Birth and adaptive evolution of a hominoid gene that supports high neurotransmitter flux.

The enzyme glutamate dehydrogenase (GDH) is important for recycling the chief excitatory neurotransmitter, glutamate, during neurotransmission. Human GDH exists in housekeeping and brain-specific isotypes encoded by the genes GLUD1 and GLUD2, respectively. Here we show that GLUD2 originated by retroposition from GLUD1 in the hominoid ancestor less than 23 million years ago. The amino acid changes responsible for the unique brain-specific properties of the enzyme derived from GLUD2 occurred during a period of positive selection after the duplication event.

Multiple origins of invasive and "native" water frogs (Pelophylax spp.) in Switzerland

The marsh frog (Pelophylax ridibundus) has been introduced in many places of Central and Western Europe due to commercial trades with Eastern Europe, and is rapidly replacing the native pool frog (P. lessonae). A large number of Pelophylax species are distributed in Eastern Europe and the strong phenotypic similarity between these species is rendering their identification hazardous. Consequently, alien populations of Pelophylax might not strictly be composed of P. ridibundus as previously suspected. In the present study, we analyzed the cytochrome b and NADH dehydrogenase subunit 3 genes of introduced and native Pelophylax from Switzerland (299 individuals), in order to properly identify the source populations of the invaders and the genetic status of the native species. Our study highlighted the occurrence of several genetic lineages of invasive frogs in western Switzerland. Unexpectedly, we also showed that several populations of the native pool frog (P. lessonae) cluster with the Italian pool frog P. bergeri from central Italy (considered by some authors as a subspecies of P. lessonae) Hence, these populations are probably also the result of introductions, meaning that the number of native P. lessonae populations is less important than expected in Switzerland. These findings have important implications concerning the conservation of the endemic pool frog populations, as the presence of multiple alien species could strongly affect their long-term subsistence.

Critical role for the lactate transporter, monocarboxylate transporter 1 (mct1), in the regeneration of peripheral nerves

Axons, and particularly regenerating axons, have high metabolic needs in order to maintain critical functions such as axon transport and membrane depolarization. Though some of the required energy likely comes form extracellular glucose and ATP generated in the soma, we and others hypothesize that some of the energy may be supplied by lactate. Unlike glucose that requires glycolytic enzymes to produce pyruvate, lactate can be converted directly to pyruvate by lactate dehydrogenase and transported into mitochondria for oxidative metabolism. In order to be transported into or out of cells, lactate requires specific monocarboxylate transporters (MCTs), the most abundant of which is MCT1. If MCT1 and lactate are critical for nerve function and regeneration, we hypothesize that MCT1 heterozygote null mice, which appear phenotypically normal despite having approximately 40% MCT1 as compared to wildtype littermate mice, would have reduced capacity for repair following nerve injury. To investigate this, adult MCT1 heterozygote null mice or wild-type mice underwent unilateral sciatic nerve crush in the proximal thigh. We found that regeneration of the sciatic nerve, as measured by recovery of compound muscle action potentials (CMAP) in the lateral plantar muscles following proximal sciatic nerve stimulation, was delayed from a median of 21 days in wildtype mice to 38.5 days in MCT1 heterozygote mice. In fact, half of the MCT1 heterozygote null mice had no recovery of CMAP by the endpoint of the study at 42 days, while all of the wild-type mice had recovered. In addition, the maximal amplitude of CMAP recovery in MCT1 heterozygote mull mice was reduced from a mean of 3 mV to 0.5 mV. As would be expected, the denervated gastrocnemius muscle of MCT1 heterozygote null mice remained atrophic at 42 days compared to wild-type mice. Our experiments show that lactate supplied through MCT1 is necessary for nerve regeneration. Experiments are underway to determine whether loss of MCT1 prevents nerve regrowth directly due to reduced energy supply to axons or indirectly by dysfunctional Schwann cells normally dependent on lactate supply through MCT1.

Selective neurodegeneration induced in rotation-mediated aggregate cell cultures by a transient switch to stationary culture conditions: a potential model to study ischemia-related pathogenic mechanisms.

