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1.
Nat Commun ; 14(1): 3261, 2023 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-37277351

RESUMO

Primary sclerosing cholangitis (PSC) is characterized by progressive biliary inflammation and fibrosis. Although gut commensals are associated with PSC, their causative roles and therapeutic strategies remain elusive. Here we detect abundant Klebsiella pneumoniae (Kp) and Enterococcus gallinarum in fecal samples from 45 PSC patients, regardless of intestinal complications. Carriers of both pathogens exhibit high disease activity and poor clinical outcomes. Colonization of PSC-derived Kp in specific pathogen-free (SPF) hepatobiliary injury-prone mice enhances hepatic Th17 cell responses and exacerbates liver injury through bacterial translocation to mesenteric lymph nodes. We developed a lytic phage cocktail that targets PSC-derived Kp with a sustained suppressive effect in vitro. Oral administration of the phage cocktail lowers Kp levels in Kp-colonized germ-free mice and SPF mice, without off-target dysbiosis. Furthermore, we demonstrate that oral and intravenous phage administration successfully suppresses Kp levels and attenuates liver inflammation and disease severity in hepatobiliary injury-prone SPF mice. These results collectively suggest that using a lytic phage cocktail shows promise for targeting Kp in PSC.


Assuntos
Colangite Esclerosante , Terapia por Fagos , Animais , Camundongos , Colangite Esclerosante/terapia , Klebsiella pneumoniae , Fígado/patologia , Inflamação/patologia
2.
Cell ; 185(16): 2879-2898.e24, 2022 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-35931020

RESUMO

Human gut commensals are increasingly suggested to impact non-communicable diseases, such as inflammatory bowel diseases (IBD), yet their targeted suppression remains a daunting unmet challenge. In four geographically distinct IBD cohorts (n = 537), we identify a clade of Klebsiella pneumoniae (Kp) strains, featuring a unique antibiotics resistance and mobilome signature, to be strongly associated with disease exacerbation and severity. Transfer of clinical IBD-associated Kp strains into colitis-prone, germ-free, and colonized mice enhances intestinal inflammation. Stepwise generation of a lytic five-phage combination, targeting sensitive and resistant IBD-associated Kp clade members through distinct mechanisms, enables effective Kp suppression in colitis-prone mice, driving an attenuated inflammation and disease severity. Proof-of-concept assessment of Kp-targeting phages in an artificial human gut and in healthy volunteers demonstrates gastric acid-dependent phage resilience, safety, and viability in the lower gut. Collectively, we demonstrate the feasibility of orally administered combination phage therapy in avoiding resistance, while effectively inhibiting non-communicable disease-contributing pathobionts.


Assuntos
Bacteriófagos , Colite , Microbioma Gastrointestinal , Doenças Inflamatórias Intestinais , Animais , Colite/terapia , Humanos , Inflamação/terapia , Doenças Inflamatórias Intestinais/terapia , Klebsiella pneumoniae , Camundongos
3.
Bioinformatics ; 38(12): 3288-3290, 2022 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-35551337

RESUMO

SUMMARY: Next-Generation Sequencing is widely used as a tool for identifying and quantifying microorganisms pooled together in either natural or designed samples. However, a prominent obstacle is achieving correct quantification when the pooled microbes are genetically related. In such cases, the outcome mostly depends on the method used for assigning reads to the individual targets. To address this challenge, we have developed Exodus-a reference-based Python algorithm for quantification of genomes, including those that are highly similar, when they are sequenced together in a single mix. To test Exodus' performance, we generated both empirical and in silico next-generation sequencing data of mixed genomes. When applying Exodus to these data, we observed median error rates varying between 0% and 0.21% as a function of the complexity of the mix. Importantly, no false negatives were recorded, demonstrating that Exodus' likelihood of missing an existing genome is very low, even if the genome's relative abundance is low and similar genomes are present in the same mix. Taken together, these data position Exodus as a reliable tool for identifying and quantifying genomes in mixed samples. Exodus is open source and free to use at: https://github.com/ilyavs/exodus. AVAILABILITY AND IMPLEMENTATION: Exodus is implemented in Python within a Snakemake framework. It is available on GitHub alongside a docker containing the required dependencies: https://github.com/ilyavs/exodus. The data underlying this article will be shared on reasonable request to the corresponding author. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Software , Genoma , Algoritmos , Projetos de Pesquisa
4.
Pharmaceuticals (Basel) ; 14(4)2021 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-33918942

