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Background The impact of preoperative breast MRI on the long-term outcomes in patients with breast cancer who are 35 years and younger has not been established. Purpose To evaluate the impact of preoperative breast MRI on recurrence-free survival (RFS) and overall survival (OS) in women with breast cancer who are 35 years and younger by using propensity score matching. Materials and Methods A total of 708 women who were 35 years and younger (mean age, 32 years ± 3 [SD]) and diagnosed with breast cancer from 2007 to 2016 were retrospectively identified. Patients who underwent preoperative MRI (MRI group) were matched with those who did not (no MRI group) according to 23 patient and tumor characteristics. RFS and OS were compared using the Kaplan-Meier method. Cox proportional hazards regression analysis was used to estimate the hazard ratios (HRs). Results Of 708 women, 125 patient pairs were matched. In the MRI group versus the no MRI group, the mean follow-up time was 82 months ± 32 versus 106 months ± 42, and the rates of total recurrence and death were 22% (104 of 478 patients) versus 29% (66 of 230 patients) and 5% (25 of 478 patients) versus 12% (28 of 230 patients), respectively. The time to recurrence was 44 months ± 33 in the MRI group and 56 months ± 42 in the no MRI group. After propensity score matching, the MRI and no MRI groups did not show significant differences in total recurrence (HR, 1.0; P = .99), local-regional recurrence (HR, 1.3; P = .42), contralateral breast recurrence (HR, 0.7; P = .39), or distant recurrence (HR, 0.9; P = .79). The MRI group showed a tendency toward better OS, but this was not statistically significant (HR, 0.47; P = .07). In the entire unmatched cohort, MRI was not an independent significant factor for predicting RFS or OS. Conclusion Preoperative breast MRI was not a significant prognostic factor for recurrence-free survival in women 35 years and younger with breast cancer. A tendency toward better overall survival was observed in the MRI group, but this was not significant. © RSNA, 2023 Supplemental material is available for this article. See also the editorial by Kim and Moy in this issue.
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Neoplasias de la Mama , Humanos , Femenino , Adulto , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/cirugía , Neoplasias de la Mama/patología , Estudios Retrospectivos , Mama/diagnóstico por imagen , Mama/cirugía , Mama/patología , Imagen por Resonancia Magnética/métodos , Radiografía , Recurrencia Local de Neoplasia/patologíaRESUMEN
Background Background parenchymal enhancement (BPE) is a known risk factor for breast cancer. However, studies on the association between BPE and second breast cancer risk are still lacking. Purpose To investigate whether BPE at surveillance breast MRI is associated with subsequent second breast cancer risk in women with a personal history of breast cancer. Materials and Methods A retrospective search of the imaging database of an academic medical center identified consecutive surveillance breast MRI examinations performed between January 2008 and December 2017 in women who underwent surgery for primary breast cancer and had no prior diagnosis of second breast cancer. BPE at surveillance breast MRI was qualitatively assessed using a four-category classification of minimal, mild, moderate, or marked. Future second breast cancer was defined as ipsilateral breast tumor recurrence or contralateral breast cancer diagnosed at least 1 year after each surveillance breast MRI examination. Factors associated with future second breast cancer risk were evaluated using the multivariable Fine-Gray subdistribution hazard model. Results Among the 2668 women (mean age at baseline surveillance breast MRI, 49 years ± 8 [SD]), 109 developed a second breast cancer (49 ipsilateral, 58 contralateral, and two ipsilateral and contralateral) at a median follow-up of 5.8 years. Mild, moderate, or marked BPE at surveillance breast MRI (hazard ratio [HR], 2.1 [95% CI: 1.4, 3.1]; P < .001), young age (<45 years) at initial breast cancer diagnosis (HR, 3.4 [95% CI: 1.7, 6.4]; P < .001), positive results from a BRCA1/2 genetic test (HR, 6.5 [95% CI: 3.5, 12.0]; P < .001), and negative hormone receptor expression in the initial breast cancer (HR, 1.6 [95% CI: 1.1, 2.6]; P = .02) were independently associated with an increased risk of future second breast cancer. Conclusion Background parenchymal enhancement at surveillance breast MRI was associated with future second breast cancer risk in women with a personal history of breast cancer. © RSNA, 2022 Online supplemental material is available for this article. See also the editorial by Niell in this issue.
