Decreased glutamine synthetase,increased citrulline–nitric oxide cycle activities,and oxidative stress in different regions of brain in epilepsy rat model

To understand their role in epilepsy, the nitric oxide synthetase (NOS), argininosuccinate synthetase (AS), argininosuccinate lyase (AL), glutamine synthetase (GS), and arginase activities, along with the concentration of nitrate/nitrite (NOx), thiobarbituric acid reactive substances (TBARS), and total antioxidant status (TAS), were estimated in different regions of brain in rats subjected to experimental epilepsy induced by subcutaneous administration of kainic acid (KA). The short-term (acute) group animals were killed after 2 h and the long term (chronic) group animals were killed after 5 days of single injection of KA (15 mg/kg body weight). After decapitation of rats, the brain regions were separated and in their homogenates, the concentration of NOx, TBARS and TAS and the activities of NOS, AS, AL, arginase and glutamine synthetase were assayed by colorimetric methods. The results of the study demonstrated the increased activity of NOS and formation of NO in acute and chronic groups epilepsy. The activities of AS and AL were increased and indicate the effective recycling of citrulline to arginine. The activity of glutamine synthetase was decreased in acute and chronic groups of epilepsy compared to control group and indicate the modulation of its activity by NO in epilepsy. The activity of arginase was not changed in acute group; however it was decreased in chronic group and may favor increased production of NO in this condition. The concentration TBARS were increased and TAS decreased in acute and chronic groups of epilepsy and supports the oxidative stress in epilepsy.     

Beta-carotene supplementation: a good thing, a bad thing, or nothing?

Available data from several completed large-scale randomized trials indicate that beta-carotene supplementation for durations up to 12 years has no overall benefit in well-nourished populations on the incidence of cardiovascular disease or the middle-to-late stages of carcinogenesis. Several important questions, however, remain unanswered. The post-trial follow-up of completed trials, together with the results of several ongoing trials of beta-carotene supplementation, will contribute reliable information to the totality of evidence from basic research, animal studies, observational epidemiologic studies, and completed trials, thus allowing more rational clinical decisions for individual patients and policy decisions for the health of the general public.     

Could the improvements in the alcoholic fermentation of high glucose concentrations by yeast immobilization be explained by media supplementation?

Summary The maximal concentration of ethanol produced during the fermentation of 320 g/l glucose bySaccharomyces bayanus was higher when the yeast cells were immobilized either by adsorption on celite or by entrapment in k-carrageenan beads (from 10.5% with free cells up to 14.5% and 13.1% (v/v) respectively). This increase was due to medium supplementation with the compounds present in the immobilization supports.     

Metabolic fate of a high concentration of glutamine and glutamate in rat brain slices: a ¹³C NMR study

This study was performed to analyze the metabolic fate of a high concentration (5 mM) of glutamine and glutamate in rat brain slices and the participation of these amino acids in the glutamine-glutamate cycle. For this, brain slices were incubated for 60 min with [3-13C]glutamine or [3-13C]glutamate. Tissue plus medium extracts were analyzed by enzymatic and 13C NMR measurements and fluxes through pathways of glutamine and glutamate metabolism were calculated. We demonstrate that both substrates were utilized and oxidized at high rates by rat brain slices and served as precursors of neurotransmitters, tricarboxylic acid (TCA) cycle intermediates and alanine. In order to determine the participation of glutamine synthetase in the appearance of new glutamine molecules with glutamine as substrate, brain slices were incubated with [3-13C]glutamine in the presence of methionine sulfoximine, a specific inhibitor of glutamine synthetase. Our results indicate that 36.5% of the new glutamine appeared was glutamine synthetase-dependent and 63.5% was formed from endogenous substrates. Flux through glutamic acid decarboxylase was higher with glutamine than with glutamate as substrate whereas fluxes from α-ketoglutarate to glutamate and through glutamine synthetase, malic enzyme, pyruvate dehydrogenase, pyruvate carboxylase and citrate synthase were in the same range with both substrates.     

Effects of leucine and citrulline versus non-essential amino acids on muscle protein synthesis in fasted rat: a common activation pathway?

