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1.
Nano Lett ; 23(5): 1904-1913, 2023 03 08.
Artículo en Inglés | MEDLINE | ID: mdl-36801829

RESUMEN

Cancer vaccines have received tremendous attention in cancer immunotherapy due to their capability to induce a tumor-specific immune response. However, their effectiveness is compromised by the insufficient spatiotemporal delivery of antigens and adjuvants in the subcellular level to induce a robust CD8+ T cell response. Herein, a cancer nanovaccine G5-pBA/OVA@Mn is prepared through multiple interactions of manganese ions (Mn2+), benzoic acid (BA)-modified fifth generation polyamidoamine (G5-PAMAM) dendrimer, and the model protein antigen ovalbumin (OVA). In the nanovaccine, Mn2+ not only exerts a structural function to assist OVA loading as well as its endosomal escape, but works as an adjuvant of stimulator of interferon genes (STING) pathway. These collaboratively facilitate the orchestrated codelivery of OVA antigen and Mn2+ into cell cytoplasm. Vaccination with G5-pBA/OVA@Mn not only shows a prophylactic effect, but also significantly inhibits growth against B16-OVA tumors, indicating its great potential for cancer immunotherapy.


Asunto(s)
Vacunas contra el Cáncer , Nanopartículas , Neoplasias , Humanos , Animales , Ratones , Manganeso , Antígenos , Adyuvantes Inmunológicos/uso terapéutico , Neoplasias/terapia , Inmunoterapia , Ratones Endogámicos C57BL , Nanopartículas/química , Células Dendríticas
2.
Arch Insect Biochem Physiol ; 108(2): e21836, 2021 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-34288123

RESUMEN

In Asian rice systems, Cyrtorhinus lividipennis Reuter is an important predator that preys on rice planthopper eggs and young nymphs, as a primary food source. Alanine aminotransferase (ALT) acts in many physiological and biochemical processes in insects. We cloned the full-length complementary DNA of C. lividipennis ClALT. Expression analysis showed higher expression in the fat body and midgut compared to other tissues. It is expressed in all C. lividipennis developmental stages and at least four organs. Silencing of ClALT by RNA interference significantly decreased the ClALT enzyme activity and ClALT expression compared to dsGFP-treated controls at 2 days after emergence (DAE). Silencing of ClALT influenced free hemolymph amino acid compositions, resulting in a reduction of Aspartic acid (Asp) and Alanine (Ala) proportions, and increased Cysteine (Cys) and Valine (Val) proportions in females at 2 DAE. dsClALT treatments led to decreased soluble total protein concentrations in ovary and fat body, and to lower reduced vitellogenin (Vg) expression, body weight, and the numbers of laid eggs. The double-stranded RNA viruse treatments also led to prolonged preoviposition periods and hindered ovarian development. Western blot analysis indicated that silencing ClALT also led to reduced fat body Vg protein abundance at 2 DAE. These data support our hypothesis that ClALT influences amino acid metabolism and fecundity in C. lividipennis.


Asunto(s)
Alanina Transaminasa , Aminoácidos/metabolismo , Fertilidad , Heterópteros , Alanina Transaminasa/genética , Alanina Transaminasa/metabolismo , Aminoácidos/genética , Animales , Hemolinfa/metabolismo , Heterópteros/genética , Heterópteros/metabolismo , Heterópteros/fisiología , Proteínas de Insectos/metabolismo , Interferencia de ARN , Vitelogeninas/metabolismo
3.
Pestic Biochem Physiol ; 153: 176-184, 2019 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-30744893

RESUMEN

Hexokinase is a rate-limiting enzyme that plays pivotal roles in glucose homeostasis and energy metabolism via glucose (Glc) phosphorylation and Glc signaling mediation. Previous investigations have revealed the modulatory role of Hexokinase (Hex) genes involved in proper glucose regulation during insect diapause and embryo development, whereas whether it functions in insect fecundity remains largely unknown. We aimed to explore the relationship between Triazophos (TZP)-induced Hex-1 and fecundity of female Nilaparvata lugens. In this study, Hex-1 expression were characterized at different developmental stages and in various tissues of N. lugens, with the highest expression registered in brain tissues and 5th instar nymph. The present findings indicated that TZP + dsHex-1 silencing significantly reduced protein synthesis, including the fat body and ovarian protein content of female adults. Meanwhile, the glycometabolism with respect to the soluble sugar, trehalose and glucose content in female adults were strikingly influenced as a result of Hex-1 knockdown. The relative transcript level of Hex-1, vitellogenin (NlVg) and vitellogenin receptor (NlVgR) considerably decreased in TZP + dsHex-1 treated females compared to TZP and TZP + dsGFP-treated groups. More importantly, TZP + dsHex-1 silencing led to reduced number of eggs laid and vitellogenin (Vg) accumulation as well as retarded ovary development compared with TZP-treated and TZP + dsGFP-treated groups. Taken together, it is proposed that Hex-1 implicates in N. lugens fecundity by exerting profound effects on glycometabolism, protein sythesis and NlVg expression.