RESUMO

Bacteriophages ("phages") infect and multiply within specific bacterial strains, causing lysis of their target. Due to the specific nature of these interactions, phages allow a high-precision approach for therapy which can also be exploited for the detection of phage-sensitive pathogens associated with chronic diseases due to gut microbiome imbalance. As rapid phage-mediated detection assays becoming standard-of-care diagnostic tools, they will advance the more widespread application of phage therapy in a precision approach. Using a conventional method and a new cloning approach to develop luminescent phages, we engineered two phages that specifically detect a disease-associated microbial strain. We performed phage sensitivity assays in liquid culture and in fecal matrices and tested the stability of spiked fecal samples stored under different conditions. Different reporter gene structures and genome insertion sites were required to successfully develop the two nluc-reporter phages. The reporter phages detected spiked bacteria in five fecal samples with high specificity. Fecal samples stored under different conditions for up to 30 days did not display major losses in reporter-phage-based detection. Luminescent phage-based diagnostics can provide a rapid co-diagnostic tool to guide the growing field of phage therapy, particularly for a precision-based approach to chronic diseases treatment.

5.
Pharmacol Res Perspect ; 6(3): e00397, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29721322

RESUMO

Some biologics can modulate cytokines that may lead to changes in expression of drug-metabolizing enzymes and cause drug-drug interactions (DDI). DDI potential of TV-1106-an albumin-fused growth hormone (GH)-was investigated. In this study, human blood was exposed to recombinant human growth hormone (rhGH) or TV-1106, followed by isolation of the plasma and its application to human hepatocytes. While the treatment of blood with rhGH increased multiple cytokines, treatment of blood with TV-1106 had no effect on any of the nine cytokines tested. The interleukin (IL)-6 concentration was higher in the rhGH then in the TV-1106-treated plasma (P < .05). While rhGH had little or no effect on CYP1A2 or CYP2C19 mRNA but increased CYP3A4 mRNA twofold, TV-1106 had little or no effect on cytochrome P450 (CYP) mRNAs in hepatocytes. Although the plasma from rhGH-treated blood lowered CYP1A2 activity, the TV-1106 plasma had no effect on CYP activities. The CYP1A2 activity was lower in the rhGH- then in the TV-1106-plasma treated hepatocytes (P < .05). The results indicated that fusing GH with albumin made TV-1106 an unlikely participant of CYP1A2, CYP2C19 or CYP3A4-facilitated, direct or cytokine-driven DDI.


Assuntos
Produtos Biológicos/farmacologia , Sistema Enzimático do Citocromo P-450/genética , Hepatócitos/metabolismo , Hormônio do Crescimento Humano/farmacologia , Interleucina-6/genética , Proteínas Recombinantes de Fusão/farmacologia , Albumina Sérica Humana/farmacologia , Adulto , Células Cultivadas , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP3A/genética , Feminino , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hormônio do Crescimento Humano/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Albumina Sérica Humana/metabolismo
6.
Clin Pharmacol Drug Dev ; 6(4): 331-342, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27489211

RESUMO

TV-1106 is a human serum albumin genetically fused to recombinant human growth hormone, designed to provide a long-acting alternative to daily growth hormone (GH) injections in patients with GH deficiency. This study investigated the pharmacokinetics, pharmacodynamics, and safety of single subcutaneous doses of TV-1106 (7.5, 15, 50, and 100 mg) in Japanese (n = 44) and caucasian (n = 44) healthy subjects. TV-1106 pharmacokinetics and pharmacodynamics were comparable in Japanese and caucasian populations. TV-1106 demonstrated relatively slow absorption (median tmax , 10-30 hours) and a mean elimination half-life of 26-36 hours. Apparent clearance and volume of distribution decreased with increasing TV-1106 doses in both populations and appeared to increase more than dose proportionality across the tested doses. Insulin-like growth factor-1 (IGF-1) and IGF binding protein-3 (IGFBP-3) increased in a dose-related manner, with maximum responses observed at 33-96 and 42-109 hours, respectively. IGF-1 and IGFBP-3 returned to baseline values at 168 hours following 7.5 and 15 mg of TV-1106, and 336 hours following 50 and 100 mg of TV-1106. TV-1106 appeared safe in both populations. There was no evidence of differences in pharmacokinetics, pharmacodynamics, or safety of TV-1106 between Japanese and caucasian populations. The data also demonstrate long-acting growth hormone properties of TV-1106 and support its potential for once-weekly dosing.


