Effect of Subthalamic Stimulation and Electrode Implantation in the Striatal Microenvironment in a Parkinson's Disease Rat Model.

Deep brain stimulation (DBS) of the subthalamic nucleus (STN) is considered the gold-standard treatment for PD; however, underlying therapeutic mechanisms need to be comprehensively elucidated, especially in relation to glial cells. We aimed to understand the effects of STN-microlesions and STN-DBS on striatal glial cells, inflammation, and extracellular glutamate/GABAergic concentration in a 6-hydroxydopamine (6-OHDA)-induced PD rat model. Rats with unilateral striatal 6-OHDA and electrodes implanted in the STN were divided into two groups: DBS OFF and DBS ON (5 days/2 h/day). Saline and 6-OHDA animals were used as control. Akinesia, striatal reactivity for astrocytes, microglia, and inflammasome, and expression of cytokines, cell signaling, and excitatory amino acid transporter (EAAT)-2 were examined. Moreover, striatal microdialysis was performed to evaluate glutamate and GABA concentrations. The PD rat model exhibited akinesia, increased inflammation, glutamate release, and decreased glutamatergic clearance in the striatum. STN-DBS (DBS ON) completely abolished akinesia. Both STN-microlesion and STN-DBS decreased striatal cytokine expression and the relative concentration of extracellular glutamate. However, STN-DBS inhibited morphological changes in astrocytes, decreased inflammasome reactivity, and increased EAAT2 expression in the striatum. Collectively, these findings suggest that the beneficial effects of DBS are mediated by a combination of stimulation and local microlesions, both involving the inhibition of glial cell activation, neuroinflammation, and glutamate excitotoxicity.

Characterization of In Vivo Function(s) of Members of the Plant Mitochondrial Carrier Family.

Although structurally related, mitochondrial carrier family (MCF) proteins catalyze the specific transport of a range of diverse substrates including nucleotides, amino acids, dicarboxylates, tricarboxylates, cofactors, vitamins, phosphate and H+. Despite their name, they do not, however, always localize to the mitochondria, with plasma membrane, peroxisomal, chloroplast and thylakoid and endoplasmic reticulum localizations also being reported. The existence of plastid-specific MCF proteins is suggestive that the evolution of these proteins occurred after the separation of the green lineage. That said, plant-specific MCF proteins are not all plastid-localized, with members also situated at the endoplasmic reticulum and plasma membrane. While by no means yet comprehensive, the in vivo function of a wide range of these transporters is carried out here, and we discuss the employment of genetic variants of the MCF as a means to provide insight into their in vivo function complementary to that obtained from studies following their reconstitution into liposomes.

Dietary methionine level alters growth, digestibility, and gene expression of amino acid transporters in meat-type chickens.

Imbalance in nutrients can affect digestibility of amino acids by altering gene expression of amino acid transporters. We investigated digestibility and molecular transporters of essential amino acids in chickens fed a methionine-deficient diet. A total of 40 chicks (23 D old) were randomly assigned to either a control (0.49% methionine) or a deficient (0.28%) diet until 41 D when they were sampled for Pectoralis (P.) major, kidney, ileum, and hypothalamus for mRNA expression analysis. The ileal content was collected for apparent ileal digestibility (AID) analysis. Birds fed the deficient diet had reduced growth and worse feed efficiency compared to control. The AID of methionine was similar between both groups. The AID of other essential amino acids was higher in the deficient group than control. mRNA expression of b0,+ AT and LAT4 were upregulated in the ileum and kidney but LAT1 was downregulated only in kidney of the deficient group compared to control. In the P. major, SNAT1, SNAT2, and CAT1 were upregulated in the deficient group compared to control. A diet deficiency in methionine affects digestibility of essential amino acids and cysteine, but not the digestibility of methionine. The change in digestibility is reflected in the mRNA expression of amino acid transporters across different tissues.

Novel function of transcription factor Uga3 as an activator of branched-chain amino acid permease BAP2 gene expression.

