Astrocyte-neuron signalling by synaptic stimulation in the ventrobasal thalamus

In the Ventrobasal (VB) thalamus, astrocytes are known to elicit NMDA-receptor mediated slow inward currents (SICs) spontaneously in neurons. Fluorescence imaging of astrocytes and patch clamp recordings from the thalamocortical (TC) neurons in the VB of 6-23 day old Wistar rats were performed. TC neurons exhibit spontaneous SICs at low frequencies (~0.0015Hz) that were inhibited by NMDA-receptor antagonists D-AP5 (50µM), and were insensitive to TTX (1µM) suggesting a non-neuronal origin. The effect of corticothalamic (CT) and sensory (Sen) afferent stimulation on astrocyte signalling was assessed by varying stimulus parameters. Moderate synaptic stimulation elicited astrocytic Ca2+ increases, but did not affect the incidence of spontaneous SICs. Prolonged synaptic stimulation induced a 265% increase in SIC frequency. This increase lasted over one hour after the cessation of synaptic stimulation, so revealing a Long Term Enhancement (LTE) of astrocyte-neuron signalling. LTE induction required group I mGluR activation. LTE SICs targeted NMDA-receptors located at extrasynaptic sites. LTE showed a developmental profile: from weeks 1-3, the SIC frequency was increased by an average 50%, 240% and 750% respectively. Prolonged exposure to glutamate (200µM) increased spontaneous SIC frequency by 1800%. This “chemical” form of LTE was prevented by the broad-spectrum excitatory amino acid transporter (EAAT) inhibitor TBOA (300µM) suggesting that glutamate uptake was a critical factor. My results therefore show complex glutamatergic signalling interactions between astrocytes and neurons. Furthermore, two previously unrecognised mechanisms of enhancing SIC frequency are described. The synaptically induced LTE represents a form of non-synaptic plasticity and a glial “memory” of previous synaptic activity whilst enhancement after prolonged glutamate exposure may represent a pathological glial signalling mechanism.

Synthesis, X-ray analysis and DFT study of 2-amino-3-(N-oxipiridin-4-ilsulfanil)-propionic acid dihydrate

The title compound, C(8)H(14)N(2)O(5)S 2(H(2)O), 2-amino-3-(N-oxipiridin-4-ilsulfanil)-propionic acid dihydrate, is obtained by the reaction of cysteine and 4-nitropyridine N-oxide in dimethylformamide, removing the NO(2) group from the benzene ring and releasing nitrous acid into the solution. The molecule exists as a Zwitterion. Hydrogen bond interactions involving the title molecule and water molecules allow the formation of R(5)(5)(23) edge fused rings parallel to (010). Water molecules are connected independently, forming infinite chains (wires), in square wave form, along the b-axis. The chirality of the cysteine molecule used in the synthesis is retained in the title molecule. A density functional theory (DFT) optimized structure at the B3LYP/6-311G(3df,2p) level allows comparison of calculated and experimental IR spectra.

ANIBAL, stable isotope-based quantitative proteomics by aniline and benzoic acid labeling of amino and carboxylic groups

Identification and relative quantification of hundreds to thousands of proteins within complex biological samples have become realistic with the emergence of stable isotope labeling in combination with high throughput mass spectrometry. However, all current chemical approaches target a single amino acid functionality (most often lysine or cysteine) despite the fact that addressing two or more amino acid side chains would drastically increase quantifiable information as shown by in silico analysis in this study. Although the combination of existing approaches, e.g. ICAT with isotope-coded protein labeling, is analytically feasible, it implies high costs, and the combined application of two different chemistries (kits) may not be straightforward. Therefore, we describe here the development and validation of a new stable isotope-based quantitative proteomics approach, termed aniline benzoic acid labeling (ANIBAL), using a twin chemistry approach targeting two frequent amino acid functionalities, the carboxylic and amino groups. Two simple and inexpensive reagents, aniline and benzoic acid, in their (12)C and (13)C form with convenient mass peak spacing (6 Da) and without chromatographic discrimination or modification in fragmentation behavior, are used to modify carboxylic and amino groups at the protein level, resulting in an identical peptide bond-linked benzoyl modification for both reactions. The ANIBAL chemistry is simple and straightforward and is the first method that uses a (13)C-reagent for a general stable isotope labeling approach of carboxylic groups. In silico as well as in vitro analyses clearly revealed the increase in available quantifiable information using such a twin approach. ANIBAL was validated by means of model peptides and proteins with regard to the quality of the chemistry as well as the ionization behavior of the derivatized peptides. A milk fraction was used for dynamic range assessment of protein quantification, and a bacterial lysate was used for the evaluation of relative protein quantification in a complex sample in two different biological states

