Folic acid metabolism in vitamin B12-deficient sheep. Depletion of liver folates

1. Metabolism of folate was studied in six ewes in an advanced state of vitamin B(12) deficiency as judged by voluntary food intake and in their pair-fed controls receiving vitamin B(12). A group of four animals that were maintained throughout the experiment at pasture was also studied. 2. After 34-40 weeks on the cobalt-deficient diet urinary excretion of formiminoglutamate by four deficient animals was about 3.2mmol/day and this was not significantly decreased by injection of three of them with about 4.5mug of [2-(14)C]folate/kg body weight per day for 5 days. Three days after the last injection retention of [2-(14)C]folate by the livers of the deficient animals (5.5% of the dose) was lower than that of their pair-fed controls (26% of the dose) but there was no evidence of net retention of injected folate in the livers of either group. Urinary excretion of (14)C indicated that renal clearance of folate may have been impaired in very severe vitamin B(12) deficiency. 3. As estimated by microbiological assays total folates in the livers of animals at pasture (12.9mug/g) included about 24% of 5-methyltetrahydrofolate as compared with about 72% of a total of 12.5mug/g in three further ewes fed on a stock diet of wheaten hay-chaff and lucerne-chaff. Liver folates of vitamin B(12)-deficient animals (0.5mug/g) included about 88% of 5-methyltetrahydrofolate as compared with about 51% of a total of 5.2mug/g in pair-fed animals treated with vitamin B(12). 4. Chromatography of liver folates of the pair-fed animals permitted quantitative estimates of the pteroylglutamates present. The results showed that the vitamin B(12)-deficient livers were more severely depleted of tetrahydrofolates and formyltetrahydrofolates than of methyltetrahydrofolates and that as the deficiency developed they were more severely depleted of the higher polyglutamates than of the monoglutamate within each of these classes. Results from animals injected with [2-(14)C]folate indicated an impairment of the exchange between pteroylmonoglutamates and pteroylpolyglutamates in the livers of deficient animals. 5. In vitamin B(12)-deficient animals with food intakes below 200g/day some of the liver folates were not completely reduced and some degradation of pteroylpolyglutamates was detected. The latter condition may have been associated with fatty liver. 6. The results are discussed in relation to current theories of vitamin B(12)-folate interactions.     

Hydrogel microchip as a tool for studying exosomes in human serum

Exosomes are cell-derived vesicles that are secreted by both normal and cancer cells. Over the last decade, a few studies have revealed that exosomes cross talk and/or influence major tumor-related pathways such as angiogenesis and metastasis involving many cell types within the tumor microenvironment. The protein composition of the membrane of an exosome reflects that of the membrane of the cell of origin. Because of this, tumor-derived exosomes differ from exosomes that are derived from normal cells. The detection of tumor exosomes and analysis of their molecular composition hold promise for diagnosis and prognosis of cancer. Here, we present hydrogel microarrays (biochips), which contain a panel of immobilized antibodies that recognize tetraspanins (CD9, CD63, CD81) and prognostic markers for colorectal cancer (A33, CD147). These biochips make it possible to analyze the surface proteins of either isolated exosomes or exosomes that are present in the serum samples without isolation. These biochips were successfully used to analyze the surface proteins of exosomes from serum that was collected from a colorectal cancer patient and healthy donor. Biochip-guided immunofluorescent analysis of the exosomes has made it possible for us to detect the A33 antigen and CD147 in the serum sample of the colorectal cancer patient with normal levels of CEA and CA19-9.     

Peroxidase activity as a tool for studying the folding of c-type cytochromes

The peroxidase activity of c-type cytochromes increases substantially by unfolding. This phenomenon was used to study the equilibrium unfolding of ferricytochrome c. The peroxidase activity is already enhanced at low denaturant concentrations. The lowest free energy folding intermediate is easily detected by this method, while it is invisible using fluorescence or optical spectroscopy. The free energy difference between this folding intermediate and the native state depends on the strength of the sixth ligand of the heme-iron and the increase in peroxidase activity upon unfolding is shown to be a sensitive indicator of the strength of this ligand. Under fully denaturing conditions, the peroxidase activity is inhibited by protein-based ligands. It is shown that at least three different ligand groups can be responsible for this inhibition, and that at neutral or alkaline pH, the predominant ligand is not histidine. The use of peroxidase activity assays as a method to study the unfolding of cytochrome c is evaluated.     

