Developments in dairy cow fertility research.

Accurate mass and MS/MS fragmentation data for (a) kynurenine, (b) melatonin, and (c) tryptophan. (TIF 191 kb

Recombinant clotting factor VIII concentrates: Heterogeneity and high-purity evaluation

Factor VIII is an important glycoprotein involved in hemostasis. Insertion of expression vectors containing either the full-length cDNA sequence of human factor VIII (FLrFVIII) or B-domain deleted (BDDrFVIII) into mammalian cell lines results in the production of recombinant factor VIII (rFVIII) for therapeutic usage. Three commercially available rFVIII concentrates (Advates, Helixate NexGens and Refactos), either FLrFVIII or BDDrFVIII, were investigated by 1- and 2-DE and MS. The objective of this study was to compare the heterogeneity and the high purity of both rFVIII preparations before and after thrombin digestion. In particular, the 2-D gel was optimized to better highlight the presence of contaminants and many unexpected proteins. Recombinant strategies consisting of insertion of expression vectors containing BDDrFVIII and FLrFVIII resulted in homogeneous and heterogeneous protein products, respectively, the latter consisting in a heterogeneous mixture of various B-domain-truncated forms of the molecule. Thrombin digestion of all the three rFVIII gave similar final products, plus one unexpected fragment of A2 domain missing 11 amino acids. Regarding the contaminants, Helixate NexGens showed the presence of impurities, such as Hsp70 kDa, haptoglobin and proapolipoprotein; Refactos showed glutathione S-transferase and b-lactamase, whereas Advates apparently did not contain any contaminants. The proteomic approach will contribute to improving the quality assurance and manufacturing processes of rFVIII concentrates. In this view, the 2-DE is mandatory for revealing the presence of contaminants.L'artcoo è disponibile sul sito dell'editore http://onlinelibrary.wiley.com

Proteomics, pigment composition, and organization of thylakoid membranes in iron-deficient spinach leaves

The changes induced in the photosynthetic apparatus of spinach (Spinacia oleracea L.) seedlings exposed to iron deficiency shortly after germination were characterized with two proteomic approaches coupled with chlorophyll and xanthophyll analysis and in vivo measurements of photosynthesis. During the first 10 d of iron deficiency the concentrations of chlorophyll b and violaxanthin were greatly reduced, but all xanthophylls recovered after 13-17 d of iron deficiency, when both chlorophylls were negatively affected. No new protein was formed in iron-deficient leaves, and no protein disappeared altogether. Photosystem I (PSI) proteins were largely reduced, but the stoichiometry of the antenna composition of PSI was not compromised. On the contrary, PSII proteins were less affected by the stress, but the specific antennae Lhcb4 and Lhcb6, Lhcb2 and its isoform Lhcb1.1 were all reduced, while the concentration of Lhcb3 increased. A strong reduction in thylakoid bending and an altered distribution pattern for the reduced PSI and PSII complexes were observed microscopically in iron-deficient leaves. Supercomplex organization was also affected by the stress. The trimeric organization of Lhcb and the dimerization of Lhca were reduced, while monomerization of Lhcb increased. However, the trimerization of Lhcb was partially recovered after 13-17 d of iron deficiency. In iron-deficient leaves, photosynthesis was strongly inhibited at different light intensities, and a high de-epoxidation status of the xanthophylls was observed, in association with a strong impairment of photochemical efficiency and an increase of heat dissipation as monitored by the non-photochemical quenching of fluorescence. All these negative effects of iron deficiency were attenuated but not fully reversed after again supplying iron to iron-deficient leaves for 7-13 d. These results indicate that iron deficiency has a strong impact on the proteomic structure of spinach photosystems and suggest that, in higher plants, adaptive mechanisms common in lower organisms, which allow rapid changes of the photosystem structure to cope with iron stress, are absent. It is speculated that the observed changes in the monomer-trimer equilibrium of major PSII antennae, which is possibly the result of xanthophyll fluctuations, is a first adaptative adjustment to iron deficiency, and may eventually play a role in light dissipation mechanisms

Comparison of Milk Fat Globule Membrane (MFGM) Proteins of Chianina and Holstein Cattle Breed Milk Samples Through Proteomics Methods

Identification of proteins involved in milk production is important to understand the biology of lactation. Many studies have advanced the understanding of mammary function and milk secretion, but the critical molecular mechanisms implicated in milk fat secretion is still incomplete. Milk Fat Globules are secreted from the apical surface of the mammary cells, surrounded by a thin membrane bilayer, the Milk Fat Globule Membrane (MFGM), formed by proteins which have been suggested to be holesterolemia-lowering factors, inhibitors of cancer cell growth, vitamin binders, bactericidal, suppressors of multiple sclerosis. Using a proteomic approach, we compared MFGM from milk samples of individuals belonging to two different cattle breeds, Chianina and Holstein,representative of selection for milk and meat traits, respectively. We were able to isolate some of the major MFGM proteins in the examined samples and to identify differences between the protein fractions of the two breeds. We detected differences in the amount of proteins linked to mammary gland development and lipid droplets formation, as well as host defence mechanisms. We have shown that proteomics is a suitable, unbiased method for the study of milk fractions proteins and a powerful tool in nutritional genomics

Metabolic effect of TAp63? enhanced glycolysis and pentose phosphate pathway, resulting in increased antioxidant defense

TAp63? is a member of the p53 family, which plays a central role in epithelial cancers. Recently, a role has emerged for p53 family members in cancer metabolic modulation.In order to assess whether TAp63? plays a role in cancer metabolism, we exploited p53-null osteosarcoma Tet-On Saos-2 cells, in which the expression of TAp63? was dependent on doxycycline supplementation to the medium. Metabolomics labeling experiments were performed by incubating the cells in 13C-glucose or 13C15N-glutamine-labeled culture media, as to monitor metabolic fluxes upon induced expression of TAp63?.Induced expression of TAp63? resulted in cell cycle arrest at the G1 phase. From a metabolic standpoint, expression of Tap63? promoted glycolysis and the pentose phosphate pathway, which was uncoupled from nucleotide biosynthesis, albeit prevented oxidative stress in the form of oxidized glutathione. Double 13C-glucose and 13C15N-glutamine metabolic labeling confirmed that induced expression of TAp63? corresponded to a decreased flux of pyruvate to the Krebs cycle and decreased utilization of glutamine for catabolic purposes in the TCA cycle. Results were not conclusive in relation to anabolic utilization of labeled glutamine, since it is unclear to what extent the observed minor TAp63?-dependent increases of glutamine-derived labeling in palmitate could be tied to increased rates of reductive carboxylation and de novo synthesis of fatty acids. Finally, bioinformatics elaborations highlighted a link between patient survival rates and the co-expression of p63 and rate limiting enzymes of the pentose phosphate pathway, G6PD and PGD