Gene regulation in yeast occurs at the transcription level, i.e. the basal level of expression is very low and increased transcription requires gene-specific transcription factors allowing the recruitment of basal transcriptional machinery. Saccharomyces cerevisiae BAP2 gene encodes the permease responsible for most uptake of leucine, valine and isoleucine, amino acids that this yeast can use as nitrogen sources. Moreover, BAP2 expression is known to be induced by the presence of amino acids such as leucine. In this context, the results presented in this paper show that BAP2 is an inducible gene in the presence of nitrogen-non-preferred source proline but exhibits high constitutive non-inducible expression in nitrogen-preferred source ammonium. BAP2 expression is regulated by the SPS sensor system and transcription factors Leu3, Gcn4 and Dal81. This can be achieved or not through a direct binding to the promoter depending on the quality of the nitrogen source. We further demonstrate here that an interaction occurs in vivo between Uga3 ‒ the transcriptional activator responsible for γ-aminobutyric acid (GABA)-dependent induction of the GABA genes ‒ and the regulatory region of the BAP2 gene, which leads to an increase in BAP2 transcription.

Amino Acid and Polyamine Membrane Transporters in Trypanosoma cruzi: Biological Function and Evaluation as Drug Targets.

Amino acids and polyamines are involved in relevant processes for the parasite Trypanosoma cruzi, like protein synthesis, stress resistance, life cycle progression, infection establishment and redox balance, among others. In addition to the biosynthetic routes of amino acids, T. cruzi possesses transport systems that allow the active uptake from the extracellular medium; and in the case of polyamines, the uptake is the unique way to obtain these compounds. The TcAAAP protein family is absent in mammals and its members are responsible for amino acid and derivative uptake, thus the TcAAAP permeases are not only interesting and promising therapeutic targets but could also be used to direct the entry of toxic compounds into the parasite. Although there is a treatment available for Chagas disease, its limited efficacy in the chronic stage of the disease, as well as the side effects reported, highlight the urgent need to develop new therapies. Discovery of new drugs is a slow and cost-consuming process, and even during clinical trials the drugs can fail. In this context, drug repositioning is an interesting and recommended strategy by the World Health Organization since costs and time are significantly reduced. In this article, amino acids and polyamines transport and their potential as therapeutic targets will be revised, including examples of synthetic drugs and drug repurposing.

Detrimental Effects of Induced Antibodies on Aedes aegypti Reproduction.

Aedes aegypti (Linnaeus) (Diptera: Culicidae) is the main vector of viruses causing dengue, chikungunya, Zika, and yellow fever, worldwide. This report focuses on immuno-blocking four critical proteins in the female mosquito when fed on blood containing antibodies against ferritin, transferrin, one amino acid transporter (NAAT1), and acetylcholinesterase (AchE). Peptides from these proteins were selected, synthetized, conjugated to carrier proteins, and used as antigens to immunize New Zealand rabbits. After rabbits were immunized, a minimum of 20 female mosquitos were fed on each rabbit, per replicate. No effect in their viability was observed after blood-feeding; however, the number of infertile females was 20% higher than the control when fed on AchE-immunized rabbits. The oviposition period was significantly longer in females fed on immunized rabbits than those fed on control (non-immunized) rabbits. Fecundity (eggs/female) of treated mosquitoes was significantly reduced (about 50%) in all four treatments, as compared with the control. Fertility (hatched larvae) was also significantly reduced in all four treatments, as compared with the control, being the effect on AchE and transferrin the highest, by reducing hatching between 70 and 80%. Survival to the adult stage of the hatched larvae showed no significant effect, as more than 95% survival was observed in all treatments, including the control. In conclusion, immuno-blocking of these four proteins caused detrimental effects on the mosquito reproduction, being the effect on AchE the most significant.

Amino acid permeases in Cryptococcus neoformans are required for high temperature growth and virulence; and are regulated by Ras signaling.

