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1.
Int J Mol Sci ; 20(17)2019 Aug 26.
Artículo en Inglés | MEDLINE | ID: mdl-31454904

RESUMEN

A large number of mutations causing PMM2-CDG, which is the most frequent disorder of glycosylation, destabilize phosphomannomutase2. We looked for a pharmacological chaperone to cure PMM2-CDG, starting from the structure of a natural ligand of phosphomannomutase2, α-glucose-1,6-bisphosphate. The compound, ß-glucose-1,6-bisphosphate, was synthesized and characterized via 31P-NMR. ß-glucose-1,6-bisphosphate binds its target enzyme in silico. The binding induces a large conformational change that was predicted by the program PELE and validated in vitro by limited proteolysis. The ability of the compound to stabilize wild type phosphomannomutase2, as well as frequently encountered pathogenic mutants, was measured using thermal shift assay. ß-glucose-1,6-bisphosphate is relatively resistant to the enzyme that specifically hydrolyses natural esose-bisphosphates.


Asunto(s)
Trastornos Congénitos de Glicosilación/genética , Trastornos Congénitos de Glicosilación/metabolismo , Glucosa-6-Fosfato/análogos & derivados , Mutación , Fosfotransferasas (Fosfomutasas)/deficiencia , Trastornos Congénitos de Glicosilación/tratamiento farmacológico , Glucosa-6-Fosfato/química , Glucosa-6-Fosfato/metabolismo , Glucosa-6-Fosfato/farmacología , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Molecular , Fosfotransferasas (Fosfomutasas)/genética , Unión Proteica
2.
mBio ; 10(3)2019 05 14.
Artículo en Inglés | MEDLINE | ID: mdl-31088917

RESUMEN

Actinobacteria have long been the main source of antibiotics, secondary metabolites with tightly controlled biosynthesis by environmental and physiological factors. Phosphorylation of exogenous glucosamine has been suggested as a mechanism for incorporation of this extracellular material into secondary metabolite biosynthesis, but experimental evidence of specific glucosamine kinases in Actinobacteria is lacking. Here, we present the molecular fingerprints for the identification of a unique family of actinobacterial glucosamine kinases. Structural and biochemical studies on a distinctive kinase from the soil bacterium Streptacidiphilus jiangxiensis unveiled its preference for glucosamine and provided structural evidence of a phosphoryl transfer to this substrate. Conservation of glucosamine-contacting residues across a large number of uncharacterized actinobacterial proteins unveiled a specific glucosamine binding sequence motif. This family of kinases and their genetic context may represent the missing link for the incorporation of environmental glucosamine into the antibiotic biosynthesis pathways in Actinobacteria and can be explored to enhance antibiotic production.IMPORTANCE The discovery of novel enzymes involved in antibiotic biosynthesis pathways is currently a topic of utmost importance. The high levels of antibiotic resistance detected worldwide threaten our ability to combat infections and other 20th-century medical achievements, namely, organ transplantation or cancer chemotherapy. We have identified and characterized a unique family of enzymes capable of phosphorylating glucosamine to glucosamine-6-phosphate, a crucial molecule directly involved in the activation of antibiotic production pathways in Actinobacteria, nature's main source of antimicrobials. The consensus sequence identified for these glucosamine kinases will help establish a molecular fingerprint to reveal yet-uncharacterized sequences in antibiotic producers, which should have an important impact in biotechnological and biomedical applications, including the enhancement and optimization of antibiotic production.


Asunto(s)
Actinobacteria/enzimología , Actinobacteria/genética , Glucosamina/análogos & derivados , Glucosa-6-Fosfato/análogos & derivados , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Antibacterianos/biosíntesis , Dermatoglifia del ADN , Glucosamina/metabolismo , Glucosa-6-Fosfato/metabolismo , Fosforilación , Unión Proteica , ARN Ribosómico 16S/genética , Microbiología del Suelo
3.
Hematol Oncol ; 37(2): 193-201, 2019 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-30821017

RESUMEN

The detection rates of whole-body combined [18 F]NaF/[18 F]FDG positron emission tomography combined with computed tomography (PET/CT), CT alone, whole-body magnetic resonance imaging (WB-MRI), and X-ray were prospectively studied in patients with treatment-requiring plasma cell disorders The detection rates of imaging techniques were compared, and focal lesions were classified according to their anatomic location. Twenty-six out of 30 initially included patients were assessable. The number of focal lesions detected in newly diagnosed patients (n = 13) and in relapsed patients (n = 13) were 296 and 234, respectively. The detection rate of PET/CT was significantly higher than those of WB-MRI (P < 0.05) and CT (P < 0.0001) both in patients with newly diagnosed and in those with relapsed multiple myeloma (MM). The X-ray detection rate was significantly lower than those of all other techniques, while CT detected more lesions compared with WB-MRI at diagnosis (P = 0.025). With regard to the infiltration patters, relapsed patients presented more diffuse patterns, and more focal lesions located in the limbs compared with newly diagnosed patients. In conclusion, the detection rate of [18 F]NaF/[18 F]FDG PET/CT was significantly higher than those of CT, MRI, and X-ray, while the detection rate of X-rays was significantly lower than those of all other imaging techniques except for focal lesions located in the skull.