Asunto(s)
Fertilidad/efectos de los fármacos , Hemípteros/efectos de los fármacos , Hexoquinasa/genética , Proteínas de Insectos/genética , Insecticidas/toxicidad , Organotiofosfatos/toxicidad , Triazoles/toxicidad , Animales , Femenino , Fertilidad/genética , Hemípteros/fisiología , Proteínas de Insectos/metabolismo , Masculino , Interferencia de ARN
4.
Artículo en Inglés | MEDLINE | ID: mdl-29114912

RESUMEN

Pyruvate kinase (PYK) operates in the glycolytic pathway, responsible for regulating the balance between glycolysis and gluconeogenesis. The previous work indicates PYK acts in development of Drosophila embryos and in embryonic muscle growth, from which it may be inferred that PYK acts in insect fecundity. More to the point, as a central enzyme in the glycolytic pathway, PYK acts in many energy-spending functions in most organisms. On the background findings that triazophos (TZP) stimulates fecundity via increase activities of several genes in brown planthoppers, Nilaparvata lugens, we investigated the combined influence of TZP and silencing a N. lugens PYK (NlPYK) on reproduction-linked biological performance parameters. Here, we report that TZP+dsNlPYK treatments led to reduced (by 26%) ovarian, but not fat body, protein content relative to controls. Ovarian (35%) and fat body (54%) soluble sugar contents were reduced. TZP+dsNlPYK treatments also led to reduced (by about 24%) fecundity, expressed as numbers of eggs laid. These data show directly that NlPYK acts in insect fecundity, probably via increases in glucose metabolism.


Asunto(s)
Hemípteros/enzimología , Óvulo/crecimiento & desarrollo , Piruvato Quinasa/metabolismo , Animales , Metabolismo de los Hidratos de Carbono , Femenino , Fertilidad , Proteínas de Insectos/metabolismo , Masculino , Organotiofosfatos , Interferencia de ARN , Reproducción , Triazoles
5.
Chem Biodivers ; 14(11)2017 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-28672071

RESUMEN

Citrus essential oils (CEOs) are important flavors in the food and confectionary industries. A lipase process was proposed for enhancing the flavor profiles and increasing the proportions of esters in CEOs. The effects of the enzymatic process were explored by detecting the constituents of the CEOs of American sweet orange oil (ASO) and Brazil mandarin oil (BMO) through GC/MS and sensory evaluation by a trained panel, and positive effects were confirmed by both methods. A further eleven kinds of CEOs were treated via the lipase process and increments of 10 - 1170% were achieved in the proportions of esters, which were mostly ethyl esters. Enhancement in fruity odor, especially the top note, was demonstrated by all CEOs after enzymatic processing. All CEOs were tested for antimicrobial activities, and only ASO displayed fairly ideal antimicrobial activities. Meanwhile, modified ASO showed a certain increase in antimicrobial activities. This methodology might be considered a sustainable route for acquiring 'natural' essential oils with enhanced flavor profiles and simultaneously enhancing the comprehensive utilization of citrus fruits.


Asunto(s)
Citrus/química , Citrus/enzimología , Ésteres/análisis , Lipasa/metabolismo , Aceites Volátiles/análisis , Aceites Volátiles/metabolismo , Antiinfecciosos/química , Antiinfecciosos/metabolismo , Antiinfecciosos/farmacología , Citrus/metabolismo , Ésteres/química , Ésteres/metabolismo , Aromatizantes/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Aceites Volátiles/farmacología , Análisis de Componente Principal
6.
Bioprocess Biosyst Eng ; 40(11): 1689-1699, 2017 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-28836017

