Glutamine Supplementation Ameliorates Chronic Stress-induced Reductions in Glutamate and Glutamine Transporters in the Mouse Prefrontal Cortex

Chronic immobilization stress (CIS) induces low levels of glutamate (Glu) and glutamine (Gln) and hypoactive glutamatergic signaling in the mouse prefrontal cortex (PFC), which is closely related to the Glu-Gln cycle. A Gln-supplemented diet ameliorates CIS-induced deleterious changes. Here, we investigated the effects of CIS and Gln supplementation on Glu-Gln cycle-related proteins to characterize the underlying mechanisms. Using the CIS-induced depression mouse model, we examined the expression of 11 proteins involved in the Glu-Gln cycle in the PFC. CIS decreased levels of glutamate transporter 1 (GLT1) and sodium-coupled neutral amino acid transporter (SNAT) 1, SANT2, SNAT3, and SNAT5. Gln supplementation did not affect the non-stressed group but significantly increased GLT1 and SNATs of the stressed group. By immunohistochemical analysis, we confirmed that SNAT1 and SNAT2 were decreased in neurons and GLT1, SNAT3, and SNAT5 were decreased in astrocytes in the medial PFC of the stressed group, but Gln-supplemented diet ameliorated these decrements. Collectively, these results suggest that CIS may cause depressive-like behaviors by decreasing Glu and Gln transportation in the PFC and that a Gln-supplemented diet could prevent the deleterious effects of CIS.

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.

JPH203, a selective L-type amino acid transporter 1 inhibitor, induces mitochondria-dependent apoptosis in Saos2 human osteosarcoma cells

Most normal cells express L-type amino acid transporter 2 (LAT2). However, L-type amino acid transporter 1 (LAT1) is highly expressed in many tumor cells and presumed to support their increased growth and proliferation. This study examined the effects of JPH203, a selective LAT1 inhibitor, on cell growth and its mechanism for cell death in Saos2 human osteosarcoma cells. FOB human osteoblastic cells and Saos2 cells expressed LAT1 and LAT2 together with their associating protein 4F2 heavy chain, but the expression of LAT2 in the Saos2 cells was especially weak. JPH203 and BCH, a non-selective L-type amino acid transporter inhibitor, potently inhibited L-leucine uptake in Saos2 cells. As expected, the intrinsic ability of JPH203 to inhibit L-leucine uptake was far more efficient than that of BCH in Saos2 cells. Likewise, JPH203 and BCH inhibited Saos2 cell growth with JPH203 being superior to BCH in this regard. Furthermore, JPH203 increased apoptosis rates and formed DNA ladder in Saos2 cells. Moreover, JPH203 activated the mitochondria-dependent apoptotic signaling pathway by upregulating pro-apoptotic factors, such as Bad, Bax, and Bak, and the active form of caspase-9, and downregulating anti-apoptotic factors, such as Bcl-2 and Bcl-xL. These results suggest that the inhibition of LAT1 activity via JPH203, which may act as a potential novel anti-cancer agent, leads to apoptosis mediated by the mitochondria-dependent intrinsic apoptotic signaling pathway by inducing the intracellular depletion of neutral amino acids essential for cell growth in Saos2 human osteosarcoma cells.

Overexpression of Ubiquitin and Amino Acid Permease Genes in Association with Antimony Resistance in Leishmania tropica Field Isolates

The mainstay therapy against leishmaniasis is still pentavalent antimonial drugs; however, the rate of antimony resistance is increasing in endemic regions such as Iran. Understanding the molecular basis of resistance to antimonials could be helpful to improve treatment strategies. This study aimed to recognize genes involved in antimony resistance of Leishmania tropica field isolates. Sensitive and resistant L. tropica parasites were isolated from anthroponotic cutaneous leishmaniasis patients and drug susceptibility of parasites to meglumine antimoniate (Glucantime(R)) was confirmed using in vitro assay. Then, complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) and real-time reverse transcriptase-PCR (RT-PCR) approaches were utilized on mRNAs from resistant and sensitive L. tropica isolates. We identified 2 known genes, ubiquitin implicated in protein degradation and amino acid permease (AAP3) involved in arginine uptake. Also, we identified 1 gene encoding hypothetical protein. Real-time RT-PCR revealed a significant upregulation of ubiquitin (2.54-fold), and AAP3 (2.86-fold) (P<0.05) in a resistant isolate compared to a sensitive one. Our results suggest that overexpression of ubiquitin and AAP3 could potentially implicated in natural antimony resistance.

