Establishing an optimized method for the separation of low and high abundance blood plasma proteins

The study tested the efficiency and reproducibility of a method for optimal separation of low and high abundant proteins in blood plasma. Firstly, three methods for the separation and concentration of eluted (E: low abundance), or bound (B: high abundance) proteins were investigated: TCA protein precipitation, the ReadyPrep™ 2-D cleanup Kit and Vivaspin Turbo 4, 5 kDa ultrafiltration units. Secondly, the efficiency and reproducibility of a Seppro column or a ProteoExtract Albumin/IgG column were assessed by quantification of E and B proteins. Thirdly, the efficiency of two elution buffers, containing either 25% or 10% glycerol for elution of the bound protein, was assessed by measuring the remaining eluted volume and the final protein concentration. Compared to the samples treated with TCA protein precipitation and the ReadyPrep™ 2-D cleanup Kit, the E and B proteins concentrated by the Vivaspin4, 5 kDa ultrafiltration unit were separated well in both 1-D and 2-D gels. The depletion efficiency of abundant protein in the Seppro column was reduced after 15 cycles of sample processing and regeneration and the average ratio of E/(B + E) × 100% was 37 ± 11(%) with a poor sample reproducibility as shown by a high coefficient of variation (CV = 30%). However, when the ProteoExtract Albumin/IgG column was used, the ratio of E/(B + E) × 100% was 43 ± 3.1% (n = 6) and its CV was 7.1%, showing good reproducibility. Furthermore, the elution buffer containing 10% (w/v) glycerol increased the rate of B protein elution from the ProteoExtract Albumin/IgG column, and an appropriate protein concentration (3.5 µg/µl) for a 2-D gel assay could also be obtained when it was concentrated with Vivaspin Turbo 4, 5 kDa ultrafiltration unit. In conclusion, the ProteoExtract Albumin/IgG column shows good reproducibility of preparation of low and high abundance blood plasma proteins when using the elution buffer containing 10% (w/v) glycerol. The optimized method of preparation of low/high abundance plasma proteins was when plasma was eluted through a ProteoExtract Albumin/IgG removal column, the column was further washed with elution buffer containing 10% glycerol. The first and second elution containing the low and high abundance plasma proteins, respectively, were further concentrated using Vivaspin® Turbo 4, 5 kDa ultrafiltration units for 1 or 2-D gel electrophoresis

Differential effects of nutritional folic acid deficiency and moderate hyperhomocysteinemia on aortic plaque formation and genome-wide DNA methylation in vascular tissue from ApoE-/- mice

Low folate intake is associated with vascular disease. Causality has been attributed to hyperhomocysteinemia. However, human intervention trials have failed to show the benefit of homocysteine-lowering therapies. Alternatively, low folate may promote vascular disease by deregulating DNA methylation. We investigated whether folate could alter DNA methylation and atherosclerosis in ApoE null mice. Mice were fed one of six diets (n = 20 per group) for 16 weeks. Basal diets were either control (C; 4% lard) or high fat (HF; 21% lard and cholesterol, 0.15%) with different B-vitamin compositions: (1) folic acid and B-vitamin replete, (2) folic acid deficient (−F), (3) folic acid, B6 and B12 deficient (−F−B). −F diets decreased plasma (up to 85%; P < 0.05), whole blood (up to 70%; P < 0.05), and liver folate (up to 65%; P < 0.05) and hepatic SAM/SAH (up to 80%; P < 0.05). −F−B diets reduced plasma (up to 76%; P < 0.05), whole blood (up to 72%; P < 0.05), and liver B12 (up to 39%; P < 0.05) and hepatic SAM/SAH (up to 90%; P < 0.05). −F increased homocysteine 2-fold, while −F−B increased homocysteine 3.6- and 6.8-fold in the C and HF groups (P < 0.05). Plaque formation was increased 2-fold (P < 0.0001) in mice fed a HF diet. Feeding a HF–F diet increased lesion formation by 17% (P < 0.05). There was no change in 5-methyldeoxycytidine in liver or vascular tissue (aorta, periadventitial tissue and heart). These data suggest that atherogenesis is not associated with genome-wide epigenetic changes in this animal model

Extensive spontaneous plasticity of corticospinal projections after primate spinal cord injury.