RESUMEN

Lipase-displaying yeast cells are a promising alternative to the conventional immobilised lipases for organic bioconversions. However, the hydrophilic characteristics of the yeast cell surface may impede efficient immobilisation. Herein, we tested three methods to enhance the hydrophobicity of the surface of Candida antarctica lipase B-displaying Pichia pastoris cells, co-displaying a fungal hydrophobin, coating with ionic liquids, and adding decane as a hydrophobic carbon source during fermentation. Modified cells showed higher surface hydrophobicity and superior esterification of C6-C18 saturated fatty acids in hydrophobic solvents. When used for biodiesel synthesis, modified cells exhibited an improved initial reaction rate and equilibrium fatty acid methyl ester yield. We systematically discuss the influence of cell surface hydrophobicity on the catalytic properties, and the results provide guidance for improving the catalytic efficiency and operational characteristics of lipase-displaying yeast cells for organic bioconversions.


Asunto(s)
Candida/enzimología , Lipasa/metabolismo , Pichia/enzimología , Catálisis , Esterificación , Fermentación , Interacciones Hidrofóbicas e Hidrofílicas , Especificidad por Sustrato , Propiedades de Superficie
7.
Anal Biochem ; 450: 46-8, 2014 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-24406905

RESUMEN

We describe a method for quantification of displayed lipase on yeast cell surface. The strategy uses an organophosphonate ester to irreversibly inhibit the active lipase and release a detectable fluorescent group. The amount of displayed lipase can be represented as "g/g cell" or "molecules/cell". The results obtained correlated well with those obtained by existing methods. Therefore, this method is credible and will provide a powerful tool to promote research of lipase yeast surface display.


Asunto(s)
Técnicas de Visualización de Superficie Celular/métodos , Proteínas Fúngicas/genética , Lipasa/genética , Saccharomyces cerevisiae/citología , Inhibidores Enzimáticos/farmacología , Proteínas Fúngicas/antagonistas & inhibidores , Lipasa/antagonistas & inhibidores , Organofosfonatos/farmacología , Pichia/genética
8.
J Ind Microbiol Biotechnol ; 41(4): 711-20, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24519503

RESUMEN

Aspergillus niger is a recognized workhorse used to produce food processing enzymes because of its extraordinarily high protein-producing capacity. We have developed a new cell surface display system de novo in A. niger using expression elements from generally recognized as safe certified microorganisms. Candida antarctica lipase B (CALB), a widely used hydrolase, was fused to an endogenous cell wall mannoprotein, CwpA, and functionally displayed on the cell surface. Localization of CALB was confirmed by enzymatic assay and immunofluorescence analysis using laser scanning confocal microscopy. After induction by maltose for 45 h, the hydrolytic activity and synthesis activity of A. niger mycelium-surface displayed CALB (AN-CALB) reached 400 and 240 U/g dry cell, respectively. AN-CALB was successfully used as a whole-cell catalyst for the enzymatic production of ethyl esters from a series of fatty acids of different chain lengths and ethanol. In a solvent-free system, AN-CALB showed great synthetic activity and afforded high substrate mole conversions, which amounted to 87 % for ethyl hexanoate after 2 h, 89 % for ethyl laurate after 2 h, and 84 % for ethyl stearate after 3 h. These results suggested that CwpA can act as an efficient anchoring motif for displaying enzyme on A. niger, and AN-CALB is a robust, green, and cost-effective alternative food-grade whole-cell catalyst to commercial lipase.


Asunto(s)
Aspergillus niger/genética , Técnicas de Visualización de Superficie Celular/métodos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Lipasa/genética , Lipasa/metabolismo , Aspergillus niger/metabolismo , Biocatálisis , Esterificación , Industria de Procesamiento de Alimentos , Proteínas Fúngicas/análisis , Lipasa/análisis , Glicoproteínas de Membrana/genética , Micelio/química
9.
Int J Ophthalmol ; 17(2): 282-288, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38371261