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Neoplasias de la Mama , Femenino , Humanos , Persona de Mediana Edad , Neoplasias de la Mama/patología , Estudios Retrospectivos , Recurrencia Local de Neoplasia/patología , Mama/patología , Imagen por Resonancia Magnética/métodosRESUMEN
Background Few studies have compared abbreviated breast MRI with full-protocol MRI in women with a personal history of breast cancer (PHBC), and they have not adjusted for confounding variables. Purpose To compare abbreviated breast MRI with full-protocol MRI in women with PHBC by using propensity score matching to adjust for confounding variables. Materials and Methods In this single-center retrospective study, women with PHBC who underwent full-protocol MRI (January 2008-August 2017) or abbreviated MRI (September 2017-April 2019) were identified. With use of a propensity score-matched cohort, screening performances were compared between the two MRI groups with the McNemar test or a propensity score-adjusted generalized estimating equation. The coprimary analyses were sensitivity and specificity. The secondary analyses were the cancer detection rate, interval cancer rate, positive predictive value for biopsies performed (PPV3), and Breast Imaging Reporting and Data System (BI-RADS) category 3 short-term follow-up rate. Results There were 726 women allocated to each MRI group (mean age ± SD, 50 years ± 8 for both groups). Abbreviated MRI and full-protocol MRI showed comparable sensitivity (15 of 15 cancers [100%; 95% CI: 78, 100] vs nine of 13 cancers [69%; 95% CI: 39, 91], respectively; P = .17). Abbreviated MRI showed higher specificity than full-protocol MRI (660 of 711 examinations [93%; 95% CI: 91, 95] vs 612 of 713 examinations [86%; 95% CI: 83, 88], respectively; P < .001). The cancer detection rate (21 vs 12 per 1000 examinations), interval cancer rate (0 vs five per 1000 examinations), and PPV3 (61% [14 of 23 examinations] vs 41% [nine of 22 examinations]) were comparable (all P < .05). The BI-RADS category 3 short-term follow-up rate of abbreviated MRI was less than half that of full-protocol MRI (5% [36 of 726 examinations] vs 12% [84 of 726 examinations], respectively; P < .001). Ninety-three percent (14 of 15) of cancers detected at abbreviated MRI were node-negative T1-invasive cancers (n = 6) or ductal carcinoma in situ (n = 8). Conclusion Abbreviated breast MRI showed comparable sensitivity and superior specificity to full-protocol MRI in breast cancer detection in women with a personal history of breast cancer. © RSNA, 2022 Online supplemental material is available for this article.
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Neoplasias de la Mama , Imagen por Resonancia Magnética , Biopsia , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/diagnóstico por imagen , Carcinoma Intraductal no Infiltrante/diagnóstico , Carcinoma Intraductal no Infiltrante/diagnóstico por imagen , Femenino , Humanos , Imagen por Resonancia Magnética/métodos , Persona de Mediana Edad , Estudios Retrospectivos , Sensibilidad y EspecificidadRESUMEN
Background The role of preoperative MRI in women 35 years of age or younger with breast cancer remains controversial. Purpose To determine the association between preoperative MRI and surgical outcomes in women aged 35 years or younger with breast cancer by using propensity score (PS) analysis to investigate the impact of preoperative MRI. Materials and Methods Women 35 years of age or younger diagnosed with breast cancer between 2007 and 2017 who had or had not undergone preoperative breast MRI were retrospectively identified. The MRI detection rate of additional suspicious lesions was analyzed, and changes in surgical management were recorded. Inverse probability weighting (IPW) and PS matching were used to adjust 19 variables and to create a balance between the two groups. Surgical outcomes were compared by using univariable logistic regression. Results Among 964 women (mean age ± standard deviation, 32 years ± 3), 665 (69%) had undergone preoperative MRI (MRI group; mean age, 32 years ± 3) and 299 (31%) had not (no-MRI group; mean age, 32 years ± 3). In the MRI group, additional suspicious lesions were found in 178 of the 665 women (27%), with 88 of those 178 women (49%) having malignant lesions. The surgical management was changed in 99 of the 665 women (15%) due to MRI findings, which was appropriate for 62 of those 99 women (63%). In the IPW analysis, the MRI group showed lower odds of repeat surgery (odds ratio [OR], 0.13; 95% CI: 0.07, 0.21; P < .001) and higher odds of initial mastectomy (OR, 1.62; 95% CI: 1.17, 2.25; P = .004). However, there was no difference in the overall mastectomy rate (OR, 1.24; 95% CI: 0.91, 1.68; P = .17) compared with the no-MRI group. These results were consistent when using the PS matching method. Conclusion Preoperative MRI in young women with breast cancer is useful for detecting additional malignancy and improving surgical outcomes by reducing the repeat surgery rate, with a similar likelihood of overall mastectomy. © RSNA, 2021 Online supplemental material is available for this article.