Cryptococcosis is an Invasive Fungal Infection (IFI) caused by Cryptococcus neoformans, mainly in immunocompromised patients. Therapeutic failure due to pathogen drug resistance, treatment inconstancy and few antifungal options is a problem. The study of amino acid biosynthesis and uptake represents an opportunity to explore possible development of novel antifungals. C. neoformans has 10 amino acids permeases, two of them (Aap3 and Aap7) not expressed at the conditions tested, and five were studied previously (Aap2, Aap4, Aap5, Mup1 and Mup3). Our previous results showed that Aap4 and Aap5 are major permeases with overlapping functions. The aap4Δ/aap5Δ double mutant fails to grow in amino acids as sole nitrogen source and is avirulent in animal model. Here, we deleted the remaining amino acid permeases (AAP1, AAP6, AAP8) that showed gene expression modulation by nutritional condition and created a double mutant (aap1Δ/aap2Δ). We studied the virulence attributes of these mutants and explored the regulatory mechanism behind amino acid uptake in C. neoformans. The aap1Δ/aap2Δ strain had reduced growth at 37°C in L-amino acids, reduced capsule production and was hypovirulent in the Galleria mellonella animal model. Our data, along with previous studies, (i) complement the analysis for all 10 amino acid permeases mutants, (ii) corroborate the idea that these transporters behave as global permeases, (iii) are required during heat and nutritional stress, and (iv) are important for virulence. Our study also indicates a new possible link between Ras1 signaling and amino acids uptake.

Combination of surveillance tools reveals that Yellow Fever virus can remain in the same Atlantic Forest area at least for three transmission seasons

BACKGROUND In Brazil, the Yellow Fever virus (YFV) is endemic in the Amazon, from where it eventually expands into epidemic waves. Coastal south-eastern (SE) Brazil, which has been a YFV-free region for eight decades, has reported a severe sylvatic outbreak since 2016. The virus spread from the north toward the south of the Rio de Janeiro (RJ) state, causing 307 human cases with 105 deaths during the 2016-2017 and 2017-2018 transmission seasons. It is unclear, however, whether the YFV would persist in the coastal Atlantic Forest of RJ during subsequent transmission seasons. OBJECTIVES To conduct a real-time surveillance and assess the potential persistence of YFV in the coastal Atlantic Forest of RJ during the 2018-2019 transmission season. METHODS We combined epizootic surveillance with fast diagnostic and molecular, phylogenetic, and evolutionary analyses. FINDINGS Using this integrative strategy, we detected the first evidence of YFV re-emergence in the third transmission season (2018-2019) in a dying howler monkey from the central region of the RJ state. The YFV detected in 2019 has the molecular signature associated with the current SE YFV outbreak and exhibited a close phylogenetic relationship with the YFV lineage that circulated in the same Atlantic Forest fragment during the past seasons. This lineage circulated along the coastal side of the Serra do Mar mountain chain, and its evolution seems to be mainly driven by genetic drift. The potential bridge vector Aedes albopictus was found probing on the recently dead howler monkey in the forest edge, very close to urban areas. MAIN CONCLUSIONS Collectively, our data revealed that YFV transmission persisted at the same Atlantic Forest area for at least three consecutive transmission seasons without the need of new introductions. Our real-time surveillance strategy permitted health authorities to take preventive actions within 48 h after the detection of the sick non-human primate. The local virus persistence and the proximity of the epizootic forest to urban areas reinforces the concern with regards to the risk of re-urbanisation and seasonal re-emergence of YFV, stressing the need for continuous effective surveillance and high vaccination coverage in the SE region, particularly in RJ, an important tourist location.

Skeletal muscle: from birth to old age, routes to mechanical and metabolic failure. / Músculo esquelético: del nacimiento a la vejez, rutas hacia la falla mecánica y metabólica.

Skeletal muscle (SM) is the most abundant tissue and the largest reservoir of protein in the body. It transports glucose in an insulin dependent manner by the glucose transporter type 4 (GLUT4) and contributes in the maintenance of serum amino acids concentration. By its mass and energetic requirements, it is fundamental for the systemic metabolic balance. In the present work, we present the effect of gestational undernourishment (GU) on the mechanical and metabolic properties of SM at birth and in old age in an animal model. Mechanical studies were performed on isolated muscles, while the GLUT4, amino acid transporters LAT2, SNAT2 and insulin receptors (IR) determination were performed on isolated transverse-tubule membranes (TT). The GU in offspring at birth, results in low muscle mass with increased contraction force and resistance to fatigue. However, in two-years old rats, there was muscle hypotrophy and sarcopenia, the force decreased between 50 and 70% in control rats and rats with GU respectively, accompanied by a lower expression of LAT2, SNAT2 and IR in TT. In conclusion, GU irreversibly affects the SM, an effect that could be similar in humans, which help us to understand the events that associate the GU with the metabolic debacle of SM and the metabolic diseases of human adulthood.

