Effects of supplementing two levels of magnesium aspartate and transportation stress on pork quality and gene expression of 𝛍-calpain and calpastatin of finishing pigs

The objective of this study was to investigate the effects of supplementing swine finishing diets with two levels of magnesium aspartate (MgAsp) and short-term transportation stress on blood parameters, pork quality and the mRNA abundance of μ-calpain and calpastatin in muscles of finishing pigs. Thirty-six crossbred finishing pigs (mean BW 90 kg) were assigned randomly to 0, 1000, or 2000 mg supplemental Mg from MgAsp per kg of diet for five days before slaughter. Then six pigs from each dietary treatment were subjected either to no transportation stress (NTS) or 2 h of transportation stress (TS). Transportation stress resulted in higher concentrations (p < 0.01) of serum calcium, glucose and cortisol, lower pH (p < 0.01), higher Warner-Bratzler shear force (WBSF) (p < 0.05) and higher calpastatin mRNA abundance (p = 0.05) of longissimus muscle (LM) compared with NTS treatments. Supplementation of MgAsp in TS treatments increased serum Mg concentration (p < 0.05) at 2000 mg of Mg/kg, reduced drip loss (p < 0.05) and improved pork quality colour (p < 0.05) at 2000 mg of Mg/kg, and decreased 1-day and 3-day WBSF (p < 0.05) at 1000 mg of Mg/kg compared with TS treatments fed the control diet. It is concluded that supplementation of MgAsp improves water-holding capacity and pork colour, and alleviates the negative effects of transportation stress on meat tenderness.     

Mixed planting with a leguminous plant outperforms bacteria in promoting growth of a metal remediating plant through histidine synthesis

The effectiveness of plant growth promoting bacteria (PGPB) in improving metal phytoremediation is still limited by stunted plant growth under high soil metal concentrations. Meanwhile, mixed planting with leguminous plants is known to improve yield in nutrient deficient soils but the use of a metal tolerant legume to enhance metal tolerance of a phytoremediator has not been explored. We compared the use of Pseudomonas brassicacearum, Rhizobium leguminosarum, and the metal tolerant leguminous plant Vicia sativa to promote the growth of Brassica juncea in soil contaminated with 400 mg Zn kg^–1, and used synchrotron based microfocus X-ray absorption spectroscopy to probe Zn speciation in plant roots. B. juncea grew better when planted with V. sativa than when inoculated with PGPB. By combining PGPB with mixed planting, B. juncea recovered full growth while also achieving soil remediation efficiency of >75%, the maximum ever demonstrated for B. juncea. μXANES analysis of V. sativa suggested possible root exudation of the Zn chelates histidine and cysteine were responsible for reducing Zn toxicity. We propose the exploration of a legume-assisted-phytoremediation system as a more effective alternative to PGPB for Zn bioremediation.     

Metabolic Enzyme Alterations and Astrocyte Dysfunction in a Murine Model of Alexander Disease With Severe Reactive Gliosis

Alexander disease (AxD) is a rare and fatal neurodegenerative disorder caused by mutations in the gene encoding glial fibrillary acidic protein (GFAP). In this report, a mouse model of AxD (GFAP^Tg;Gfap^+/R236H) was analyzed that contains a heterozygous R236H point mutation in murine Gfap as well as a transgene with a GFAP promoter to overexpress human GFAP. Using label-free quantitative proteomic comparisons of brain tissue from GFAP^Tg;Gfap^+/R236H versus wild-type mice confirmed upregulation of the glutathione metabolism pathway and indicated proteins were elevated in the peroxisome proliferator-activated receptor (PPAR) signaling pathway, which had not been reported previously in AxD. Relative protein-level differences were confirmed by a targeted proteomics assay, including proteins related to astrocytes and oligodendrocytes. Of particular interest was the decreased level of the oligodendrocyte protein, 2-hydroxyacylsphingosine 1-beta-galactosyltransferase (Ugt8), since Ugt8-deficient mice exhibit a phenotype similar to GFAP^Tg;Gfap^+/R236H mice (e.g., tremors, ataxia, hind-limb paralysis). In addition, decreased levels of myelin-associated proteins were found in the GFAP^Tg;Gfap^+/R236H mice, consistent with the role of Ugt8 in myelin synthesis. Fabp7 upregulation in GFAP^Tg;Gfap^+/R236H mice was also selected for further investigation due to its uncharacterized association to AxD, critical function in astrocyte proliferation, and functional ability to inhibit the anti-inflammatory PPAR signaling pathway in models of amyotrophic lateral sclerosis (ALS). Within Gfap⁺ astrocytes, Fabp7 was markedly increased in the hippocampus, a brain region subjected to extensive pathology and chronic reactive gliosis in GFAP^Tg;Gfap^+/R236H mice. Last, to determine whether the findings in GFAP^Tg;Gfap^+/R236H mice are present in the human condition, AxD patient and control samples were analyzed by Western blot, which indicated that Type I AxD patients have a significant fourfold upregulation of FABP7. However, immunohistochemistry analysis showed that UGT8 accumulates in AxD patient subpial brain regions where abundant amounts of Rosenthal fibers are located, which was not observed in the GFAP^Tg;Gfap^+/R236H mice.     