Asunto(s)
Radioisótopos de Flúor/administración & dosificación , Glucosa-6-Fosfato/análogos & derivados , Imagen por Resonancia Magnética , Mieloma Múltiple/diagnóstico por imagen , Tomografía de Emisión de Positrones , Neoplasias Craneales/diagnóstico por imagen , Fluoruro de Sodio/administración & dosificación , Tomografía Computarizada por Rayos X , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Glucosa-6-Fosfato/administración & dosificación , Humanos , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Estudios Prospectivos
4.
Hematol Oncol ; 37(2): 168-175, 2019 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-30734341

RESUMEN

We evaluated the role of 18-fluoro-2-deoxy-d-glucose positron emission tomography ([18F] FDG-PET) with computed tomography (CT) (PET/CT) as a diagnostic and prognostic tool in newly diagnosed marginal zone lymphoma (MZL) patients. This is a retrospective cohort study of patients with newly diagnosed MZL, treated with immunotherapy, chemotherapy regimens, surgery, or Helicobacter pylori eradication between 2008 and 2016 in a single tertiary center. Only patients who had a pretreatment PET/CT (P-PET/CT) were included. P-PET/CT, interim (I-PET/CT), and end-of-treatment PET/CT (E-PET/CT) studies were reviewed. P-PET/CT results were reported using two methods of evaluation, qualitative and semi quantitative: visual assessment (VAS) and maximal standardized uptake value (SUVmax), and I-PET and E-PET results were reported by Deauville 5-point score (DS) evaluation as well. Avidity of PET/CT was defined as abnormal uptake in any of these methods. The primary outcome was the prognostic role of P-PET/CT, I-PET/CT, and E-PET/CT on progression-free survival (PFS) and overall survival (OS). Data of 196 patients with MZL were identified, 110 of which had P-PET/CT and were included in this analysis. Median age was 67 years (range 18-93). The median follow-up period was 63 months (range 3-278). The median OS and PFS for the whole cohort were 63 (interquartile range 39-85) and 60 (interquartile range 37-76) months, respectively. The avidity of PET at baseline for the whole cohort was 70% (77/110 patients), for MALT lymphoma, 62.5% (40/64 patients), for NMZL, 76.4% (13/17 patients), and for SMZL, 82.7% (24/29 patients). When adjusted for IPI, sex, and comorbidities, positive E-PET/CT was associated with reduced PFS with a hazard ratio (HR) of 3.4 (95% CI, 1.27-9.14, P = 0.02). Positive E-PET/CT did not correlate with OS. However, there were only three events. P-PET/CT was not predictive of PFS or OS. Our study demonstrates that above 70% of MZL are FDG avid. Positive E-PET/CT is a strong prognostic factor for PFS.


Asunto(s)
Glucosa-6-Fosfato/análogos & derivados , Infecciones por Helicobacter , Helicobacter pylori , Linfoma de Células B de la Zona Marginal , Tomografía de Emisión de Positrones , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Supervivencia sin Enfermedad , Femenino , Glucosa-6-Fosfato/administración & dosificación , Infecciones por Helicobacter/diagnóstico por imagen , Infecciones por Helicobacter/mortalidad , Infecciones por Helicobacter/terapia , Humanos , Linfoma de Células B de la Zona Marginal/diagnóstico por imagen , Linfoma de Células B de la Zona Marginal/mortalidad , Linfoma de Células B de la Zona Marginal/terapia , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Estudios Retrospectivos , Tasa de Supervivencia
5.
Cell Mol Neurobiol ; 39(3): 415-434, 2019 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-30771196

RESUMEN

Glucose and glutamine are two essential ingredients for cell growth. Glycolysis and glutaminolysis can be linked by glutamine: fructose-6-phosphate aminotransferase (GFAT, composed of GFAT1 and GFAT2) that catalyzes the synthesis of glucosamine-6-phosphate and glutamate by using fructose-6-phosphate and glutamine as substrates. The role of mammalian target of rapamycin (MTOR, composed of MTOR1 and MTOR2) in regulating glycolysis has been explored in human cancer cells. However, whether MTOR can interact with GFAT to regulate glucosamine-6-phosphate is poorly understood. In this study, we report that GFAT1 is essential to maintain the malignant features of GBM cells. And MTOR2 rather than MTOR1 plays a robust role in promoting GFAT1 protein activity, and accelerating the progression of glucosamine-6-phosphate synthesis, which is not controlled by the PI3K/AKT signaling. Intriguingly, high level of glucose or glutamine supply promotes MTOR2 protein activity. In turn, up-regulating glycolytic and glutaminolytic metabolisms block MTOR dimerization, enhancing the release of MTOR2 from the MTOR complex. As a transcriptional factor, C-MYC, directly targeted by MTOR2, promotes the relative mRNA expression level of GFAT1. Notably, our data reveal that GFAT1 immunoreactivity is positively correlated with the malignant grades of glioma patients. Kaplan-Meier assay reveals the correlations between patients' 5-year survival and high GFAT1 protein expression. Taken together, we propose that the MTOR2/C-MYC/GFAT1 axis is responsible for the modulation on the crosstalk between glycolysis and glutaminolysis in GBM cells. Under the condition of accelerated glycolytic and/or glutaminolytic metabolisms, the MTOR2/C-MYC/GFAT1 axis will be up-regulated in GBM cells.