Leucine (LEU) is recognized as a major regulator of muscle protein synthesis (MPS). Citrulline (CIT) is emerging as a potent new regulator. The aim of our study was to compare MPS modulation by CIT and LEU in food-deprived rats and to determine whether their action was driven by similar mechanisms. Rats were either freely fed (F, n?=?10) or food deprived for 18?h. Food-deprived rats were randomly assigned to one of four groups and received per os, i.e., gavage, saline (S, n?=?10), L: -leucine (1.35?g/kg, LEU, n?=?10), L: -citrulline (1.80?g/kg CIT, n?=?10) or isonitrogenous non-essential amino acids (NEAA, n?=?10). After gavage, the rats were injected with a flooding dose of [(13)C] valine to determine MPS. The rats were killed 50?min after the injection of the flooding dose. Blood was collected for amino acid, glucose and insulin determinations. Tibialis anterior muscles were excised for determination of MPS and for Western blot analyses of the PI3K/Akt, mTORC1, ERK1/2/MAPK pathways and AMP kinase component. MPS was depressed by 61% in starved rats (Saline vs. Fed, P??CIT). LEU but not CIT increased the phosphorylation of rpS6 at serine 235/236. Our findings clearly demonstrated that both CIT and LEU were able to stimulate MPS, but this effect was likely related to the nitrogen load. LEU, CIT and NEAA may have different actions on MPS in this model as they share different mTORC1 regulation capacities.     

Netrin-1 role in angiogenesis: To be or not to be a pro-angiogenic factor?

Angiogenesis designates the formation of new vessels from preexisting ones, and occurs mainly during development. Tight control of this process is a prerequisite to avoid excess/defect in angiogenesis that are the underlying causes of several diseases conditions. Growing lines of evidences have indicated that some guidance cues are involved in regulation of vascular system elaboration, in addition to their role during nervous system development. In this way, netrin-1 has been involved in control of angiogenesis. However, a controversy has emerged regarding its action, since it was concluded from different studies that this protein was either a pro or an anti-angiogenic factor. Thus, netrin-1 role is still unclear. The aim of this review is to propose clues to explain previously reported discrepancies, in light of the dependence receptors model. Indeed, netrin-1 could likely favor angiogenesis, notably by blocking apoptosis induced by its unbound UNC5B receptor.     

Changes in dialysate concentrations of glutamate and GABA in the brain: an index of volume transmission mediated actions?

Brain microdialysis has become a frequently used method to study the extracellular concentrations of neurotransmitters in specific areas of the brain. For years, and this is still the case today, dialysate concentrations and hence extracellular concentrations of neurotransmitters have been interpreted as a direct index of the neuronal release of these specific neurotransmitter systems. Although this seems to be the case for neurotransmitters such as dopamine, serotonin and acetylcholine, the extracellular concentrations of glutamate and GABA do not provide a reliable index of their synaptic exocytotic release. However, many microdialysis studies show changes in extracellular concentrations of glutamate and GABA under specific pharmacological and behavioural stimuli that could be interpreted as a consequence of the activation of specific neurochemical circuits. Despite this, we still do not know the origin and physiological significance of these changes of glutamate and GABA in the extracellular space. Here we propose that the changes in dialysate concentrations of these two neurotransmitters found under specific treatments could be an expression of the activity of the neurone-astrocyte unit in specific circuits of the brain. It is further proposed that dialysate changes of glutamate and GABA could be used as an index of volume transmission mediated actions of these two neurotransmitters in the brain. This hypothesis is based firstly on the assumption that the activity of neurones is functionally linked to the activity of astrocytes, which can release glutamate and GABA to the extracellular space; secondly, on the existence of extrasynaptic glutamate and GABA receptors with functional properties different from those of GABA receptors located at the synapse; and thirdly, on the experimental evidence reporting specific electrophysiological and neurochemical effects of glutamate and GABA when their levels are increased in the extracellular space. According to this concept, glutamate and GABA, once released into the extracellular compartment, could diffuse and have long-lasting effects modulating glutamatergic and/or GABAergic neurone-astrocytic networks and their interactions with other neurotransmitter neurone networks in the same areas of the brain.     

Genetic and phenotypic divergence in an island bird: isolation by distance,by colonization or by adaptation?