Although axonal regeneration after CNS injury is limited, partial injury is frequently accompanied by extensive functional recovery. To investigate mechanisms underlying spontaneous recovery after incomplete spinal cord injury, we administered C7 spinal cord hemisections to adult rhesus monkeys and analyzed behavioral, electrophysiological and anatomical adaptations. We found marked spontaneous plasticity of corticospinal projections, with reconstitution of fully 60% of pre-lesion axon density arising from sprouting of spinal cord midline-crossing axons. This extensive anatomical recovery was associated with improvement in coordinated muscle recruitment, hand function and locomotion. These findings identify what may be the most extensive natural recovery of mammalian axonal projections after nervous system injury observed to date, highlighting an important role for primate models in translational disease research

Cumulative mutagenesis of the basic residues in the 201-218 region of insulin-like growth factor (IGF)-binding protein-5 results in progressive loss of both IGF-I binding and inhibition of IGF-I biological action

We have reported previously that mutation of two conserved nonbasic amino acids (G203 and Q209) within the highly basic 201–218 region in the C-terminal domain of IGF-binding protein-5 (IGFBP-5) decreases binding to IGFs. This study reveals that cumulative mutagenesis of the 10 basic residues in this region, to create the C-Term series of mutants, ultimately results in a 15-fold decrease in the affinity for IGF-I and a major loss in heparin binding. We examined the ability of mutants to inhibit IGF-mediated survival of MCF-7 cells and were able to demonstrate that this depended not only upon the affinity for IGF-I, but also the kinetics of this interaction, because IGFBP-5 mutants with similar affinity constants (KD) values, but with different association (Ka) and dissociation (Kd) rate values, had markedly different inhibitory properties. In contrast, the affinity for IGF-I provided no predictive value in terms of the ability of these mutants to enhance IGF action when bound to the substratum. Instead, these C-Term mutants appeared to enhance the actions of IGF-I by a combination of increased dissociation of IGF-IGFBP complexes from the substratum, together with dissociation of IGF-I from IGFBP-5 bound to the substratum. These effects of the IGFBPs were dependent upon binding to IGF-I, because a non-IGF binding mutant (N-Term) was unable to inhibit or enhance the actions of IGF-I. These results emphasize the importance of the kinetics of association/dissociation in determining the enhancing or inhibiting effects of IGFBP-5 and demonstrate the ability to generate an IGFBP-5 mutant with exclusively IGF-enhancing activity

Acute dietary zinc deficiency in rats exacerbates myocardial ischaemia-reperfusion injury through depletion of glutathione.

Zinc (Zn) plays an important role in maintaining the anti-oxidant status within the heart, and helps to counter the acute redox stress that occurs during myocardial ischaemia and reperfusion. Individuals with low zinc (Zn) levels are at greater risk of developing an acute myocardial infarction; however, the impact of this on the extent of myocardial injury is unknown. The present study aimed to compare the effects of dietary zinc depletion with in vitro removal of Zn (TPEN) on the outcome of acute myocardial infarction and vascular function. Male Sprague-Dawley rats were fed either a zinc adequate (ZA; 35mg Zn/kg diet) or zinc deficient (ZD; < 1mg Zn/kg diet) diet for two weeks prior to heart isolation. Perfused hearts were subjected to a thirty-minute ischaemia/two-hour reperfusion (I/R) protocol, during which time ventricular arrhythmias were recorded and after which infarct size was measured, along with markers of anti-oxidant status. In separate experiments hearts were challenged with the Zn chelator TPEN (10μM) prior to ischaemia onset. Both dietary and TPEN-induced Zn depletion significantly extended infarct size; dietary Zn depletion was associated with reduced total cardiac glutathione (GSH) levels, while TPEN decreased cardiac SOD-1 levels. TPEN, but not dietary Zn depletion also suppressed ventricular arrhythmias and depressed vascular responses to nitric oxide (NO). These findings demonstrate that both modes of zinc depletion worsen the outcome from I/R but through different mechanisms. Dietary Zn deficiency, resulting in reduced cardiac GSH, is the most appropriate model for determining the role of endogenous Zn in I/R injury

Nutritional B vitamin deficiency alters the expression of key proteins associated with vascular smooth muscle cell proliferation and migration in the aorta of atherosclerotic apolipoprotein E null mice.