RESUMEN

AIM: To define the predictive factors of severe retinopathy of prematurity (ROP) and develop a nomogram for predicting severe ROP in southeast China. METHODS: Totally 554 infants diagnosed with ROP hospitalized in the Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University and hospitalized in Taizhou Women and Children's Hospital were included. Clinical data and 43 candidate predictive factors of ROP infants were collected retrospectively. Logistic regression model was used to identify predictive factors of severe ROP and to propose a nomogram for individual risk prediction, which was compared with WINROP model and Digirop-Birth model. RESULTS: Infants from the Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University (n=478) were randomly allocated into training (n=402) and internal validation group (n=76). Infants from Taizhou Women and Children's Hospital were set as external validation group (n=76). Severe ROP were found in 52 of 402 infants, 12 of 76 infants, and 7 of 76 infants in training group, internal validation group, and external validation group, respectively. Birth weight [odds ratio (OR), 0.997; 95% confidence interval (CI), 0.996-0.999; P<0.001], multiple births (OR, 1.885; 95%CI, 1.013-3.506; P=0.045), and non-invasive ventilation (OR, 0.288; 95%CI, 0.146-0.570; P<0.001) were identified as predictive factors for the prediction of severe ROP, by univariate analysis and multivariate analysis. For predicting severe ROP based on the internal validation group, the areas under receiver operating characteristic curve (AUC) was 78.1 (95%CI, 64.2-92.0) for the nomogram, 32.9 (95%CI, 15.3-50.5) for WINROP model, 70.2 (95%CI, 55.8-84.6) for Digirop-Birth model. In external validation group, AUC of the nomogram was also higher than that of WINROP model and Digirop-Birth model (80.2 versus 51.1 and 63.4). The decision curve analysis of the nomogram demonstrated better clinical efficacy than that of WINROP model and Digirop-Birth model. The calibration curves demonstrated a good consistency between the actual severe ROP incidence and the predicted probability. CONCLUSION: Birth weight, multiple births, and non-invasive ventilation are independent predictors of severe ROP. The nomogram has a good ability to predict severe ROP and performed well on internal validation and external validation in southeast China.

10.
Biotechnol Lett ; 35(3): 367-74, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23143175

RESUMEN

A new approach is described to quantify the number of enzyme molecules, such as Candia antarctica lipase B, that are displayed on the cell surface of Pichia pastoris. Enhanced green fluorescent protein (EGFP) and Candida antarctica lipase B (CALB) were fused and displayed on the surface of P. pastoris by linking to the anchor flocculation functional domain of FLO1p from Saccharomyces cerevisiae. Confocal laser scanning microscopy, flow cytometry, and fluorescence spectrophotometry were used to monitor the fluorescence intensity of fused EGFP. Combined with the corresponding protein concentration detected in the medium, a standard curve describing the relationship between the fusion protein concentration and fluorescence intensity were obtained and could be used to number CALB displayed on the cell surface. The results showed that approx. 10(4) molecules of CALB molecules were immobilized on the single P. pastoris cell wall based on FS anchor system.


Asunto(s)
Biotecnología/métodos , Candida/enzimología , Técnicas de Visualización de Superficie Celular/métodos , Proteínas Fúngicas/análisis , Lipasa/análisis , Pichia/enzimología , Candida/genética , Estudios de Evaluación como Asunto , Fluorescencia , Proteínas Fúngicas/genética , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Lipasa/genética , Lectinas de Unión a Manosa/análisis , Lectinas de Unión a Manosa/genética , Pichia/genética , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/análisis , Proteínas de Saccharomyces cerevisiae/genética
11.
Zhonghua Yi Xue Za Zhi ; 93(41): 3280-3, 2013 Nov 05.
Artículo en Zh | MEDLINE | ID: mdl-24401623

RESUMEN

OBJECTIVE: To evaluate the effects of accommodation on lens morphological characters. METHODS: From January 2011 to June 2011, magnetic resonance images of eyes were acquired from 30 subjects aged 20 to 24 years during accommodation and at rest. The optimal images were analyzed by Autocad 2010 to obtain the total lens cross-sectional area (CSA) and CSA of anterior and posterior portions of lens, anterior chamber depth, lens thickness, lens diameter, vitreous chamber depth and axial length during accommodation and at rest. Paired-t test was performed. RESULTS: The anterior curvature radius (mm), posterior curvature radius (mm), CSA of anterior portion (mm(2)), CSA of posterior portion (mm(2)), total lens CSA (mm(2)) was (8.7 ± 0.8), (6.2 ± 0.5), (7.5 ± 2.1), (12.0 ± 2.6), (20 ± 4) during relaxed accommodation; anterior curvature radius (mm), posterior curvature radius (mm), CSA of anterior portion (mm(2)), CSA of posterior portion (mm(2)), total lens CSA (mm(2)) was (7.1 ± 1.3), (5.6 ± 0.5), (14.7 ± 2.9), (12.2 ± 2.1) and (27 ± 4) during accommodation. The total lens CSA (t = -11.556, P < 0.01) and CSA of anterior portion (t = -15.653, P < 0.01) both increased in accommodative states. The CSA of posterior portion of lens (t = -0.437, P > 0.05) under a statistically independent accommodative state. There was significant difference in the anterior chamber depth (t = 4.366, P < 0.01), lens thickness (t = -5.456, P < 0.01) and lens diameter (t = 4.597, P < 0.01) in accommodative states. There were insignificant differences both in vitreous chamber depth (t = 0.428, P > 0.05) and axial length (t = 0.418, P > 0.05) under accommodative states. CONCLUSION: During accommodation, the anterior chamber depth decreases, lens thickness increases and diameter of lens decreases while anterior portions and total lens CSA increase. There are insignificant changes in posterior portions of lens CSA, vitreous chamber depth and axial length. The accommodative changes in CSA indicate that the anterior portion of lens may be related with the properties of anterior capsule and lens material, the position of zonular attachments and the location of fetal nucleus. Helmholtz theory is supported.