Discerning the relative roles of adaptive and nonadaptive processes in generating differences among populations and species, as well as how these processes interact, is a fundamental aim in biology. Both genetic and phenotypic divergence across populations can be the product of limited dispersal and gradual genetic drift across populations (isolation by distance), of colonization history and founder effects (isolation by colonization) or of adaptation to different environments preventing migration between populations (isolation by adaptation). Here, we attempt to differentiate between these processes using island populations of Berthelot's pipit (Anthus berthelotii), a passerine bird endemic to three Atlantic archipelagos. Using microsatellite markers and approximate Bayesian computation, we reveal that the northward colonization of this species ca. 8500 years ago resulted in genetic bottlenecks in the colonized archipelagos. We then show that high levels of genetic structure exist across archipelagos and that these are consistent with a pattern of isolation by colonization, but not with isolation by distance or adaptation. Finally, we show that substantial morphological divergence also exists and that this is strongly concordant with patterns of genetic structure and bottleneck history, but not with environmental differences or geographic distance. Overall, our data suggest that founder effects are responsible for both genetic and phenotypic changes across archipelagos. Our findings provide a rare example of how founder effects can persist over evolutionary timescales and suggest that they may play an important role in the early stages of speciation.     

Consumption of flavonoid-rich foods and increased plasma antioxidant capacity in humans: cause, consequence, or epiphenomenon?

Increased fruit and vegetable consumption is associated with a decreased incidence of cardiovascular diseases, cancer, and other chronic diseases. The beneficial health effects of fruits and vegetables have been attributed, in part, to antioxidant flavonoids present in these foods. Large, transient increases in the total antioxidant capacity of plasma have often been observed after the consumption of flavonoid-rich foods by humans. These observations led to the hypothesis that dietary flavonoids play a significant role as antioxidants in vivo, thereby reducing chronic disease risk. This notion, however, has been challenged recently by studies on the bioavailability of flavonoids, which indicate that they reach only very low concentrations in human plasma after the consumption of flavonoid-rich foods. In addition, most flavonoids are extensively metabolized in vivo, which can affect their antioxidant capacity. Furthermore, fruits and vegetables contain many macro- and micronutrients, in addition to flavonoids, that may directly or through their metabolism affect the total antioxidant capacity of plasma. In this article, we critically review the published research in this field with the goal to assess the contribution of dietary flavonoids to the total antioxidant capacity of plasma in humans. We conclude that the large increase in plasma total antioxidant capacity observed after the consumption of flavonoid-rich foods is not caused by the flavonoids themselves, but is likely the consequence of increased uric acid levels.     

Decreases in the relative concentrations of specific hepatocyte plasma membrane proteins during liver regeneration: down-regulation or dilution?

Antibodies were used to quantify seven domain-specific integral proteins of the rat hepatocyte plasma membrane during rat liver regeneration in response to two-thirds hepatectomy. Quantitative immunoblotting revealed that a subset of the plasma membrane proteins exhibited transient 30-70% decreases in relative concentration during the period of hepatocyte proliferation. The list of affected proteins included at least one representative from each of the plasma membrane domains: the apical protein HA 4, the lateral protein HA 321, and the basolateral receptors for epidermal growth factor and asialoglycoproteins. In contrast, the relative concentrations of three other plasma membrane proteins, the basolateral protein CE 9 and the two apical proteins dipeptidylpeptidase IV and aminopeptidase N, remained unchanged throughout liver regeneration. The decreases in the relative concentrations of the plasma membrane proteins were observed even when the synthesis of hepatocyte DNA was blocked by hydroxyurea, suggesting that the signalling for these two delayed consequences of two-thirds hepatectomy occurred along parallel, dependent pathways. Pulse and pulse-chase metabolic radiolabeling studies revealed that the decreases in the concentrations of the PM proteins were accomplished through protein-selective decreases in the rates of synthesis of the high-mannose precursors of the affected proteins, but not through the accelerated degradation of the mature plasma membrane proteins.     

Determination of micafungin and anidulafungin in human plasma: UV- or mass spectrometric quantification?