Asunto(s)
Glioblastoma/metabolismo , Glucosamina/análogos & derivados , Glucosa-6-Fosfato/análogos & derivados , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Glutamina/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Antígenos de Neoplasias/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/patología , Glucosamina/biosíntesis , Glucosa/metabolismo , Glucosa-6-Fosfato/biosíntesis , Humanos , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica , Fosfatidilinositol 3-Quinasas/metabolismo , Multimerización de Proteína , Proteínas Proto-Oncogénicas c-akt/metabolismo
6.
Biotechnol J ; 14(3): e1800264, 2019 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-30105781

RESUMEN

Glucosamine-6-phosphate N-acetyltransferase (GNA1) that catalyzes acetyl transfer from acetyl-coenzyme A to glucosamine-6-phosphate (GlcN-6P), and glutamine-fructose-6-phosphate aminotransferase (GlmS) that catalyzes the formation of GlcN-6P from fructose-6-phosphate (Fru-6P), are two key enzymes in Bacillus subtilis for the bioproduction of N-acetylglucosamine (GlcNAc), a nutraceutical that has various applications in healthcare. In this study, the expression of GNA1 and GlmS is fine-tuned by 5'-terminus fusion engineering to improve GlcNAc production. Specifically, the expression level of GNA1 is enhanced at the translational level via fusion of an epitope tag to the 5'-terminus of GNA1 gene and ribosome binding site (RBS) sequence engineering. Next, enhanced expression of GlmS is achieved at the transcriptional and translational levels by fusing an mRNA stabilizer to the 5'-terminus of GlmS gene. Under the control of GNA1 (fusion with cMyc tag and with the optimum RBS M-Rm) and GlmS (fusion with mRNA stabilizer ΔermC+14/7A), the GlcNAc titer and yield in the shake flask increase to 18.5 g L-1 and 0.37 g GlcNAc/g glucose, which are 2.9-fold and 2.3-fold that of the control, respectively. This synthetic pathway fine-tuning method at the transcriptional and translational levels by combinatorial modulation of regulatory elements, including epitope tag, RBS sequence, and mRNA stabilizer, might represent a general and effective approach for the construction of microbial cell factories.


Asunto(s)
Acetilglucosamina/genética , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Glucosamina 6-Fosfato N-Acetiltransferasa/genética , Sitios de Unión/genética , Glucosamina/análogos & derivados , Glucosamina/genética , Glucosa/genética , Glucosa-6-Fosfato/análogos & derivados , Glucosa-6-Fosfato/genética , Ingeniería Metabólica/métodos , Biosíntesis de Proteínas/genética , ARN Mensajero/genética , Ribosomas/genética , Transcripción Genética/genética
7.
PLoS One ; 13(12): e0208861, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30532215

RESUMEN

BACKGROUND: The aim of this study was to investigate the metabolism of the spleen, bone marrow (BM), and liver from preoperative F-18 FDG PET/CT scans for the prediction of recurrence in breast cancer. METHODS: We retrospectively included 153 patients diagnosed with invasive ductal carcinoma (IDC) of the breast who underwent preoperative F-18 FDG PET/CT scan and a curative operation. The mean standardized uptake value (SUVmean) of the spleen, liver, and BM and maximum SUV (SUVmax) of primary tumors were measured. The relationships between spleen, BM, and liver metabolism and clinicopathologic parameters were evaluated, and possible prognostic parameters predicting recurrence were assessed using disease-free survival (DFS). RESULTS: Spleen SUVmean was significantly correlated with primary tumor SUVmax, pathologic T (pT) stage, and histologic grade of primary tumor. BM SUVmean also showed a positive correlation with primary tumor SUVmax. Spleen SUVmean were significantly associated with recurrence from binary logistic regression analysis (P = 0.004). Spleen, BM, liver, and primary tumor SUVs were all significant prognostic factors for DFS in univariate Cox regression analysis (all P<0.024). Among all PET parameters analyzed, spleen SUVmean ≥ 2.21 (P = 0.032) was in the multivariable analysis the powerful poor prognostic factor predicting DFS that was independent of other clinicopathological features like T stage (pT >2; P = 0.009) and estrogen receptor (ER) status (ER negativity; P = 0.001). CONCLUSION: Splenic metabolism together with pT stage and ER status was an independent prognostic factor for predicting recurrence in breast cancer. Metabolic activity of reticuloendothelial system such as spleen, liver or BM on preoperative F-18 FDG PET/CT can be a meritorious imaging factor for discriminating patients with IDC that require adjunctive therapy to prevent recurrence.


Asunto(s)
Neoplasias de la Mama , Carcinoma Ductal de Mama , Glucosa-6-Fosfato/análogos & derivados , Sistema Mononuclear Fagocítico , Recurrencia Local de Neoplasia , Tomografía de Emisión de Positrones , Tomografía Computarizada por Rayos X , Adulto , Anciano , Médula Ósea/diagnóstico por imagen , Médula Ósea/metabolismo , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Carcinoma Ductal de Mama/diagnóstico por imagen , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/mortalidad , Femenino , Glucosa-6-Fosfato/administración & dosificación , Glucosa-6-Fosfato/farmacocinética , Humanos , Hígado/diagnóstico por imagen , Hígado/metabolismo , Persona de Mediana Edad , Sistema Mononuclear Fagocítico/diagnóstico por imagen , Sistema Mononuclear Fagocítico/metabolismo , Metástasis de la Neoplasia , Recurrencia Local de Neoplasia/diagnóstico por imagen , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/mortalidad , Valor Predictivo de las Pruebas , Cuidados Preoperatorios , Bazo/diagnóstico por imagen , Bazo/metabolismo
8.
Sci Rep ; 8(1): 16949, 2018 11 16.
Artículo en Inglés | MEDLINE | ID: mdl-30446667

