Carbamylation of proteins in 2-D electrophoresis--myth or reality?

Carbamylation is widely quoted as being a problem in 2-D gel analysis and the associated sample preparation steps. This modification occurs when iso-cyanate, a urea break-down product, covalently modifies lysine residues, thus inducing a change in isoelectric point. Urea is used at up to 9 M concentrations in sample preparation and 2-D gels because of its ability to disrupt protein structure and effect denaturation without the need for ionic surfactants such as SDS. We have studied carbamylation using 7 M urea and 2 M thiourea, under a range of experimental temperatures to establish when, and if, it occurs and what can be done to minimize the modification. The actual time required for protein extraction from a tissue is usually short compared to the time required for procedures such as reduction and alkylation and IPG rehydration and focusing. Therefore, it is the temperature during these post-extraction procedures that is the most critical factor. Our experiments have shown that carbamylation does not occur during electrophoresis in the presence of urea, even with prolonged run-times. However, under poorly controlled sample preparation and storage conditions, it can become a major event.     

Age and sex differences in human skeletal muscle: role of reactive oxygen species

Previous studies, conducted on experimental animals, have indicated that reactive oxygen species (ROS) are involved in the aging process. The objective of this work was to study the relationship between oxidative damage and human skeletal muscle aging, measuring the activity of the main antioxidant enzymes superoxide dismutase (total and MnSOD), glutathione peroxidase (GPx) and catalase in the skeletal muscle of men and women in the age groups: young (17-40 years), adult (41-65 years) and aged (66-91 years). We also measured glutathione and glutathione disulfide (GSH and GSSG) levels and the redox index; lipid peroxidation and protein carbonyl content. Total SOD activity was lower in the 66-91 year-old vs. the 17-40 year-old men; MnSOD activity was significantly greater in 66-91 year-old vs. 17-40 year-old women. GPx activity remained unchanged. The activity of catalase was lower in adults than in young men but higher in the aged. We observed no changes in GSH levels and significantly higher GSSG levels only in aged men vs. adult men, and a significant decrease in aged women vs. aged men. The protein carbonyl content increased significantly in the 41-65 and 66-91 year-old vs. the 17-40 year-old men. Finally, young women have lower lipid peroxidation levels than young men. Significantly higher lipid peroxidation levels were observed in aged men vs. both young and adult men, and the same trend was noticed for women. We conclude that oxidative damage may play a crucial role in the decline of functional activity in human skeletal muscle with normal aging in both sexes; and that men appear to be more subject to oxidative stress than women.     

Hepcidin assay in serum by SELDI-TOF-MS and other approaches

Hepcidin, a liver peptide hormone, is the central regulator of iron homeostasis. Hepcidin synthesis is modulated by iron stores, so that iron repletion increases its levels to prevent pathological overload, while iron deficiency strongly inhibits hepcidin to allow an increase in iron absorption from duodenal cells. The emerging pivotal role of hepcidin in iron homeostasis, along with its important links with basic pathways like inflammation, makes the availability of an accurate hepcidin assay as a potentially powerful investigative tool to improve our understanding as well as our diagnostic/prognostic capabilities in many human diseases. There has been a great interest worldwide in developing a reliable and widely applicable assay of the hormone in biological fluids. Being optimal for low-molecular-weight biomarkers, SELDI-TOF-MS has emerged as a valid tool for hepcidin assay. Here we review recent results obtained with this technique, as well as with other Mass Spectrometry-based and immunological methods.     

Nutrient uptake and management under saline conditions in the xerohalophyte: Tecticornia indica (Willd.) subsp. indica

In the present investigation, we studied uptake and management of the major cations in the xerohalophyte, Tecticornia indica (Willd.) subsp. indica as subjected to salinity. Plants were grown under greenhouse conditions at various salinity levels (0, 100, 200 and 400 mM NaCl) over 110 days. At harvest, they were separated into shoots and roots then analyzed for water contents, dry weights (DW), and Na+, K+, Ca2+, and Mg2+ contents. Plants showed a growth optimum at 200 mM NaCl and much better tissue hydration under saline than non-saline conditions. At this salt concentration (200 mM NaCl), shoot Na+ content reached its highest value (7.9 mmol · g-?1 DW). In spite of such stressful conditions, salt-treated plants maintained adequate K+, Ca2+, and Mg2+ status even under severe saline conditions. This was mainly due to their aptitude to selectively acquire these essential cations and efficiently use them for biomass production.     