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Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/cirugía , Imagen por Resonancia Magnética/métodos , Mastectomía/métodos , Cuidados Preoperatorios/métodos , Adolescente , Adulto , Mama/diagnóstico por imagen , Mama/cirugía , Femenino , Humanos , Puntaje de Propensión , Estudios Retrospectivos , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND: Lactulose, a synthetic disaccharide, has received increasing interest due to its role as a prebiotic, specifically proliferating Bifidobacilli and Lactobacilli and enhancing absorption of calcium and magnesium. The use of cellobiose 2-epimerase (CE) is considered an interesting alternative for industrial production of lactulose. CE reversibly converts D-glucose residues into D-mannose residues at the reducing end of unmodified ß-1,4-linked oligosaccharides, including ß-1,4-mannobiose, cellobiose, and lactose. Recently, a few CE 3D structure were reported, revealing mechanistic details. Using this information, we redesigned the substrate binding site of CE to extend its activity from epimerization to isomerization. RESULTS: Using superimposition with 3 known CE structure models, we identified 2 residues (Tyr114, Asn184) that appeared to play an important role in binding epilactose. We modified these residues, which interact with C2 of the mannose moiety, to prevent epimerization to epilactose. We found a Y114E mutation led to increased release of a by-product, lactulose, at 65 °C, while its activity was low at 37 °C. Notably, this phenomenon was observed only at high temperature and more reliably when the substrate was increased. Using Y114E, isomerization of lactose to lactulose was investigated under optimized conditions, resulting in 86.9 g/l of lactulose and 4.6 g/l of epilactose for 2 h when 200 g/l of lactose was used. CONCLUSION: These results showed that the Y114E mutation increased isomerization of lactose, while decreasing the epimerization of lactose. Thus, a subtle modification of the active site pocket could extend its native activity from epimerization to isomerization without significantly impairing substrate binding. While additional studies are required to scale this to an industrial process, we demonstrated the potential of engineering this enzyme based on structural analysis.
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Carbohidrato Epimerasas/química , Carbohidrato Epimerasas/metabolismo , Celobiosa/química , Celobiosa/metabolismo , Bacterias Grampositivas/enzimología , Ingeniería de Proteínas/métodos , Proteínas Bacterianas/metabolismo , Sitios de Unión , Bacterias Grampositivas/genética , Bacterias Grampositivas/metabolismo , Calor , Microbiología Industrial/métodos , Isomerismo , Lactosa/genética , Lactosa/metabolismo , Lactulosa/biosíntesis , Lactulosa/química , Lactulosa/metabolismo , Manosa/metabolismo , Oligosacáridos/metabolismo , Prebióticos , Dominios Proteicos , Especificidad por SustratoRESUMEN
Since Pt-based catalysts have the disadvantages of high cost, large overpotential loss, and limited long-term stability, there have been various promising alternatives to Pt-based catalysts to improve the catalytic activity towards the oxygen reduction reaction (ORR). We have synthesized iron-nitrogen-doped mesoporous tungsten carbide catalysts (WC-m-FT) by pyrolysis of well-ordered mesoporous tungsten carbides with iron porphyrin. WC-m-FT exhibits excellent ORR catalytic activity in an alkaline medium, i.e. a high electron-transfer number as well as superior stability and methanol tolerance. The improved activity and stability of WC-m-FT are ascribed to iron-containing catalytic active sites surrounded by nitrogen species and the well-defined mesoporous tungsten carbide structure.