RESUMEN

Saccharomyces cerevisiae can transport and phosphorylate glucosamine, but cannot grow on this amino sugar. While an enzyme catalyzing the reaction from glucosamine-6-phosphate to fructose-6-phosphate, necessary for glucosamine catabolism, is present in yeasts using N-acetylglucosamine as carbon source, a sequence homology search suggested that such an enzyme is absent from Saccharomyces cerevisiae. The gene YlNAG1 encoding glucosamine-6-phosphate deaminase from Yarrowia lipolytica was introduced into S. cerevisiae and growth in glucosamine tested. The constructed strain grew in glucosamine as only carbon and nitrogen source. Growth on the amino sugar required respiration and caused an important ammonium excretion. Strains overexpressing YlNAG1 and one of the S. cerevisiae glucose transporters HXT1, 2, 3, 4, 6 or 7 grew in glucosamine. The amino sugar caused catabolite repression of different enzymes to a lower extent than that produced by glucose. The availability of a strain of S. cerevisiae able to grow on glucosamine opens new possibilities to investigate or manipulate pathways related with glucosamine metabolism in a well-studied organism.


Asunto(s)
Carbono/metabolismo , Glucosamina/metabolismo , Ingeniería Metabólica/métodos , Nitrógeno/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Acetilglucosamina/metabolismo , Isomerasas Aldosa-Cetosa/genética , Isomerasas Aldosa-Cetosa/metabolismo , Amino Azúcares/metabolismo , Fructosafosfatos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glucosamina/análogos & derivados , Glucosa-6-Fosfato/análogos & derivados , Glucosa-6-Fosfato/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Yarrowia/enzimología , Yarrowia/genética
9.
Molecules ; 23(10)2018 Oct 06.
Artículo en Inglés | MEDLINE | ID: mdl-30301217

RESUMEN

A series of novel pyridine and fused pyridine derivatives have been prepared starting from 6-(3,4-dimethylphenyl)-2-hydrazinyl-4-(thiophen-2-yl)-pyridine-3-carbonitrile 1 which on treatment with appropriate formic acid, acetic acid/ acetic anhydride, benzoyl chloride and/or carbon disulfide afforded the corresponding triazolopyridine derivatives 2⁻5. Also, treatment of hydrazide 1 with diethyloxalate, chloroacetyl chloride, chloroacetic acid and/or 1,2-dichloroethane yielded the corresponding pyridotriazine derivatives 7⁻10. Further transformation of compound 1 with a different active methylene group, namely acetyl acetone, diethylmalonate, ethyl cyanoacetate, ethyl benzoylacetate and/or ethyl acetoacetate, produced the pyridine⁻pyrazole hybrid derivatives 11⁻15. These newly synthesized compounds (1⁻15) were subjected to in silico molecular docking screenings towards GlcN-6-P synthase as the target protein. The results revealed moderate to good binding energies of the ligands on the target protein. All the newly prepared products exhibited antimicrobial and antioxidant activity.


Asunto(s)
Antibacterianos/síntesis química , Antiinfecciosos/síntesis química , Pirazoles/síntesis química , Piridinas/química , Ácido Acético/síntesis química , Ácido Acético/química , Antibacterianos/química , Antibacterianos/farmacología , Antiinfecciosos/química , Antiinfecciosos/farmacología , Benzoatos/química , Disulfuro de Carbono/síntesis química , Disulfuro de Carbono/química , Formiatos/síntesis química , Formiatos/química , Glucosamina/análogos & derivados , Glucosamina/química , Glucosa-6-Fosfato/análogos & derivados , Glucosa-6-Fosfato/química , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Pirazoles/química , Pirazoles/farmacología , Piridinas/síntesis química , Piridinas/farmacología , Relación Estructura-Actividad
10.
PLoS One ; 13(9): e0204918, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30265736

RESUMEN

PURPOSE: To evaluate the potential role of 18F-fluorodeoxyglucose-positron emission tomography/computerized tomography (FDG-PET/CT) for predicting treatment response after radiotherapy (RT) in patients with spinal metastases. MATERIALS AND METHODS: A retrospective analysis was performed of 42 patients with spinal metastases who received RT from January 2010 to December 2014. All patients underwent FDG-PET/CT before and after treatment. Changes in metabolic responses, expressed as the maximum, mean, peak standardized uptake values (SUVmax, SUVmean, SUVpeak), metabolic tumor volume (MTV) and total lesion glycolysis (TLG) were analyzed to determine their association with clinical outcomes. RESULTS: The median age at the time of spinal metastasis diagnosis was 58 years. Median progression-free survival (PFS) and overall survival after RT were 15 months and 22.4 months, respectively. RT produced a significant decrease in SUVmean (2.27 to 1.41), SUVmax (6.87 to 2.99), SUVpeak (5.75 to 2.33) and TLG (52.84 to 24.17) when compared with the baseline values (p<0.001). The mean pain score decreased from 3.86 before RT to 0.79 after RT (p<0.001). There were significant linear relationships between maximum SUV and pain scores at baseline (r = 0.321, p = 0.038) and after treatment (r = 0.369, p = 0.016) as well as TLG at baseline (r = 0.428, p = 0.005) and after treatment (r = 0.403, p = 0.009). Local progression after treatment was identified in 12 patients (28.6%). Univariate analyses showed that >70% reduction in maximum SUV after treatment was independently associated with good PFS (p = 0.036). CONCLUSIONS: RT is an effective treatment for patients with spinal metastases, and there were significant changes in PET parameters compared with baseline. The metabolic response measured by SUV and TLG changes in FDG-PET/CT correlated with the clinical outcomes, especially with shorter PFS in patients who had higher residual maximum SUV after treatment.


