Sample preparation for two-dimensional gel electrophoresis.

The choice of sample preparation protocol is a critical influential factor for isoelectric focusing which in turn affects the two-dimensional gel result in terms of quality and protein species distribution. The optimal protocol varies depending on the nature of the sample for analysis and the properties of the constituent protein species (hydrophobicity, tendency to form aggregates, copy number) intended for resolution. This review explains the standard sample buffer constituents and illustrates a series of protocols for processing diverse samples for two-dimensional gel electrophoresis, including hydrophobic membrane proteins. Current methods for concentrating lower abundance proteins, by removal of high abundance proteins, are also outlined. Finally, since protein staining is becoming increasingly incorporated into the sample preparation procedure, we describe the principles and applications of current (and future) pre-electrophoretic labelling methods.

Transport and metabolism of [214-C]abscisic acid in maize root

The tips of intact maize (cv. LG 11) roots, maintained vertically, were pretreated with a droplet of buffer solution or a bead of anion exchange resin, both containing [214-C]abscisic acid (ABA). A significant basipetal ABA movement was observed and two metabolites of ABA (possibly phaseic acid and dihydrophaseic acid) were found. ABA pretreatment enhanced the gravireaction of 10 mm apical root segments kept both in the dark and in the light. The possibility that ABA could be one of the endogenous growth inhibitors produced or released by the cap cells is discussed.

Cadmium distribution in soils covering Jurassic oolitic limestone with high Cd contents in the Swiss Jura

Oolitic carbonates belonging to the Hauptrogenstein Formation of Bajocian (Middle Jurassic) age have been shown to be anomalously enriched in cadmium (Cd) throughout the Jura Mountains. Soils associated with this type of rock substratum may be naturally polluted with regards to Cd. At Schleifenberg (Canton Basel Land, Switzerland) the Hauptrogenstein Formation is almost entirely exposed along a trail on its SW flank. Cadmium concentrations were systematically measured throughout this formation and Cd enrichments in rocks are shown to occur to a maximum content of 4.9 mg kg(-1). We investigated associated soils, which cover the entire outcrop, and show that they have been formed through the weathering of the underlying bedrock and through the uptake of colluvial limestone fragments from the same and older formations. Cadmium contents in the soils reach a maximum value of 2.0 mg kg(-1), thereby exceeding the official Swiss indicative guideline value for soils fixed at 0.8 mg.kg(-1). Mineralogical analyses on the soils and associated bedrock suggest that no allochthonous component related to aeolian transport is present. Sequential extractions applied to selected soil samples show that about half of the Cd resides in the carbonate fraction coming from the fractured parent-rock, while the Cd released from the weathered carbonates is associated either with organic matter (over 10%) or with Fe and Mn-oxihydroxides (approximately 30%). No exchangeable Cd phase was found and this, together with the buffer capacity of this calcareous soil, suggests that the amount of mobile Cd is quite negligible in this soil, which also greatly reduces the amount of bioavailable

L'exploitation des eaux souterraines en Syrie centrale : construction de diagnostics et politiques d'intervention