Aggregating brain cell cultures at an advanced maturational stage (20-21 days in vitro) were subjected for 1-3 h to anaerobic (hypoxic) and/or stationary (ischemic) conditions. After restoration of the normal culture conditions, cell loss was estimated by measuring the release of lactate dehydrogenase as well as the irreversible decrease of cell type-specific enzyme activities, total protein and DNA content. Ischemia for 2 h induced significant neuronal cell death. Hypoxia combined with ischemia affected both neuronal and glial cells to different degrees (GABAergic neurons>cholinergic neurons>astrocytes). Hypoxic and ischemic conditions greatly stimulated the uptake of 2-deoxy-D-glucose, indicating increased glucose consumption. Furthermore, glucose restriction (5.5 mM instead of 25 mM) dramatically increased the susceptibility of neuronal and glial cells to hypoxic and ischemic conditions. Glucose media concentrations below 2 mM caused selective neuronal cell death in otherwise normal culture conditions. GABAergic neurons showed a particularly high sensitivity to glucose restriction, hypoxia, and ischemia. The pattern of ischemia-induced changes in vitro showed many similarities to in vivo findings, suggesting that aggregating brain cell cultures provide a useful in vitro model to study pathogenic mechanisms related to brain ischemia.

Target Molecular Size and Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Analysis of the ATP-and Pyrophosphate-Dependent Proton Pumps from Maize Root Tonoplast.

Tonoplast-enriched membranes were prepared from maize (Zea mays L. cv LG 11) primary roots, using sucrose nonlinear gradients. The functional molecular size of the tonoplast ATP-and PPi-dependent proton pumps were analyzed by radiation inactivation. Glucose-6-phosphate dehydrogenase (G6PDH) was added as an internal standard. Frozen samples (-196 degrees C) of the membranes were irradiated with (60)Co for different periods of time. After thawing the samples, the activities of G6PDH, ATPase, and PPase were tested. By applying target theory, the functional sizes of the ATPase and PPase in situ were found to be around 540 and 160 kilodaltons, respectively. The two activities were solubilized and separated by gel filtration chromatography. The different polypeptides copurifying with the two pumps were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two bands (around 59 and 65 kilodaltons) were associated with the ATPase activity, whereas a double band (around 40 kilodaltons) was recovered with the PPase activity.

The relationship between monocarboxylate transporters 1 and 4 expression in skeletal muscle and endurance performance in athletes

The purpose of this study was to examine the relationship between skeletal muscle monocarboxylate transporters 1 and 4 (MCT1 and MCT4) expression, skeletal muscle oxidative capacity and endurance performance in trained cyclists. Ten well-trained cyclists (mean +/- SD; age 24.4 +/- 2.8 years, body mass 73.2 +/- 8.3 kg, VO(2max) 58 +/- 7 ml kg(-1) min(-1)) completed three endurance performance tasks [incremental exercise test to exhaustion, 2 and 10 min time trial (TT)]. In addition, a muscle biopsy sample from the vastus lateralis muscle was analysed for MCT1 and MCT4 expression levels together with the activity of citrate synthase (CS) and 3-hydroxyacyl-CoA dehydrogenase (HAD). There was a tendency for VO(2max) and peak power output obtained in the incremental exercise test to be correlated with MCT1 (r = -0.71 to -0.74; P < 0.06), but not MCT4. The average power output (P (average)) in the 2 min TT was significantly correlated with MCT4 (r = -0.74; P < 0.05) and HAD (r = -0.92; P < 0.01). The P (average) in the 10 min TT was only correlated with CS activity (r = 0.68; P < 0.05). These results indicate the relationship between MCT1 and MCT4 as well as cycle TT performance may be influenced by the length and intensity of the task.

Quantification of typical antipsychotics in human plasma by ultra-high performance liquid chromatography tandem mass spectrometry for therapeutic drug monitoring.

