Increased vulnerability to kainic acid-induced epileptic seizures in mice underexpressing the scaffold protein Islet-Brain 1/JIP-1.

Islet-Brain 1, also known as JNK-interacting protein-1 (IB1/JIP-1) is a scaffold protein mainly involved in the regulation of the pro-apoptotic signalling cascade mediated by c-Jun-N-terminal kinase (JNK). IB1/JIP-1 organizes JNK and upstream kinases in a complex that facilitates JNK activation. However, overexpression of IB1/JIP-1 in neurons in vitro has been reported to result in inhibition of JNK activation and protection against cellular stress and apoptosis. The occurrence and the functional significance of stress-induced modulations of IB1/JIP-1 levels in vivo are not known. We investigated the regulation of IB1/JIP-1 in mouse hippocampus after systemic administration of kainic acid (KA), in wild-type mice as well as in mice hemizygous for the gene MAPK8IP1, encoding for IB1/JIP-1. We show here that IB1/JIP-1 is upregulated transiently in the hippocampus of normal mice, reaching a peak 8 h after seizure induction. Heterozygous mutant mice underexpressing IB1/JIP-1 showed a higher vulnerability to the epileptogenic properties of KA, whereas hippocampal IB1/JIP-1 levels remained unchanged after seizure induction. Subsequently, an increasing activation of JNK in the 8 h following seizure induction was observed in IB1/JIP-1 haploinsufficient mice, which also underwent more severe excitotoxic lesions in hippocampal CA3, as assessed histologically 3 days after KA administration. Taken together, these data indicate that IB1/JIP-1 in hippocampus participates in the regulation of the neuronal response to excitotoxic stress in a level-dependent fashion.

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.

Synthesis and characterization of new amino acyl-4-thiazolidones

A series of heterocyclic compounds with a 4-thiazolidone nucleus and amino acyl moiety were synthesized by protection reaction of thiosemicarbazide using the symmetrical anhydride (Boc)2O and cyclization with chloroacetic acid under mild conditions. Trifluoroacetic acid was used to obtain 4-thiazolidone and the alpha-amino acid condensation reactions were carried out using strategies for peptide synthesis. The characterization of this new class of compounds was performed using IR and ¹H-NMR spectroscopy.

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.

Plasma amino acids in pregnancy, placental intervillous space and preterm newborn infants

Plasma amino acid levels have never been studied in the placental intervillous space of preterm gestations. Our objective was to determine the possible relationship between plasma amino acids of maternal venous blood (M), of the placental intervillous space (PIVS) and of the umbilical vein (UV) of preterm newborn infants. Plasma amino acid levels were analyzed by ion-exchange chromatography in M from 14 parturients and in the PIVS and UV of their preterm newborn infants. Mean gestational age was 34 ± 2 weeks, weight = 1827 ± 510 g, and all newborns were considered adequate for gestational age. The mean Apgar score was 8 and 9 at the first and fifth minutes. Plasma amino acid values were significantly lower in M than in PIVS (166%), except for aminobutyric acid. On average, plasma amino acid levels were significantly higher in UV than in M (107%) and were closer to PIVS than to M values, except for cystine and aminobutyric acid (P < 0.05). Comparison of the mean plasma amino acid concentrations in the UV of preterm to those of term newborn infants previously studied by our group showed no significant difference, except for proline (P < 0.05), preterm > term. These data suggest that the mechanisms of active amino acid transport are centralized in the syncytiotrophoblast, with their passage to the fetus being an active bidirectional process with asymmetric efflux. PIVS could be a reserve amino acid space for the protection of the fetal compartment from inadequate maternal amino acid variations.

Oxidative stress protection of Trypanosomes requires selenophosphate synthase

Selenoproteins are characterized by the incorporation of at least one amino acid selenocysteine (Sec-U) encoded by in-frame UGA stop codons. These proteins, as well as the components of the Sec synthesis pathway, are present in members of the bacteria, archaea and eukaryote domains. Although not a ubiquitous pathway in all organisms, it was also identified in several protozoa, including the Kinetoplastida. Genetic evidence has indicated that the pathway is non-essential to the survival of Trypanosoma growing in non-stressed conditions. By analyzing the effects of RNA interference of the Trypanosoma brucei selenophosphate synthetase SPS2, we found a requirement under sub-optimal growth conditions. The present work shows that SPS2 is involved in oxidative stress protection of the parasite and its absence severely hampers the parasite survival in the presence of an oxidizing environment that results in an apoptotic-like phenotype and cell death. (C) 2011 Elsevier B.V. All rights reserved.