Syrian dry areas have been for several millennia a place of interaction between human populations and the environment. If environmental constraints and heterogeneity condition the human occupation and exploitation of resources, socio-political, economic and historical elements play a fundamental role. Since the late 1980s, Syrian dry areas are viewed as suffering a serious water crisis, due to groundwater overdraft. The Syrian administration and international development agencies believe that groundwater overexploitation is also leading to a decline of agricultural activities and to poverty increase. Action is thus required to address these problems.However, the overexploitation diagnosis needs to be reviewed. The overexploitation discourse appears in the context of Syria's opening to international organizations and to the market economy. It echoes the international discourse of "global water crisis". The diagnosis is based on national indicators recycling old Soviet data that has not been updated. In the post-Soviet era, the Syrian national water policy seems to abandon large surface water irrigation projects in favor of a strategy of water use rationalization and groundwater conservation in crisis regions, especially in the district of Salamieh.This groundwater conservation policy has a number of inconsistencies. It is justified for the administration and also probably for international donors, since it responds to an indisputable environmental emergency. However, efforts to conserve water are anecdotal or even counterproductive. The water conservation policy appears a posteriori as an extension of the national policy of food self-sufficiency. The dominant interpretation of overexploitation, and more generally of the water crisis, prevents any controversary approach of the status of resources and of the agricultural system in general and thus destroys any attempt to discuss alternatives with respect to groundwater management, allocation, and their inclusion in development programs.A revisited diagnosis of the situation needs to take into account spatial and temporal dimensions of the groundwater exploitation and to analyze the co-evolution of hydrogeological and agricultural systems. It should highlight the adjustments adopted to cope with environmental and economic variability, changes of water availability and regulatory measures enforcements. These elements play an important role for water availability and for the spatial, temporal, sectoral allocation of water resource. The groundwater exploitation in the last century has obviously had an impact on the environment, but the changes are not necessarily catastrophic.The current groundwater use in central Syria increases the uncertainty by reducing the ability of aquifers to buffer climatic changes. However, the climatic factor is not the only source of uncertainty. The high volatility of commodity prices, fuel, land and water, depending on the market but also on the will (and capacity) of the Syrian State to preserve social peace is a strong source of uncertainty. The research should consider the whole range of possibilities and propose alternatives that take into consideration the risks they imply for the water users, the political will to support or not the local access to water - thus involving a redefinition of the economic and social objectives - and finally the ability of international organizations to reconsider pre-established diagnoses.

Evaluation of the direct agglutination test and the rK39 dipstick test for the sero-diagnosis of visceral leishmaniasis

The direct agglutination test (DAT) based on a freeze-dried antigen and the rK39 dipstick test were evaluated for the sero-diagnosis of visceral leishmaniasis (VL). The sensitivity and specificity of both tests were determined using sera from confirmed VL patients (n = 21), healthy controls (n = 19) and from patients with other confirmed infectious diseases (n = 42). The DAT had a sensitivity and a specificity of 100%. The rK39 had a sensitivity of 85.7% and a specificity of 82%. Both tests were also used to screen blood samples of confirmed VL patients (n = 15) and serum samples of VL suspects (n = 61). The DAT found all blood samples of confirmed VL patients positive and tested 98.4% of the serum samples of the VL suspects positive. In contrast, rK39 detected in 9/15 blood samples (60%) antibodies against Leishmania chagasi and found 85.3% of the serum samples of the suspected patients positive. Although the rK39 dipstick is more rapid and user friendlier than the DAT, the latter has a superior sensitivity and specificity. Furthermore, the reagents used for DAT do not require cold storage, whereas the buffer of the rK39 must be stored at 4ºC. Therefore, the DAT is the most suitable test for the sero-diagnosis of VL under field conditions.

Effects of tetrodotoxin and ion replacements on the short-circuit current induced by Escherichiacoli heat stable enterotoxin across small intestine of the gerbil (Gerbillus cheesmani)

The effects of mucosally added Escherichia coli heat stable enterotoxin (STa 30 ng ml-1) on the basal short-circuit current (Isc in µA cm-2) across stripped and unstripped sheets of jejuna and ilea taken from fed, starved (4 days, water ad lib) and undernourished (50% control food intake for 21 days) gerbil (Gerbillus cheesmani) were investigated. The effect of neurotoxin tetrodotoxin (TTX 10 µM) and the effects of replacing chloride by gluconate or the effects of removing bicarbonate from bathing buffers on the maximum increase in Isc induced by STa were also investigated. The maximum increase in Isc which resulted from the addition of STa were significantly higher in jejuna and ilea taken from starved and undernourished gerbils when compared with the fed control both using stripped and unstripped sheets. In the two regions of the small intestine taken from fed and starved animals TTX reduced the maximum increase in Isc induced by STa across unstripped sheets only. Moreover in jejuna and ilea taken from undernourished gerbils TTX reduced significantly the maximum increase in Isc induced by STa across stripped and unstripped sheets. Replacing chloride by gluconate decreased the maximum increase in Isc induced by STa across jejuna and ilea taken from undernourished gerbils only. Removing bicarbonates from bathing buffer decreased the maximum increase in Isc across the jejuna and ilea taken from starved and undernourished gerbils.