A selective and sensitive method was developed for the simultaneous quantification of seven typical antipsychotic drugs (cis-chlorprothixene, flupentixol, haloperidol, levomepromazine, pipamperone, promazine and zuclopenthixol) in human plasma. Ultra-high performance liquid chromatography (UHPLC) was used for complete separation of the compounds in less than 4.5min on an Acquity UPLC BEH C18 column (2.1mm×50mm; 1.7μm), with a gradient elution of ammonium formate buffer pH 4.0 and acetonitrile at a flow rate of 400μl/min. Detection was performed on a tandem quadrupole mass spectrometer (MS/MS) equipped with an electrospray ionization interface. A simple protein precipitation procedure with acetonitrile was used for sample preparation. Thanks to the use of stable isotope-labeled internal standards for all analytes, internal standard-normalized matrix effects were in the range of 92-108%. The method was fully validated to cover large concentration ranges of 0.2-90ng/ml for haloperidol, 0.5-90ng/ml for flupentixol, 1-450ng/ml for levomepromazine, promazine and zuclopenthixol and 2-900ng/ml for cis-chlorprothixene and pipamperone. Trueness (89.1-114.8%), repeatability (1.8-9.9%), intermediate precision (1.9-16.3%) and accuracy profiles (<30%) were in accordance with the latest international recommendations. The method was successfully used in our laboratory for routine quantification of more than 500 patient plasma samples for therapeutic drug monitoring. To the best of our knowledge, this is the first UHPLC-MS/MS method for the quantification of the studied drugs with a sample preparation based on protein precipitation.

Repercussion of a deficiency in mitochondrial ß-oxidation on the carbon flux of short-chain fatty acids to the peroxisomal ß-oxidation cycle in Aspergillus nidulans.

The fungus Aspergillus nidulans contains both a mitochondrial and peroxisomal ß-oxidation pathway. This work was aimed at studying the influence of mutations in the foxA gene, encoding a peroxisomal multifunctional protein, or in the scdA/echA genes, encoding a mitochondrial short-chain dehydrogenase and an enoyl-CoA hydratase, respectively, on the carbon flux to the peroxisomal ß-oxidation pathway. A. nidulans transformed with a peroxisomal polyhydroxyalkanoate (PHA) synthase produced PHA from the polymerization of 3-hydroxyacyl-CoA intermediates derived from the peroxisomal ß-oxidation of external fatty acids. PHA produced from erucic acid or heptadecanoic acid contained a broad spectrum of monomers, ranging from 5 to 14 carbons, revealing that the peroxisomal ß-oxidation cycle can handle both long and short-chain intermediates. While the ∆foxA mutant grown on erucic acid or oleic acid synthesized 10-fold less PHA compared to wild type, the same mutant grown on octanoic acid or heptanoic acid produced 3- to 6-fold more PHA. Thus, while FoxA has an important contribution to the degradation of long-chain fatty acids, the flux of short-chain fatty acids to peroxisomal ß-oxidation is actually enhanced in its absence. While no change in PHA was observed in the ∆scdA∆echA mutant grown on erucic acid or oleic acid compared to wild type, there was a 2- to 4-fold increased synthesis of PHA in ∆scdA∆echA cells grown in octanoic acid or heptanoic acid. These results reveal that a compensatory mechanism exists in A. nidulans that increases the flux of short-chain fatty acids towards the peroxisomal ß-oxidation cycle when the mitochondrial ß-oxidation pathway is defective.

A threefold dose intensity treatment with ifosfamide, carboplatin, and etoposide for patients with small cell lung cancer: a randomized trial.

BACKGROUND: The dose intensity of chemotherapy can be increased to the highest possible level by early administration of multiple and sequential high-dose cycles supported by transfusion with peripheral blood progenitor cells (PBPCs). A randomized trial was performed to test the impact of such dose intensification on the long-term survival of patients with small cell lung cancer (SCLC). METHODS: Patients who had limited or extensive SCLC with no more than two metastatic sites were randomly assigned to high-dose (High, n = 69) or standard-dose (Std, n = 71) chemotherapy with ifosfamide, carboplatin, and etoposide (ICE). High-ICE cycles were supported by transfusion with PBPCs that were collected after two cycles of treatment with epidoxorubicin at 150 mg/m(2), paclitaxel at 175 mg/m(2), and filgrastim. The primary outcome was 3-year survival. Comparisons between response rates and toxic effects within subgroups (limited or extensive disease, liver metastases or no liver metastases, Eastern Cooperative Oncology Group performance status of 0 or 1, normal or abnormal lactate dehydrogenase levels) were also performed. RESULTS: Median relative dose intensity in the High-ICE arm was 293% (range = 174%-392%) of that in the Std-ICE arm. The 3-year survival rates were 18% (95% confidence interval [CI] = 10% to 29%) and 19% (95% CI = 11% to 30%) in the High-ICE and Std-ICE arms, respectively. No differences were observed between the High-ICE and Std-ICE arms in overall response (n = 54 [78%, 95% CI = 67% to 87%] and n = 48 [68%, 95% CI = 55% to 78%], respectively) or complete response (n = 27 [39%, 95% CI = 28% to 52%] and n = 24 [34%, 95% CI = 23% to 46%], respectively). Subgroup analyses showed no benefit for any outcome from High-ICE treatment. Hematologic toxicity was substantial in the Std-ICE arm (grade > or = 3 neutropenia, n = 49 [70%]; anemia, n = 17 [25%]; thrombopenia, n = 17 [25%]), and three patients (4%) died from toxicity. High-ICE treatment was predictably associated with severe myelosuppression, and five patients (8%) died from toxicity. CONCLUSIONS: The long-term outcome of SCLC was not improved by raising the dose intensity of ICE chemotherapy by threefold.