Low B vitamin status is linked with human vascular disease. We employed a proteomic and biochemical approach to determine whether nutritional folate deficiency and/or hyperhomocysteinemia altered metabolic processes linked with atherosclerosis in ApoE null mice. Animals were fed either a control fat (C; 4 % w/w lard) or a high-fat [HF; 21 % w/w lard and cholesterol (0/15 % w/w)] diet with different B vitamin compositions for 16 weeks. Aorta tissue was prepared and global protein expression, B vitamin, homocysteine and lipoprotein status measured. Changes in the expression of aorta proteins were detected in response to multiple B vitamin deficiency combined with a high-fat diet (P < 0.05) and were strongly linked with lipoprotein concentrations measured directly in the aorta adventitia (P < 0.001). Pathway analysis revealed treatment effects in the aorta-related primarily to cytoskeletal organisation, smooth muscle cell adhesion and invasiveness (e.g., fibrinogen, moesin, transgelin, vimentin). Combined B vitamin deficiency induced striking quantitative changes in the expression of aorta proteins in atherosclerotic ApoE null mice. Deregulated expression of these proteins is associated with human atherosclerosis. Cellular pathways altered by B vitamin status included cytoskeletal organisation, cell differentiation and migration, oxidative stress and chronic inflammation. These findings provide new insight into the molecular mechanisms through which B vitamin deficiency may accelerate atherosclerosis

Zinc deficiency decreased cell viability both in endothelial EA.hy926 cells and mouse aortic culture ex vivo and its implication for anti-atherosclerosis

Zinc plays a protective role in anti-atherosclerosis but the clear mechanism has not been proposed yet. In the present study, we evaluated whether zinc modulates atherosclerotic markers, VACM-1 and ICAM-1 and cell viability both in endothelial cells in vitro and mouse aortic cell viability ex vivo. In study 1, as in vitro model, endothelial EA.hy926 cells were treated with TNFα for 5 hours for inducing oxidative stress, and then treated with Zn-adequacy (15 µM Zn) or Zn-deficiency (0 µM Zn) for 6 hours. Pro-atherosclerosis factors, VCAM-1 and ICAM-1 mRNA expression and cell viability was measured. In study 2, as ex vivo model, mouse aorta ring was used. Mourse aorta was removed and cut in ring then, cultured in a 96-well plate. Aortic ring was treated with various TNFα (0-30 mg/ml) and intracellular zinc chelator, N, N, N', N', -tetrakis (2-pyridylmethyl) ethylenediamine (TPEN, 0-30 µM) for cellular zinc depletion for 2 days and then cell viability was measured. The results showed that in in vitro study, Zn-adequate group induced more VCAM-1 & ICAM-1 mRNA expression than Zn-deficient group during 6-hour zinc treatment post-5 hour TNF-α treatment, unexpectedly. These results might be cautiously interpreted that zinc would biologically induce the early expression of anti-oxidative stress through the increased adhesion molecule expression for reducing atherosclerotic action, particularly under the present 6-hour zinc treatment. In ex vivo, mouse aortic ring cell viability was decreased as TNF-α and TPEN levels increased, which suggests that mouse aortic blood vessel cell viability was decreased, when oxidative stress increases and cellular zinc level decreases. Taken together, it can be suggested that zinc may have a protective role in anti-atherosclerosis by cell viability in endothelial cells and aorta tissue. Further study is needed to clarify how pro-atherosclerosis molecule expression is modulated by zinc

A DNA nanoswitch incorporating the fluorescent base analogue 2-aminopurine detects single nucleotide mismatches in unlabelled targets

DNA nanoswitches can be designed to detect unlabelled nucleic acid targets and have been shown to discriminate between targets which differ in the identity of only one base. This paper demonstrates that the fluorescent base analogue 2-aminopurine (AP) can be used to discriminate between nanoswitches with and without targets and to discriminate between matched and mismatched targets. In particular, we have used both steady-state and time-resolved fluorescence spectroscopy to determine differences in AP environment at the branchpoint of nanoswitches assembled using complementary targets and targets which incorporate single base mismatches