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.

Ki-1/57 And Cgi-55 Ectopic Expression Impact Cellular Pathways Involved In Proliferation And Stress Response Regulation.

Ki-1/57 (HABP4) and CGI-55 (SERBP1) are regulatory proteins and paralogs with 40.7% amino acid sequence identity and 67.4% similarity. Functionally, they have been implicated in the regulation of gene expression on both the transcriptional and mRNA metabolism levels. A link with tumorigenesis is suggested, since both paralogs show altered expression levels in tumor cells and the Ki-1/57 gene is found in a region of chromosome 9q that represents a haplotype for familiar colon cancer. However, the target genes regulated by Ki-1/57 and CGI-55 are unknown. Here, we analyzed the alterations of the global transcriptome profile after Ki-1/57 or CGI-55 overexpression in HEK293T cells by DNA microchip technology. We were able to identify 363 or 190 down-regulated and 50 or 27 up-regulated genes for Ki-1/57 and CGI-55, respectively, of which 20 were shared between both proteins. Expression levels of selected genes were confirmed by qRT-PCR both after protein overexpression and siRNA knockdown. The majority of the genes with altered expression were associated to proliferation, apoptosis and cell cycle control processes, prompting us to further explore these contexts experimentally. We observed that overexpression of Ki-1/57 or CGI-55 results in reduced cell proliferation, mainly due to a G1 phase arrest, whereas siRNA knockdown of CGI-55 caused an increase in proliferation. In the case of Ki-1/57 overexpression, we found protection from apoptosis after treatment with the ER-stress inducer thapsigargin. Together, our data give important new insights that may help to explain these proteins putative involvement in tumorigenic events.

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.

Effective Tityus serrulatus anti-venom produced using the Ts1 component

Scorpion stings are a public health problem in Brazil, with most incidents involving the species Tityus serrulatus. Some T serrulatus toxins may act as immunogens for the production of a specific anti-venom, but many of the component toxins remain poorly characterized. Here, we describe the immunological characteristics of the toxin Ts1 (also known as TsVII and Ts-gamma) and evaluate production of neutralizing antibodies against the crude venom of T serrulatus. Recombinant Ts1 with one copy (Ts1((1))) or two copies in tandem (Ts1((2))) was expressed in BL21 (DE3) cells. Rabbits and mice were immunized with the recombinant proteins (inclusion bodies) and then tested for production of neutralizing antibodies. Neutralization assays showed that anti-Ts1((1)) and anti-Ts1((2)) protected animals challenged with T serrulatus crude venom and native Ts1 Thus, Ts1 could be used in a mixed ""cocktail"" of immunogens for T serrulatus anti-venom production. (C) 2008 Elsevier Ltd. All rights reserved.

Functional characterization of the Aspergillus nidulans methionine sulfoxide reductases (msrA and msrB)

Proteins are subject to modification by reactive oxygen species (ROS), and oxidation of specific amino acid residues can impair their biological function, leading to an alteration in cellular homeostasis. Sulfur-containing amino acids as methionine are the most vulnerable to oxidation by ROS, resulting in the formation of methionine sulfoxide [Met(O)] residues. This modification can be repaired by methionine sulfoxide reductases (Msr). Two distinct classes of these enzymes, MsrA and MsrB, which selectively reduce the two methionine sulfoxide epimers, methionine-S-sulfoxide and methionine-R-sulfoxide, respectively, are found in virtually all organisms. Here. we describe the homologs of methionine sulfoxide reductases, msrA and msrB, in the filamentous fungus Aspergillus nidulans. Both single and double inactivation mutants were viable, but more sensitive to oxidative stress agents as hydrogen peroxide, paraquat, and ultraviolet light. These strains also accumulated more carbonylated proteins when exposed to hydrogen peroxide indicating that MsrA and MsrB are active players in the protection of the cellular proteins from oxidative stress damage. (C) 2009 Elsevier Inc. All rights reserved.