Effect of whole body vibration training on lower limb performance in selected high-level ballet students

The aim of this study was to examine the effects of 8 weeks of whole body vibration (WBV) training on vertical jump ability (CMJ) and knee-extensor performance at selected external loads (50, 70, and 100 kg; leg-press exercise) in elite ballerinas. Twenty-two (age, 21.25 +/- 1.5 years) full-time ballerinas were assigned randomly to the experimental (E, n = 11) and control (C, n = 11) groups. The experimental group was submitted to WBV training 3 times per week before ballet practice. During the training period, the E and C groups undertook the same amount of ballet practice. Posttraining CMJ performance significantly increased in E group (6.3 +/- 3.8%, p < 0.001). Furthermore, E group showed significant (p < 0.05-0.001) posttraining average leg-press power and velocity improvements at all the external loads considered. Consequently, the force-velocity and power-velocity relationship shifted to the right after WBV training in the E group. The results of the present study show that WBV training is an effective short-term training methodology for inducing improvements in knee-extensor explosiveness in elite ballerinas.     

Interaction between lysine 102 and aspartate 338 in the insect amino acid cotransporter KAAT1

KAAT1 is a lepidopteran neutral amino acid transporter belonging to the NSS super family (SLC6), which has an unusual cation selectivity, being activated by K⁺ and Li⁺ in addition to Na⁺. We have previously demonstrated that Asp338 is essential for KAAT1 activation by K⁺ and for the coupling of amino acid and driver ion fluxes. By comparing sequences of NSS family members, site-directed mutagenesis, and expression in Xenopus laevis oocytes, we identified Lys102 as a residue likely to interact with Asp338. Compared with wild type, the single mutants K102V and D338E each showed altered leucine uptake and transport-associated currents in the presence of both Na⁺ and K⁺. However, in K102V/D338E double mutant, the K102V mutation reversed both the inhibition of Na⁺-dependent transport and the block in K⁺-dependent transport that characterize the D338E mutant. K⁺-dependent leucine currents were not observed in any mutants with D338E. In the presence of the oxidant Cu(II) (1,10-phenanthroline)₃, we observed specific and reversible inhibition of K102C/D338C mutant, but not of the corresponding single cysteine mutants, suggesting that these residues are sufficiently close to form a disulfide bond. Thus both structural and functional evidence suggests that these two residues interact. Similar results have been obtained mutating the bacterial transporter homolog TnaT. Asp338 corresponds to Asn286, a residue located in the Na⁺ binding site in the recently solved crystal structure of the NSS transporter LeuT_Aa (41). Our results suggest that Lys102, interacting with Asp338, could contribute to the spatial organization of KAAT1 cation binding site and permeation pathway. residue interaction; oxidants; tertiary structure     

Effect of training intensity distribution on aerobic fitness variables in elite soccer players: a case study

The aim of this article was to quantify the distribution of training intensities and its effect on aerobic fitness in professional elite soccer players. Fourteen professional soccer players were observed during the prechampionship training period (6 weeks). Treadmill running speed and heart rates (HRs) at 2 and 4 mmol · L(-1) blood-lactate concentrations were assessed pre and posttraining. Training intensities were categorized using 3 HR zones: low intensity (

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HR 4 mmol · L(-1)). Analysis of the 504 individual training sessions showed that 73 ± 2.5, 19 ± 2.8, and 8 ± 1.4% of the total training time was spent at low, moderate, and high intensity, respectively (p < 0.001). Speed at 2 and 4 mmol · L(-1) significantly improved posttraining (5 and 7%, respectively, p < 0.01). Training spent at high intensity was significantly related to relative speed improvements at 2 mmol · L(-1) (r = 0.84, p < 0.001;) and 4 mmol · L(-1) (r = 0.65, p = 0.001). Players spent almost two-thirds of their training time at low intensities. However, only the time spent at high intensity (>90% of maximal HR) was related to changes in aerobic fitness. These results support the usefulness of the quantification of aerobic training load using HR. Furthermore, it stresses the effectiveness of the high-intensity training in soccer.     

