Does selenium exert cardioprotective effects against oxidative stress in myocardial ischemia?

In the mid-1960s, a small number of scientists postulated the role of oxidative stress and oxygen-derived free radicals in the pathophysiological mechanisms underlying ischemic heart disease. However, because of the technical difficulty of measuring free radicals and quantitating oxidative damage, it was very difficult to prove that free radicals could contribute to cell pathology. The role of oxidative stress in biological systems was not definitely recognized until the early 1980s when measurement of short-lived oxygen-derived reactive species was made possible by the advent of sophisticated techniques such as EPR spectroscopy or fluorescent probes. These enabled both the study of free radical biochemistry and the acquisition of useful information about the nature and consequences of free radical-induced protein and lipid oxidation. The hypothesis that reactive oxygen species mediate cellular damage produced upon reperfusion of ischemic myocardium has gained considerable support during the past 10-15 years. Several experimental studies indicated that the administration of antioxidant enzymes or non-enzymatic antioxidants offers a significant degree of protection against ischemic damage, improving functional recovery and reducing morphological alterations to cardiomyocytes. In this context, selenium, as an essential component of glutathione peroxidase, plays a critical role in protecting aerobic tissues from oxygen radical-initiated cell injury.     

Clinical Quantitation of Prostate-specific Antigen Biomarker in the Low Nanogram/Milliliter Range by Conventional Bore Liquid Chromatography-Tandem Mass Spectrometry (Multiple Reaction Monitoring) Coupling and Correlation with ELISA Tests

