral concentrations and losses in urine and colostrum. Supplementing with calcidiol increased plasma concentrations of 25-hydroxyvitamin D3, 3-epi 25-hydroxyvitamin D3, 25-hydroxyvitamin D2, 1,25-dihydroxyvitamin D3, and 24,25-dihydroxyvitamin D3 compared with supplementing with cholecalciferol. Cows fed the diet with negative DCAD had lesser concentrations of vitamin D metabolites before and after calving than cows fed the diet with positive DCAD, except for 25-hydroxyvitamin D2. Feeding the diet with negative DCAD induced a compensated metabolic acidosis that attenuated the decline in blood ionized Ca (iCa) and serum total Ca (tCa) around calving, particularly in parous cows, whereas cows fed the diet with positive DCAD and supplemented with calcidiol had the greatest 1,25-dihydroxyvitamin D3 concentrations and the lowest iCa and tCa concentrations on d 1 and 2 postpartum. The acidogenic diet or calcidiol markedly increased urinary losses of tCa and tMg, and feeding calcidiol tended to increase colostrum yield and increased losses of tCa and tMg in colostrum. Cows fed the diet with negative DCAD had increased concentrations of serotonin and C-terminal telopeptide of type 1 collagen prepartum compared with cows fed the diet with positive DCAD. Concentrations of undercarboxylated and carboxylated osteocalcin and those of adiponectin did not differ with treatment. These results provide evidence that dietary manipulations can induce metabolic adaptations that improve mineral homeostasis with the onset of lactation that might explain some of the improvements observed in health and production when cows are fed diets with negative DCAD or supplemented with calcidiol.

Effects of prepartum dietary cation-anion difference and source of vitamin D in dairy cows: Health and reproductive responses.

The objectives of the experiment were to evaluate the effects of feeding diets with distinct dietary cation-anion difference (DCAD) levels and supplemented with 2 sources of vitamin D during the prepartum transition period on postpartum health and reproduction in dairy cows. The hypotheses were that feeding acidogenic diets prepartum would reduce the risk of hypocalcemia and other diseases, and the benefits of a negative DCAD treatment on health would be potentiated by supplementing calcidiol compared with cholecalciferol. Cows at 252 d of gestation were blocked by parity (28 nulliparous and 52 parous cows) and milk yield within parous cows, and randomly assigned to 1 of 4 treatments arranged as a 2 × 2 factorial, with 2 levels of DCAD, positive (+130 mEq/kg) or negative (-130 mEq/kg), and 2 sources of vitamin D, cholecalciferol or calcidiol, fed at 3 mg for each 11 kg of diet dry matter. The resulting treatment combinations were positive DCAD with cholecalciferol (PCH), positive DCAD with calcidiol (PCA), negative DCAD with cholecalciferol (NCH), and negative DCAD with calcidiol (NCA), which were fed from 252 d of gestation to calving. After calving, cows were fed the same lactation diet supplemented with cholecalciferol at 0.70 mg for every 20 kg of dry matter. Blood was sampled 7 d before parturition, and at 2 and 7 d postpartum to evaluate cell counts and measures of neutrophil function. Postpartum clinical and subclinical diseases and reproductive responses were evaluated. Feeding a diet with negative DCAD eliminated clinical hypocalcemia (23.1 vs. 0%) and drastically reduced the incidence and daily risk of subclinical hypocalcemia, and these effects were observed in the first 48 to 72 h after calving. The diet with negative DCAD tended to improve the intensity of oxidative burst activity of neutrophils in all cows prepartum and increased the intensity of phagocytosis in parous cows prepartum and the proportion of neutrophils with killing activity in parous cows postpartum (58.5 vs. 67.6%). Feeding calcidiol improved the proportion of neutrophils with oxidative burst activity (60.0 vs. 68.7%), reduced the incidences of retained placenta (30.8 vs. 2.5%) and metritis (46.2 vs. 23.1%), and reduced the proportion of cows with multiple diseases in early lactation. Combining the negative DCAD diet with calcidiol reduced morbidity by at least 60% compared with any of the other treatments. Cows with morbidity had lower blood ionized Ca and serum total Ca concentrations than healthy cows. Treatments did not affect the daily risk of hyperketonemia in the first 30 d of lactation. Despite the changes in cow health, manipulating the prepartum DCAD did not influence reproduction, but feeding calcidiol tended to increase the rate of pregnancy by 55%, which reduced the median days open by 19. In conclusion, feeding prepartum cows with a diet containing a negative DCAD combined with 3 mg of calcidiol benefited health in early lactation.

