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1.
J Appl Microbiol ; 128(3): 828-839, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31755153

RESUMO

AIMS: Crown gall, a phytobacteriosis characterized by the formation of tumours on plant roots was observed in recently planted vineyards of the Meknes region (Morocco). The objective of this research was to analyse the diversity of pathogenic agrobacteria isolated from grapevine in Morocco. METHODS AND RESULTS: Eighty-two isolates from 11 affected vineyards were characterized by recA sequencing and were found to belong to Agrobacterium tumefaciens genomospecies G1, G4 or G7, Rhizobium rhizogenes, and to Allorhizobium vitis. Only the All. vitis isolates appeared to be pathogenic on tomato and multilocus sequence analysis phylogenetic analyses revealed a weak genetic diversity, with the definition of only four genomic groups. Definition of the All. vitis genomic groups correlated with specific pathogenic traits: indeed, genomic groups differed with respect to the severity of hypersensitive response symptoms on tobacco leaves, the intensity of necrotic response on grapevine explants and opine profiles. Both vitopine and octopine were detected by UHPLC in tumours induced by isolates of three genomic groups, an opine signature scarcely ever reported. CONCLUSIONS: Allorhizobium vitis is the only causative agent of crown gall on grape in Morocco, pathogenic isolates can be separated into four genomic groups. SIGNIFICANCE AND IMPACT OF THE STUDY: This study of recently crown-gall-infested vineyards demonstrated that All. vitis is the only causative agent and revealed the presence of nonpathogenic Agrobacterium strain within tumours. Moreover, as the genetic diversity of the All. vitis isolates is relatively narrow, this study lays the basis for further analyses on the evolution of the disease, on the dissemination of the pTi and more globally on the fate of the different genomic groups in this newly colonized environment.


Assuntos
Agrobacterium/classificação , Agrobacterium/fisiologia , Filogenia , Vitis/microbiologia , Agrobacterium/genética , Agrobacterium/patogenicidade , Arginina/análogos & derivados , Arginina/metabolismo , Proteínas de Bactérias/genética , Variação Genética , Genoma Bacteriano/genética , Glutamina/análogos & derivados , Glutamina/metabolismo , Marrocos , Tumores de Planta/microbiologia
2.
Talanta ; 207: 120259, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31594574

RESUMO

We report a new method: biomimetic cell-cell adhesion capillary electrophoresis (BCCACE) to screen drugs targeting interactions between cell membrane receptors and ligands under an environment close to physiological conditions, in which the cell membrane receptors/ligands can maintain their natural conformations and bioactivity without being isolated and purified. Firstly, we screened twenty-one lactose derivatives by cell-immobilized capillary electrophoresis and obtained Gu-4 with the best activity (K = 3.58 ±â€¯0.22 × 104) targeting macrophage antigen-1 (Mac-1). Then, BCCACE was performed as follows: HEK 293 cells overexpressed with receptor (intercellular adhesion molecules-1, ICAM-1) were cultured and immobilized on the inner wall of capillaries as stationary phase, which simulated the endothelial cells lining on the inner surface of blood vessels. HEK 293 cells overexpressed with ligand Mac-1 as samples were used to simulate the neutrophils cells in blood vessels. And Gu-4 added into the running buffer solution as the antagonist was used to simulate the drug in blood. The results showed that Gu-4 (40 µM) could selectively inhibit cell-cell adhesion by targeting the interaction between Mac-1 and ICAM-1. Finally, the pharmaceutical efficacy assays of Gu-4 at cellular and animal levels were carried out using the concentration of 40 µM and the dose of 20 mg kg-1 respectively, which showed the anti-cancer metastasis activity of Gu-4 and the validity of the method. This method simulated a complete three-dimensional vascular model, which can easily obtain the suitable blood concentration of drugs. This system simulated the interaction between leukocytes and vascular endothelial cells in the bloodstream antagonized by drugs, and obtained the effective concentration of the antagonist. It can be used as an accuracy and efficient drug screening method and will be expected to become a new method to screen drugs targeting cell-cell adhesion.


Assuntos
Biomimética/métodos , Adesão Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Eletroforese Capilar/métodos , Glutamina/análogos & derivados , Lactose/análogos & derivados , Proteínas de Membrana/metabolismo , Relação Dose-Resposta a Droga , Glutamina/farmacologia , Células HEK293 , Humanos , Lactose/farmacologia , Ligantes , Ligação Proteica/efeitos dos fármacos , Cicatrização/efeitos dos fármacos
3.
Biomed Pharmacother ; 120: 109436, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31561068