Assuntos
Hormônio do Crescimento Humano/genética , Proteínas Recombinantes de Fusão/farmacocinética , Albumina Sérica Humana/genética , Adulto , Povo Asiático , Relação Dose-Resposta a Droga , Feminino , Voluntários Saudáveis , Hormônio do Crescimento Humano/metabolismo , Humanos , Injeções Subcutâneas , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/efeitos adversos , Albumina Sérica Humana/metabolismo , População Branca , Adulto Jovem
7.
Growth Horm IGF Res ; 30-31: 16-21, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27552669

RESUMO

PURPOSE: TV-1106 is a recombinant human albumin genetically fused to growth hormone which is intended to reduce the frequency of injections for GH therapy users. We report the safety, tolerability, pharmacokinetics and pharmacodynamics of repeated subcutaneous injections of TV-1106 in Cynomolgus monkeys. METHOD: Cynomolgus monkeys received four weekly subcutaneous injections of 0, 5, 10 or 20mg/kg TV-1106 and were monitored for safety signals throughout the study. Serum levels of TV-1106 and insulin-like growth factor 1 (IGF-1) were assayed. RESULTS: Treated animals showed no adverse effects or histopathological changes. TV-1106 serum concentrations showed sustained exposure to the drug. Exposure increased in a dose-dependent manner with peak concentrations at approximately 24h post-dosing and elimination half-lives in the range of 12 to 24h. IGF-1 serum concentrations were elevated throughout the entire study duration, indicative of the pharmacological response. There was a clear correlation between change in IGF-1 levels and dose or exposure to TV-1106. CONCLUSIONS: The safety, pharmacokinetic and pharmacodynamic findings support the further development of TV-1106 as a once-weekly administered treatment for patients with GHD.


Assuntos
Hormônio do Crescimento Humano/farmacologia , Fator de Crescimento Insulin-Like I/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Albumina Sérica Humana/farmacologia , Animais , Feminino , Hormônio do Crescimento Humano/efeitos adversos , Hormônio do Crescimento Humano/metabolismo , Humanos , Injeções Subcutâneas , Fator de Crescimento Insulin-Like I/metabolismo , Macaca fascicularis , Masculino , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/metabolismo , Albumina Sérica Humana/efeitos adversos , Albumina Sérica Humana/metabolismo
8.
Eur J Endocrinol ; 173(5): 541-51, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26286586

RESUMO

BACKGROUND: TV-1106 (Teva Pharmaceuticals) is a genetically fused recombinant protein of human GH (hGH) and human serum albumin, in development for treatment of GH deficiency (GHD). TV-1106 is expected to have an extended duration of action compared to daily GH treatment and may enable a reduction in the frequency of injections and improve compliance and quality of life for adults and children requiring GHD therapy. OBJECTIVE: To assess the safety, local tolerability, pharmacokinetics and pharmacodynamics of TV-1106 following single s.c. injections in healthy male volunteers. METHODS: Subjects (n=56) were assigned to one of seven ascending dose groups (3-100 mg) and received either a single dose of TV-1106 (n=6) or placebo (n=2) by s.c. injection. RESULTS: Eighteen subjects reported 43 adverse effects (AEs), which were mild to moderate; no serious AEs (SAEs) occurred. In 50, 70 and 100 mg groups there were mild to moderate increases in heart rate and systolic blood pressure that significantly correlated with higher levels of IGF1. TV-1106 showed pharmacokinetic characteristics of a long-acting hGH as demonstrated by a terminal elimination half-life of 23-35 h, delayed time of peak concentration, and systemic levels seen up to 7 days after dosing. IGF1 levels increased in a dose-dependent manner, before reaching a plateau, with levels above baseline extending beyond 7 days post dose. CONCLUSION: Single administration of TV-1106 up to 100 mg was safe in healthy volunteers. Pharmacokinetics and pharmacodynamics support once-weekly administration in patients with GHD.