L-arginine availability and arginase activity: Characterization of amino acid permease 3 in Leishmania amazonensis.

BACKGROUND: Leishmania uses the amino acid L-arginine as a substrate for arginase, enzyme that produces urea and ornithine, last precursor of polyamine pathway. This pathway is used by the parasite to replicate and it is essential to establish the infection in the mammalian host. L-arginine is not synthesized by the parasite, so its uptake occurs through the amino acid permease 3 (AAP3). AAP3 is codified by two copies genes (5.1 and 4.7 copies), organized in tandem in the parasite genome. One copy presents the expression regulated by L-arginine availability. METHODOLOGY/PRINCIPAL FINDINGS: RNA-seq data revealed 14 amino acid transporters differentially expressed in the comparison of La-WT vs. La-arg- promastigotes and axenic amastigotes. The 5.1 and 4.7 aap3 transcripts were down-regulated in La-WT promastigotes vs. axenic amastigotes, and in La-WT vs. La-arg- promastigotes. In contrast, transcripts of other transporters were up-regulated in the same comparisons. The amount of 5.1 and 4.7 aap3 mRNA of intracellular amastigotes was also determined in sample preparations from macrophages, obtained from BALB/c and C57BL/6 mice and the human THP-1 lineage infected with La-WT or La-arg-, revealing that the genetic host background is also important. We also determined the aap3 mRNA and AAP3 protein amounts of promastigotes and axenic amastigotes in different environmental growth conditions, varying pH, temperature and L-arginine availability. Interestingly, the increase of temperature increased the AAP3 level in plasma membrane and consequently the L-arginine uptake, independently of pH and L-arginine availability. In addition, we demonstrated that besides the plasma membrane localization, AAP3 was also localized in the glycosome of L. amazonensis promastigotes and axenic amastigotes. CONCLUSIONS/SIGNIFICANCE: In this report, we described the differential transcriptional profiling of amino acids transporters from La-WT and La-arg- promastigotes and axenic amastigotes. We also showed the increased AAP3 levels under amino acid starvation or its decrease in L-arginine supplementation. The differential AAP3 expression was determined in the differentiation of promastigotes to amastigotes conditions, as well as the detection of AAP3 in the plasma membrane reflecting in the L-arginine uptake. Our data suggest that depending on the amino acid pool and arginase activity, Leishmania senses and could use an alternative route for the amino acid transport in response to stress signaling.

Amino Acid Permeases and Virulence in Cryptococcus neoformans.

Fungal opportunistic pathogens colonize various environments, from plants and wood to human and animal tissue. Regarding human pathogens, one great challenge during contrasting niche occupation is the adaptation to different conditions, such as temperature, osmolarity, salinity, pressure, oxidative stress and nutritional availability, which may constitute sources of stress that need to be tolerated and overcome. As an opportunistic pathogen, C. neoformans faces exactly these situations during the transition from the environment to the human host, encountering nutritional constraints. Our previous and current research on amino acid biosynthetic pathways indicates that amino acid permeases are regulated by the presence of the amino acids, nitrogen and temperature. Saccharomyces cerevisiae and Candida albicans have twenty-four and twenty-seven genes encoding amino acid permeases, respectively; conversely, they are scarce in number in Basidiomycetes (C. neoformans, Coprinopsis cinerea and Ustilago maydis), where nine to ten permease genes can be found depending on the species. In this study, we have demonstrated that two amino acid permeases are essential for virulence in C. neoformans. Our data showed that C. neoformans uses two global and redundant amino acid permeases, Aap4 and Aap5 to respond correctly to thermal and oxidative stress. Double deletion of these permeases causes growth arrest in C. neoformans at 37°C and in the presence of hydrogen peroxide. The inability to uptake amino acid at a higher temperature and under oxidative stress also led to virulence attenuation in vivo. Our data showed that thermosensitivity caused by the lack of permeases Aap4 and Aap5 can be remedied by alkaline conditions (higher pH) and salinity. Permeases Aap4 and Aap5 are also required during fluconazole stress and they are the target of the plant secondary metabolite eugenol, a potent antifungal inhibitor that targets amino acid permeases. In summary, our work unravels (i) interesting physiological property of C. neoformans regarding its amino acid uptake system; (ii) an important aspect of virulence, which is the need for amino acid permeases during thermal and oxidative stress resistance and, hence, host invasion and colonization; and (iii) provides a convenient prototype for antifungal development, which are the amino acid permeases Aap4/Aap5 and their inhibitor.