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OBJECTIVE: To investigate whether reader training improves the performance and agreement of radiologists in interpreting unenhanced breast magnetic resonance imaging (MRI) scans using diffusion-weighted imaging (DWI). MATERIALS AND METHODS: A study of 96 breasts (35 cancers, 24 benign, and 37 negative) in 48 asymptomatic women was performed between June 2019 and October 2020. High-resolution DWI with b-values of 0, 800, and 1200 sec/mm² was performed using a 3.0-T system. Sixteen breast radiologists independently reviewed the DWI, apparent diffusion coefficient maps, and T1-weighted MRI scans and recorded the Breast Imaging Reporting and Data System (BI-RADS) category for each breast. After a 2-h training session and a 5-month washout period, they re-evaluated the BI-RADS categories. A BI-RADS category of 4 (lesions with at least two suspicious criteria) or 5 (more than two suspicious criteria) was considered positive. The per-breast diagnostic performance of each reader was compared between the first and second reviews. Inter-reader agreement was evaluated using a multi-rater κ analysis and intraclass correlation coefficient (ICC). RESULTS: Before training, the mean sensitivity, specificity, and accuracy of the 16 readers were 70.7% (95% confidence interval [CI]: 59.4-79.9), 90.8% (95% CI: 85.6-94.2), and 83.5% (95% CI: 78.6-87.4), respectively. After training, significant improvements in specificity (95.2%; 95% CI: 90.8-97.5; P = 0.001) and accuracy (85.9%; 95% CI: 80.9-89.8; P = 0.01) were observed, but no difference in sensitivity (69.8%; 95% CI: 58.1-79.4; P = 0.58) was observed. Regarding inter-reader agreement, the κ values were 0.57 (95% CI: 0.52-0.63) before training and 0.68 (95% CI: 0.62-0.74) after training, with a difference of 0.11 (95% CI: 0.02-0.18; P = 0.01). The ICC was 0.73 (95% CI: 0.69-0.74) before training and 0.79 (95% CI: 0.76-0.80) after training (P = 0.002). CONCLUSION: Brief reader training improved the performance and agreement of interpretations by breast radiologists using unenhanced MRI with DWI.
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Neoplasias de la Mama , Femenino , Humanos , Neoplasias de la Mama/diagnóstico por imagen , Imagen de Difusión por Resonancia Magnética , Imagen por Resonancia Magnética , Mama/diagnóstico por imagen , RadiólogosRESUMEN
Immunotherapy has a number of advantages over traditional anti-tumor therapy but can cause severe adverse reactions due to an overactive immune system. In contrast, a novel metabolic treatment approach can induce metabolic vulnerability through multiple cancer cell targets. Here, we show a therapeutic effect by inducing nucleotide imbalance and apoptosis in triple negative breast cancer cells (TNBC), by treating with cytosolic thymidylate 5'-phosphohydrolase (CT). We show that a sustained consumption of dTMP by CT could induce dNTP imbalance, leading to apoptosis as tricarboxylic acid cycle intermediates were depleted to mitigate this imbalance. These cytotoxic effects appeared to be different, depending on substrate specificity of the 5' nucleotide or metabolic dependency of the cancer cell lines. Using representative TNBC cell lines, we reveal how the TNBC cells were affected by CT-transfection through extracellular acidification rate (ECAR)/oxygen consumption rate (OCR) analysis and differential transcription/expression levels. We suggest a novel approach for treating refractory TNBC by an mRNA drug that can exploit metabolic dependencies to exacerbate cell metabolic vulnerability.
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Neoplasias de la Mama Triple Negativas , Humanos , Neoplasias de la Mama Triple Negativas/patología , Timidina Monofosfato , Línea Celular Tumoral , Apoptosis , Monoéster Fosfórico HidrolasasRESUMEN
This study aimed to evaluate the imaging and pathological findings in axillary lymph nodes in patients with breast cancer who received concurrent ipsilateral coronavirus disease 2019 (COVID-19) vaccination. Of the 19 women with breast cancer who received concurrent COVID-19 vaccination shot in the arm ipsilateral to breast cancer, axillary lymphadenopathy was observed in 84.2% (16 of 19) of patients on ultrasound (US) and 71.4% (10 of 14) of patients on magnetic resonance imaging (MRI), and 21.0% (4 of 19) of patients were diagnosed with metastasis. Abnormal US and MRI findings of cortical thickening, effacement of the fatty hilum, round shape, and asymmetry in the number or size relative to the contralateral side were noted in more than half of the non-metastatic and metastatic lymph nodes; however, statistical significance was not noted. Axillary lymphadenopathy is commonly observed in patients with breast cancer who receive concurrent ipsilateral COVID-19 vaccination without specific differential imaging features. Thus, understanding the limitations of axillary imaging and cautious interpretation is necessary to avoid overestimation or underestimation of the axillary disease burden.