RESUMO

Guhong Injection (GHI), composed of aceglutamide and safflower aqueous extract, has been applied to the clinical treatment of orthopedic diseases, but the relevant mechanism by which GHI exerts effects on bone remodeling has not been reported. In the present study, we investigated the effects of various concentrations of GHI (2.5, 5 and 10 ml/kg) in accelerating rat tibia healing progress by observing haematoxylin and eosin (HE) stained sections, detecting the activity of bone metabolism biochemical markers such as bone morphogenetic protein-2 (BMP-2), transforming growth factor-beta (TGF-beta), osteocalcin (OC) and C-terminal crosslinking telopeptide of type Ⅰ collagen (CTX-1) in rat serum, as well as measuring the expressions of collagen I (COL-1) and collagen II (COL-2) in rat tibia. Also, we investigated the effects of different concentrations of GHI (30, 60 and 90 µl/ml) on the proliferation and differentiation of osteoblasts (OBs) through proliferating cell nuclear antigen (PCNA), alkaline phosphatase (ALP) and type I collagen (COL-1). At the same time, the expression of important factors of Wnt/beta-catenin signaling pathway including Wnt-3a, beta-catenin, disheveled-1 (Dvl-1), glycogen synthase kinases-3beta (GSK-3beta), lymphoid enhancing factor-1 (LEF-1) and axis inhibition protein-2 (Axin-2) after GHI intervention was detected by quantitative real-time PCR (q-PCR), immunohistochemistry and Western blotting. In vivo, rats of tibia fracture model treated with intraperitoneal injection (ip) of GHI had more mature fibroblasts, as well as shorter period formation of new bone. The levels of BMP-2, TGF-beta and OC in rat serum were significantly up-regulated, while the level of CTX was down-regulated. After 4 weeks of drug treatment, the level of COL-1 in the rat tibia increased, but there was no significant change in the level of COL-2. In vitro, after drug intervention, the number of OBs increased significantly, the activities of PCNA, ALP and COL-1 were enhanced. Treatment with GHI increased the mRNA and protein expression of Wnt-3a, beta-catenin, Dvl-1 and LEF-1, and decreased the expression of mRNA of Axin-2 and GSK-3beta. All results demonstrate that GHI accelerates the proliferation of OBs and shortens the recovery time of bone structure, and the Wnt/beta-catenin signaling pathway is involved in the regulation process.


Assuntos
Consolidação da Fratura/efeitos dos fármacos , Glutamina/análogos & derivados , Osteoblastos/efeitos dos fármacos , Extratos Vegetais/administração & dosagem , Tíbia/efeitos dos fármacos , Fraturas da Tíbia/tratamento farmacológico , Via de Sinalização Wnt/efeitos dos fármacos , Proteína Wnt3/metabolismo , beta Catenina/metabolismo , Animais , Biomarcadores/sangue , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Glutamina/administração & dosagem , Injeções Intraperitoneais , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteogênese/efeitos dos fármacos , Ratos Sprague-Dawley , Tíbia/lesões , Tíbia/metabolismo , Tíbia/fisiopatologia , Fraturas da Tíbia/metabolismo , Fraturas da Tíbia/patologia , Fraturas da Tíbia/fisiopatologia , Proteína Wnt3/genética , beta Catenina/genética
4.
J Pharm Biomed Anal ; 176: 112798, 2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31394303

RESUMO

PURPOSE: Salts of phenylacetic acid (PAA) and phenylbutyric acid (PBA) have been used for nitrogen elimination as a treatment for hyperammonaemia caused by urea cycle disorders (UCD). A new analytical method for PBA measurement in urine which helps to evaluate the drug adherence has been implemented. METHODS: Urine specimens from UCD patients receiving PBA were analysed by tandem mass spectrometry to measure urine phenylacetylglutamine (PAGln). Some clinical and biochemical data for each patient were collected. RESULTS: Our study included 87 samples from 40 UCD patients. The PAGln levels did not correlate with height, weight or age. However, the PAGln values showed correlation with PBA dose (r = 0.383, P = 0.015). Plasma glutamine and ammonia levels presented a positive correlation (r = 0.537, P < 0.001). The stability for PAGln in urine was determined at different storage temperatures. CONCLUSIONS: We have developed a simple method for the determination of PAGln in urine, which acts as useful biomarker of effective drug delivery. PAGln in urine is stable at room temperature at least for 15 days, and for several months when frozen at -20 °C. This procedure is useful for the optimization and monitorization of the drug dose allowing the use of spot urine samples.