Light receptor action is critical for maintaining plant biomass at warm ambient temperatures.

The ability to withstand environmental temperature variation is essential for plant survival. Former studies in Arabidopsis revealed that light signalling pathways had a potentially unique role in shielding plant growth and development from seasonal and daily fluctuations in temperature. In this paper we describe the molecular circuitry through which the light receptors cry1 and phyB buffer the impact of warm ambient temperatures. We show that the light signalling component HFR1 acts to minimise the potentially devastating effects of elevated temperature on plant physiology. Light is known to stabilise levels of HFR1 protein by suppressing proteasome-mediated destruction of HFR1. We demonstrate that light-dependent accumulation and activity of HFR1 are highly temperature dependent. The increased potency of HFR1 at warmer temperatures provides an important restraint on PIF4 that drives elongation growth. We show that warm ambient temperatures promote the accumulation of phosphorylated PIF4. However, repression of PIF4 activity by phyB and cry1 (via HFR1) is critical for controlling growth and maintaining physiology as temperatures rise. Loss of this light-mediated restraint has severe consequences for adult plants which have greatly reduced biomass.

N-Terminal Modifications Improve the Receptor Affinity and Pharmacokinetics of Radiolabeled Peptidic Gastrin-Releasing Peptide Receptor Antagonists: Examples of 68Ga- and 64Cu-Labeled Peptides for PET Imaging.

Gastrin-releasing peptide receptors (GRPrs) are overexpressed on a variety of human cancers, providing the opportunity for peptide receptor targeting via radiolabeled bombesin-based peptides. As part of our ongoing investigations into the development of improved GRPr antagonists, this study aimed at verifying whether and how N-terminal modulations improve the affinity and pharmacokinetics of radiolabeled GRPr antagonists. METHODS: The potent GRPr antagonist MJ9, Pip-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 (Pip, 4-amino-1-carboxymethyl-piperidine), was conjugated to 1,4,7-triazacyclononane, 1-glutaric acid-4,7 acetic acid (NODAGA), and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and radiolabeled with (68)Ga and (64)Cu. The GRPr affinity of the corresponding metalloconjugates was determined using (125)I-Tyr(4)-BN as a radioligand. The labeling efficiency of (68)Ga(3+) was compared between NODAGA-MJ9 and NOTA-MJ9 in acetate buffer, at room temperature and at 95°C. The (68)Ga and (64)Cu conjugates were further evaluated in vivo in PC3 tumor xenografts by biodistribution and PET imaging studies. RESULTS: The half maximum inhibitory concentrations of all the metalloconjugates are in the high picomolar-low nanomolar range, and these are the most affine-radiolabeled GRPr antagonists we have studied so far in our laboratory. NODAGA-MJ9 incorporates (68)Ga(3+) nearly quantitatively (>98%) at room temperature within 10 min and at much lower peptide concentrations (1.4 × 10(-6) M) than NOTA-MJ9, for which the labeling yield was approximately 45% under the same conditions and increased to 75% at 95°C for 5 min. Biodistribution studies showed high and specific tumor uptake, with a maximum of 23.3 ± 2.0 percentage injected activity per gram of tissue (%IA/g) for (68)Ga-NOTA-MJ9 and 16.7 ± 2.0 %IA/g for (68)Ga-NODAGA-MJ9 at 1 h after injection. The acquisition of PET images with the (64)Cu-MJ9 conjugates at later time points clearly showed the efficient clearance of the accumulated activity from the background already at 4 h after injection, whereas tumor uptake still remained high. The high pancreas uptake for all radiotracers at 1 h after injection was rapidly washed out, resulting in an increased tumor-to-pancreas ratio at later time points. CONCLUSION: We have developed 2 GRPr antagonistic radioligands, which are improved in terms of binding affinity and overall biodistribution profile. Their promising in vivo pharmacokinetic performance may contribute to the improvement of the diagnostic imaging of tumors overexpressing GRPr.

Single-stranded oligonucleotide-mediated in vivo gene repair in the rd1 retina.