Solution structure of the R3H domain from human Smubp-2

The R3H domain is a conserved sequence motif, identified in over 100 proteins, that is thought to be involved in polynucleotide-binding, including DNA, RNA and single-stranded DNA. In this work the 3D structure of the R3H domain from human Smubp-2 was determined by NMR spectroscopy. It is the first 3D structure determination of an R3H domain. The fold presents a small motif, consisting of a three-stranded antiparallel beta-sheet and two alpha-helices, which is related to the structures of the YhhP protein and the C-terminal domain of the translational initiation factor IF3. The similarities are non-trivial, as the amino acid identities are below 10%. Three conserved basic residues cluster on the same face of the R3H domain and could play a role in nucleic acid recognition. An extended hydrophobic area at a different site of the molecular surface could act as a protein-binding site. A strong correlation between conservation of hydrophobic amino acids and side-chain solvent protection indicates that the structure of the Smubp-2 R3H domain is representative of R3H domains in general.     

Protein kinase D: a family affair

The protein kinase D family of enzymes consists of three isoforms: PKD1/PKCmu PKD2 and PKD3/PKCnu. They all share a similar architecture with regulatory sub-domains that play specific roles in the activation, translocation and function of the enzymes. The PKD enzymes have recently been implicated in very diverse cellular functions, including Golgi organization and plasma membrane directed transport, metastasis, immune responses, apoptosis and cell proliferation.     

The use of Quantitative Agarose Gel Electrophoresis for rapid analysis of the integrity of protein-DNA complexes

Recent biochemical studies evaluated the affinity of histones to DNA in the context of nucleosome core particle (NCP). These have indicated a concentration-dependence for nucleosome stability. However, when studying chromatin the preferred templates are nucleosome arrays (NA) and not the NCP. Biochemical methods are poorly suited for structural analysis of chromatin. To overcome that technical hindrance, and investigate the effect of concentration on stability of the histone-DNA interactions, we have applied the multigel Quantitative Agarose Gel Electrophoresis (QAGE) method to in vitro-assembled nucleosomal arrays. The results demonstrated the method to be extremely valuable for the evaluation of the effect of low concentration on NA. However, QAGE is a fairly time-demanding and complex method. To maximize the efficiency of use of this technology, we devised a protocol that allowed for multiple sets of templates to be analyzed simultaneously. Briefly, samples can be loaded at regular intervals and analyzed individually for their molecular composition. The technique presented in this study describes the calibration steps and proof of concept necessary to validate the use of multiple loading of multigel to evaluate the composition of nucleosomal arrays as a function of concentration.     

Regioselective phosphorylation of branched cyclodextrins with cyclo-mono-mu-imidotriphosphate

The phosphorylation of the branched cyclodextrins, mono-6-O-(alpha-D-glucopyranosyl)cyclomaltohexaose, mono-6-O-(alpha-D-maltosyl)cyclomaltohexaose, mono-6-O-(alpha-D-glucopyranosyl)cyclomaltoheptaose, and mono-6-O-(alpha-D-maltosyl)cyclomaltoheptaose, in aqueous solution by sodium cyclo-mono-mu-imidotriphosphate (cMITP) was examined. In these reactions, only the 2-OH group of a single alpha-D-glucopyranosyl residue of the cyclodextrin ring was phosphorylated, in a maximum yield of 67%. A possible mechanism for the phosphorylation is discussed.     