Assuntos
Benzoatos/farmacocinética , Monitoramento de Medicamentos/métodos , Glutamina/análogos & derivados , Fenilbutiratos/farmacocinética , Distúrbios Congênitos do Ciclo da Ureia/tratamento farmacológico , Adolescente , Adulto , Benzoatos/uso terapêutico , Biomarcadores/urina , Criança , Pré-Escolar , Cromatografia Líquida de Alta Pressão/métodos , Feminino , Glutamina/metabolismo , Glutamina/urina , Humanos , Lactente , Recém-Nascido , Masculino , Adesão à Medicação , Fenilbutiratos/uso terapêutico , Espectrometria de Massas em Tandem/métodos , Distúrbios Congênitos do Ciclo da Ureia/urina , Adulto Jovem
5.
Biomed Chromatogr ; 33(9): e4559, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31016738

RESUMO

A novel chiral method was developed and validated to determine N-acetyl-glutamine (NAG) enantiomers by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Enantioseparation was achieved on a Chiralpak QD-AX column (150 × 4.6 mm i.d., 5 µm) using methanol-water (50 mm ammonium formate, pH 4.3; 70:30, v/v) at a flow rate of 500 µL/min. The detection was operated with an electrospray ionization source interface in positive mode. The ion transition for NAG enantiomers was m/z 189.0 → 130.0. The retention time of N-acetyl-l-glutamine and N-acetyl-d-glutamine were 15.2 and 17.0 min, respectively. Calibration curves were linear over the range of 0.02-20 µg/mL with r > 0.99. The deviation of accuracy and the coefficient of variation of within-run and between-run precision were within 10% for both enantiomers, except for the lower limit of quantification (20 ng/mL), where they deviated <15%. The recovery was >88% and no obvious matrix effect was observed. This method was successfully applied to investigate the plasma protein binding of NAG enantiomers in rats. The results showed that the plasma protein binding of NAG enantiomers was stereoselective. The assay method also exhibited good application prospects for the clinical monitoring of free drugs in plasma.


Assuntos
Proteínas Sanguíneas/metabolismo , Cromatografia Líquida/métodos , Glutamina/análogos & derivados , Espectrometria de Massas em Tandem/métodos , Animais , Proteínas Sanguíneas/análise , Estabilidade de Medicamentos , Glutamina/análise , Glutamina/química , Glutamina/isolamento & purificação , Glutamina/metabolismo , Limite de Detecção , Modelos Lineares , Masculino , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Estereoisomerismo
6.
Bioorg Med Chem Lett ; 29(9): 1047-1050, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30871772

RESUMO

We report the preparation of a novel glutamine derivative, (2S,4S)-2,5-diamino-4-(4-(2-fluoroethoxy)benzyl)-5-oxopentanoic acid, (2S, 4S)4-[18F]FEBGln ([18F]4), through efficient organic and radiosyntheses. In vitro assays of [18F]4 using MCF-7 cells showed that it entered cells via multiple amino acid transporter systems including system L and ASC2 transporters but not through the system A transporter. [18F]4 showed promising properties for tumor imaging and may serve as a lead compound for further optimizing and targeting the system L transporter associated with enhanced glutamine metabolism in cancer cells.


Assuntos
Glutamina/análogos & derivados , Compostos Radiofarmacêuticos/síntese química , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Éteres de Coroa/química , Radioisótopos de Flúor/química , Glutamina/síntese química , Glutamina/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo
7.
J Labelled Comp Radiopharm ; 62(5): 209-214, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30861162

RESUMO

We report initial experience in synthesis of (2S,4R)-4-[18 F]fluoroglutamine, [18 F]FGln, which has been used as a tool for monitoring glutamine metabolism in cancer patients. [18 F]FGln was prepared by a fully automated PET-MF-2V-IT-I synthesizer under GMP-compliant conditions for routine clinical studies. The total radiosynthesis time was about 65 minutes, the decay-corrected radiochemical yield was 18.0 ± 4.2% (n = 59; failure n = 15), and the radiochemical purity was greater than 90%. In some situations, the yields were low (less than 5%), and the most likely cause of this problem is the initial fluorination step; the fluoride ion might not have been fully activated. In other occasions, low final radiochemical purity was often associated with the failure of the second step-removal of protection groups by anhydrous trifluoroacetic acid. A trace amount of water led to production of undesired 4-[18 F]fluoroglutamic acid. Knowledge learned from the successes and failures of synthesis may be helpful to identify critical steps and pitfalls for preparation of this clinically useful metabolic probe, [18 F]FGln, for imaging glutamine utilization in tumor of cancer patients.


Assuntos
Glutamina/análogos & derivados , Técnicas de Química Sintética , Ciclotrons , Glutamina/síntese química , Glutamina/química , Humanos , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons/instrumentação , Controle de Qualidade , Radioquímica
8.
Clin Chem ; 65(3): 406-418, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30647123