The Pseudomonas aeruginosa antimetabolite L -2-amino-4-methoxy-trans-3-butenoic acid (AMB) is made from glutamate and two alanine residues via a thiotemplate-linked tripeptide precursor.

The Pseudomonas aeruginosa toxin L-2-amino-4-methoxy-trans-3-butenoic acid (AMB) is a non-proteinogenic amino acid which is toxic for prokaryotes and eukaryotes. Production of AMB requires a five-gene cluster encoding a putative LysE-type transporter (AmbA), two non-ribosomal peptide synthetases (AmbB and AmbE), and two iron(II)/α-ketoglutarate-dependent oxygenases (AmbC and AmbD). Bioinformatics analysis predicts one thiolation (T) domain for AmbB and two T domains (T1 and T2) for AmbE, suggesting that AMB is generated by a processing step from a precursor tripeptide assembled on a thiotemplate. Using a combination of ATP-PPi exchange assays, aminoacylation assays, and mass spectrometry-based analysis of enzyme-bound substrates and pathway intermediates, the AmbB substrate was identified to be L-alanine (L-Ala), while the T1 and T2 domains of AmbE were loaded with L-glutamate (L-Glu) and L-Ala, respectively. Loading of L-Ala at T2 of AmbE occurred only in the presence of AmbB, indicative of a trans loading mechanism. In vitro assays performed with AmbB and AmbE revealed the dipeptide L-Glu-L-Ala at T1 and the tripeptide L-Ala-L-Glu-L-Ala attached at T2. When AmbC and AmbD were included in the assay, these peptides were no longer detected. Instead, an L-Ala-AMB-L-Ala tripeptide was found at T2. These data are in agreement with a biosynthetic model in which L-Glu is converted into AMB by the action of AmbC, AmbD, and tailoring domains of AmbE. The importance of the flanking L-Ala residues in the precursor tripeptide is discussed.

Modulating the electronic structure of amino acids: interaction of model lewis acids with anthranilic acid

On the basis of theoretical B3LYP calculations, Yáñez and co-workers (J. Chem. Theory Comput. 2012, 8, 2293) illustrated that beryllium ions are capable of significantly modulating (changing) the electronic structures of imidazole. In this computational organic chemistry study, the interaction of this β-amino acid and five model Lewis acids (BeF1+, Be2+, AlF2(1+), AlF2+, and Al3+) were investigated. Several aspects were addressed: natural bond orbitals, including second order perturbation analysis of intra-molecular charge delocalization and the natural population analysis atomic charges; molecular geometries; selected infrared stretching frequencies (C-N, C-O, and N-H), and selected ¹H-NMR chemical shifts. The data illustrate that this interaction can weaken the H-O bond and goes beyond strengthening the intra-molecular hydrogen bond (N...H-O) to cause a spontaneous transfer of the proton to the nitrogen atom in five cases generating zwitterion structures. Many new features are observed. Most importantly, the zwitterion structures include a stabilizing hydrogen bond (N-H...O) that varies in relative strength according to the Lewis acid. These findings explain the experimental observations of α-amino acids (for example: J. Am. Chem. Soc. 2001, 123, 3577) and are the first reported fundamental electronic structure characterization of β-amino acids in zwitterion form.