PURPOSE: The aim of this study was to test whether oligonucleotide-targeted gene repair can correct the point mutation in genomic DNA of PDE6b(rd1) (rd1) mouse retinas in vivo. METHODS: Oligonucleotides (ODNs) of 25 nucleotide length and complementary to genomic sequence subsuming the rd1 point mutation in the gene encoding the beta-subunit of rod photoreceptor cGMP-phosphodiesterase (beta-PDE), were synthesized with a wild type nucleotide base at the rd1 point mutation position. Control ODNs contained the same nucleotide bases as the wild type ODNs but with varying degrees of sequence mismatch. We previously developed a repeatable and relatively non-invasive technique to enhance ODN delivery to photoreceptor nuclei using transpalpebral iontophoresis prior to intravitreal ODN injection. Three such treatments were performed on C3H/henJ (rd1) mouse pups before postnatal day (PN) 9. Treatment outcomes were evaluated at PN28 or PN33, when retinal degeneration was nearly complete in the untreated rd1 mice. The effect of treatment on photoreceptor survival was evaluated by counting the number of nuclei of photoreceptor cells and by assessing rhodopsin immunohistochemistry on flat-mount retinas and sections. Gene repair in the retina was quantified by allele-specific real time PCR and by detection of beta-PDE-immunoreactive photoreceptors. Confirmatory experiments were conducted using independent rd1 colonies in separate laboratories. These experiments had an additional negative control ODN that contained the rd1 mutant nucleotide base at the rd1 point mutation site such that the sole difference between treatment with wild type and control ODN was the single base at the rd1 point mutation site. RESULTS: Iontophoresis enhanced the penetration of intravitreally injected ODNs in all retinal layers. Using this delivery technique, significant survival of photoreceptors was observed in retinas from eyes treated with wild type ODNs but not control ODNs as demonstrated by cell counting and rhodopsin immunoreactivity at PN28. Beta-PDE immunoreactivity was present in retinas from eyes treated with wild type ODN but not from those treated with control ODNs. Gene correction demonstrated by allele-specific real time PCR and by counts of beta-PDE-immunoreactive cells was estimated at 0.2%. Independent confirmatory experiments showed that retinas from eyes treated with wild type ODN contained many more rhodopsin immunoreactive cells compared to retinas treated with control (rd1 sequence) ODN, even when harvested at PN33. CONCLUSIONS: Short ODNs can be delivered with repeatable efficiency to mouse photoreceptor cells in vivo using a combination of intravitreal injection and iontophoresis. Delivery of therapeutic ODNs to rd1 mouse eyes resulted in genomic DNA conversion from mutant to wild type sequence, low but observable beta-PDE immunoreactivity, and preservation of rhodopsin immunopositive cells in the outer nuclear layer, suggesting that ODN-directed gene repair occurred and preserved rod photoreceptor cells. Effects were not seen in eyes treated with buffer or with ODNs having the rd1 mutant sequence, a definitive control for this therapeutic approach. Importantly, critical experiments were confirmed in two laboratories by several different researchers using independent mouse colonies and ODN preparations from separate sources. These findings suggest that targeted gene repair can be achieved in the retina following enhanced ODN delivery.

Thyroid hormone enhances transected axonal regeneration and muscle reinnervation following rat sciatic nerve injury.

Improvement of nerve regeneration and functional recovery following nerve injury is a challenging problem in clinical research. We have already shown that following rat sciatic nerve transection, the local administration of triiodothyronine (T3) significantly increased the number and the myelination of regenerated axons. Functional recovery is a sum of the number of regenerated axons and reinnervation of denervated peripheral targets. In the present study, we investigated whether the increased number of regenerated axons by T3-treatment is linked to improved reinnervation of hind limb muscles. After transection of rat sciatic nerves, silicone or biodegradable nerve guides were implanted and filled with either T3 or phosphate buffer solution (PBS). Neuromuscular junctions (NMJs) were analyzed on gastrocnemius and plantar muscle sections stained with rhodamine alpha-bungarotoxin and neurofilament antibody. Four weeks after surgery, most end-plates (EPs) of operated limbs were still denervated and no effect of T3 on muscle reinnervation was detected at this stage of nerve repair. In contrast, after 14 weeks of nerve regeneration, T3 clearly enhanced the reinnervation of gastrocnemius and plantar EPs, demonstrated by significantly higher recovery of size and shape complexity of reinnervated EPs and also by increased acetylcholine receptor (AChRs) density on post synaptic membranes compared to PBS-treated EPs. The stimulating effect of T3 on EP reinnervation is confirmed by a higher index of compound muscle action potentials recorded in gastrocnemius muscles. In conclusion, our results provide for the first time strong evidence that T3 enhances the restoration of NMJ structure and improves synaptic transmission.