RESUMO

BACKGROUND: Clinical practice guidelines recommend estimation of glomerular filtration rate (eGFR) using validated equations based on serum creatinine (eGFRcr), cystatin C (eGFRcys), or both (eGFRcr-cys). However, when compared with the measured GFR (mGFR), only eGFRcr-cys meets recommended performance standards. Our goal was to develop a more accurate eGFR method using a panel of metabolites without creatinine, cystatin C, or demographic variables. METHODS: An ultra-performance liquid chromatography-tandem mass spectrometry assay for acetylthreonine, phenylacetylglutamine, pseudouridine, and tryptophan was developed, and a 20-day, multiinstrument analytical validation was conducted. The assay was tested in 2424 participants with mGFR data from 4 independent research studies. A new GFR equation (eGFRmet) was developed in a random subset (n = 1615) and evaluated in the remaining participants (n = 809). Performance was assessed as the frequency of large errors [estimates that differed from mGFR by at least 30% (1 - P30); goal <10%]. RESULTS: The assay had a mean imprecision (≤10% intraassay, ≤6.9% interassay), linearity over the quantitative range (r 2 > 0.98), and analyte recovery (98.5%-113%). There was no carryover, no interferences observed, and analyte stability was established. In addition, 1 - P30 in the validation set for eGFRmet (10.0%) was more accurate than eGFRcr (13.1%) and eGFRcys (12.0%) but not eGFRcr-cys (8.7%). Combining metabolites, creatinine, cystatin C, and demographics led to the most accurate equation (7.0%). Neither equation had substantial variation among population subgroups. CONCLUSIONS: The new eGFRmet equation could serve as a confirmatory test for GFR estimation.


Assuntos
Cromatografia Líquida/métodos , Taxa de Filtração Glomerular , Espectrometria de Massas em Tandem/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Glutamina/análogos & derivados , Glutamina/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Pseudouridina/sangue , Reprodutibilidade dos Testes , Treonina/análogos & derivados , Treonina/sangue , Triptofano/sangue
9.
Int J Biol Macromol ; 125: 970-978, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30576731

RESUMO

The Mycobacterium tuberculosis (Mtb) Rv2747 gene encodes for a functional protein known as ArgA, which plays an important role in the first step of the l-arginine biosynthesis pathway. ArgA transfers the acetyl group from the acetyl-CoA to either l-glutamate or l-glutamine, which are the known substrates. Here, we present two crystal structures of ArgA: one complexed with CoA and product bound N-acetylglutamine and the other complexed with acetyl-CoA and the inhibitor l-arginine at 2.3 and 3.0 Šresolution respectively. The Mtb ArgA protomer was found to have a "V" cleft and a "ß" bulge, archetypal of a classical GCN5-related N-acetyltransferase superfamily of proteins. The product bound form implies that ArgA can also acetylate l-glutamine like l-glutamate. The active site is strongly inhibited by l-arginine resulting in a closed conformation of ArgA and both l-arginine and N-acetylglutamine were found to occupy at the same active site. Together with structural analysis, molecular docking studies, microscale thermophoresis and enzyme inhibition assays, we conclude that l-glutamine, l-glutamate and l-arginine, all occupy at the same active site of ArgA. Furthermore in case of Mtb ArgA, l-arginine does not act as an allosteric inhibitor unlike other N-acetylglutamate synthase family of proteins.


Assuntos
Acetilcoenzima A/química , Acetiltransferases/química , Arginina/química , Proteínas de Bactérias/química , Ácido Glutâmico/química , Glutamina/química , Mycobacterium tuberculosis/química , Acetilcoenzima A/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Arginina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Ácido Glutâmico/metabolismo , Glutamina/análogos & derivados , Glutamina/metabolismo , Cinética , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/enzimologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
10.
Mol Nutr Food Res ; 63(1): e1700834, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29468821

RESUMO

SCOPE: The impact of meat consumption on human health is widely examined in nutritional epidemiological studies, especially due to the connection between the consumption of red and processed meat and the risk of colon cancer. Food questionnaires do not assess the exposure to different methods of meat cooking. This study aimed to identify biomarkers of the acute ingestion of bovine meat cooked with two different processes. METHODS AND RESULTS: Non-targeted UPLC-MS metabolite profiling was done on urine samples obtained from 24 healthy volunteers before and 8 h after the ingestion of a single meal composed of intrinsically 15 N labelled bovine meat, either cooked at 55 °C for 5 min or at 90 °C for 30 min. A discriminant analysis extension of independent components analysis was applied to the mass spectral data. After meat ingestion, the urinary excretion of 1-methylhistidine, phenylacetylglutamine, and short- and medium-chained acylcarnitines was observed. 15 N labelling was detected in these metabolites, thus confirming their origin from ingested meat. However, no difference was observed in urinary metabolomic profiles according to the meat cooking process used. CONCLUSION: Meat ingestion led to the excretion of several nitrogen-containing compounds, but although a metabolic signature was detected for meat ingestion, the impact of the cooking process was not detectable at the level of urinary metabolic signature in our experimental conditions.