Increasing the carbon flux toward synthesis of short-chain-length--medium-chain-length polyhydroxyalkanoate in the peroxisome of Saccharomyces cerevisiae through modification of the beta-oxidation cycle.

Short-chain-length-medium-chain-length polyhydroxyalkanoates were synthesized in Saccharomyces cerevisiae from intermediates of the beta-oxidation cycle by expressing the polyhydroxyalkanoate synthases from Aeromonas caviae and Ralstonia eutropha in the peroxisomes. The quantity of polymer produced was increased by using a mutant of the beta-oxidation-associated multifunctional enzyme with low dehydrogenase activity toward R-3-hydroxybutyryl coenzyme A.

Proteomic analysis of peach fruit mesocarp softening and chilling injury using difference gel electrophoresis (DIGE)

Background: Peach fruit undergoes a rapid softening process that involves a number of metabolic changes. Storing fruit at low temperatures has been widely used to extend its postharvest life. However, this leads to undesired changes, such as mealiness and browning, which affect the quality of the fruit. In this study, a 2-D DIGE approach was designed to screen for differentially accumulated proteins in peach fruit during normal softening as well as under conditions that led to fruit chilling injury. Results:The analysis allowed us to identify 43 spots -representing about 18% of the total number analyzed- that show statistically significant changes. Thirty-nine of the proteins could be identified by mass spectrometry. Some of the proteins that changed during postharvest had been related to peach fruit ripening and cold stress in the past. However, we identified other proteins that had not been linked to these processes. A graphical display of the relationship between the differentially accumulated proteins was obtained using pairwise average-linkage cluster analysis and principal component analysis. Proteins such as endopolygalacturonase, catalase, NADP-dependent isocitrate dehydrogenase, pectin methylesterase and dehydrins were found to be very important for distinguishing between healthy and chill injured fruit. A categorization of the differentially accumulated proteins was performed using Gene Ontology annotation. The results showed that the 'response to stress', 'cellular homeostasis', 'metabolism of carbohydrates' and 'amino acid metabolism' biological processes were affected the most during the postharvest. Conclusions: Using a comparative proteomic approach with 2-D DIGE allowed us to identify proteins that showed stage-specific changes in their accumulation pattern. Several proteins that are related to response to stress, cellular homeostasis, cellular component organization and carbohydrate metabolism were detected as being differentially accumulated. Finally, a significant proportion of the proteins identified had not been associated with softening, cold storage or chilling injury-altered fruit before; thus, comparative proteomics has proven to be a valuable tool for understanding fruit softening and postharvest.

A large deletion in the adRP gene PRPF31: evidence that haploinsufficiency is the cause of disease.

PURPOSE: To report a large deletion that encompasses more than 90% of PRPF31 gene and two other neighboring genes in their entirety in an adRP pedigree that appears to show only the typical clinical features of retinitis pigmentosa. METHODS: To identify PRPF31 mutation in a dominant RP family (ADRP2) previously linked to the RP11 locus, the 14 exons of PRPF31 were screened for mutations by direct sequencing. To investigate the possibility of a large deletion, microsatellite markers near PRPF31 gene were analyzed by non-denaturing PAGE. RESULTS: Initial screening of PRPF31 gene in the ADRP2 family did not reveal an obvious mutation. A large deletion was however suspected due to lack of heterozygosity for nearly all PRPF31 intragenic single nucleotide polymorphysm (SNPs). In order to estimate the size of the deletion, SNPs and microsatellite markers spanning and flanking PRPF31 were analyzed in the entire ADRP2 family. Haplotype analysis with the above markers suggested a deletion of approximately 30 kb that included the putative promoter region of a novel gene OSCAR, the entire genomic content of genes NDUFA3, TFPT and more than 90% of PRPF31 gene. Sequence analysis of the region flanking the potential deletion showed a high presence of Alu elements implicating Alu mediated recombination as the mechanism responsible for this event. CONCLUSIONS: This mutation provides evidence that haploinsufficiency rather than aberrant function of mutated proteins is the cause of disease in these adRP patients with mutations in PRPF31 gene.