Proteomics discovery leads to a list of potential protein biomarkers that have to be subsequently verified and validated with a statistically viable number of patients. Although the most sensitive, the development of an ELISA test is time-consuming when antibodies are not available and need to be conceived. Mass spectrometry analysis driven in quantitative multiple reaction monitoring mode is now appearing as a promising alternative to quantify proteins in biological fluids. However, all the studies published to date describe limits of quantitation in the low μg/ml range when no immunoenrichment of the target protein is applied, whereas the concentration of known clinical biomarkers is usually in the ng/ml range. Using prostate-specific antigen as a model biomarker, we now provide proof of principle that mass spectrometry enables protein quantitation in a concentration range of clinical interest without immunoenrichment. We have developed and optimized a robust sample processing method combining albumin depletion, trypsin digestion, and solid phase extraction of the proteotypic peptides starting from only 100 μl of serum. For analysis, mass spectrometry was coupled to a conventional liquid chromatography system using a 2-mm-internal diameter reverse phase column. This mass spectrometry-based strategy was applied to the quantitation of prostate-specific antigen in sera of patients with either benign prostate hyperplasia or prostate cancer. The quantitation was performed against an external calibration curve by interpolation, and results showed good correlation with existing ELISA tests applied to the same samples. This strategy might now be implemented in any clinical laboratory or certified company for further evaluation of any putative biomarker in the low ng/ml range of serum or plasma.Used for years across a wide range of pathologies, proteomics studies based on semiquantitative mass spectrometry of proteins have already led to the discovery of numerous protein biomarker candidates. Often tens of putative biomarkers have been described for a single disease, but the subsequent phase of clinical evaluation on large cohorts for each candidate is clearly the bottleneck as revealed by the meager number of newly approved biomarkers for clinical use. One of the critical limitations of discovery work flows arises when no antibody is available to initiate an immunoassay because 1–3 years are required to conceive de novo a reliable immunoassay. Such a delay is a serious drawback when tens of putative markers are concerned. Quantitation of small organic molecules by mass spectrometry has been used extensively for years in the field of environmental contaminant analysis or pharmacokinetic profiling of drug candidates during clinical studies. More recently, absolute quantitation of proteins using mass spectrometry by single (SRM)¹ or multiple reaction monitoring (MRM) and stable isotope dilution has thus naturally emerged as an alternative to immunoassays. Basically the absolute quantitation of a protein is provided by the integration of the specific MRM signals corresponding, respectively, to a proteotypic peptide (1) obtained from enzymatic hydrolysis of the target protein (usually by trypsin) and to its synthetic stable isotope-labeled isotopomer (2). The validation criteria of an MS-based method in terms of accuracy and precision are relatively easy to fulfill when addressing small molecules or proteins below 10 kDa in plasma or serum. Indeed they may be easily extracted from the bulk of high molecular mass proteins simply by selective precipitation. The quantitation of higher molecular mass proteins has proven to be more challenging because of the complexity and large dynamic range of proteins in e.g. plasma. In a pioneering study Anderson and Hunter (3) successfully demonstrated generation of a multiplexed assay for proteins covering high (tens to hundreds of micrograms/milliliter) to medium (hundreds of nanograms/milliliter to a few micrograms/milliliter) abundance ranges in plasma when combined with immunodepletion. However, these ranges remain problematic because clinically relevant biomarkers are usually present in plasma or serum in the low nanogram/milliliter range or below. To significantly improve the limit of quantitation (LOQ) of LC-MRM mass spectrometry, Keshishian et al. (4) evaluated a combination of immunodepletion of the most abundant plasma or serum proteins with strong cation exchange (SCX) chromatography for sample preparation prior to LC-MRM analysis. LOQs in the 1–10 ng/ml range were obtained with a coefficient of variation from 3 to 15% for five exogenous non-human proteins and the human prostate-specific antigen (PSA) protein spiked together into immunodepleted plasma from a healthy female donor. Very recently, a new approach using product ion scanning on a linear ion trap was proposed by Diamandis and co-workers (5) that allowed reaching a limit of quantitation of 1 ng/ml for PSA spiked into control plasma. This study marked a gain in sensitivity compared with previous attempts by others on similar instrumentation (6–9) but applied immunopurification of the target protein.Interestingly all the strategies published to date have been dealing with analytical development of work flows for the validation of biomarker candidates using microbore, nanoflow chromatography. Nanoflow is without any doubt appealing over conventional microflow during the proteomics discovery phase when the amount of biological material, for instance from a tumor biopsy, is often limited. Nonetheless this technique inherently still suffers from a lack of robustness and requires skilled personnel to be operational on a daily basis. As a consequence, nanoflow chromatography is not easily adaptable for the high throughput analysis environment encountered in clinical laboratories or good laboratory practice-certified or contract research organization companies where hundreds of samples are handled per day. In such organizations only microflow separations using 1- or 2-mm-internal diameter HPLC columns are compatible with the requirements of robustness and sample throughput.Therefore, the present work was centered on how a simple work flow could, in the near future, enable the large scale verification phase of putative biomarkers in the ng/ml of plasma range by the use of conventional LC equipment, i.e. using a 2-mm-internal diameter HPLC column. To address this question, we have considered that the absolute quantitation of PSA in true clinical samples could represent a challenging model. Combining immunodepletion of serum albumin and peptide fractionation simply by solid phase extraction (SPE), we were able to demonstrate for the first time the absolute quantitation of PSA by LC-MRM mass spectrometry in clinical serum samples of patients with benign prostate hyperplasia (BPH) or prostate cancer (PCa) within concentrations ranging from 4 to 30 ng/ml. Furthermore a good correlation was observed between the clinical ELISA tests and the mass spectrometry-based assays. We believe that these results are an unprecedented demonstration that the clinical relevance of putative biomarkers issued from proteomics investigation may now be confidently evaluated in the ng/ml range by robust coupling between conventional bore LC and mass spectrometry.     