Micafungin and anidulafungin are two newer antifungal drugs from the echinocandine class. They are used as monotherapy or in combination with azole-antifungal drugs. The optimized clinical treatment course for the echinocandin drugs with regard to the different infection types and patient subgroups (renal or hepatic impairment, overweight) is still under debate. Therefore, an easy and rugged assay for these two drugs is highly desirable. We here present a method for the quantification of micafungin or anidulafungin in human plasma, applying protein precipitation as sample preparation, reversed phase separation of the analytes and UV-detection and simultaneous tandem mass spectrometry. Anidulafungin served as I.S. for micafungin quantification and vice versa. The method was validated in the calibration ranges from 0.1 μg/ml to 20 μg/ml for both substances. Intra-day precision and accuracies recorded with the UV-detector were 1.80% and 2.65% for micafungin and 4.30% and 10.44% for anidulafungin at the 0.1 μg/ml level. The respective data at the 1 μg/ml level were 2.25% and -0.83% for micafungin and 4.35% and -1.85% for anidulafungin and at the 20 μg/ml level 0.97% and -2.98% for micafungin and 1.04% and 4.74% for anidulafungin, respectively. With the mass spectrometer, because of the unique properties of the analyte molecules, no acceptable validation results could be achieved. Therefore, the mass spectrometric chromatograms served only as identity confirmation of the observed UV-peaks.     

Flatfishes, Turtles, and Bolyerine Snakes: Evolution by Small Steps or Large, or Both?

Step size in evolution is rapidly becoming a major subject of interest to biologists. Traditionally, small changes alone were seen as significant in evolution, and any large differences between an ancestor and its descendant could be explained entirely by the accumulation of many small steps. Whether the evolution of major adaptations is mainly a matter of amassing such small increments of change, or if a significant proportion occurs through the inclusion of one or more large steps, is vital to the understanding of how evolution works. This paper begins by examining four “classic” examples of animals—flatfishes, turtles, bolyerine snakes, and gastropod molluscs—each of which has seemed difficult to interpret as resulting from a continuous series of tiny increments. Recently, however, arguments have appeared to contradict those views, and to propose instead that each of these animal groups arose through continuous adaptive modifications. While some of these arguments are more plausible than others, the view of the present author is that in none of them is there convincing evidence to conclude that the groups arose solely through gradual changes. More specifically, there is no basis to reject an evolutionary origin of the major traits in each group that included at least one principal inovation, occurring in a large, discontinuous step, preceded and followed by relatively small, more gradual changes. The broader question of how discontinuous evolution may occur is addressed through several means. These include considerations of epigenetic (sensu Waddington) development, comparative embryology, population genetics, and other approaches. Overall the phenomenon of adaptive accommodation is seen as of major importance, and its implications for ameliorating possible deleterious attributes of novel phenotypes are discussed. In the course of these analyses the doctrine that all adaptive evolution is driven by external environmental change is reviewed. It is suggested that while small adaptive changes are often closely related to shifts in specific environmental facets, the modifications associated with more major adaptations may be less so.     

Determination of vinpocetine and its primary metabolite,apovincaminic acid,in rat plasma by liquid chromatography–tandem mass spectrometry

A precise and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for simultaneous determination of vinpocetine (VP) and its primary metabolite, apovincaminic acid (AVA), in rat plasma was developed and validated. The analytes and the internal standard-dimenhydrinate were extracted from 50 μL aliquots of rat plasma via solid–liquid extraction. Chromatographic separation was achieved in a run time of 3.5 min on a C₁₈ column under isocratic conditions. Detection of analytes and IS was done by tandem mass spectrometry, operating in positive ion and multiple reaction monitoring (MRM) acquisition mode. The protonated precursor to product ion transitions monitored for VP, AVA and IS were m/z 351.4 → 280.2, 323.2 → 280.2 and 256.2 → 167.3 respectively. The method was fully validated for its sensitivity, selectivity, accuracy and precision, matrix effect, stability study and dilution integrity. A linear dynamic range of 0.5–500 ng/mL for both VP and AVA was evaluated with mean correlation coefficient (r) of 0.9970 and 0.9984 respectively. The precision of the assay (RSD%) was less than 8.55% at all concentrations levels for both VP and AVA. This method was successfully applied to a pharmacokinetic study of VP in rats after intravenous (1 mg/kg) and oral (1 mg/kg) administration.