Respiratory effects of kynurenic acid microinjected into the ventromedullary surface of the rat

Several studies demonstrate that, within the ventral medullary surface (VMS), excitatory amino acids are necessary components of the neural circuits involved in the tonic and reflex control of respiration and circulation. In the present study we investigated the cardiorespiratory effects of unilateral microinjections of the broad spectrum glutamate antagonist kynurenic acid (2 nmol/200 nl) along the VMS of urethane-anesthetized rats. Within the VMS only one region was responsive to this drug. This area includes most of the intermediate respiratory area, partially overlapping the rostral ventrolateral medulla (IA/RVL). When microinjected into the IA/RVL, kynurenic acid produced a respiratory depression, without changes in mean arterial pressure or heart rate. The respiratory depression observed was characterized by a decrease in ventilation, tidal volume and mean inspiratory flow and an increase in respiratory frequency. Therefore, the observed respiratory depression was entirely due to a reduction in the inspiratory drive. Microinjections of vehicle (200 nl of saline) into this area produced no significant changes in breathing pattern, blood pressure or heart rate. Respiratory depression in response to the blockade of glutamatergic receptors inside the rostral VMS suggests that neurons at this site have an endogenous glutamatergic input controlling the respiratory cycle duration and the inspiratory drive transmission.

The neurochemically diverse intermedius nucleus of the medulla as a source of excitatory and inhibitory synaptic input to the nucleus tractus solitarii

Sensory afferent signals from neck muscles have been postulated to influence central cardiorespiratory control as components of postural reflexes, but neuronal pathways for this action have not been identified. The intermedius nucleus of the medulla (InM) is a target of neck muscle spindle afferents and is ideally located to influence such reflexes but is poorly investigated. To aid identification of the nucleus, we initially produced three-dimensional reconstructions of the InM in both mouse and rat. Neurochemical analysis including transgenic reporter mice expressing green fluorescent protein in GABA-synthesizing neurons, immunohistochemistry, and in situ hybridization revealed that the InM is neurochemically diverse, containing GABAegric and glutamatergic neurons with some degree of colocalization with parvalbumin, neuronal nitric oxide synthase, and calretinin. Projections from the InM to the nucleus tractus solitarius (NTS) were studied electrophysiologically in rat brainstem slices. Electrical stimulation of the NTS resulted in antidromically activated action potentials within InM neurons. In addition, electrical stimulation of the InM resulted in EPSPs that were mediated by excitatory amino acids and IPSPs mediated solely by GABA(A) receptors or by GABA(A) and glycine receptors. Chemical stimulation of the InM resulted in (1) a depolarization of NTS neurons that were blocked by NBQX (2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonoamide) or kynurenic acid and (2) a hyperpolarization of NTS neurons that were blocked by bicuculline. Thus, the InM contains neurochemically diverse neurons and sends both excitatory and inhibitory projections to the NTS. These data provide a novel pathway that may underlie possible reflex changes in autonomic variables after neck muscle spindle afferent activation.

Lipoic acid and ascorbic acid affect plasma free amino acids selectively in the teleost fish pacu (Piaractus mesopotamicus)

Most studies on the antioxidants, lipoic acid (LA) and ascorbic acid (AA), focused on species that, unlike teleost fish, are not scurvy-prone, and are able to synthesize AA. The antioxidant properties of LA may make it useful in aquaculture nutrition, but several effects must first be investigated, and we address here plasma free amino acids (FAA). In mammals, LA and AA in high doses were claimed to alter plasma FAA profile; to our knowledge, however, no data are available in fish. We therefore studied the effects of dietary LA and AA on plasma FAA in the South American teleost fish pacu, which is being used increasingly in aquaculture. LA treatment decreased concentrations of 18 of 23 individual FAA; specifically, dispensable and total FAA were significantly affected. Ornithine was elevated (+26%) in LA-treated fish and significantly decreased ratios of plasma [Arg]/[Orn] and other individual [FAA]/[Orn] were observed. LA and AA both affected sulfur FAA concentrations. Plasma cystine levels were significantly increased in the LA-supplemented groups. AA had little effect on most amino acids, and no interaction with LA was detected. AA supplementation did, however, significantly lower taurine (-42%) and cystathionine (-31%) levels in plasma. No effect on the branched chain:aromatic amino acid ratios was observed. The data indicate that at the dietary level studied, LA and AA independently affect selected plasma FAA in pacu, and suggest that any use of LA in particular as a dietary supplement should take into account an altered plasma FAA profile.