A peptide inhibitor of c-Jun N-terminal kinase for the treatment of endotoxin-induced uveitis.

PURPOSE: To evaluate the effect of XG-102 (formerly D-JNKI1), a TAT-coupled dextrogyre peptide that selectively inhibits the c-Jun N-terminal kinase, in the treatment of endotoxin-induced uveitis (EIU). METHODS: EIU was induced in Lewis rats by LPS injection. XG-102 was administered at the time of LPS challenge. The ocular biodistribution of XG-102 was evaluated using immunodetection at 24 hours after either 20 microg/kg IV (IV) or 0.2 microg/injection intravitreous (IVT) administrations in healthy or uveitic eyes. The effect of XG-102 on EIU was evaluated using clinical scoring, infiltration cell quantification, inducible nitric oxide synthase (iNOS) expression and immunohistochemistry, and cytokines and chemokines kinetics at 6, 24, and 48 hours using multiplex analysis on ocular media. Control EIU eyes received vehicle injection IV or IVT. The effect of XG-102 on c-Jun phosphorylation in EIU was evaluated by Western blot in eye tissues. RESULTS: After IVT injection, XG-102 was internalized in epithelial cells from iris/ciliary body and retina and in glial and microglial cells in both healthy and uveitic eyes. After IV injection, XG-102 was concentrated primarily in inflammatory cells of uveitic eyes. Using both routes of administration, XG-102 significantly inhibited clinical signs of EIU, intraocular cell infiltration, and iNOS expression together with reduced phosphorylation of c-Jun. The anti-inflammatory effect of XG-102 was mediated by iNOS, IFN-gamma, IL-2, and IL-13. CONCLUSIONS: This is the first evidence that interfering with the JNK pathway can reduce intraocular inflammation. Local administration of XG-102, a clinically evaluated peptide, may have potential for treating uveitis.

CO-EXPRESSION OF GCDH AND OAT1 IN NEURONS AND PROXIMAL TUBULE CELLS

Tissue-specific expression studies of Glutaryl-CoA dehydrogenase (Gcdh) in adult rats revealed expression in the whole rat brain, almost exclusively in neurons, and surprisingly high expression in the juxtamedullar cortex of the kidney. The organic anion transporter 1 (OAT1) mediates basolateral uptake of glutarate derivatives from proximal tubule cells and contributes to their renal clearance. In brain, OAT1 is expressed at the choroid plexus, in neurons of cortex and hippocampus. We hypothesized that Gcdh and Oat1 are co-expressed in the same cells in kidney and brain and analyzed their mRNA expression by in situ hybridization on cryosections of adult rat brain, kidney and liver. In brain, Gcdh and Oat1 were found co-expressed in most neurons. Only the Purkinje neurons of the cerebellum were found to be Oat1 negative. In the kidney Gcdh and Oat1 are widely co-expressed with a specific high expression in proximal tubule cells. In conclusion there seems to be a functional coupling of Gcdh and Oat1 on a renal and neuronal level. Further studies are ongoing to confirm these findings in human tissues.

Nephrotoxicity Of Polymyxin B: Experimental Study In Cells And Implications For Nursing Practice

The aim of the study was to characterize the cell damage mechanisms involved in the pathophysiology of cytotoxicity of polymyxin B in proximal tubular cells (LLC - PK1) and discuss about the nurses interventions to identify at risk patients and consider prevention or treatment of nephrotoxicity acute kidney injury. This is a quantitative experimental in vitro study, in which the cells were exposed to 375μM polymyxin B sulfate concentration. Cell viability was determined by exclusion of fluorescent dyes and morphological method with visualization of apoptotic bodies for fluorescence microscopy. Cells exposed to polymyxin B showed reduced viability, increased number of apoptotic cells and a higher concentration of the enzyme lactate dehydrogenase. The administration of polymyxin B in vitro showed the need for actions to minimize adverse effects such as nephrotoxicity.