Biochemical Characterization of Branched Chain Amino Acids Uptake in Trypanosoma cruzi.

Trypanosoma cruzi is the etiological agent of Chagas disease. During its life cycle, it alternates among vertebrate and invertebrate hosts. Metabolic flexibility is a main biochemical characteristic of this parasite, which is able to obtain energy by oxidizing a variety of nutrients that can be transported from the extracellular medium. Moreover, several of these metabolites, more specifically amino acids, have a variety of functions beyond being sources of energy. Branched chain amino acids (BCAA), beyond their role in ATP production, are involved in sterol biosynthesis; for example, leucine is involved as a negative regulator of the parasite differentiation process occurring in the insect midgut. BCAA are essential metabolites in most nonphotosynthetic eukaryotes, including trypanosomes. In view of this, the metabolism of BCAA in T. cruzi depends mainly on their transport into the cell. In this work, we kinetically characterized the BCAA transport in T. cruzi epimastigotes. Our data point to BCAA as being transported by a single saturable transport system able to recognize leucine, isoleucine and valine. In view of this, we used leucine to further characterize this system. The transport increased linearly with temperature from 10 to 45 °C, allowing the calculation of an activation energy of 51.30 kJ/mol. Leucine uptake was an active process depending on ATP production and a H(+) gradient, but not on a Na(+) or K(+) gradient at the cytoplasmic membrane level.

Expression of genes coding for selected amino acid transporters in small intestine, liver, and skeletal muscle of pigs fed excess branched-chain amino acids.

Excess Leu reduces the availability of Ile and Val in pigs likely by reducing absorption of the latter amino acids (AA). Twenty-four crossbred pigs were used to evaluate the effect of excess Leu alone or with surplus Ile and Val on the expression of cationic (b(0),(+)AT and CAT1) and neutral (B(0)AT1) AA transporters in the small intestine, liver, and skeletal muscle. Dietary treatments included: 1) basal diet; 2) basal plus 0.43% L-Leu (excess Leu); 3) basal plus 0.43% Leu, 0.20% L-Ile, and 0.25% L-Val (excess Leu, Ile, and Val). The basal diet contained 0.90% standardized ileal digestible Lys, as well as crystalline L-Lys, L-Thr, DL-Met, L-Trp, L-Leu, L-Ile, L-His, and L-Val. Diets 2 and 3 contained 52% more Leu and diet 3 contained 42% more Ile and Val compared with the basal diet. Excess Leu or excess Leu, Ile, and Val reduced b(0),(+)AT expression in the jejunum (P < 0.05) but had no effect in the ileum and liver. Excess Leu increased CAT1 expression in the ileum but reduced expression in the liver (P < 0.05). Excess Leu, Ile, and Val increased (P < 0.05) B(0)AT1 expression in the jejunum and tended to increase (P = 0.10) expression in the ileum. In general, b(0),(+)AT expression was higher but CAT1 expression was lower in the jejunum than in the ileum; B0AT1 was similarly expressed in the jejunum and ileum. Excess Leu or any branched-chain AA affects AA transporter expression, which may affect the absorption and availability of AA for animal growth.

Effect of high-concentrate diet on amino acid transporter expression and milk quality in Holstein dairy cows.