Rational drug design via intrinsically disordered protein

Despite substantial increases in research funding by the pharmaceutical industry, drug discovery rates seem to have reached a plateau or perhaps are even declining, suggesting the need for new strategies. Protein-protein interactions have long been thought to provide interesting drug discovery targets, but the development of small molecules that modulate such interactions has so far achieved a low success rate. In contrast to this historic trend, a few recent successes raise hopes for routinely identifying druggable protein-protein interactions. In this Opinion article, we point out the importance of coupled binding and folding for protein-protein signalling interactions generally, and from this and associated observations, we develop a new strategy for identifying protein-protein interactions that would be particularly promising targets for modulation by small molecules. This novel strategy, based on intrinsically disordered protein, has the potential to increase significantly the discovery rate for new molecule entities.     

Paradoxical effects of endurance training and chronic hypoxia on myofibrillar ATPase activity

This study aimed to determine the changes in soleus myofibrillar ATPase (m-ATPase) activity and myosin heavy chain (MHC) isoform expression after endurance training and/or chronic hypoxic exposure. Dark Agouti rats were randomly divided into four groups: control, normoxic sedentary (N; n = 14), normoxic endurance trained (NT; n = 14), hypoxic sedentary (H; n = 10), and hypoxic endurance trained (HT; n = 14). Rats lived and trained in normoxia at 760 mmHg (N and NT) or hypobaric hypoxia at 550 mmHg (approximately 2,800 m) (H and HT). m-ATPase activity was measured by rapid flow quench technique; myosin subunits were analyzed with mono- and two-dimensional gel electrophoresis. Endurance training significantly increased m-ATPase (P < 0.01), although an increase in MHC-I content occurred (P < 0.01). In spite of slow-to-fast transitions in MHC isoform distribution in chronic hypoxia (P < 0.05) no increase in m-ATPase was observed. The rate constants of m-ATPase were 0.0350 +/- 0.0023 s(-1) and 0.047 +/- 0.0050 s(-1) for N and NT and 0.033 +/- 0.0021 s(-1) and 0.038 +/- 0.0032 s(-1) for H and HT. Thus, dissociation between variations in m-ATPase and changes in MHC isoform expression was observed. Changes in fraction of active myosin heads, in myosin light chain isoform (MLC) distribution or in MLC phosphorylation, could not explain the variations in m-ATPase. Myosin posttranslational modifications or changes in other myofibrillar proteins may therefore be responsible for the observed variations in m-ATPase activity.     

Does Liebig's law of the minimum scale up from species to communities?

Liebig's law of the minimum, which states that only one element limits the growth of organisms at any given time, is widely used in ecology. This principle is routinely applied to organisms, populations and communities, but can it really be applied indistinguishably across these different scales? Here we show, by prediction of a resource ratio conceptual model and with an experimental test carried out in microcosms with bacteria that, unlike single species, communities are likely to adjust their stoichiometry to that of their resources. This adjustment results from competitive exclusion and coexistence mechanisms, and is sensitive to the overall diversity of species in the community. It guaranties co‐limitation, i.e. simultaneous limitation by multiple resources, at the community scale and optimal use of resources and maximization of community biomass for wide ranges of resource ratios. These results question the applicability of the Liebig's law of the minimum at the community level, and the relevance of ecosystem models relying on this principle.     

A Synoptic Ecopath model of biomass flows during two different static ecological situations in Lake Nakuru (Kenya)

Trophic relationships inside the Lake Nakuru ecosystem during two different phases are described by using the ECOPATH model. This allows a partial explanation of the ecological changes observed in the lake and to show that this apparently unstable ecosystem is characterized by constant transfer efficiencies within the existing trophic levels.     

The influence of clone and morph on the parameters of intrinsic rate of increase in the cereal aphids Sitobion avenae and Rhopalosiphum padi

This study aimed at evaluating the influence of intrinsic factors (clone, morph) on the parameters of the intrinsic rate of increase of Sitobion avenae (F.) and Rhopalosiphum padi (L.). For each species, apterous and alate exules of three clones originating from French oceanic regions were compared at 20 °C.The clonal factor had a significant effect on both time from birth until onset of reproduction (TBR) and intrinsic rate of increase (r). This effect was lower than that of the morph. Moreover, in the case of R. padi, a strong clone-morph interaction was observed.Both apterous and alate morphs were shown to have a higher rate of increase in R. padi than in S. avenae.Various models were fitted to aphid daily rate of reproduction. Both types of model and quality of fit depended mainly on the morph.