Assuntos
Biomarcadores/urina , Carne Vermelha , Urina/química , Acetilcarnitina/urina , Adulto , Animais , Bovinos , Cromatografia Líquida de Alta Pressão/métodos , Culinária , Ingestão de Alimentos , Feminino , Glutamina/análogos & derivados , Glutamina/urina , Voluntários Saudáveis , Humanos , Masculino , Metaboloma , Metilistidinas/urina , Isótopos de Nitrogênio/urina , Espectrometria de Massas em Tandem/métodos
11.
Asia Pac J Clin Nutr ; 27(5): 1067-1076, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30272855

RESUMO

BACKGROUND AND OBJECTIVES: Obesity is linked to metabolic diseases characterized by insulin resistance, such as diabetes and cardiovascular disease. In this study, we investigated the metabolic disorders of uncomplicated obesity to identify early alterations in biological systems. METHODS AND STUDY DESIGN: Metabolic differences between overweight/obese (n=36) and normal-weight (n=35) young Chinese men without known metabolic disorders were assessed. Metabolic profiling of the serum and urine was performed using ultra-performance liquidchromatography quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS). Partial least squares discriminant analysis (PLS-DA) was undertaken to reveal and classify the differences between the two groups. RESULTS: Compared to normal-weight men, obese men had higher levels of the serum metabolites phenylalanine, Phe-Phe, and L-tryptophan, whereas those of p-cresol sulfate and p-cresol were less in obesity. Urinary metabolites phenylacetamide, L-glutamine, phenylacetylglutamine, indoxyl sulfate, p-cresol, and p-cresol sulfate were greater in obese men. CONCLUSIONS: These findings indicate that disorders involving aromatic amino acids and the tricarboxylic acid cycle (TCA) have microbiomic involvement in the uncomplicated phase of obesity.


Assuntos
Metabolômica/métodos , Sobrepeso/sangue , Sobrepeso/urina , Adulto , Biomarcadores/sangue , Biomarcadores/urina , Cromatografia Líquida de Alta Pressão , Cresóis/sangue , Cresóis/urina , Análise Discriminante , Glutamina/análogos & derivados , Glutamina/urina , Humanos , Indicã/urina , Masculino , Espectrometria de Massas , Obesidade/sangue , Obesidade/urina , Fenilalanina/sangue , Triptofano/sangue , Adulto Jovem
12.
Clin Nucl Med ; 43(11): e392-e399, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30179907

RESUMO

PURPOSE: There is a need for an alternative PET probe, which does not show normal brain tissue uptake in the evaluation of metastasis to the brain. Therefore, we investigate the feasibility of F-labeled glutamine analog, F-(2S,4R)-4-fluoroglutamine (F-FGln), as a new metabolic probe to detect brain metastasis. METHODS: Patients (7 men and 7 women; age, 25-67 years) with suspected brain metastasis were enrolled for this study. All patients were imaged first with F-FGln PET (3 patients for 1-hour dynamic whole-body PET/CT scans, and 11 patients for static whole-body scans at 30 ± 10 minutes after injection), followed by a whole-body F-FDG PET performed in the same week. The characteristics of F-FGln PET imaging in brain metastasis patients were compared with that of F-FDG PET and/or contrast-enhanced MRI patient-by-patient. A composite of all functional and anatomic imaging studies served as the imaging comparator. RESULTS: Initial study in 3 patients using 1-hour dynamic scan showed that 30 ± 10 minutes after injection is optimal for identifying brain metastasis with a high-contrast ratio. All patients were positive for brain metastasis on this studies that demonstrated 38 lesions in 6 anatomic regions on the imaging comparator. The per-lesion detection rates for F-FGln PET and F-FDG PET were 81.6% and 36.8%, respectively. The average tumor-to-normal brain ratio of F-FGln PET was significantly better than that of F-FDG PET in all patients (4.97 ± 2.23 vs 1.22 ± 0.69, P < 0.05). Furthermore, our results suggest that F-FGln uptake in brain metastasis appeared to be independent of tumor size and peripheral edema. In addition, in 14 brain metastatic lesions visualized by both F-FDG PET and F-FGln PET imaging, a positive correlation of SUVmax was observed (r = 0.780, P < 0.01). As to the extracranial metastasis, both tracers showed a concordant increased radioactive uptake except in liver and bone. CONCLUSIONS: The initial imaging of F-FGln presenting a promising new PET radiotracer for patients with brain metastasis and its utility in the liver and bone metastatic lesions may require more caution due to uptake in normal structures.


Assuntos
Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/secundário , Glutamina/análogos & derivados , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons , Adulto , Idoso , Feminino , Fluordesoxiglucose F18 , Humanos , Imagem por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Imagem Corporal Total
13.
Int Immunopharmacol ; 63: 94-100, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30077058