Biology of SLAC1-type anion channels - from nutrient uptake to stomatal closure.

Contents 46 I. 46 II. 47 III. 50 IV. 53 V. 56 VI. 57 58 58 References 58 SUMMARY: Stomatal guard cells control leaf CO2 intake and concomitant water loss to the atmosphere. When photosynthetic CO2 assimilation is limited and the ratio of CO2 intake to transpiration becomes suboptimal, guard cells, sensing the rise in CO2 concentration in the substomatal cavity, deflate and the stomata close. Screens for mutants that do not close in response to experimentally imposed high CO2 atmospheres identified the guard cell-expressed Slowly activating anion channel, SLAC1, as the key player in the regulation of stomatal closure. SLAC1 evolved, though, before the emergence of guard cells. In Arabidopsis, SLAC1 is the founder member of a family of anion channels, which comprises four homologues. SLAC1 and SLAH3 mediate chloride and nitrate transport in guard cells, while SLAH1, SLAH2 and SLAH3 are engaged in root nitrate and chloride acquisition, and anion translocation to the shoot. The signal transduction pathways involved in CO2 , water stress and nutrient-sensing activate SLAC/SLAH via distinct protein kinase/phosphatase pairs. In this review, we discuss the role that SLAC/SLAH channels play in guard cell closure, on the one hand, and in the root-shoot continuum on the other, along with the molecular basis of the channels' anion selectivity and gating.

Structural basis for anion conduction in the calcium-activated chloride channel TMEM16A.

The calcium-activated chloride channel TMEM16A is a member of a conserved protein family that comprises ion channels and lipid scramblases. Although the structure of the scramblase nhTMEM16 has defined the architecture of the family, it was unknown how a channel has adapted to cope with its distinct functional properties. Here we have addressed this question by the structure determination of mouse TMEM16A by cryo-electron microscopy and a complementary functional characterization. The protein shows a similar organization to nhTMEM16, except for changes at the site of catalysis. There, the conformation of transmembrane helices constituting a membrane-spanning furrow that provides a path for lipids in scramblases has changed to form an enclosed aqueous pore that is largely shielded from the membrane. Our study thus reveals the structural basis of anion conduction in a TMEM16 channel and it defines the foundation for the diverse functional behavior in the TMEM16 family.

Observation of the seleno bis-(S-glutathionyl) arsinium anion in rat bile.

Certain arsenic and selenium compounds show a remarkable mutual cancelation of toxicities, where a lethal dose of one can be voided by an equimolar and otherwise lethal dose of the other. It is now well established that the molecular basis of this antagonism is the formation and biliary excretion of seleno bis-(S-glutathionyl) arsinium anion [(GS)2AsSe](-). Previous work has definitively demonstrated the presence of [(GS)2AsSe](-) in rabbit bile, but only in the presence of other arsenic and selenium species. Rabbits have a gall bladder, which concentrates bile and lowers its pH; it seems likely that this may be responsible for the breakdown of biliary [(GS)2AsSe](-). Since rats have no gall bladder, the bile proceeds directly through the bile duct from the hepatobiliary tree. In the present work we have shown that the primary product of biliary co-excretion of arsenic and selenium in rats is [(GS)2AsSe](-), with essentially 100% of the arsenic and selenium present as this species. The chemical plausibility of the X-ray absorption spectroscopy-derived structural conclusions of this novel arsenic and selenium co-excretion product is supported by density functional theory calculations. These results establish the biomolecular basis to further explore the use of selenium dietary supplements as a possible palliative for chronic low-level arsenic poisoning of human populations.