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Résumé Cette étude a eu pour but d'évaluer l'importance de facteurs intrinsèques (clone et morphe) sur les paramètres du taux d'accroissement de Sitobion avenae (F.) et Rhopalosiphum padi (L.).Pour chaque espèce, trois clones originaires de régions océaniques françaises ont été comparés à 20 °C pour les morphes exules ailés et exules aptères.L'effet du clone est significatif sur l'âge reproducteur (durée comprise entre la naissance du puceron et le début de sa phase de reproduction) et sur le taux intrinsèque d'accroissement naturel. Il est plus faible que celui du morphe avec lequel il intéragit dans le cas de R. padi.Les différences interspécifiques relevées mettent en évidence les capacités d'accroissement supérieures de R. padi par rapport à celles de S. avenae, dans le cas des ailés comme dans celui des aptères.Enfin, différents modèles ont été appliqués aux données de reproduction journalière des pucerons. Le type et la qualité des ajustements varient essentiellement en fonction du morphe.

     

BTX modification of Na channels in squid axons. I. State dependence of BTX action

The state dependence of Na channel modification by batrachotoxin (BTX) was investigated in voltage-clamped and internally perfused squid giant axons before (control axons) and after the pharmacological removal of the fast inactivation by pronase, chloramine-T, or NBA (pretreated axons). In control axons, in the presence of 2-5 microM BTX, a repetitive depolarization to open the channels was required to achieve a complete BTX modification, characterized by the suppression of the fast inactivation and a simultaneous 50-mV shift of the activation voltage dependence in the hyperpolarizing direction, whereas a single long-lasting (10 min) depolarization to +50 mV could promote the modification of only a small fraction of the channels, the noninactivating ones. In pretreated axons, such a single sustained depolarization as well as the repetitive depolarization could induce a complete modification, as evidenced by a similar shift of the activation voltage dependence. Therefore, the fast inactivated channels were not modified by BTX. We compared the rate of BTX modification of the open and slow inactivated channels in control and pretreated axons using different protocols: (a) During a repetitive depolarization with either 4- or 100-ms conditioning pulses to +80 mV, all the channels were modified in the open state in control axons as well as in pretreated axons, with a similar time constant of approximately 1.2 s. (b) In pronase-treated axons, when all the channels were in the slow inactivated state before BTX application, BTX could modify all the channels, but at a very slow rate, with a time constant of approximately 9.5 min. We conclude that at the macroscopic level BTX modification can occur through two different pathways: (a) via the open state, and (b) via the slow inactivated state of the channels that lack the fast inactivation, spontaneously or pharmacologically, but at a rate approximately 500-fold slower than through the main open channel pathway.     

PBX1 intracellular localization is independent of MEIS1 in epithelial cells of the developing female genital tract

While studies have highlighted the role of HOXA9-13 and PBX1 homeobox genes during the development of the female genital tract, the molecular mechanisms triggered by these genes are incompletely elucidated. In several developmental pathways, PBX1 binds to MEINOX family members in the cytoplasm to be imported into the nucleus where they associate with HOX proteins to form a higher complex that modulates gene expression. This concept has been challenged by a recent report showing that in some cell cultures, PBX1 nuclear localization might be regulated independently of MEINOX proteins (Kilstrup-Nielsen et al., 2003). Our work gives the first illustration of this alternative mechanism in an organogenesis process. Indeed, we show that PBX1 is mostly cytoplasmic in epithelial endometrial cells of the developing female genital tract despite the nuclear localization of MEIS1. We thus provide evidence for a control of PBX1 intracellular distribution which is independent of MEINOX proteins, but is cell cycle correlated.