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A recombinant beta-galactosidase from Caldicellulosiruptor saccharolyticus was purified with a specific activity of 211 U mg(-1) by using heat treatment and His-trap affinity chromatography. The native enzyme was an 80-kDa trimer with a molecular mass of 240 kDa. Maximum activity was observed at pH 6.0 and 80 degrees C, and the half-life at 70 degrees C was 48 h. The enzyme exhibited hydrolytic activity for p-nitrophenyl-beta-D: -galactopyranoside (pNPGal), oNPGal, or lactose, whereas no activity for p-nitrophenyl-beta-D: -glucopyranoside (pNPGlu), oNPGlu, or cellobiose. The catalytic residues E150 and E311 of beta-galactosidase from C. saccharolyticus were completely conserved in all aligned glycoside hydrolase family 42 beta-galactosidases. The results indicated that the enzyme was a beta-galactosidase. Galactose uncompetitively inhibited the enzyme. Glucose inhibition of the enzyme was the lowest among beta-galactosidases. When 50 g l(-1) galactose was added, the enzyme activity for pNPGal was reduced to 26%. When 400 g l(-1) glucose instead of galactose was added, the activity was reduced to 82%. When adding galactose (200 g l(-1)), only 14% of the lactose was hydrolyzed after 180 min. In contrast, the addition of glucose (400 g l(-1)) did not affect lactose hydrolysis, and more than 99% of the lactose was hydrolyzed after 120 min.
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Galactosa/farmacología , Glucosa/farmacología , Bacterias Grampositivas/enzimología , Lactosa/metabolismo , beta-Galactosidasa/metabolismo , Secuencia de Aminoácidos , Bacterias Anaerobias/enzimología , Bacterias Anaerobias/genética , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Catálisis , Cromatografía de Afinidad , Clonación Molecular , Estabilidad de Enzimas , Escherichia coli/genética , Bacterias Grampositivas/genética , Semivida , Calor , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Datos de Secuencia Molecular , Peso Molecular , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , beta-Galactosidasa/antagonistas & inhibidores , beta-Galactosidasa/genética , beta-Galactosidasa/aislamiento & purificaciónRESUMEN
Galacto-oligosaccharides have become the focus of a great deal of attention in the field of functional foods, owing to their known health benefits and potential to improve the quality of many foods. Because of these properties, they are currently used as low-calorie sweeteners in fermented milk products, confectioneries, breads, and beverages. In this article, the characterization and product inhibition of many microbial beta-galactosidases with transgalactosylation activities, their galacto-oligosaccharide production from lactose, and the application of galacto-oligosaccharides are reviewed. A method for increasing galacto-oligosaccharide production by reducing product inhibition properties via the immobilization and structure modification of the enzymes is also introduced herein.
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Lactosa/metabolismo , Oligosacáridos/metabolismo , beta-Galactosidasa/metabolismo , Bacterias/enzimología , Catálisis , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacocinética , Hongos/enzimología , Galactosa/metabolismo , Galactosa/farmacología , Glucosa/metabolismo , Glucosa/farmacología , Glicosilación , Cinética , Oligosacáridos/química , Temperatura , beta-Galactosidasa/antagonistas & inhibidoresRESUMEN
A recombinant putative beta-galactosidase from Thermoplasma acidophilum was purified as a single 57 kDa band of 82 U mg(-1). The molecular mass of the native enzyme was 114 kDa as a dimer. Maximum activity was observed at pH 6.0 and 90 degrees C. The enzyme was unstable below pH 6.0: at pH 6 its half-life at 75 degrees C was 28 days but at pH 4.5 was only 13 h. Catalytic efficiencies decreased as p-nitrophenyl(pNP)-beta-D-fucopyranoside (1067) > pNP-beta-D-glucopyranoside (381) > pNP-beta-D-galactopyranoside (18) > pNP-beta-D-mannopyranoside (11 s(-1) mM(-1)), indicating that the enzyme was a beta-glycosidase.