RESUMO

Agonists of nucleotide oligomerization domain (NOD) 1 and NOD2 receptors represent a promising class of immunostimulants and immunological adjuvants. Here, we describe a cell-based test system to assess their pharmacokinetics. In this system, NOD1 and NOD2 agonist concentrations in sera are determined using a reporter cell line, 293Luc, which contains an NF-κB-inducible luciferase reporter construct and naturally expresses NOD1 and NOD2. The 293Luc cells dose-dependently respond to different NOD1 and NOD2 agonists in the nanomolar to low-micromolar concentration range. To verify that the NF-κB-inducing activity of serum samples is due to the administered agonist and not to secondarily induced endogenous molecules, a 293Luc-derived NOD1/NOD2 double-knockout clone is used. Within-run and between-run precisions of the system are <15% and <20%, respectively. Applicability of the novel assay is illustrated by studying pharmacokinetics of two specific NOD2 agonists (N­acetyl­d­glucosaminyl­N­acetyl­d­muramyl­l­alanyl­d­isoglutamine and N­glycolyl­d­muramyl­l­alanyl­d­isoglutamine) and a specific NOD1 agonist (N­acetyl­d­glucosaminyl­N­acetyl­d­sorbitolamine­d­lactoyl­l­alanyl­d­isoglutamyl­meso­diaminopimelic acid). In summary, the test system described here can potentially be used to assess pharmacokinetics of NOD1 and NOD2 agonists in different animal species.


Assuntos
Bioensaio , Glutamina/análogos & derivados , Glutamina/farmacocinética , Proteína Adaptadora de Sinalização NOD1/agonistas , Proteína Adaptadora de Sinalização NOD2/agonistas , Animais , Linhagem Celular , Humanos , Masculino , Coelhos
14.
Sci Rep ; 8(1): 12953, 2018 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-30154570

RESUMO

The peptidoglycan of Staphylococcus aureus is highly amidated. Amidation of α-D-isoglutamic acid in position 2 of the stem peptide plays a decisive role in the polymerization of cell wall building blocks. S. aureus mutants with a reduced degree of amidation are less viable and show increased susceptibility to methicillin, indicating that targeting the amidation reaction could be a useful strategy to combat this pathogen. The enzyme complex that catalyzes the formation of α-D-isoglutamine in the Lipid II stem peptide was identified recently and shown to consist of two subunits, the glutamine amidotransferase-like protein GatD and the Mur ligase homolog MurT. We have solved the crystal structure of the GatD/MurT complex at high resolution, revealing an open, boomerang-shaped conformation in which GatD is docked onto one end of MurT. Putative active site residues cluster at the interface between GatD and MurT and are contributed by both proteins, thus explaining the requirement for the assembled complex to carry out the reaction. Site-directed mutagenesis experiments confirm the validity of the observed interactions. Small-angle X-ray scattering data show that the complex has a similar conformation in solution, although some movement at domain interfaces can occur, allowing the two proteins to approach each other during catalysis. Several other Gram-positive pathogens, including Streptococcus pneumoniae, Clostridium perfringens and Mycobacterium tuberculosis have homologous enzyme complexes. Combined with established biochemical assays, the structure of the GatD/MurT complex provides a solid basis for inhibitor screening in S. aureus and other pathogens.


Assuntos
Proteínas de Bactérias/metabolismo , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/metabolismo , Parede Celular/metabolismo , Complexos Multienzimáticos/metabolismo , Peptidoglicano/metabolismo , Staphylococcus aureus/metabolismo , Amidas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/química , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Domínio Catalítico , Cristalografia por Raios X , Glutamina/análogos & derivados , Glutamina/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas , Proteínas Recombinantes/metabolismo
15.
Cell Host Microbe ; 24(1): 109-119.e6, 2018 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-30008290

RESUMO

Animal-microbe facultative symbioses play a fundamental role in ecosystem and organismal health. Yet, due to the flexible nature of their association, the selection pressures that act on animals and their facultative symbionts remain elusive. Here we apply experimental evolution to Drosophila melanogaster associated with its growth-promoting symbiont Lactobacillus plantarum, representing a well-established model of facultative symbiosis. We find that the diet of the host, rather than the host itself, is a predominant driving force in the evolution of this symbiosis. Furthermore, we identify a mechanism resulting from the bacterium's adaptation to the diet, which confers growth benefits to the colonized host. Our study reveals that bacterial adaptation to the host's diet may be the foremost step in determining the evolutionary course of a facultative animal-microbe symbiosis.


Assuntos
Adaptação Fisiológica , Drosophila melanogaster/microbiologia , Evolução Molecular , Interações entre Hospedeiro e Microrganismos , Lactobacillus plantarum/genética , Simbiose , Acetato Quinase/genética , Acetato Quinase/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Glutamina/análogos & derivados , Glutamina/metabolismo , Lactobacillus plantarum/crescimento & desenvolvimento , Larva/microbiologia , Microbiota , Mutação
16.
Mol Pharm ; 15(8): 3448-3455, 2018 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-29985631