Quantification of nicotine, cotinine, trans-3'-hydroxycotinine and varenicline in human plasma by a sensitive and specific UPLC-tandem mass-spectrometry procedure for a clinical study on smoking cessation.

A sensitive and specific ultra performance liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of nicotine, its metabolites cotinine and trans-3'-hydroxycotinine and varenicline in human plasma was developed and validated. Sample preparation was realized by solid phase extraction of the target compounds and of the internal standards (nicotine-d4, cotinine-d3, trans-3'-hydroxycotinine-d3 and CP-533,633, a structural analog of varenicline) from 0.5mL of plasma, using a mixed-mode cation exchange support. Chromatographic separations were performed on a hydrophilic interaction liquid chromatography column (HILIC BEH 2.1×100mm, 1.7μm). A gradient program was used, with a 10mM ammonium formate buffer pH 3/acetonitrile mobile phase at a flow of 0.4mL/min. The compounds were detected on a triple quadrupole mass spectrometer, operated with an electrospray interface in positive ionization mode and quantification was performed using multiple reaction monitoring. Matrix effects were quantitatively evaluated with success, with coefficients of variation inferior to 8%. The procedure was fully validated according to Food and Drug Administration guidelines and to Société Française des Sciences et Techniques Pharmaceutiques. The concentration range was 2-500ng/mL for nicotine, 1-1000ng/mL for cotinine, 2-1000ng/mL for trans-3'-hydroxycotinine and 1-500ng/mL for varenicline, according to levels usually measured in plasma. Trueness (86.2-113.6%), repeatability (1.9-12.3%) and intermediate precision (4.4-15.9%) were found to be satisfactory, as well as stability in plasma. The procedure was successfully used to quantify nicotine, its metabolites and varenicline in more than 400 plasma samples from participants in a clinical study on smoking cessation.

Speciation of antimony (III) and antimony (V) using hydride generation for meglumine antimoniate pharmaceutical formulationsquality control

The pentavalent antimonies, mainly the meglumine antimoniate, are recommends as first-choice medicines for leishmaniasis therapy. In this work we described the development of formulations of meglumine antimoniate injectable medication, as well as the analytical methodology used in the selective determination of Sb(III) and Sb(Total) by hydride generation - inductively coupled plasma atomic emission spectrometry (HG-ICP-AES) and ICP-AES, respectively. On that purpose the analytical methodology was developed focusing on the HG-ICP-AES technique. The formulations using propylene glycol/water as vehicles in a 20:80 proportion were more appropriate for subsequent use in industrial scale. These formulations also showed a lower variation on Sb(III) percentage, no need of buffer solution to stabilize the formulation and no influence of the autoclaving in the quality of the product. The results of the development of the analytical methodology point out the proposed method as an efficient alternative for the determination of Sb(III) in the presence of large quantities of Sb(V) in injectable solutions of meglumine antimoniate, in a selective, linear, accurate and precise manner. In addition, the method showed a low limit of quantification, less interference of the matrix, and more resilience than batch techniques proposed in the Brazilian Pharmacopeia.

Bovine tuberculosis in Doñana Biosphere Reserve: the role of wild ungulates as disease reservoirs in the last Iberian lynx strongholds.

