Effects of folic acid supplementation to rations differing in the concentrate to roughage ratio on ruminal fermentation, nutrient flow at the duodenum, and on serum and milk variables of dairy cows

The present study was undertaken to determine the effects of dietary folic acid (FOL) supplementation on ruminal fermentation, duodenal nutrient flow, serum and milk variables, and on B-vitamin concentration in serum. The study was divided into two experiments: in Exp. 1 the forage to concentrate (F:C) ratio of the diet (DM basis) was 34:66 (high concentrate, HC), while in Exp. 2 the F:C ratio was 66:34 (high forage, HF). In addition, the cows received 0 or 1 g FOL/d. In Exp. 1, two German Holstein cows equipped with cannulas in the dorsal sac of the rumen and in the proximal duodenum were dry and five were lactating (186 +/- 144 days in milk); in Exp. 2 four cows were dry and four were lactating (165 +/- 57 days in milk). In cows fed the HC diet, FOL supplementation decreased the ruminally-fermented organic matter. Thus, less energy was available for ruminal microorganisms, which resulted in a reduced microbial crude protein flow at the duodenum. Feeding the HF diet, FOL supplementation only increased the apparent ruminal digestibility of acid detergent fibre (ADF). With the HF diet, FOL had no influence on the serum levels of glucose, non-esterified fatty acids, beta-hydroxybutyrate, urea, thiamine, riboflavin, pyridoxal-5'-phosphate, pyridoxic acid, pyridoxal, pyridoxine, pyridoxamine, pantothenic acid, nicotinamide or nicotinic acid, whereas supplementing FOL to the HC diet lowered the serum glucose and riboflavin levels. In both experiments, the supplementation of FOL had no effects on milk composition. Folic acid supplementation to both diets increased the concentrations of serum 5-methyl-tetrahydrofolate. However, no beneficial effects to dairy cows were obvious. Therefore, to achieve certain results, studies with a higher number of non-fistulated cows would be necessary.     

Probing diffusion laws within cellular membranes by Z-scan fluorescence correlation spectroscopy

The plasma membrane of various mammalian cell types is heterogeneous in structure and may contain microdomains, which can impose constraints on the lateral diffusion of its constituents. Fluorescence correlation spectroscopy (FCS) can be used to investigate the dynamic properties of the plasma membrane of living cells. Very recently, Wawrezinieck et al. (Wawrezinieck, L., H. Rigneault, D. Marguet, and P. F. Lenne. 2005. Biophys. J. 89:4029-4042) described a method to probe the nature of the lateral microheterogeneities of the membrane by varying the beam size in the FCS instrument. The dependence of the width of the autocorrelation function at half-maximum, i.e., the diffusion time, on the transverse area of the confocal volume gives information on the nature of the imposed confinement. We describe an alternative approach that yields essentially the same information, and can readily be applied on commercial FCS instruments by measuring the diffusion time and the particle number at various relative positions of the cell membrane with respect to the waist of the laser beam, i.e., by performing a Z-scan.     

Limited Compatibility of Polymerase Subunit Interactions in Influenza A and B Viruses

Despite their close phylogenetic relationship, natural intertypic reassortants between influenza A (FluA) and B (FluB) viruses have not been described. Inefficient polymerase assembly of the three polymerase subunits may contribute to this incompatibility, especially because the known protein-protein interaction domains, including the PA-binding domain of PB1, are highly conserved for each virus type. Here we show that substitution of the FluA PA-binding domain (PB1-A_1–25) with that of FluB (PB1-B_1–25) is accompanied by reduced polymerase activity and viral growth of FluA. Consistent with these findings, surface plasmon resonance spectroscopy measurements revealed that PA of FluA exhibits impaired affinity to biotinylated PB1-B_1–25 peptides. PA of FluB showed no detectable affinity to biotinylated PB1-A_1–25 peptides. Consequently, FluB PB1 harboring the PA-binding domain of FluA (PB1-AB) failed to assemble with PA and PB2 into an active polymerase complex. To regain functionality, we used a single amino acid substitution (T6Y) known to confer binding to PA of both virus types, which restored polymerase complex formation but surprisingly not polymerase activity for FluB. Taken together, our results demonstrate that the conserved virus type-specific PA-binding domains differ in their affinity to PA and thus might contribute to intertypic exclusion of reassortants between FluA and FluB viruses.     

Visualizing spatial lipid distribution in porcine lens by MALDI imaging high-resolution mass spectrometry

The intraocular lens contains high levels of both cholesterol and sphingolipids, which are believed to be functionally important for normal lens physiology. The aim of this study was to explore the spatial distribution of sphingolipids in the ocular lens using mass spectrometry imaging (MSI). Matrix-assisted laser desorption/ionization (MALDI) imaging with ultra high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) was used to visualize the lipid spatial distribution. Equatorially-cryosectioned, 12 μm thick slices of tissue were thaw-mounted to an indium-tin oxide (ITO) glass slide by soft-landing to an ethanol layer. This procedure maintained the tissue integrity. After the automated MALDI matrix deposition, the entire lens section was examined by MALDI MSI in a 150 μm raster. We obtained spatial- and concentration-dependent distributions of seven lens sphingomyelins (SM) and two ceramide-1-phosphates (CerP), which are important lipid second messengers. Glycosylated sphingolipids or sphingolipid breakdown products were not observed. Owing to ultra high resolution MS, all lipids were identified with high confidence, and distinct distribution patterns for each of them are presented. The distribution patterns of SMs provide an understanding of the physiological functioning of these lipids in clear lenses and offer a novel pathophysiological means for understanding diseases of the lens.