Asunto(s)
Glucosa-6-Fosfato/análogos & derivados , Tomografía de Emisión de Positrones , Neoplasias de la Columna Vertebral , Tomografía Computarizada por Rayos X , Adulto , Anciano , Anciano de 80 o más Años , Supervivencia sin Enfermedad , Femenino , Glucosa-6-Fosfato/administración & dosificación , Humanos , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Estudios Retrospectivos , Neoplasias de la Columna Vertebral/diagnóstico por imagen , Neoplasias de la Columna Vertebral/mortalidad , Neoplasias de la Columna Vertebral/radioterapia , Neoplasias de la Columna Vertebral/secundario , Tasa de Supervivencia
11.
ACS Synth Biol ; 7(10): 2423-2435, 2018 10 19.
Artículo en Inglés | MEDLINE | ID: mdl-30138558

RESUMEN

Bacillus subtilis is a typical industrial microorganism and is widely used in industrial biotechnology, particularly for nutraceutical production. There are many studies on the static metabolic engineering of B. subtilis, whereas there are few reports on dynamic metabolic engineering due to the lack of appropriate elements. Here, we established a dynamic reprogramming strategy for reconstructing metabolic networks in B. subtilis, using a typical nutraceutical, N-acetylglucosamine (GlcNAc), as a model product and the glmS (encoding glucosamine-6-phosphate synthase) ribozyme as an engineering element. First, a trp terminator was introduced to effectively release the glmS ribozyme feedback inhibition. Further, we engineered the native glucosamine-6-phosphate (GlcN6P) responsive glmS ribozyme switch to dynamically control the metabolic flux in B. subtilis for overproduction of GlcNAc. With GlcN6P as a ligand, the native sensor glmS ribozyme is integrated at the 5'- of phosphoglucosamine mutase and 6-phosphofructokinase genes to decrease the flux dynamically toward the peptidoglycan synthesis and glycolysis pathway, respectively. The glmS ribozyme mutant M5 ( glmS ribozyme cleavage site AG → GG) with decreased ribozyme activity is integrated at the 5'- of glucose-6-phosphate isomerase gene to increase the flux dynamically toward the GlcNAc synthesis pathway. This strategy increased the GlcNAc titer from 9.24 to 18.45 g/L, and the specific GlcNAc productivity from 0.53 to 1.21 g GlcNAc/g cell. Since GlcN6P is involved in the biosynthesis of various products, here the developed strategy for multiple target dynamic engineering of metabolic pathways can be generally used in B. subtilis and other industrial microbes for chemical production.


Asunto(s)
Acetilglucosamina/metabolismo , Bacillus subtilis/metabolismo , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/genética , Ingeniería Metabólica/métodos , ARN Catalítico/genética , Acetilglucosamina/análisis , Bacillus subtilis/genética , Cromatografía Líquida de Alta Presión , Glucosamina/análogos & derivados , Glucosamina/metabolismo , Glucosa-6-Fosfato/análogos & derivados , Glucosa-6-Fosfato/metabolismo , Glucosa-6-Fosfato Isomerasa/genética , Glucosa-6-Fosfato Isomerasa/metabolismo , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Peptidoglicano/biosíntesis , ARN Catalítico/metabolismo
12.
PLoS One ; 13(8): e0202482, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30125303

RESUMEN

PURPOSE: Previously, fluorodeoxy glucose conjugated magnetite nanoparticles (FDG-mNPs) injected into cancer cells in conjunction with the application of magnetic hyperthermia have shown promise in new FDG-mNPs applications. The aim of this study was to determine potential toxic or unwanted effects involving both tumour cells and normal tissue in other organs when FDG-mNPs are administered intravenously or intratumourally in mice. MATERIALS AND METHODS: FDG-mNPs were synthesized. A group of six prostate-tumour bearing mice were injected with 23.42 mg/ml FDG-mNPs (intravenous injection, n = 3; intratumoural injection into the prostate tumour, n = 3). Mice were euthanized and histological sampling of tissue was conducted for the prostate tumour, as well as for lungs, lymph nodes, liver, kidneys, spleen, and brain, at 1 hour (n = 2) and 7 days (n = 4) post-injection. A second group of two normal (non-cancerous) mice received the same injection intravenously into the tail vein and were euthanised at 3 and 6 months post-injection, respectively, to investigate if FDG-mNPs remained in organs at those time points. RESULTS: In prostate-tumour bearing mice, FDG-mNPs concentrated in the prostate tumour, while relatively small amounts were found in the organs of other tissues, particularly the spleen and the liver; FDG-mNP concentrations decreased over time in all tissues. In normal mice, no detrimental effects were found in either mouse at 3 or 6 months. CONCLUSION: Intravenous or intratumoural FDG-mNPs can be safely administered for effective cancer cell destruction. Further research on the clinical utility of FDG-mNPs will be conducted by applying hyperthermia in conjunction with FDG-mNPs in mice.