Assuntos
Hormônio do Crescimento Humano , Adulto , Voluntários Saudáveis , Hormônio do Crescimento Humano/administração & dosagem , Hormônio do Crescimento Humano/efeitos adversos , Hormônio do Crescimento Humano/deficiência , Hormônio do Crescimento Humano/farmacocinética , Humanos , Masculino , Proteínas Recombinantes/administração & dosagem , Albumina Sérica/administração & dosagem , Adulto Jovem
9.
J Clin Psychopharmacol ; 35(4): 396-405, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26082975

RESUMO

UNLABELLED: Cocaine dependence presents a major public health issue, and to date, no pharmacotherapies are approved for its treatment. TV-1380 is a novel recombinant albumin-fused mutated butyrylcholinesterase (Albu-BChE) that has increased catalytic efficiency for cocaine compared with wild-type BChE and therefore has the potential to facilitate abstinence in cocaine-dependent subjects by decreasing exposure to cocaine and its reinforcing effects. METHODS: This randomized, double-blind, placebo-controlled, parallel-group study in nondependent cocaine users was conducted to evaluate the effect of a single intramuscular dose of Albu-BChE (50, 100, and 300 mg) on the pharmacokinetic and metabolic profile of intravenous cocaine infusions (40 mg) administered at baseline and at 24, 96, and 168 hours after Albu-BChE dosing, to assess safety of coadministering Albu-BChE and cocaine, and to explore the subjective responses to cocaine infusions after Albu-BChE dosing. RESULTS: Administration of Albu-BChE resulted in significant dose-dependent reductions in cocaine exposure (maximum concentration, area under the curve) and half-life. Effects were greatest at 24 hours after Albu-BChE dose, but were sustained up to 168 hours. Spearman correlations indicated a significant negative relationship between Albu-BChE concentration and cocaine clearance and exposure. Consistent with its mechanism of action, Albu-BChE also shifted cocaine metabolism toward preferential formation of ecgonine methyl ester. Administration of Albu-BChE was associated with modest decreases in subjective reports of feeling high and willingness to take cocaine again after cocaine infusion. Coadministration of Albu-BChE and cocaine was safe and well tolerated. CONCLUSIONS: Administration of Albu-BChE at single doses of 50, 100, and 300 mg safely resulted in long-lasting decreases in cocaine exposure in recreational cocaine users.


Assuntos
Albuminas/administração & dosagem , Butirilcolinesterase/administração & dosagem , Butirilcolinesterase/sangue , Cocaína/administração & dosagem , Cocaína/sangue , Drogas Ilícitas/sangue , Adolescente , Adulto , Albuminas/metabolismo , Relação Dose-Resposta a Droga , Método Duplo-Cego , Interações Medicamentosas/fisiologia , Feminino , Humanos , Injeções Intravenosas , Masculino , Pessoa de Meia-Idade , Adulto Jovem
10.
J Clin Pharmacol ; 55(5): 573-83, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25524052

RESUMO

Human plasma butyrylcholinesterase (BChE) contributes to cocaine metabolism and has been considered for use in treating cocaine addiction and cocaine overdose. TV-1380 is a recombinant protein composed of the mature form of human serum albumin fused at its amino terminus to the carboxy-terminus of a truncated and mutated BChE. In preclinical studies, TV-1380 has been shown to rapidly eliminate cocaine in the plasma thus forestalling entry of cocaine into the brain and heart. Two randomized, blinded phase I studies were conducted to evaluate the safety, pharmacokinetics, and pharmacodynamics of TV-1380, following single and multiple administration in healthy subjects. TV-1380 was found to be safe and well tolerated with a long half-life (43-77 hours) and showed a dose-proportional increase in systemic exposure. Consistent with preclinical results, the ex vivo cocaine hydrolysis, TV-1380 activity clearly increased upon treatment in a dose-dependent manner. In addition, there was a direct relationship between ex vivo cocaine hydrolysis (kel ) and TV-1380 serum concentrations. There was no evidence that TV-1380 affected heart rate, the uncorrected QT interval, or the heart-rate-corrected QTcF interval. TV-1380, therefore, offers a safe once-weekly therapy to increase cocaine hydrolysis.