Doñana National Park (DNP) in southern Spain is a UNESCO Biosphere Reserve where commercial hunting and wildlife artificial feeding do not take place and traditional cattle husbandry still exists. Herein, we hypothesized that Mycobacterium bovis infection prevalence in wild ungulates will depend on host ecology and that variation in prevalence will reflect variation in the interaction between hosts and environmental risk factors. Cattle bTB reactor rates increased in DNP despite compulsory testing and culling of infected animals. In this study, 124 European wild boar, 95 red deer, and 97 fallow deer were sampled from April 2006 to April 2007 and analyzed for M. bovis infection. Modelling and GIS were used to identify risk factors and intra and inter-species relationships. Infection with M. bovis was confirmed in 65 (52.4%) wild boar, 26 (27.4%) red deer and 18 (18.5%) fallow deer. In the absence of cattle, wild boar M. bovis prevalence reached 92.3% in the northern third of DNP. Wild boar showed more than twice prevalence than that in deer (p<0.001). Modelling revealed that M. bovis prevalence decreased from North to South in wild boar (p<0.001) and red deer (p<0.01), whereas no spatial pattern was evidenced for fallow deer. Infection risk in wild boar was dependent on wild boar M. bovis prevalence in the buffer area containing interacting individuals (p<0.01). The prevalence recorded in this study is among the highest reported in wildlife. Remarkably, this high prevalence occurs in the absence of wildlife artificial feeding, suggesting that a feeding ban alone would have a limited effect on wildlife M. bovis prevalence. In DNP, M. bovis transmission may occur predominantly at the intra-species level due to ecological, behavioural and epidemiological factors. The results of this study allow inferring conclusions on epidemiological bTB risk factors in Mediterranean habitats that are not managed for hunting purposes. Our results support the need to consider wildlife species for the control of bTB in cattle and strongly suggest that bTB may affect animal welfare and conservation.

Guidelines for specialized nutritional and metabolic support in the critically-ill patient. Update. Consensus SEMICYUC-SENPE: Acute renal failure

Nutritional support in acute renal failure must take into account the patient's catabolism and the treatment of the renal failure. Hypermetabolic failure is common in these patients, requiring continuous renal replacement therapy or daily hemodialysis. In patients with normal catabolism (urea nitrogen below 10 g/day) and preserved diuresis, conservative treatment can be attempted. In these patients, relatively hypoproteic nutritional support is essential, using proteins with high biological value and limiting fluid and electrolyte intake according to the patient's individual requirements. Micronutrient intake should be adjusted, the only buffering agent used being bicarbonate. Limitations on fluid, electrolyte and nitrogen intake no longer apply when extrarenal clearance techniques are used but intake of these substances should be modified according to the type of clearance. Depending on their hemofiltration flow, continuous renal replacement systems require high daily nitrogen intake, which can sometimes reach 2.5 g protein/kg. The amount of volume replacement can induce energy overload and therefore the use of glucose-free replacement fluids and glucose-free dialysis or a glucose concentration of 1 g/L, with bicarbonate as a buffer, is recommended. Monitoring of electrolyte levels (especially those of phosphorus, potassium and magnesium) and of micronutrients is essential and administration of these substances should be individually-tailored.

Synthesis and transport of creatine in the CNS: importance for cerebral functions.

J. Neurochem. (2010) 10.1111/j.1471-4159.2010.06935.x Abstract Apart of its well known function of 'energetic buffer' through the creatine/phosphocreatine/creatine kinase system allowing the regeneration of ATP, creatine has been recently suggested as a potential neuromodulator of even true neurotransmitter. Moreover, the recent discovery of primary creatine deficiency syndromes, due to deficiencies in l-arginine : glycine amidinotransferase or guanidinoacetate methyltransferase (the two enzymes allowing creatine synthesis) or in the creatine transporter, has shed new light on creatine synthesis, metabolism and transport, in particular in CNS which appears as the main tissue affected by these creatine deficiencies. Recent data suggest that creatine can cross blood-brain barrier but only with a poor efficiency, and that the brain must ensure parts of its needs in creatine by its own endogenous synthesis. Finally, the recent years have demonstrated the interest to use creatine as a neuroprotective agent in a growing number of neurodegenerative diseases, including Parkinson's and Huntington's diseases. This article aims at reviewing the latest data on creatine metabolism and transport in the brain, in relation to creatine deficiencies and to the potential use of creatine as neuroprotective molecule. Emphasis is also given to the importance of creatine for cerebral function.