A Fluorescence-Based Method to Measure ADP/ATP Exchange of Recombinant Adenine Nucleotide Translocase in Liposomes

Several mitochondrial proteins, such as adenine nucleotide translocase (ANT), aspartate/glutamate carrier, dicarboxylate carrier, and uncoupling proteins 2 and 3, are suggested to have dual transport functions. While the transport of charge (protons and anions) is characterized by an alteration in membrane conductance, investigating substrate transport is challenging. Currently, mainly radioactively labeled substrates are used, which are very expensive and require stringent precautions during their preparation and use. We present and evaluate a fluorescence-based method using Magnesium Green (MgGrTM), a Mg2+-sensitive dye suitable for measurement in liposomes. Given the different binding affinities of Mg2+ for ATP and ADP, changes in their concentrations can be detected. We obtained an ADP/ATP exchange rate of 3.49 ± 0.41 mmol/min/g of recombinant ANT1 reconstituted into unilamellar liposomes, which is comparable to values measured in mitochondria and proteoliposomes using a radioactivity assay. ADP/ATP exchange calculated from MgGrTM fluorescence solely depends on the ANT1 content in liposomes and is inhibited by the ANT-specific inhibitors, bongkrekic acid and carboxyatractyloside. The application of MgGrTM to investigate ADP/ATP exchange rates contributes to our understanding of ANT function in mitochondria and paves the way for the design of other substrate transport assays

Quality of Milk from Lactating Dairy Cattle Fed Dried Distillers Grains with Solubles

Healthy mid-lactation Holstein dairy cows (n=24) were fed total mixed rations containing dried distillers grains with solubles (DDGS). The objective of this study was to examine the effect of feeding DDGS to lactating dairy cows on production parameters and flavor and oxidative stability of milk. Cows were assigned to two groups and fed one of three treatment diets (0% DDGS, 10% DDGS, 25% DDGS by dry matter (DM)) as a total mixed ration. Each group was fed all three of the diets after a wash-out period of 7 days. Milk yield was unaffected by both the 0% and 10% DDGS diets but decreased significantly when fed the 25% DDGS diet. Rumen volatile fatty acids were unaffected by treatment. Milk protein and solids-not-fat (SNF) increased with increasing inclusion of DDGS, but milk fat decreased concomitantly. Milk fatty acid composition was affected with milk fat from cows fed higher concentrations of DDGS producing milk with higher concentrations of unsaturated fatty acids. Milk oxidative stability was unaffected by dietary treatment, and milk flavor, as determined by a trained sensory panel, also was unaffected. The results of this study indicate that feeding of DDGS to lactating dairy cows, under controlled conditions, does not have negative effects on milk oxidative stability or flavor; however, feeding 25% DDGS did negatively impact milk production and changed milk fatty acid profile

Aquaporins with anion/monocarboxylate permeability: mechanisms, relevance for pathogen–host interactions

Classically, aquaporins are divided based on pore selectivity into water specific, orthodox aquaporins and solute-facilitating aquaglyceroporins, which conduct, e.g., glycerol and urea. However, more aquaporin-passing substrates have been identified over the years, such as the gasses ammonia and carbon dioxide or the water-related hydrogen peroxide. It became apparent that not all aquaporins clearly fit into one of only two subfamilies. Furthermore, certain aquaporins from both major subfamilies have been reported to conduct inorganic anions, such as chloride, or monoacids/monocarboxylates, such as lactic acid/lactate. Here, we summarize the findings on aquaporin anion transport, analyze the pore layout of such aquaporins in comparison to prototypical non-selective anion channels, monocarboxylate transporters, and formate-nitrite transporters. Finally, we discuss in which scenarios anion conducting aquaporins may be of physiological relevance

Lactational Performance of Holstein Dairy Cows Fed Two Levels of Full-fat Corn Dried Distillers Grains with Solubles

The lactational performance of 30 healthy multiparous Holstein dairy cows was tested when cows were fed 0, 10, and 20% dried distillers grains with solubles (DDGS) that contained 13.6% fat on a dry matter (DM) basis. Cows fed 10% DDGS on a DM basis experienced a reduction in milk fat percentage of 0.5 percentage points, but did not perform differently in other measures than did cows not fed DDGS. When cows were fed 20% DDGS on a DM basis, every common measure of feed utilization was impacted negatively

Dihydroxyacetone and methylglyoxal as permeants of the Plasmodium aquaglyceroporin inhibit parasite proliferation

AbstractThe aquaglyceroporin of Plasmodium falciparum (PfAQP) is a bi-functional channel with permeability for water and solutes. Its functions supposedly are in osmotic protection of parasites and in facilitation of glycerol permeation for glycerolipid biosynthesis. Here, we show PfAQP permeability for the glycolysis-related metabolites methylglyoxal, a cytotoxic byproduct, and dihydroxyacetone, a ketotriose. AQP3, the red cell aquaglyceroporin, also passed dihydroxacetone but excluded methylglyoxal. Proliferation of malaria parasites was inhibited by methylglyoxal with an IC50 around 200 μM. Surprisingly, also dihydroxyacetone, which is an energy source in human cells, was antiproliferative in chloroquine-sensitive and resistant strains with an IC50 around 3 mM. We expressed P. falciparum glyceraldehyde 3-phosphate dehydrogenase (PfGAPDH) to examine whether it is inhibited by either carbonyl compound. Methylglyoxal did not affect PfGAPDH on incubation with 2.5 mM for 20 h. Treatment with 2.5 mM dihydroxyacetone, however, abolished PfGAPDH activity within 6 h. Aquaglyceroporin permeability for glycolytic metabolites may thus be of physiological significance