Asunto(s)
Glucosa-6-Fosfato/análogos & derivados , Hipertermia Inducida , Nanopartículas de Magnetita/uso terapéutico , Neoplasias Experimentales/terapia , Neoplasias de la Próstata/terapia , Animales , Glucosa-6-Fosfato/farmacocinética , Glucosa-6-Fosfato/farmacología , Masculino , Ratones , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Especificidad de Órganos , Proyectos Piloto , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología
13.
Biochem J ; 475(15): 2547-2557, 2018 08 16.
Artículo en Inglés | MEDLINE | ID: mdl-29967067

RESUMEN

N-acetylphosphoglucosamine mutase (AGM1) is a key component of the hexosamine biosynthetic pathway that produces UDP-GlcNAc, an essential precursor for a wide range of glycans in eukaryotes. AGM belongs to the α-d-phosphohexomutase metalloenzyme superfamily and catalyzes the interconversion of N-acetylglucosamine-6-phosphate (GlcNAc-6P) to N-acetylglucosamine-1-phosphate (GlcNAc-1P) through N-acetylglucosamine-1,6-bisphosphate (GlcNAc-1,6-bisP) as the catalytic intermediate. Although there is an understanding of the phosphoserine-dependent catalytic mechanism at enzymatic and structural level, the identity of the requisite catalytic base in AGM1/phosphoglucomutases is as yet unknown. Here, we present crystal structures of a Michaelis complex of AGM1 with GlcNAc-6P and Mg2+, and a complex of the inactive Ser69Ala mutant together with glucose-1,6-bisphosphate (Glc-1,6-bisP) that represents key snapshots along the reaction co-ordinate. Together with mutagenesis, these structures reveal that the phosphate group of the hexose-1,6-bisP intermediate may act as the catalytic base.


Asunto(s)
Acetilglucosamina/análogos & derivados , Aspergillus fumigatus/enzimología , Proteínas Fúngicas/química , Glucosa-6-Fosfato/análogos & derivados , Fosfoglucomutasa/química , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Catálisis , Proteínas Fúngicas/metabolismo , Glucosa-6-Fosfato/química , Glucosa-6-Fosfato/metabolismo , Fosfoglucomutasa/metabolismo
14.
Ann Biomed Eng ; 46(7): 1001-1012, 2018 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-29644496

RESUMEN

Inter-subject networks are used to model correlations between brain regions and are particularly useful for metabolic imaging techniques, like 18F-2-deoxy-2-(18F)fluoro-D-glucose (FDG) positron emission tomography (PET). Since FDG PET typically produces a single image, correlations cannot be calculated over time. Little focus has been placed on the basic properties of inter-subject networks and if they are affected by group size and image normalization. FDG PET images were acquired from rats (n = 18), normalized by whole brain, visual cortex, or cerebellar FDG uptake, and used to construct correlation matrices. Group size effects on network stability were investigated by systematically adding rats and evaluating local network connectivity (node strength and clustering coefficient). Modularity and community structure were also evaluated in the differently normalized networks to assess meso-scale network relationships. Local network properties are stable regardless of normalization region for groups of at least 10. Whole brain-normalized networks are more modular than visual cortex- or cerebellum-normalized network (p < 0.00001); however, community structure is similar at network resolutions where modularity differs most between brain and randomized networks. Hierarchical analysis reveals consistent modules at different scales and clustering of spatially-proximate brain regions. Findings suggest inter-subject FDG PET networks are stable for reasonable group sizes and exhibit multi-scale modularity.


Asunto(s)
Cerebelo/diagnóstico por imagen , Glucosa-6-Fosfato/análogos & derivados , Modelos Neurológicos , Red Nerviosa/diagnóstico por imagen , Tomografía de Emisión de Positrones , Corteza Visual/diagnóstico por imagen , Animales , Cerebelo/fisiología , Glucosa-6-Fosfato/farmacología , Red Nerviosa/fisiología , Ratas , Corteza Visual/fisiología
15.
Theranostics ; 8(7): 1956-1965, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29556367

RESUMEN

Although metabolic modulation in the tumor microenvironment (TME) is one of the key mechanisms of cancer immune escape, there is a lack of understanding of the comprehensive immune landscape of the TME and its association with tumor metabolism based on clinical evidence. We aimed to investigate the relationship between the immune landscape in the TME and tumor glucose metabolism in lung adenocarcinoma. Methods: Using RNA sequencing and image data, we developed a transcriptome-based tumor metabolism estimation model. The comprehensive TME cell types enrichment scores and overall immune cell enrichment (ImmuneScore) were estimated. Subjects were clustered by cellular heterogeneity in the TME and the clusters were characterized by tumor glucose metabolism and immune cell composition. Moreover, the prognostic value of ImmuneScore, tumor metabolism and the cell type-based clusters was also evaluated. Results: Four clusters were identified based on the cellular heterogeneity in the TME. They showed distinct immune cell composition, different tumor metabolism, and close relationship with overall survival. A cluster with high regulatory T cells showed relative hypermetabolism and poor prognosis. Another cluster with high mast cells and CD4+ central memory T cells showed relative hypometabolism and favorable prognosis. A cluster with high ImmuneScore showed favorable prognosis. Multivariate Cox analysis demonstrated that ImmuneScore was a predictive prognostic factor independent of other clinical features. Conclusions: Our results showed the association between predicted tumor metabolism and immune cell composition in the TME. Our studies also suggest that tumor glucose metabolism and immune cell infiltration in the TME can be clinically applicable for developing a prognostic stratification model.