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Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Thermoplasma/aislamiento & purificación , beta-Glucosidasa/química , beta-Glucosidasa/metabolismo , Proteínas Arqueales/aislamiento & purificación , Dimerización , Estabilidad de Enzimas , Semivida , Concentración de Iones de Hidrógeno , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Temperatura , beta-Glucosidasa/aislamiento & purificaciónRESUMEN
CRM197, which retains the same inflammatory and immune-stimulant properties as diphtheria toxin but with reduced toxicity, has been used as a safe carrier in conjugated vaccines. Expression of recombinant CRM197 in E. coli is limited due to formation of inclusion bodies. Soluble expression attempts in Bacillus subtilis, P. fluorescens, Pichia pastoris, and E. coli were partially unsuccessful or did not generate yields sufficient for industrial scale production. Multiple approaches have been attempted to produce CRM197 in E. coli, which has attractive features such as high yield, simplicity, fast growth, etc., including expression of oxidative host, concurrent expression of chaperones, or periplasmic export. Recently, alternative methods for recovery of insoluble proteins expressed in E. coli were reported. Compared to traditional denaturation/refolding, these methods used the non-denaturing solubilization agent, N-lauroylsarkosine to obtain higher recovery yields of native proteins. Based on this work, here, we focused on solubilization of CRM197 from E. coli inclusion bodies. First, CRM197 was expressed as inclusion bodies by high-level expression of recombinant CRM197 in E. coli (126.8 mg/g dcw). Then bioactive CRM197 was isolated from these inclusion bodies with high yield (108.1 mg/g dcw) through solubilization with N-lauroylsarkosine including Triton X-100 and CHAPS, and purified by Ni-affinity chromatography and size-exclusion chromatography. In this study, we present a cost-effective alternative for the production of bioactive CRM197 and compare our recovery yield with yields in other production processes.
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Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/citología , Escherichia coli/genética , Cuerpos de Inclusión/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Bacterianas/química , Clonación Molecular , Expresión Génica , Proteínas Recombinantes/química , SolubilidadRESUMEN
ß-Glucosidase, which hydrolyzes cellobiose into two glucoses, plays an important role in the process of saccharification of the lignocellulosic biomass. In this study, we optimized the activity of ß-glucosidase of brown-rot fungus Fomitopsis pinicola KCTC 6208 using the response surface methodology (RSM) with various concentrations of glucose, yeast extract and ascorbic acid, which are the most significant nutrients for activity of ß-glucosidase. The highest activity of ß-glucosidase was achieved 3.02% of glucose, 4.35% of yeast extract, and 7.41% ascorbic acid where ascorbic acid was most effective. The maximum activity of ß-glucosidase predicted by the RSM was 15.34 U/mg, which was similar to the experimental value 14.90 U/mg at the 16th day of incubation. This optimized activity of ß-glucosidase was 23.6 times higher than the preliminary activity value, 0.63 U/mg, and was also much higher than previous values reported in other fungi strains. Therefore, a simplified medium supplemented with a cheap vitamin source, such as ascorbic acid, could be a cost effective mean of increasing ß-glucosidase activity.
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A ß-glucosidase from Penicillium italicum was purified with a specific activity of 61.8 U/mg, using a chromatography system. The native form of the enzyme was an 88.5-kDa tetramer with a molecular mass of 354 kDa. Optimum activity was observed at pH 4.5 and 60â, and the half-lives were 1,737, 330, 34, and 1 hr at 50, 55, 60, and 65â, respectively. Its activity was inhibited by 47% by 5 mM Ni(2+). The enzyme exhibited hydrolytic activity for p-nitrophenyl-ß-D-glucopyranoside (pNP-Glu), p-nitrophenyl-ß-D-cellobioside, p-nitrophenyl-ß-D-xyloside, and cellobiose, however, no activity was observed for p-nitrophenyl-ß-D-lactopyranoside, p-nitrophenyl-ß-D-galactopyranoside, carboxymetyl cellulose, xylan, and cellulose, indicating that the enzyme was a ß-glucosidase. The k(cat)/K(m) (s(-1) mM(-1)) values for pNP-Glu and cellobiose were 15,770.4 mM and 6,361.4 mM, respectively. These values were the highest reported for ß-glucosidases. Non-competitive inhibition of the enzyme by both glucose (K(i) = 8.9 mM) and glucono-δ-lactone (K(i) = 11.3 mM) was observed when pNP-Glu was used as the substrate. This is the first report of non-competitive inhibition of ß-glucosidase by glucose and glucono-δ-lactone.