RESUMO

Sustaining the growth of tumor cells requires extra energy and metabolic building blocks. In addition to consuming glucose, glutamine may play the role as an alternative source of nutrient for growth and survival. We aim to characterize a glutamine analog, 18F-(2 S,4 R)4-fluoroglutamine (18F-(2 S,4 R)4-FGln), as an imaging agent for interrogating the role of glutamine from the in vitro study of tumor cells to clinical manifestation in breast cancer patients. Purity was measured by radio-high-performance liquid chromatography (radio-HPLC), and the stability after production was evaluated in phosphate buffer saline (PBS), saline, and mouse and human serum buffers. The presence of Myc expression in MCF-7 and U87 cells was conducted using qPCR. In vitro cell uptake of 18F-(2 S,4 R)4-FGln in MCF-7 and U87 cells was directly compared with 18F-fluorodeoxyglucose (18F-FDG). In vivo biodistribution and micro-PET imaging of 18F-(2 S,4 R)4-FGln in MCF-7 bearing BALB/c nude mice were performed. PET/CT imaging of 18F-(2 S,4 R)4-FGln was compared with 18F-FDG in the same group of breast cancer patients ( n = 10). We successfully synthesized 18F-(2 S,4 R)4-FGln with a high radiochemical purity (>98%), and the radiochemical purity was unchanged in PBS and saline buffers during a 2 h incubation. In vitro cell uptake studies of 18F-(2 S,4 R)4-FGln displayed a rapid and higher uptake in MCF-7 and U87 cells as compared with 18F-FDG. Biodistribution and micro-PET images showed excellent tumor accumulation of 18F-(2 S,4 R)4-FGln in the MCF-7-implanted mice tumor model. In a preliminary clinical study, 18F-(2 S,4 R)4-FGln/PET detected more lesions in breast cancer patients than 18F-FDG/PET (90% vs 80%). Additionally, in one patient with breast lobular carcinoma, there was a lesion mean standardized uptake value (SUVmean) and maximum standardized uptake value (SUVmax) for 18F-(2 S,4 R)4-FGln higher than those obtained by 18F-FDG, as determined by PET imaging. 18F-(2 S,4 R)4-FGln may be a useful glutamine-targeting metabolic probe for noninvasive imaging of breast cancer.


Assuntos
Neoplasias da Mama/diagnóstico por imagem , Radioisótopos de Flúor/farmacocinética , Glutamina/análogos & derivados , Compostos Radiofarmacêuticos/farmacocinética , Adulto , Animais , Neoplasias da Mama/patologia , Feminino , Radioisótopos de Flúor/administração & dosagem , Fluordesoxiglucose F18/administração & dosagem , Fluordesoxiglucose F18/farmacocinética , Glutamina/administração & dosagem , Glutamina/metabolismo , Glutamina/farmacocinética , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/administração & dosagem , Distribuição Tecidual , Microtomografia por Raio-X/métodos , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Mol Pharm ; 15(8): 3032-3045, 2018 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-29939755

RESUMO

It is not efficient enough using the current approaches for tumor-selective drug delivery based on the EPR effect and ligand-receptor interactions, and they have largely failed to translate into the clinic. Therefore, it is urgent to explore an enhanced strategy for effective delivery of anticancer agents. Clinically, many cancers require large amounts of glutamine for their continued growth and survival, resulting in circulating glutamine extraction by the tumor being much greater than that for any organs, behaving as a "glutamine trap". In the present study, we sought to elucidate whether the glutamine-trap effect could be exploited to deliver therapeutic agents to selectively kill cancer cells. Here, a macromolecular glutamine analogue, glutamine-functionalized branched polyethylenimine (GPI), was constructed as the carrier to deliver anti-CD47 siRNA for the blockage of CD47 "don't eat me" signals on cancer cells. The GPI/siRNA glutamine-rich polyplexes exhibited remarkably high levels of cellular uptake by glutamine-dependent lung cancer cells, wild-type A549 cells (A549WT), and its cisplatin-resistant cells (A549DDP), specifically under glutamine-depleted conditions. It was noted that the glutamine transporter ASCT2 was highly expressed both on A549WT and A549DDP but with almost no expression in normal human lung fibroblasts cells. Inhibition of ASCT2 significantly prevented the internalization of GPI polyplexes. These findings raised the intriguing possibility that the glutamine-rich GPI polyplexes utilize the ASCT2 pathway to selectively facilitate their cellular uptake by cancer cells. GPI further delivered anti-CD47 siRNA efficiently both in vitro and in vivo to downregulate the intratumoral mRNA and protein expression levels of CD47. CD47 functions as a "don't eat me" signal and binds to the immunoreceptor SIRPα inducing evasion of phagocytic clearance. GPI/anti-CD47 siRNA polyplexes achieved significant antitumor activities both on A549WT and A549DDP tumor-bearing nude mice. Notably, it had no adverse effect on CD47-expressing red blood cells and platelets, likely because of selective delivery. Therefore, the glutamine-rich carrier GPI driven by the glutamine-trap effect provides a promising new strategy for designing anticancer drug delivery systems.