Feeding Lactating Holstein Dairy Cows Reduced-Fat Dried Distillers Grains with Solubles: Quality of Baby Swiss Cheese

In this experiment, feedingreduced-fat distillers grains(RF-DDGS)as 20% DM of a total mixed ration (TMR)supplemented with lysine did not negatively influence flavor attributes of baby Swiss cheese.Eye appearancein all cheeseswas atypical,but was not related to diet. Any defects in appearance of Baby Swiss cheese appeared in both control and RF-DDGS fed cows and therefore the defects observed can be attributed to the make procedure itself rather than to of feeding RF-DDGS. The results indicate that lactating Holstein dairy cows can be fed RF-DDGS as 20% DM of a TMR without negatively affecting usability of milk forproduction of Baby Swiss cheese

Feeding Lactating Holstein Dairy Cows Reduced-Fat Dried Distillers Grains with Solubles: Milk Composition and Feed Efficiency

In this experiment, feeding reduced-fat distillers grains with solubles (RF-DDGS)as 20% DM of a TMR supplemented with lysine did not negatively influence production parameters related to milk composition or nutritional physiology of the cow. Milk urea nitrogen (MUN) was, however, decreased,and milk protein percentage was increased. Total milk solids werenot influenced by inclusion of RF-DDGS. Additionally, RF-DDGS did cause a decrease in fat-corrected milk (FCM)efficiency as a result of an increase in DMI. When energy-corrected milk (ECM)efficiency was calculated (accounting for fat, protein, and lactose concentration in milk),no difference in feed efficiency resulted(p \u3e 0.05). These data indicate that RF-DDGS can effectively be included in rations of multiparous lactating dairy cows, at least when supplemented with lysine. Additionally, decreased milk urea nitrogen (MUN)and increased milk protein percentage indicate thatdietaryprotein utilization may be improved by including RF-DDGS as a protein source in the ration, presumably because DDGS are generally considered to be a good source of rumen undegradable protein. Taken together, these results indicate that RF-DDGS may be an attractive feed ingredient for inclusion in lactating ruminant diets

Degraded Arabinogalactans and Their Binding Properties to Cancer-Associated Human Galectins

Galectins represent β-galactoside-binding proteins with numerous functions. Due to their role in tumor progression, human galectins-1, -3 and -7 (Gal-1, -3 and -7) are potential targets for cancer therapy. As plant derived glycans might act as galectin inhibitors, we prepared galactans by partial degradation of plant arabinogalactan-proteins. Besides commercially purchased galectins, we produced Gal-1 and -7 in a cell free system and tested binding capacities of the galectins to the galactans by biolayer-interferometry. Results for commercial and cell-free expressed galectins were comparable confirming functionality of the cell-free produced galectins. Our results revealed that galactans from Echinacea purpurea bind to Gal-1 and -7 with KD values of 1-2 µM and to Gal-3 slightly stronger with KD values between 0.36 and 0.70 µM depending on the sensor type. Galactans from the seagrass Zostera marina with higher branching of the galactan and higher content of uronic acids showed stronger binding to Gal-3 (0.08-0.28 µM) compared to galactan from Echinacea. The results contribute to knowledge on interactions between plant polysaccharides and galectins. Arabinogalactan-proteins have been identified as a new source for production of galactans with possible capability to act as galectin inhibitors

Direct-Fed Microbials Decreases Dry Matter Intake and Increases Feed Efficiency When Fed to Lactating Holstein Dairy Cows

Dry matter intake, milk production, and milk production efficiency were evaluated in 84 Holstein dairy cows fed either a control diet or a control diet plus the direct-fed microbial Bovamine. Neither milk nor ECM production were affected by feeding Bovamine. Feeding Bovamine, however, decreased DMI by 3.59%, resulting in an improvement in milk production efficiency and ECM production efficiency of 6.1% and 5.3%, respectively. Inclusion of Bovamine in dairy cattle diets should be considered to increase milk production efficiency

Fluorescence Cross-Correlation Spectroscopy Yields True Affinity and Binding Kinetics of Plasmodium Lactate Transport Inhibitors

Blocking lactate export in the parasitic protozoan Plasmodium falciparum is a novel strategy to combat malaria. We discovered small drug-like molecules that inhibit the sole plasmodial lactate transporter, PfFNT, and kill parasites in culture. The pentafluoro-3-hydroxy-pent-2-en-1-one BH296 blocks PfFNT with nanomolar efficiency but an in vitro selected PfFNT G107S mutation confers resistance against the drug. We circumvented the mutation by introducing a nitrogen atom as a hydrogen bond acceptor site into the aromatic ring of the inhibitor yielding BH267.meta. The current PfFNT inhibitor efficiency values were derived from yeast-based lactate transport assays, yet direct affinity and binding kinetics data are missing. Here, we expressed PfFNT fused with a green fluorescent protein in human embryonic kidney cells and generated fluorescent derivatives of the inhibitors, BH296 and BH267.meta. Using confocal imaging, we confirmed the location of the proposed binding site at the cytosolic transporter entry site. We then carried out fluorescence cross-correlation spectroscopy measurements to assign true Ki-values, as well as kon and koff rate constants for inhibitor binding to PfFNT wildtype and the G107S mutant. BH296 and BH267.meta gave similar rate constants for binding to PfFNT wildtype. BH296 was inactive on PfFNT G107S, whereas BH267.meta bound the mutant protein albeit with weaker affinity than to PfFNT wildtype. Eventually, using a set of PfFNT inhibitor compounds, we found a robust correlation of the results from the biophysical FCCS binding assay to inhibition data of the functional transport assay