In order to evaluate the effect of high-concentrate diet supplementation on milk protein content, six Holstein dairy cows were assigned into high-concentrate diet (HC) or low-concentrate diet (LC) groups (N = 3/group) for 50 days. With regard to milk protein, HC feeding significantly reduced the percentage of milk protein (P < 0.01), and milk protein yield also reduced. The milk somatic cell count numbers and N-acetyl-D-glucosaminidase activity was significantly higher (P < 0.01) in the HC group than in the LC group. A pre-column derivatization procedure of o-phthalaldehyde was used to analyze the milk amino acid profile, the contents of Asp, Gln, Ala, Ile, Leu, and Lys were significantly lower in milk (P < 0.05), but Arg and Phe were significantly higher (P < 0.05) in the HC group than in the LC group. The mRNA abundance for amino acid transporters SLC7A8, SLC7A10 (P < 0.05), SLC1A3 (P < 0.05), and SLC16A10 (P < 0.05) were decreased in the HC group. These data indicate that expression of amino acid transporters alters regulation of amino acid utilization and decreases milk quality in dairy cows.

SLC6A14 and 5-HTR2C polymorphisms are associated with food intake and nutritional status in children.

BACKGROUND: Serotonin plays a critical role in the regulation of food intake. The solute carrier family 6 member 14 (SLC6A14) and serotonin receptor 2C (5-HTR2C) genes are involved in the bioavailability and action of this neurotransmitter. OBJECTIVE: Evaluation of the association of six polymorphisms in these genes with food intake and nutritional status in children followed to 7-8years of age. DESIGN: Blood samples and the biodemographic data of 344 children were collected at three development stages, in a cross-sectional study undertaken with the cohort from a randomized trial. Polymorphisms were analyzed using polymerase chain reaction-based techniques. RESULTS: At 7 to 8years of age, carriers of the A alleles for both the SLC6A14 rs2312054 and SLC6A14 rs12391221 polymorphisms showed higher food intake, except for the sugar-dense food (SDF) intake parameter, than T/T and C/C homozygotes, respectively. Boy carriers of the C allele of rs2071877 had a higher sum of triceps and subscapular folds than T allele carriers at 7 to 8years old. For 5-HTR2C gene variants, A allele carriers (rs3813928) and T allele carriers (rs3813929) had higher food intake at 3 to 4years old than G/G and C/C homozygotes, respectively, except for SDF. At this age, the intake of energy-dense foods was higher in carriers of the T allele (rs3813929) than in C/C homozygotes. CONCLUSION: This study provides evidence that genetic variants of these proteins might be involved in the determination of food intake and nutritional status in children.

Involvement of the ornithine decarboxylase gene in acid stress response in probiotic Lactobacillus delbrueckii UFV H2b20.

Amino acid decarboxylation is important for the maintenance of intracellular pH under acid stress. This study aims to carry out phylogenetic and expression analysis by real-time PCR of two genes that encode proteins involved in ornithine decarboxylation in Lactobacillus delbrueckii UFV H2b20 exposed to acid stress. Sequencing and phylogeny analysis of genes encoding ornithine decarboxylase and amino acid permease in L. delbrueckii UFV H2b20 showed their high sequence identity (99%) and grouping with those of L. delbrueckii subsp. bulgaricus ATCC 11842. Exposure of L. delbrueckii UFV H2b20 cells in MRS pH 3.5 for 30 and 60 min caused a significant increase in expression of the gene encoding ornithine decarboxylase (up to 8.1 times higher when compared to the control treatment). Increased expression of the ornithine decarboxylase gene demonstrates its involvement in acid stress response in L. delbrueckii UFV H2b20, evidencing that the protein encoded by that gene could be involved in intracellular pH regulation. The results obtained show ornithine decarboxylation as a possible mechanism of adaptation to an acidic environmental condition, a desirable and necessary characteristic for probiotic cultures and certainly important to the survival and persistence of the L. delbrueckii UFV H2b20 in the human gastrointestinal tract.

Promoter characterization and role of CRE in the basal transcription of the rat SNAT2 gene.