Assuntos
Antígeno CD47/antagonistas & inibidores , Portadores de Fármacos/química , Neoplasias Pulmonares/tratamento farmacológico , RNA Interferente Pequeno/administração & dosagem , Células A549 , Sistema ASC de Transporte de Aminoácidos/antagonistas & inibidores , Sistema ASC de Transporte de Aminoácidos/metabolismo , Animais , Antígeno CD47/genética , Dipeptídeos/farmacologia , Fibroblastos , Glutamina/análogos & derivados , Glutamina/metabolismo , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Antígenos de Histocompatibilidade Menor/metabolismo , Polietilenoimina/química , RNA Interferente Pequeno/genética , Cloridrato de Raloxifeno/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Zhongguo Zhong Yao Za Zhi ; 43(9): 1940-1945, 2018 May.
Artigo em Chinês | MEDLINE | ID: mdl-29902908

RESUMO

To investigate the pharmacokinetic characteristics of active constituents of Guhong injection in rats with cerebral ischemia reperfusion injury. The middle cerebral artery occlusion (MCAO) model was established in our studies, and then all the rats received iv administration of Guhong injection (2.1 mL·kg⁻¹). The blood concentrations of aceglutamide and hydroxysafflor yellow A (HSYA) were determined by high performance liquid chromatography (HPLC) method at different time points. The concentration-time curves were drawn and pharmacokinetic data were obtained by DAS 3.2.6 software. The results showed that aceglutamide and HSYA showed good linear relationship within the ranges of 1.5-500 mg·L⁻¹ (R²=0.997 5) and 0.33-40 mg·L⁻¹ (R²=0.998 9) respectively. This quantitative method showed a high recovery rate, good precision and stability. The main pharmacokinetics parameters of t1/2α, t1/2ß, CL1, CL2, AUC0-t, AUC0-∞, Vd1, and Vd2 were (0.139±0.007) and (0.155±0.017) h, (0.803±0.046) and (2.233±0.410) h, (0.016±0) and (0.149±0.018) L·h⁻¹·kg⁻¹, (0.015±0.001) and (0.446±0.016) L·h⁻¹·kg⁻¹, (133.335±3.844) and (9.298±0.179) mg·h·L⁻¹, (143.851±3.595) and (14.464±1.451) mg·h·L⁻¹, (0.009±0.001) and (0.223±0.007) L·kg⁻¹, (0.006±0.001) and (0.212±0.032) L·kg⁻¹, respectively. The results showed that the established HPLC method was highly specific, and could be used for the simultaneous detection of aceglutamide and HSYA of Guhong injection in MCAO rats, which was conducive to pharmacokinetic studies. Pharmacokinetic data and parameters could provide reference for continuous administration and interval administration of the drug.


Assuntos
Isquemia Encefálica , Infarto da Artéria Cerebral Média , Animais , Glutamina/análogos & derivados , Extratos Vegetais , Ratos , Ratos Sprague-Dawley
20.
J Am Soc Nephrol ; 29(7): 1992-1999, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29728422

RESUMO

Background Most patients on hemodialysis are treated thrice weekly even if they have residual kidney function, in part because uncertainty remains as to how residual function should be valued and incorporated into the dialysis prescription. Recent guidelines, however, have increased the weight assigned to residual function and thus reduced the treatment time required when it is present. Increasing the weight assigned to residual function may be justified by knowledge that the native kidney performs functions not replicated by dialysis, including solute removal by secretion. This study tested whether plasma concentrations of secreted solutes are as well controlled in patients with residual function on twice weekly hemodialysis as in anuric patients on thrice weekly hemodialysis.Methods We measured the plasma concentration and residual clearance, dialytic clearance, and removal rates for urea and the secreted solutes hippurate, phenylacetylglutamine, indoxyl sulfate, and p-cresol sulfate in nine patients on twice weekly hemodialysis and nine patients on thrice weekly hemodialysis.Results Compared with anuric patients on thrice weekly dialysis with the same standard Kt/Vurea, patients on twice weekly hemodialysis had lower hippurate and phenylacetylglutamine concentrations and similar indoxyl sulfate and p-cresol sulfate concentrations. Mathematical modeling revealed that residual secretory function accounted for the observed pattern of solute concentrations.Conclusions Plasma concentrations of secreted solutes can be well controlled by twice weekly hemodialysis in patients with residual kidney function. This result supports further study of residual kidney function value and the inclusion of this function in dialysis adequacy measures.


Assuntos
Falência Renal Crônica/fisiopatologia , Falência Renal Crônica/terapia , Diálise Renal/métodos , Idoso , Idoso de 80 Anos ou mais , Cresóis/sangue , Feminino , Glutamina/análogos & derivados , Glutamina/sangue , Hipuratos/sangue , Humanos , Indicã/sangue , Falência Renal Crônica/sangue , Masculino , Pessoa de Meia-Idade , Insuficiência Renal Crônica , Ésteres do Ácido Sulfúrico/sangue , Ureia/sangue
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