Effect of mufti-gene knockout of L-tryptophan transport system on L-tryptophan production in Escherichia coli / 生物工程学报

In Escherichia coli, uptake of L-tryptophan is done by three distinct permeases, encoded by mtr, tnaB, and aroP. Based on the mtr single-gene knockout, we constructed the mtr.tnaB and mtr.aroP double-gene knockout mutants and the mtr.tnaB.aroP triple-gene knockout mutant. The fermentation results showed that the mtr.tnaB and mtr.aroP knockout mutants produced 1.38 g/L and 1.27 g/L L-tryptophan, respectively, which was 17% and 9% higher than that of the mtr knockout mutant. However, the mtr.tnaB.aroP knockout mutant was significantly affected on cell growth and only produced 0.63 g/L L-tryptophan. During the fed-batch fermentation in a 3-L fermentor, the mtr.tnaB knockout mutant produced 12.2 g/L L-tryptophan, which was 27% higher than that of the mtr knockout mutant. This study demonstrates the effect of multi-gene knockouts of L-tryptophan transport system of Escherichia coli on the biosynthesis of L-tryptophan.

Regulation of key enzymes in tryptophan biosynthesis pathway in Escherichia coli / 生物工程学报

To improve tryptophan production in Escherichia coli, key genes in the tryptophan biosynthesis pathway -aroG, trpED, trpR and tnaA were manipulated. TrpR gene was knocked out to eliminate the repression on the key genes controlling tryptophan biosynthesis and transportation on bacteria chromosome, and the tryptophan degradation was blocked by tnaA gene knockout. Then the bottleneck in tryptophan biosynthesis pathway was removed by co-expressing aroGfbr gene and trpEDfbr gene. Compared with the MG1655, the tryptophan production of trpR knockout and double-genes knockout strains was improved 10-folds and about 20-folds, respectively. After the trpEDfbr was expressed, the tryptophan production increased to 168 mg/L, and when the aroGfbr and trpEDfbr were co-expressed, the tryptophan production increased to 820 mg/L. This work laid the foundation for further construction of higher-efficient engineered strain for tryptophan production.

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.

Genes Expressed During Fruiting Body Formation of Agrocybe cylindracea

Agrocybe cylindracea, an edible mushroom belonging to Bolbitiaceae, Agaricales, is widely used as invaluable medicinal material in the oriental countries. This study was initiated to find the genes expressed during the fruiting body formation of A. cylindracea. The cDNAs expressed differentially during fruiting body morphogenesis of A. cylindracea were isolated through subtractive hybridization between vegetative mycelia and fruiting bodies. The cDNAs expressed in the fruiting body morphogenesis of A. cylindracea were cloned and twenty genes were identified. Eleven were homologous to genes of known functions, three were homologous to genes in other organism without any function known. Six were completely novel genes specific to A. cylindracea so far examined. Some genes with known functions were a pleurotolysin, a self-assembling poreforming cytolysins; Aa-Pri1 and Pir2p, specifically induced genes during fruiting initiation of other mushroom, Agrocybe aegerita; an amino acid permease; a cytochrome P450; a MADS-box gene; a peptidylprolyl isomerase; and a serine proteinase. For other clones, no clear function was annotated so far. We believe the first report of the differentially expressed genes in fruiting process of A. cylindracea will be great helps for further research.

Differential Expression of System L Amino Acid Transporters in Wound Healing Process of Rat Skin / 체질인류학회지

The continuous growth and proliferation of cells are essential for the wound healing process, and the amino acid transporters plays an important role in the continuous growing and proliferating cells. Among the amino acid transport systems, the amino acid transport system L, which is a Na+/-independent neutral amino acid transport system, is a major route for providing living cells including tumor cells with neutral amino acids including several essential amino acids. In the present study, to elucidate the role of amino acid transport system L in the wound healing process, we investigated the expression pattern of LAT1 and LAT2 in the healing process after inflicting the wound on skin of rat. The expression of LAT1 was increased at 12 hours after inflicting the wound and was similar to the control group getting closer to 7 days. The expression of LAT2 was increased at 1 day and 3 days after inflicting the wound and was similar to the control group getting closer to 7 days. These results suggest that the LAT1 and LAT2 play important roles at the early stage and at the middle stage getting closer to normal skin in the wound healing process after inflicting the wound, respectively.