LeProT1, a Transporter for Proline, Glycine Betaine, and γ-Amino Butyric Acid in Tomato Pollen

During maturation, pollen undergoes a period of dehydration accompanied by the accumulation of compatible solutes.Solute import across the pollen plasma membrane, which occurs via proteinaceous transporters, is required to support pollen development and also for subsequent germination and pollen tube growth. Analysis of the free amino acid composition of various tissues in tomato revealed that the proline content in flowers was 60 times higher than in any other organ analyzed. Within the floral organs, proline was confined predominantly to pollen, where it represented >70 of total free amino acids. Uptake experiments demonstrated that mature as well as germinated pollen rapidly take up proline. To identify proline transporters in tomato pollen, we isolated genes homologous to Arabidopsis proline transporters. LeProT1 was specifically expressed both in mature and germinating pollen, as demonstrated by RNA in situ hybridization. Expression in a yeast mutant demonstrated that LeProT1 transports proline and γ-amino butyric acid with low affinity and glycine betaine with high affinity. Direct uptake and competition studies demonstrate that LeProT1 constitutes a general transporter for compatible solutes.

Effect of eicosapentaenoic acid, protein and amino acids on protein synthesis and degradation in skeletal muscle of cachectic mice

Atrophy of skeletal muscle reduces both the quality and quantity of life of patients with cancer cachexia. Loss of muscle mass is thought to arise from a reduction in protein synthesis combined with an enhanced rate of protein degradation, and few treatments are available to counteract this process. Eicosapentaenoic acid (EPA) has been shown to attenuate the enhanced protein degradation, but to have no effect on protein synthesis. This study examines the effect of EPA combined with a protein and amino-acid supplementation on protein synthesis and degradation in gastrocnemius muscle of mice bearing the cachexia-inducing MAC16 tumour. Muscles from cachectic mice showed an 80% reduction in protein synthesis and about a 50-fold increase in protein degradation compared with muscles from nontumour-bearing mice of the same age and weight. Treatment with EPA (1 g kg-1) daily reduced protein degradation by 88%, but had no effect on protein synthesis. Combination of EPA with casein (5.35 g kg-1) also had no effect on protein synthesis, but when combined with the amino acids leucine, arginine and methionine there was almost a doubling of protein synthesis. The addition of carbohydrate (10.7 g kg-1) to stimulate insulin release had no additional effect. The combination involving the amino acids produced almost a doubling of the ratio of protein synthesis to protein degradation in gastrocnemius muscle over that of EPA alone. No treatment had a significant effect on tumour growth rate, but the inclusion of amino acids had a more significant effect on weight loss induced by the MAC16 tumour than that of EPA alone. The results suggest that combination therapy of cancer cachexia involving both inhibition of the enhanced protein degradation and stimulation of the reduced protein synthesis may be more effective than either treatment alone. © 2004 Cancer Research UK.

The role of γ -aminobutyric acid (Gaba) in somatic embryogenesis of Acca sellowiana Berg. (Myrtaceae)

The γ-aminobutyric acid (Gaba) is a non-protein amino acid found in prokaryotes and eukaryotes. Its role in plant development has not been fully established. This study reports a quantification of the levels of endogenous Gaba, as well as investigation of its role in different stages of somatic embryogenesis in Acca sellowiana Berg. (Myrtaceae). Zygotic embryos were used as explants and they were inoculated into the culture medium contained different concentrations of Gaba (0,2, 4, 6, 8 and 10 µM). The highest concentrations of endogenous Gaba were detected between the third and nine days after inoculation, reaching the value of 12.77 µmol.g-1FW. High frequency of somatic embryogenesis was observed in response to 10 µM Gaba. This treatment also resulted in a large number of normal embryos, and the lowest percentage of formation of fused somatic embryos, phenotypic characteristic of most deformed embryos in all treatments. Also, all treatments promoted the formation of the somatic embryos with positive characteristics of development resumption, which however did not originate the seedlings.