Assuntos
Albuminas/efeitos adversos , Albuminas/farmacologia , Butirilcolinesterase/efeitos adversos , Butirilcolinesterase/farmacologia , Transtornos Relacionados ao Uso de Cocaína/tratamento farmacológico , Proteínas Recombinantes de Fusão/efeitos adversos , Proteínas Recombinantes de Fusão/farmacologia , Adulto , Albuminas/farmacocinética , Área Sob a Curva , Butirilcolinesterase/farmacocinética , Cocaína/metabolismo , Relação Dose-Resposta a Droga , Método Duplo-Cego , Eletrocardiografia , Feminino , Meia-Vida , Humanos , Injeções Intramusculares , Masculino , Dose Máxima Tolerável , Taxa de Depuração Metabólica , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes
11.
J Matern Fetal Neonatal Med ; 23(7): 595-600, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19757337

RESUMO

OBJECTIVE: We sought to determine the pathogenesis of neurodevelopmental impairments in survivors of intrauterine growth retardation (IUGR). METHODS: We used an experimental rabbit vascular IUGR model. We ligated 25% of uteroplacental vessels (partial ischemia) of one-half of the fetuses on day 25 at the end of the third trimester. We then determined hemispheral DNA and protein levels, and used glial fibrillary acidic protein (GFAP) staining to count the labeled astrocytes at the superficial cortical layers. RESULTS: Ischemic fetuses were significantly smaller than control fetuses and presented a disproportionately small body and a relatively larger head compared with the normal body/head ratio, confirming the study model as that of asymmetric IUGR. Hemispheral DNA was unchanged in IUGR fetuses, but they had decreased brain weight, hemispheral protein content, and a reduced number of mature (GFAP-positive) cortical astrocytes compared with control fetuses. CONCLUSION: Vascular IUGR, as demonstrated in our asymmetric IUGR model, adversely affected brain growth, cell size, and cortical astrocytes maturation. In view of the neurotrophic and neuroprotective functions of astrocytes, a reduced number of mature astrocytes during this critical period of development may be implicated in the pathogenesis of the neurodevelopmental impairments observed in IUGR.


Assuntos
Astrócitos/patologia , Retardo do Crescimento Fetal/patologia , Complicações Cardiovasculares na Gravidez/patologia , Animais , Animais Recém-Nascidos , Astrócitos/fisiologia , Peso ao Nascer/fisiologia , Vasos Sanguíneos/patologia , Contagem de Células , Córtex Cerebral/embriologia , Córtex Cerebral/patologia , Constrição Patológica , Modelos Animais de Doenças , Feminino , Retardo do Crescimento Fetal/fisiopatologia , Ligadura , Circulação Placentária/fisiologia , Gravidez , Complicações Cardiovasculares na Gravidez/fisiopatologia , Coelhos
12.
Eur J Neurosci ; 27(1): 66-82, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18184314

RESUMO

Synaptic plasticity is implemented by the interaction of glutamate receptors with PDZ domain proteins. Glutamate transporters provide the only known mechanism of clearance of glutamate from excitatory synapses, and GLT1 is the major glutamate transporter. We show here that GLT1 interacts with the PDZ domain protein PICK1, which plays a critical role in regulating the expression of glutamate receptors at excitatory synapses. A yeast two-hybrid screen of a neuronal library using the carboxyl tail of GLT1b yielded clones expressing PICK1. The GLT1b C-terminal peptide bound to PICK1 with high affinity (K(i) = 6.5 +/- 0.4 microM) in an in vitro fluorescence polarization assay. We also tested peptides based on other variants of GLT1 and other glutamate transporters. GLT1b co-immunoprecipitated with PICK1 from rat brain lysates and COS7 cell lysates derived from cells transfected with plasmids expressing PICK1 and GLT1b. In addition, expression of GLT1b in COS7 cells changed the distribution of PICK1, bringing it to the surface. GLT1b and PICK1 co-localized with each other and with synaptic markers in hippocampal neurons in culture. Phorbol ester, an activator of protein kinase C (PKC), a known PICK1 interactor, had no effect on glutamate transport in rat forebrain neurons in culture. However, we found that exposure of neurons to a myristolated decoy peptide with sequence identical to the C-terminal sequence of GLT1b designed to block the PICK1-GLT1b interaction rendered glutamate transport into neurons responsive to phorbol ester. These results suggest that the PICK1-GLT1b interaction regulates the modulation of GLT1 function by PKC.