Expression of amino acid transport system L in the differentiation of periodontal ligament fibroblast cells / 대한치주과학회지

The periodontium is a topographically complex organ consisting of epithelial tissue, soft and mineralized tissues. Structures comprising the periodontium include the gingiva, periodontal ligament (PDL), cementum and the alveolar bone. The molecular mechanism of differentiation in PDL fibroblast cells remain unclear. Amino acid transporters play an important role in supplying nutrition to normal and cancer cells and for cell proliferation. Amino acid transport system L is a major nutrient transport system responsible for the Na+-independent transport of neutral amino acids including several essential amino acids. The system L is divided into two major subgroups, the L-type amino acid transporter 1 (LAT1) and the L-type amino acid transporter 2 (LAT2). In this study, the expression pattern of amino acid transport system L was, therefore, investigated in the differentiation of PDL fibroblast cells. To determine the expression level of amino acid transport system L participating in intracellular transport of amino acids in the differentiation of PDL fibroblast cells, it was examined by RT-PCR, observation of cell morphology, Alizaline red-S staining and uptake analysis after inducing experimental differentiation in PDL fibroblast cells isolated from mouse molar teeth. The results are as follows. 1. The LAT1 mRNA was expressed in the early stage of PDL fibroblast cell differentiation. This expression level was gradually reduced by differentiation-inducing time and it was not observed after the late stage. 2. The expression level of LAT2 mRNA was increased in time-dependent manner during differentiation induction of PDL fibroblast cells. 3. There was no changes in the expression level of 4F2hc mRNA, the cofactor of LAT1 and LAT2, during differentiation of PDL fibroblast cells. 4. The expression level of ALP mRNA was gradually increased and the expression level of Col I mRNA was decreased during differentiation of PDL fibroblast cells. 5. The L-leucine transport was reduced by time from the early stage to the late stage in PDL fibroblast cell differentiation. As the results, it is considered that among neutral amino acid transport system L in differentiation of PDL fibroblast cells, the LAT1 has a key role in cell proliferation in the early stage of cell differentiation and the LAT2 has an important role in the late stage of cell differentiation for providing cells with neutral amino acids including several essential amino acids.

Physiological, Pharmacological and Toxicological Implications of Heterodimeric Amino Acid Transporters

The heterodimeric amino acid transporter family is a subfamily of SLC7 solute transporter family which includes 14-transmembrane cationic amino acid transporters and 12-transmembrane heterodimeric amino acid transporters. The members of heterodimeric amino acid transporter family are linked via a disulfide bond to single membrane spanning glycoproteins such as 4F2hc (4F2 heavy chain) and rBAT (related to b0, +-amino acid transporter). Six members are associated with 4F2hc and one is linked to rBAT. Two additional members were identified as ones associated with unknown heavy chains. The members of heterodimeric amino acid transporter family exhibit diverse substrate selectivity and are expressed in variety of tissues. They play variety of physiological roles including epithelial transport of amino acids as well as the roles to provide cells in general with amino acids for cellular nutrition. The dysfunction or hyperfunction of the members of the heterodimeric amino acid transporter family are involved in some diseases and pathologic conditions. The genetic defects of the renal and intestinal transporters b0, +AT/BAT1 (b0, +-type amino acid transporter/b0, +-type amino acid transporter 1) and y+LAT1 (y+L-type amino acid transporter 1) result in the amino aciduria with sever clinical symptoms such as cystinuria and lysin uric protein intolerance, respectively. LAT1 is proposed to be involved in the progression of malignant tumor. xCT (x-C-type transporter) functions to protect cells against oxidative stress, while its over-function may be damaging neurons leading to the exacerbation of brain damage after brain ischemia. Because of broad substrate selectivity, system L transporters such as LAT1 transport amino acid-related compounds including L-Dopa and function as a drug transporter. System L also interacts with some environmental toxins with amino acid-related structure such as cysteine-conjugated methylmercury. Therefore, these transporter would be candidates for drug targets based on new therapeutic strategies.