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A beta-glucosidase from Phoma sp. KCTC11825BP isolated from rotten mandarin peel was purified 8.5-fold with a specific activity of 84.5 U/mg protein. The purified enzyme had a molecular mass of 440 kDa with a subunit of 110 kDa. The partial amino acid sequence of the purified beta-glucosidase evidenced high homology with the fungal beta- glucosidases belonging to glycosyl hydrolase family 3. Its optimal activity was detected at pH 4.5 and 60 degrees C, and the enzyme had a half-life of 53 h at 60 degrees C. The Km values for p-nitrophenyl-beta-D-glucopyranoside and cellobiose were 0.3 mM and 3.2 mM, respectively. The enzyme was competitively inhibited by both glucose (Ki=1.7 mM) and glucono-delta-lactone (Ki=0.1 mM) when pNPG was used as the substrate. Its activity was inhibited by 41% by 10 mM Cu2+ and stimulated by 20% by 10 mM Mg2+.
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Ascomicetos/enzimología , Citrus sinensis/microbiología , Proteínas Fúngicas/química , Proteínas Fúngicas/aislamiento & purificación , beta-Glucosidasa/química , beta-Glucosidasa/aislamiento & purificación , Ascomicetos/clasificación , Ascomicetos/genética , Ascomicetos/aislamiento & purificación , Citrus sinensis/química , Estabilidad de Enzimas , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Cinética , Datos de Secuencia Molecular , Peso Molecular , Filogenia , Especificidad por Sustrato , beta-Glucosidasa/genética , beta-Glucosidasa/metabolismoRESUMEN
We expressed a putative beta-galactosidase from Sulfolobus acidocaldarius in Escherichia coli and purified the recombinant enzyme using heat treatment and Hi-Trap ion-exchange chromatography. The resultant protein gave a single 57-kDa band by SDS-PAGE and had a specific activity of 58 U/mg. The native enzyme existed as a dimer with a molecular mass of 114 kDa by gel filtration. The maximum activity of this enzyme was observed at pH 5.5 and 90 degrees C. The half-lives of the enzyme at 70, 80, and 90 degrees C were 494, 60, and 0.2 h, respectively. The hydrolytic activity with p-nitrophenyl(pNP) substrates followed the order p-nitrophenyl-beta-D-fucopyranoside > pNP-beta-D-glucopyranoside > pNP-beta-D-galactopyranoside > pNP-beta-D-mannopyranoside > pNP-beta-D-xylopyranoside, but not toward aryl-alpha-glycosides or pNP-beta-L-arabinofuranoside. Thus, the enzyme was actually a beta-glycosidase. The beta-glycosidase exhibited transglycosylation activity with pNP-beta-D-galactopyranoside, pNP-beta-D-glucopyranoside, and pNP-beta-D-fucopyranoside in decreasing order of activity, in the reverse order of its hydrolytic activity. The hydrolytic activity was higher toward cellobiose than toward lactose, but the transglycosylation activity was lower with cellobiose than with lactose.
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Proteínas Arqueales/metabolismo , Glucosidasas/metabolismo , Proteínas Recombinantes/metabolismo , Sulfolobus acidocaldarius/enzimología , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas Arqueales/aislamiento & purificación , Celobiosa/metabolismo , Glucosidasas/química , Glucosidasas/genética , Glucosidasas/aislamiento & purificación , Glicosilación , Concentración de Iones de Hidrógeno , Hidrólisis , Lactosa/metabolismo , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , TemperaturaRESUMEN
Factor XI deficiency (also called Hemophilia C) rarely occurs among ethnicities other than Ashkenazi Jews. A boy was scheduled for frontoethmoidectomy due to bilateral chronic rhinosinusitis. He was incidentally found to have factor XI deficiency due to prolonged aPTT on preoperative laboratory finding. His medical history reveals frequent epistaxis 2 or 3 times per day and his factor XI and XII activity were 17% (normal; 60-140%) and 34% (normal; 60-140%), respectively on furthermore laboratory evaluation. He was diagnosed as hereditary factor XI deficiency. He underwent the operation with administration of the fresh frozen plasma without complication.