Actions of a Proline Analogue, L-Thiazolidine-4-Carboxylic Acid (T4C), on Trypanosoma cruzi

It is well established that L-proline has several roles in the biology of trypanosomatids. In Trypanosoma cruzi, the etiological agent of Chagas' disease, this amino acid is involved in energy metabolism, differentiation processes and resistance to osmotic stress. In this study, we analyzed the effects of interfering with L-proline metabolism on the viability and on other aspects of the T. cruzi life cycle using the proline analogue L- thiazolidine-4-carboxylic acid (T4C). The growth of epimastigotes was evaluated using different concentrations of T4C in standard culture conditions and at high temperature or acidic pH. We also evaluated possible interactions of this analogue with stress conditions such as those produced by nutrient starvation and oxidative stress. T4C showed a dose-response effect on epimastigote growth (IC(50) = 0.89+/-0.02 mM at 28 degrees C), and the inhibitory effect of this analogue was synergistic (p<0.05) with temperature (0.54+/-0.01 mM at 37 degrees C). T4C significantly diminished parasite survival (p<0.05) in combination with nutrient starvation and oxidative stress conditions. Pre-incubation of the parasites with L-proline resulted in a protective effect against oxidative stress, but this was not seen in the presence of the drug. Finally, the trypomastigote bursting from infected mammalian cells was evaluated and found to be inhibited by up to 56% when cells were treated with non-toxic concentrations of T4C (between 1 and 10 mM). All these data together suggest that T4C could be an interesting therapeutic drug if combined with others that affect, for example, oxidative stress. The data also support the participation of proline metabolism in the resistance to oxidative stress.

Nutritional Quality of Sorghum Seeds: Storage Proteins and Amino Acids

One sorghum commercial genotype (MASSA 03) and nine ICRISAT high-lysine genotypes from India were analyzed for storage protein content, distribution profile, and soluble amino acid concentrations. Storage proteins fraction were extracted and separated by SDS-PAGE. Soluble amino acids contents were determined by HPLC. Variations in intensity and appearance and disappearance of protein bands were observed among the sorghum genotypes suggesting genetic variability. Amino acid profile also indicated large variations in the amino acid concentrations. The high lysine and threonine soluble concentrations observed in the seeds of the sorghum genotypes encouraged the use of these genotypes as potential food source due to the better balanced amino acids profile.

Sequence and tissue distribution of chicken cellular nucleic acid binding protein cDNA

We sequenced cDNAs coding for chicken cellular nucleic acid binding protein (CNBP). Two slightly different variations of the open reading frame were found, each of which translates into a protein with seven zinc finger domains. The longest transcript contains an in-frame insert of 3 bp. The sequence conservation between chick CNBP cDNAs with human, rat and mouse CNBP cDNAs is extreme, especially in the coding region, where the deduced amino acid sequence identity with human, rat and mouse CNBP is 99%. CNBP-like transcripts were also found in various tissues from insect, shrimp, fish and lizard. Regions with remarkable nucleotide conservation were also found in the 3' untranslated region, indicating important functions for these regions. Quantitative reverse transcription polymerase chain reaction (RT-PCR) indicated that in the chick, CNBP is present in all tissues examined in approximately equal ratios to total RNA. RT-PCR of total RNA isolated from different phyla indicate CNBP-like proteins art widespread throughout the animal kingdom. The extraordinary level of conservation suggests an important physiological role for CNBP. (C) 1997 Elsevier Science Inc.

Growth and lactic acid production in batch culture of Lactobacillus rhamnosus in a defined medium

To facilitate metabolic analysis, batch fermentations of Lactobacillus rhamnosus were carried out in a new defined medium. Biomass at 10.5 g/l and lactic acid at 67 g/l with a Y-P/S of 0.84 were achieved. The maximum specific growth rate and the average productivity were 0.49/h and 2.48 g/l.h, respectively. These are comparable to those of this organism and related organisms in complex media. Preliminary amino acid studies were also conducted, highlighting the importance of serine, asparagine, glutamine and cysteine. Kinetic analysis revealed that lactic acid production was predominantly growth-associated with growth associated and non-growth associated lactic acid constants of 0.389 mol/g-cell and 0.0025 mol/g-cell.h, respectively. Finally a kinetic model has been included to describe the fermentation of L. rhamnosus.