Investigating serum factors promoting erythrocytic growth of Plasmodium falciparum

The elucidation of factors inducing the growth of Plasmodium falciparum can provide critical information about the developmental mechanisms of this parasite and open the way to search for novel targets for malaria chemotherapy. The ability of components of a growth-promoting factor derived from bovine serum and various related substances to sustain growth of P. falciparum was characterized. A simple total lipid fraction (GFS-C) containing non-esterified fatty acids (NEFAs) as essential factors was noted to promote the parasite's growth. Various proteins from a variety of animals were tested, indicating the importance not only of GFS-C, but also of specific proteins, such as bovine and human albumin, in the parasite growth. Several combinations of the NEFAs tested sustained low parasite growth. Among various phospholipids and lysophospholipids tested, lysophosphatidylcholine containing C-18 unsaturated fatty acids was found to sustain the complete development of the parasite in the presence of bovine albumin. Several other lysophospholipids can partially support growth of P. falciparum.     

Structural consideration of the formation of the activation complex between the staphylokinase-like streptococcal plasminogen activator PadA and bovine plasminogen

The characteristics of a streptococcal plasminogen activator (PA) displaying specificity for ruminant plasminogen (Plg) were defined using molecular approaches. The 16-kDa secreted protein PadA was found to be prevalent in Streptococcus dysgalactiae subspecies dysgalactiae isolated from cases of bovine mastitis and septic arthritis in lambs. PadA was able to activate bovine, ovine and caprine Plg, but not human Plg. Amino acid sequence analysis identified a limited level of homology to other streptococcal PAs, including streptokinase; however, PadA was found to align well with and match in size the staphylococcal PA, staphylokinase. Recombinant PadA was used to investigate interaction with bovine Plg, leading to formation of an activator complex that was capable of recruiting and converting further substrate Plg into plasmin. Individual non-overlapping peptides of PadA or bovine microplasminogen were found to block the interaction between PadA and bovine Plg, preventing the formation of the activation complex. Homology modelling based upon structures of staphylokinase complexed with human microplasminogen supported these findings by placing critical residues in close proximity to the plasmin component of the activation complex.     

Trans-channel interactions in batrachotoxin-modified rat skeletal muscle sodium channels: kinetic analysis of mutual inhibition between mu-conotoxin GIIIA derivatives and amine blockers

R13X derivatives of μ-conotoxin GIIIA bind externally to single sodium channels and block current incompletely with mean “blocked” durations of several seconds. We studied interactions between two classes of blockers (μ-conotoxins and amines) by steady state, kinetic analysis of block of BTX-modified Na channels in planar bilayers. The amines cause all-or-none block at a site internal to the selectivity filter. TPrA and DEA block single Na channels with very different kinetics. TPrA induces discrete, all-or-none, blocked events (mean blocked durations, ∼100 ms), whereas DEA produces a concentration-dependent reduction of the apparent single channel amplitude (“fast” block). These distinct modes of action allow simultaneous evaluation of block by TPrA and DEA, showing a classical, competitive interaction between them. The apparent affinity of TPrA decreases with increasing [DEA], based on a decrease in the association rate for TPrA. When an R13X μ-conotoxin derivative and one of the amines are applied simultaneously on opposite sides of the membrane, a mutually inhibitory interaction is observed. Dissociation constants, at +50 mV, for TPrA (∼4 mM) and DEA (∼30 mM) increase by ∼20%-50% when R13E (nominal net charge, +4) or R13Q (+5) is bound. Analysis of the slow blocking kinetics for the two toxin derivatives showed comparable decreases in affinity of the μ-conotoxins in the presence of an amine. Although this mutual inhibition seems to be qualitatively consistent with an electrostatic interaction across the selectivity filter, quantitative considerations raise questions about the mechanistic details of the interaction.