Lipid-impregnated filters as a tool for studying the electric current-generating proteins.

Porous filters and collodion film impregnated with decane solution of phospholipids, were used for measurements of electric potential differences generated by bacteriorhodopsin, chromatophore redox chain, H⁺-ATPase, pyrophosphatase, and mitochondrial respiratory chain. It was shown that reconstituted proteoliposomes, containing e.g., bacteriorhodopsin or natural coupling membrane vesicles, such as Rhodospirillum rubrum chromatophores, can be attached to a filter surface by means of Ca²⁺ or Mg²⁺ ions. Addition of the respective energy source was found to result in electric potential difference being generated across the filter. This effect was measured directly by $\text{Ag}\text{AgCl}$ electrodes immersed into electrolyte solutions on both sides of the filter. Using a phospholipid-impregnated collodion film one can measure electric responses as fast as 300 nsec. The phospholipid-impregnated filters turned out to be sensitive and reliable electrodes for measuring the concentration of synthetic penetrating ions, such as phenyldicarbaundecaborane, tetraphenylborate, tetrapentylammonium, and tetraphenylphosphonium. By measuring changes in the concentration of these ions in the suspension of proteoliposomes, chromatophores, mitochondria, or bacterial cells, one can follow the formation and dissipation of transmembrane potential differences in these systems. It is shown that the phospholipid-impregnated filters are much more reliable and handy than planar phospholipid membranes previously used for studying electrogenic activity of electric current-producing membrane proteins.     

Cross-linking and mass spectrometry as a tool for studying the structural biology of ribonucleoproteins

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Somatic mutations as a useful tool for studying clonal dynamics in trees

The seemingly eternal cycles of clonal growth in many tree species, with members of Populus (aspen, poplars, cottonwoods and the like) featuring most prominently, provoke a number of questions on the interface between ecology, genetics and forestry. In this issue, two groups present their approaches to clonal dynamics ( Ally et al. 2008 and Mock et al. 2008 ), using microsatellite (or simple sequence repeat, SSR) variation in P. tremuloides. Ally et al. developed and applied a model for using microsatellites to estimate clone age and infer other community characteristics. Mock et al. used fewer microsatellites but in more individuals, to examine clone size and distribution across the landscape.     

Metaproteomics as a tool for studying the protein landscape of human-gut bacterial species

Host-microbiome interactions and the microbial community have broad impact in human health and diseases. Most microbiome based studies are performed at the genome level based on next-generation sequencing techniques, but metaproteomics is emerging as a powerful technique to study microbiome functional activity by characterizing the complex and dynamic composition of microbial proteins. We conducted a large-scale survey of human gut microbiome metaproteomic data to identify generalist species that are ubiquitously expressed across all samples and specialists that are highly expressed in a small subset of samples associated with a certain phenotype. We were able to utilize the metaproteomic mass spectrometry data to reveal the protein landscapes of these species, which enables the characterization of the expression levels of proteins of different functions and underlying regulatory mechanisms, such as operons. Finally, we were able to recover a large number of open reading frames (ORFs) with spectral support, which were missed by de novo protein-coding gene predictors. We showed that a majority of the rescued ORFs overlapped with de novo predicted protein-coding genes, but on opposite strands or in different frames. Together, these demonstrate applications of metaproteomics for the characterization of important gut bacterial species.     

Molecular modelling as a tool for studying the disassembly of potentially leishmanicide-targeted dendrimer

Molecular modelling studies were carried out to analyse which carbonyl group is the most vulnerable to the disassembly of potentially leishmanicide-targeted dendrimer. The dendrimers were designed using myo-inositol (core and directing group), d-mannose (directing group), l-malic acid (spacer and dendron) and hydroxymethylnitrofurazone (NFOH) as a bioactive agent. The molecular models were built containing one, two and three branches. For this preliminary analysis, physicochemical properties, such as electronic density distribution and steric hindrance regarding the carbonyl groups were evaluated, and the carbon atoms in the following carbonyl groups were considered: near the core (C₁), close to the directing group (C₂), in l-malic acid (C₃) and near the bioactive agent (C₄). The most probable targeted dendrimers showed the carbonyl close to the core as the most susceptible to a nucleophilic attack, except those molecular systems containing two branches with d-mannose as the directing group, which displayed the carbonyl group near NFOH as the most likely ester breaking point.