The effects of a constant sprint-to-rest ratio and recovery mode on repeated sprint performance

It is unclear if a constant sprint-to-rest ratio allows full performance recovery between repeated sprints over different distances. This is important for the development of sprint-training programs. Additionally, there is conflicting evidence on whether active recovery enhances sprint performance. Three repeated sprint protocols were used (22 × 15, 13 × 30, and 8 × 50 m), with each having an active and passive recovery. Each trial was conducted with an initial sprint-to-rest ratio of 1:10. Repeated sprints were analyzed by comparing the first sprint to the last sprint. For the 15-m trials, there were no significant main effects for recovery or time and no significant interaction. For the 30-m trials, there was no main effect for recovery, but a main effect for time (F[1,10] = 15.995, p = 0.003; mean difference = 0.20 seconds, 95% confidence interval [CI] = 0.09-0.31 seconds, d = 1.4 [large effect]). There was no interaction of recovery and time in the 30-m trials. For the 50-m trials, there was no main effect for recovery, but a main effect for time (F[1,10] = 34.225, p = 0.0002; mean difference = 0.39 seconds, 95% CI = 0.24-0.55 seconds, d = 1.3 [large effect]). There was no interaction of recovery and time in the 50-m trials. The results demonstrate that a 1:10 sprint-to-rest ratio allows full performance recovery between 15-m sprints, but not between sprints of 30 or 50 m, and that recovery mode did not influence repeated sprint performance.     

How to Bring the “Unseen” Proteome to the Limelight via Electrophoretic Pre-Fractionation Techniques

The present review reports a panoply of electrophoretic methods as pre-fractionation tools in proteomic investigations in preparation for mass spectrometry or two-dimensional electrophoresis map analysis. Such electrophoretic pre-fractionation protocols include all those electrokinetic methodologies which are performed in free solution, most of them relying on isoelectric focusing steps (although some approaches based on gels and granulated media are also discussed). Devices associated with electrophoretic separations are multi-chamber apparatuses, such as the multi-compartment electrolyzers equipped with either isoelectric membranes or with isoelectric beads, Off-Gel electrophoresis in a multi-cup device and the Rotofor, an instrument also based on a multi-chamber system but exploiting the conventional technique of carrier-ampholyte-focusing. Other free-flow systems, as well as miniaturized chambers, are also described.     

Oxidative stress and phytochelatin characterisation in bread wheat exposed to cadmium excess.

In this work, we first investigated if the bread wheat (Triticum aestivum L.) cv. Albimonte can be defined as "shoot cadmium excluder"--by comparing the cadmium (Cd) content in leaves and roots and by calculating the shoot-to-root Cd concentration ratio. Furthermore, we evaluated if the exposure to Cd excess could generate oxidative stress in leaves and roots of this cv., in terms of hydrogen peroxide (H(2)O(2)) accumulation, NAD(P)H oxidation rate, and variations in reduced glutathione (GSH) content and peroxidase (POD, EC 1.11.1.7) activity. Finally, we surveyed possible quali- quantitative differences in thiol-peptide compound pattern between roots and leaves, in order to verify whether phytochelatins (PCs) and related thiol-peptides could contribute in limiting the Cd-induced oxidative stress. Unambiguous characterisation of PCs and related forms present in the root samples was obtained by electrospray ionisation mass spectrometry (ESI-MS) and ESI-tandem MS (ESI-MS/MS). Our results indicate that in leaves the stress generated by the low accumulation of Cd (due to a moderate translocation in planta) seems to be counteracted by the antioxidant response and by the PC biosynthesis. On the contrary, in roots, in spite of the elevated presence of PCs and related thiol-peptide-compounds, the excess of Cd causes a decline in the antioxidant protection of the organ, with the consequent generation of considerable amounts of H(2)O(2), a direct agent of oxidative stress.