Asunto(s)
Adenocarcinoma del Pulmón/diagnóstico por imagen , Perfilación de la Expresión Génica , Glucosa/metabolismo , Inmunidad Celular , Neoplasias Pulmonares/diagnóstico por imagen , Tomografía de Emisión de Positrones , Microambiente Tumoral , Adenocarcinoma del Pulmón/mortalidad , Adenocarcinoma del Pulmón/patología , Técnicas de Apoyo para la Decisión , Glucosa-6-Fosfato/administración & dosificación , Glucosa-6-Fosfato/análogos & derivados , Humanos , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Pronóstico , Análisis de Secuencia de ARN , Análisis de Supervivencia
16.
J Bacteriol ; 200(10)2018 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-29507091

RESUMEN

Most organisms, from Bacteria to Eukarya, synthesize UDP-N-acetylglucosamine (UDP-GlcNAc) from fructose-6-phosphate via a four-step reaction, and UDP-N-acetylgalactosamine (UDP-GalNAc) can only be synthesized from UDP-GlcNAc by UDP-GlcNAc 4-epimerase. In Archaea, the bacterial-type UDP-GlcNAc biosynthetic pathway was reported for Methanococcales. However, the complete biosynthetic pathways for UDP-GlcNAc and UDP-GalNAc present in one archaeal species are unidentified. Previous experimental analyses on enzymatic activities of the ST0452 protein, identified from the thermophilic crenarchaeon Sulfolobus tokodaii, predicted the presence of both a bacterial-type UDP-GlcNAc and an independent UDP-GalNAc biosynthetic pathway in this archaeon. In the present work, functional analyses revealed that the recombinant ST2186 protein possessed an glutamine:fructose-6-phosphate amidotransferase activity and that the recombinant ST0242 protein possessed a phosphoglucosamine-mutase activity. Along with the acetyltransferase and uridyltransferase activities of the ST0452 protein, the activities of the ST2186 and ST0242 proteins confirmed the presence of a bacterial-type UDP-GlcNAc biosynthetic pathway in S. tokodaii In contrast, the UDP-GlcNAc 4-epimerase homologue gene was not detected within the genomic data. Thus, it was expected that galactosamine-1-phosphate or galactosamine-6-phosphate (GalN-6-P) was provided by conversion of glucosamine-1-phosphate or glucosamine-6-phosphate (GlcN-6-P). A novel epimerase converting GlcN-6-P to GalN-6-P was detected in a cell extract of S. tokodaii, and the N-terminal sequence of the purified protein indicated that the novel epimerase was encoded by the ST2245 gene. Along with the ST0242 phosphogalactosamine-mutase activity, this observation confirmed the presence of a novel UDP-GalNAc biosynthetic pathway from GlcN-6-P in S. tokodaii Discovery of the novel pathway provides a new insight into the evolution of nucleotide sugar metabolic pathways.IMPORTANCE In this work, a novel protein capable of directly converting glucosamine-6-phosphate to galactosamine-6-phosphate was successfully purified from a cell extract of the thermophilic crenarchaeon Sulfolobus tokodaii Confirmation of this novel activity using the recombinant protein indicates that S. tokodaii possesses a novel UDP-GalNAc biosynthetic pathway derived from glucosamine-6-phosphate. The distributions of this and related genes indicate the presence of three different types of UDP-GalNAc biosynthetic pathways: a direct pathway using a novel enzyme and two conversion pathways from UDP-GlcNAc using known enzymes. Additionally, Crenarchaeota species lacking all three pathways were found, predicting the presence of one more unknown pathway. Identification of these novel proteins and pathways provides important insights into the evolution of nucleotide sugar biosynthesis, as well as being potentially important industrially.


Asunto(s)
Acetilgalactosamina/biosíntesis , Proteínas Arqueales/metabolismo , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Fosfoglucomutasa/metabolismo , Sulfolobus/enzimología , Uridina Difosfato N-Acetilglucosamina/biosíntesis , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Proteínas Arqueales/genética , Vías Biosintéticas , Galactosamina/análogos & derivados , Galactosamina/metabolismo , Glucosamina/análogos & derivados , Glucosamina/metabolismo , Glucosa-6-Fosfato/análogos & derivados , Glucosa-6-Fosfato/metabolismo , Glucofosfatos/metabolismo , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/genética , Fosfatos/metabolismo , Fosfoglucomutasa/genética , Sulfolobus/genética
17.
J Clin Exp Hematop ; 58(1): 10-16, 2018 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-29415976

RESUMEN

Recent studies have revealed the clinical and biological features of stage I follicular lymphoma (FL), but information about patients with stage I FL who underwent total resection after tissue biopsy is limited. Among 305 FL patients diagnosed between 2001 and 2013, clinical stage I disease was observed in 36 patients. Of these, 18 patients underwent total resection after diagnostic tissue biopsy. We used 18F-fluorodeoxyglucose positron emission CT for staging assessment in 13 of 18 patients (72.2%). The median age was 56.5 years. Six patients (33.3%) were male. The soluble interleukin-2 receptor alpha concentration was significantly lower than in patients with residual disease. Among these 18 patients, 7 patients (38.9%) were treated with a "watch-and-wait" (WW) policy, 7 (38.9%) were treated with involved-field irradiation, and 4 (22.2%) received systemic chemotherapy. Patients with resected disease were treated with significantly different strategies from those with residual disease (p = 0.0026). Five patients experienced relapse during follow-up (median follow-up: 48.2 months). All relapses were distant from the primary site, irrespective of treatment strategy. Among all stage I patients, disease resection was not a significant factor for survival (p = 0.9294). Collectively, the choice of treatment strategy was significantly influenced by patient status. Resection status was not significantly associated with survival after several treatment strategies.