Estudo comparativo de parâmetros elétricos na absorção de substratos Na+/H+-dependentes em epitélio jejunal e ileal de coelho em câmaras de ussing / Comparative study of electrical parameters on the absorption of substrates the Na + / H + -dependent in jejunal and ileal epithelium in Ussing chambers rabbit

A cólera é uma doença que se tornou endêmica no Nordeste do Brasil após a pandemia iniciada no Peru em 1991. A toxina do Vibrio cholerae (TC) causa potente ação secretória no intestino delgado, podendo levar a desequilíbrio hidro-eletrolítico e choque hipovolêmico nos pacientes com cólera. Postulamos que a administração de substratos, tais como aminoácidos e di-peptídeos a base de glutamina, possa aumentar a absorção de sódio e água no intestino, reduzindo parcialmente as perdas hidro-eletrolíticas na doença. Este trabalho tem como objetivo estudar a farmacodinâmica da absorção de substratos Na+ e/ou H+ -dependentes no intestino delgado de coelho normal e naquele pré-tratado com a TC. A administração de substratos com mecanismos de absorção Na+ -dependentes pode contrabalançar parcialmente as perdas hídricas e eletrolíticas. Outros substratos, tais como os di- e tri-peptídeos, que apresentam mecanismos de absorção próton-dependentes, também possuem propriedades semelhantes. Estas reflexões nos levaram a estudar os efeitos da toxina de cólera sobre os parâmetros elétricos de dois segmentos intestinais (jejuno e íleo). Alças de jejuno proximal e de íleo distal foram isoladas in vivo e injetadas com 5 ml de toxina de cólera (1 μ/ml) em solução fisiológica (grupo experimental) ou igual volume de solução fisiológica (grupo controle). Após 1 hora de incubação com a toxina, as alças foram retiradas, dissecadas da membrana serosa e abertas. Os fragmentos planos foram montados em câmaras de perfusão com temperatura e oxigenação controladas. O lado mucoso foi perfundido por solução sem glicose (substituída por manitol), enquanto que o lado seroso por solução glicosada. Após estabilização, foram adicionados cumulativamente substratos em concentrações que variam de 1x10-5 a 1,7x10-3 M...

Characterization of anionic amino acid transport systems in mouse mammary gland.

L-glutamate was transported into mammary tissue via Na(+)-dependent system XAG- that strongly interacted with both D- and L-isomers of aspartate but only with L-isomer of glutamate. Replacement of Cl- by gluconate from the extracellular medium did not affect the uptake of L-glutamate. Although neutral amino acids weakly inhibited the uptake of L-glutamate, there was no evidence for the heterogeneity of anionic amino acid transport system. The XAG- system was inhibited by sulfhydryl group blocking reagent N-ethylmalemide. Low pH (6) partially inhibited the uptake by L-glutamate by mammary tissue. Prior loading of mammary tissue with L-glutamate slightly down regulated its uptake. Culturing pregnant mouse mammary tissue explants in vitro in the presence of lactogenic hormones (insulin plus cortisol plus prolactin) did not affect appreciably the uptake of L-glutamate.

Functional characteristics of neutral amino acid transporter in opossum kidney (OK) cells

The characteristics of Na+/-dependent cycloleucine uptake was investigated in OK cells with regard to substrate specificity and regulation by protein kinase C (PKC). Inhibition studies with different synthetic and natural amino acids showed a broad spectrum affinity to neutral amino acids regardless of their different side chains including branched or aromatic, indicating that the Na+/-dependent cycloleucine uptake in OK cells is mediated by System B-o or System B degree -like transporter rather than the classical System A or ASC. Phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate, but not 4 alpha-PMA elicited a time-dependent biphasic stimulation of Na+/-dependent cycloleucine uptake, which produced early transient peak at 30 min and late sustained peak at 180 min. Both the early and late stimulations by PMA were due to an increase in Vmax and not due to a change in Km. PKC inhibitors blocked both the early and late stimulation by PMA, while protein synthesis inhibitors blocked the late stimulation only. These results suggest the existence and regulation by PKC of System B degree or System B degree -like broad spectrum transport system for neutral amino acids in OK cells.