Assuntos
Proteínas de Transporte/metabolismo , Transportador 2 de Aminoácido Excitatório/metabolismo , Proteínas Nucleares/metabolismo , Domínios PDZ/fisiologia , Alanina/metabolismo , Animais , Biotinilação/métodos , Encéfalo/citologia , Células Cultivadas , Chlorocebus aethiops , Proteínas do Citoesqueleto , Embrião de Mamíferos , Ácido Glutâmico/metabolismo , Imunoprecipitação/métodos , Mutação/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Estrutura Terciária de Proteína , Ratos , Ratos Long-Evans , Ratos Sprague-Dawley , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacologia
13.
Hypertens Pregnancy ; 24(3): 201-11, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16263593

RESUMO

INTRODUCTION: Hypertension in pregnancy and vascular placental insufficiency are considered common pathogenic factors in human intrauterine growth retardation (IUGR). IUGR neonates experience higher mortality, and the surviving infants have a higher incidence of neurological and intellectual impairment. METHODS: To mimic this condition, we used pregnant spontaneously hypertensive rats (SHR) and performed biometric measurements on Embryonic Day 20, postnatal developmental reflexes, and locomotor activity evaluations. RESULTS: SHR fetuses had significant decreased body weight compared to the Wistar-Kyoto control fetuses (1.51+/-0.02 g vs. 2.05+/-0.01 g, respectively; p<0.0001), and were relatively microcephalic (2.86+/-0.04 cm vs. 3.3+/-0.03 cm, respectively; p<0.0001). Their cephalization index (head circumference/body weight) was increased (1.88+/-0.03 vs. 1.62+/-0.02, respectively; p<0.0001), indicating a "brain-sparing" process. The disproportional ratio indicated that the IUGR type in this model is asymmetric. The SHR pups exhibited a significant (p<0.04) neurodevelopmental delay in the acquisition of neonatal reflexes (righting, negative geotaxis, placing), but they spontaneously caught up with the control pups after approximately 10 days. On Day 30, the SHR pups exhibited significantly increased walking speed and distance and spent less time in quadrant than the controls (p<0.002). CONCLUSION: We speculate that the model of pregnant SHR closely simulate human IUGR caused by hypertension in pregnancy and should enable investigation of mechanisms of hypertension-mediated placenta-vascular injury as well as provide a system for preclinical evaluations of future preventive neuroprotective treatments.


Assuntos
Animais Recém-Nascidos/crescimento & desenvolvimento , Retardo do Crescimento Fetal/etiologia , Análise de Variância , Animais , Biometria , Modelos Animais de Doenças , Discinesias/etiologia , Feminino , Atividade Motora , Gravidez , Ratos , Ratos Endogâmicos SHR
14.
J Neurosci ; 24(5): 1136-48, 2004 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-14762132

RESUMO

GLT1 is the major glutamate transporter of the brain and has been thought to be expressed exclusively in astrocytes. Although excitatory axon terminals take up glutamate, the transporter responsible has not been identified. GLT1 is expressed in at least two forms varying in the C termini, GLT1a and GLT1b. GLT1 mRNA has been demonstrated in neurons, without associated protein. Recently, evidence has been presented, using specific C terminus-directed antibodies, that GLT1b protein is expressed in neurons in vivo. These data suggested that the GLT1 mRNA detected in neurons encodes GLT1b and also that GLT1b might be the elusive presynaptic transporter. To test these hypotheses, we used variant-specific probes directed to the 3'-untranslated regions for GLT1a and GLT1b to perform in situ hybridization in the hippocampus. Contrary to expectation, GLT1a mRNA was the more abundant form. To investigate further the expression of GLT1 in neurons in the hippocampus, antibodies raised against the C terminus of GLT1a and against the N terminus of GLT1, found to be specific by testing in GLT1 knock-out mice, were used for light microscopic and EM-ICC. GLT1a protein was detected in neurons, in 14-29% of axons in the hippocampus, depending on the region. Many of the labeled axons formed axo-spinous, asymmetric, and, thus, excitatory synapses. Labeling also occurred in some spines and dendrites. The antibody against the N terminus of GLT1 also produced labeling of neuronal processes. Thus, the originally cloned form of GLT1, GLT1a, is expressed as protein in neurons in the mature hippocampus and may contribute significantly to glutamate uptake into excitatory terminals.


Assuntos
Transportador 2 de Aminoácido Excitatório/biossíntese , Hipocampo/metabolismo , Neurônios/metabolismo , Terminações Pré-Sinápticas/metabolismo , Animais , Especificidade de Anticorpos , Química Encefálica , Transportador 2 de Aminoácido Excitatório/genética , Heterozigoto , Hipocampo/citologia , Hipocampo/ultraestrutura , Homozigoto , Imuno-Histoquímica , Hibridização In Situ/métodos , Camundongos , Camundongos Knockout , Neurônios/ultraestrutura , Terminações Pré-Sinápticas/ultraestrutura , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Transporte Proteico/fisiologia , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley
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