Small neutral amino acid transporter 2 (SNAT2) is the most abundant and ubiquitous transporter for zwitterionic short-chain amino acids. The activity of this amino acid transporter is stimulated in vivo or in vitro by glucagon or cAMP analogs. However, it is not known whether the increase in activity at the protein level is due to an increase in SNAT2 gene transcription. Thus, the aim of the present work was to study whether cAMP was able to stimulate SNAT2 gene expression and to localize and characterize the presence of cAMP response elements (CRE) in the promoter that controls the expression of the rat SNAT2 gene. We found that consumption of a high-protein diet that increased serum glucagon concentration or the administration of glucagon or incubation of hepatocytes with forskolin increased the SNAT2 mRNA level. We then isolated the 5' regulatory region of the SNAT2 gene and determined that the transcriptional start site was located 970 bp upstream of the translation start codon. We identified two potential CRE sites located at -354 and -48 bp. Our results, using deletion analysis of the 5' regulatory region of the SNAT2 gene, revealed that the CRE site located at -48 bp was fully responsible for SNAT2 regulation by cAMP. This evidence was strongly supported by mutation of the CRE site and EMSA and ChIP analysis. Alignment of rat, mouse, and human sequences revealed that this CRE site is highly conserved among species, indicating its essential role in the regulation of SNAT2 gene expression.

Sequencing, bioinformatic characterization and expression pattern of a putative amino acid transporter from the parasitic cestode Echinococcus granulosus.

We have sequenced and partially characterized an Echinococcus granulosus cDNA, termed egat1, from a protoscolex signal sequence trap (SST) cDNA library. The isolated 1627 bp long cDNA contains an ORF of 489 amino acids and shows an amino acid identity of 30% with neutral and excitatory amino acid transporters members of the Dicarboxylate/Amino Acid Na+ and/or H+ Cation Symporter family (DAACS) (TC 2.A.23). Additional bioinformatics analysis of EgAT1, confirmed the results obtained by similarity searches and showed the presence of 9 to 10 transmembrane domains, consensus sequences for N-glycosylation between the third and fourth transmembrane domain, a highly similar hydropathy profile with ASCT1 (a known member of DAACS family), high score with SDF (Sodium Dicarboxilate Family) and similar motifs with EDTRANSPORT, a fingerprint of excitatory amino acid transporters. The localization of the putative amino acid transporter was analyzed by in situ hybridization and immunofluorescence in protoscoleces and associated germinal layer. The in situ hybridization labelling indicates the distribution of egat1 mRNA throughout the tegument. EgAT1 protein, which showed in Western blots a molecular mass of approximately 60 kD, is localized in the subtegumental region of the metacestode, particularly around suckers and rostellum of protoscoleces and layers from brood capsules. The sequence and expression analyses of EgAT1 pave the way for functional analysis of amino acids transporters of E. granulosus and its evaluation as new drug targets against cystic echinococcosis.

Trocas materno fetais da glicose, dos aminoácidos e das vitaminas / Glucose, amino acids and vitamins fetomaternal exchange

A descrição do transporte placentário envolve informações sobre mecanismos, propriedades e regulação genética das substâncias moleculares. As trocas materno-fetais se concretizam pelas células do sinciotrofoblasto (microvilosidades e camada basal da membrana plasmática) bem como o tecido conectivo e o endotélio capilar fetal. A transferência de glicose ocorre pelo mecanismo de difusão facilitada e já foram identificados na placenta humana aproximadamente 15 sistemas de transporte dos aminoácidos. A grande maioria das drogas é transportada pelo mecanismo de difusão, porém uma pequena parte depende de suas características fisicoquímicas.

Brefeldin A decreases the activity of the general amino acid permease (GAP1) and the more specific systems for L-leucine uptake in Saccharomyces cerevisiae.

Brefeldin A is a commonly used antifungal agent that reversibly blocks protein transport from the endoplasmic reticulum to the Golgi complex. In this study, we aimed to characterize L-leucine uptake in Saccharomyces cerevisiae in the presence of brefeldin A. For this purpose, we used a synthetic medium, containing L-proline and the detergent SDS, which allows the agent to permeate into the yeast cell. The results obtained with a wild type strain and a gap1 mutant indicate that BFA causes either direct or indirect modification of the transport and/or processing of L-leucine permeases. The presence of BFA affects the kinetic parameter values for L-leucine uptake and decreases not only the uptake mediated by the general system (GAP1), but also that through the specific BAP2 (S1) and/or S2 systems.