Asunto(s)
Glucosa-6-Fosfato/análogos & derivados , Linfoma Folicular , Tomografía de Emisión de Positrones , Anciano , Supervivencia sin Enfermedad , Femenino , Glucosa-6-Fosfato/administración & dosificación , Humanos , Linfoma Folicular/diagnóstico por imagen , Linfoma Folicular/mortalidad , Linfoma Folicular/cirugía , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasia Residual , Tasa de Supervivencia
19.
J Bacteriol ; 200(5)2018 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-29229699

RESUMEN

Amino sugars are good sources of both ammonia and fructose-6-phosphate, produced by the glucosamine 6-phosphate deaminase, NagB. NagB is known to be allosterically regulated by N-acetylglucosamine 6-phosphate (GlcNAc-6P) and the phosphocarrier protein of the bacterial phosphotransferase system, HPr, in Escherichia coli We provide evidence that NanE, GlcNAc-6P epimerase, and the uridylylated PII protein (U-PII) also allosterically activate NagB by direct protein-protein interactions. NanE is essential for neuraminic acid (NANA) and N-acetylmannosamine (ManNAc) utilization, and PII is known to be a central metabolic nitrogen regulator. We demonstrate that uridylylated PII (but not underivatized PII) activates NagB >10-fold at low concentrations of substrate, whereas NanE increases NagB activity >2-fold. NanE activates NagB in the absence or presence of GlcNAc-6P, but HPr and U-PII activation requires the presence of GlcNAc-6P. Activation of NagB by HPr and uridylylated PII, as well as by NanE and HPr (but not by NanE and U-PII), is synergistic, and the modeling, which suggests specific residues involved in complex formation, provides possible explanations. Specific physiological functions for the regulation of NagB by its three protein activators are proposed. Each regulatory agent is suggested to mediate signal transduction in response to a different stimulus.IMPORTANCE The regulation of amino sugar utilization is important for the survival of bacteria in a competitive environment. NagB, a glucosamine 6-phosphate deaminase in Escherichia coli, is essential for amino sugar utilization and is known to be allosterically regulated by N-acetylglucosamine 6-phosphate (GlcNAc-6P) and the histidine-phosphorylatable phosphocarrier protein, HPr. We provide evidence here that NanE, GlcNAc-6P epimerase, and the uridylylated PII protein allosterically activate NagB by direct protein-protein interactions. NanE is essential for N-acetylneuraminic acid (NANA) and N-acetylmannosamine (ManNAc) utilization, and the PII protein is known to be a central metabolic nitrogen regulator. Regulatory links between carbon and nitrogen metabolism are important for adaptation of metabolism to different growth conditions.


Asunto(s)
Acetilglucosamina/análogos & derivados , Isomerasas Aldosa-Cetosa/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Proteínas PII Reguladoras del Nitrógeno/genética , Racemasas y Epimerasas/genética , Acetilglucosamina/metabolismo , Acetilglucosamina/farmacología , Isomerasas Aldosa-Cetosa/efectos de los fármacos , Isomerasas Aldosa-Cetosa/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Glucosamina/análogos & derivados , Glucosamina/metabolismo , Glucosa-6-Fosfato/análogos & derivados , Glucosa-6-Fosfato/metabolismo , Hexosaminas/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Nitrógeno/metabolismo , Proteínas PII Reguladoras del Nitrógeno/metabolismo , Fosforilación , Mapeo de Interacción de Proteínas , Racemasas y Epimerasas/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/metabolismo
20.
Contrast Media Mol Imaging ; 2017: 3179607, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29114175

RESUMEN

Purpose: This study evaluated the potential of 68Ga-citrate positron emission tomography/computed tomography (PET/CT) for the detection of infectious foci in patients with Staphylococcus aureus bacteraemia by comparing it with 2-[18F]fluoro-2-deoxy-D-glucose (18F-FDG) PET/CT. Methods: Four patients admitted to hospital due to S. aureus bacteraemia underwent both 18F-FDG and 68Ga-citrate whole-body PET/CT scans to detect infectious foci. Results: The time from hospital admission and the initiation of antibiotic treatment to the first PET/CT was 4-10 days. The time interval between 18F-FDG and 68Ga-citrate PET/CT was 1-4 days. Three patients had vertebral osteomyelitis (spondylodiscitis) and one had osteomyelitis in the toe; these were detected by both 18F-FDG (maximum standardised uptake value [SUVmax] 6.0 ± 1.0) and 68Ga-citrate (SUVmax 6.8 ± 3.5, P = 0.61). Three patients had soft tissue infectious foci, with more intense 18F-FDG uptake (SUVmax 6.5 ± 2.5) than 68Ga-citrate uptake (SUVmax 3.9 ± 1.2, P = 0.0033). Conclusions: Our small cohort of patients with S. aureus bacteraemia revealed that 68Ga-citrate PET/CT is comparable to 18F-FDG PET/CT for detection of osteomyelitis, whereas 18F-FDG resulted in a higher signal for the detection of soft tissue infectious foci.


Asunto(s)
Bacteriemia/diagnóstico por imagen , Medios de Contraste/administración & dosificación , Gadolinio/administración & dosificación , Glucosa-6-Fosfato/análogos & derivados , Osteomielitis/diagnóstico por imagen , Tomografía de Emisión de Positrones , Infecciones Estafilocócicas/diagnóstico por imagen , Staphylococcus aureus , Anciano , Anciano de 80 o más Años , Citratos/administración & dosificación , Femenino , Glucosa-6-Fosfato/administración & dosificación , Humanos , Masculino
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