Asunto(s)
Acomodación Ocular , Cristalino/anatomía & histología , Cristalino/fisiología , Adulto , Femenino , Humanos , Imagen por Resonancia Magnética , Masculino , Adulto Joven
12.
Zhonghua Yi Xue Za Zhi ; 93(45): 3573-6, 2013 Dec 03.
Artículo en Zh | MEDLINE | ID: mdl-24534304

RESUMEN

OBJECTIVE: To investigate the variations of macular thickness and peripapillary retinal nerve fiber layer (RNFL) thickness in myopic children. METHODS: A total of 96 eyes from 48 myopic children at department of Ophthalmology, Second Affiliated Hospital of Wenzhou Medical College from September 2010 to March 2012 were enrolled in this study and divided into three groups (low, moderate and high myopia group) according to the severity of myopia. Another 33 eyes from 19 emmetropic children were recruited as control group. The macular thickness and peripapillary RNFL thickness of the myopic children measured by optical coherence tomography were compared with that of the control group. RESULTS: The mean thickness of nasal, superior and inferior regions of outer-ring macular in the high myopia group were 276 µm, 294 µm, 285 µm respectively, which were significantly lower than that in the control group (P < 0.05). The mean thickness of superior of outer-ring macular in the low and moderate groups were 302 µm, 301 µm respectively, and the inferior of outer-ring ones were 288 µm, 283 µm respectively, which were significantly lower than that in the control group (P < 0.05). The temporal region of peripapillary RNFL thickness was significantly greater, and the other six regions of RNFL thicknesses were significantly lower in the high group than in the control group (P < 0.05). The central-1 mm, superior region of inner-ring, temporal and superior region of outer-ring macular thickness had positive correlations with spherical equivalent (SE) (P < 0.05). There was a negative correlation between the temporal peripapillary RNFL thickness and SE, while positive correlations were found between other regions of peripapillary RNFL thickness and SE (P < 0.05). CONCLUSIONS: The thicknesses of macular and peripapillary RNFL of myopic children have already redistributed before apparent changes of funds.


Asunto(s)
Mácula Lútea/diagnóstico por imagen , Miopía/diagnóstico por imagen , Retina/diagnóstico por imagen , Tomografía de Coherencia Óptica/métodos , Adolescente , Estudios de Casos y Controles , Niño , Preescolar , Femenino , Humanos , Masculino , Disco Óptico , Radiografía
13.
ACS Nano ; 17(16): 15905-15917, 2023 08 22.
Artículo en Inglés | MEDLINE | ID: mdl-37565626

RESUMEN

Metal-organic frameworks (MOFs) show tremendous promise for drug delivery due to their structural and functional versatility. However, MOFs are usually used as biologically inert carriers in most cases. The creation of intrinsically immunostimulatory MOFs remains challenging. In this study, a facile and green synthesis method is proposed for the preparation of a manganese ion (Mn2+)-based immunostimulatory MOF (ISAMn-MOF) for cancer metalloimmunotherapy. ISAMn-MOF significantly facilitates the activation of cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) related genes and signaling pathways in bone-marrow-derived dendritic cells (BMDCs). BMDCs treated with ISAMn-MOF secrete 4-fold higher type I interferon and 2- to 16-fold higher proinflammatory cytokines than those treated with equivalent MnCl2. ISAMn-MOF alone or its combination with immune checkpoint antibodies significantly suppresses tumor growth and metastasis and prolongs mouse survival. Mechanistic studies indicate that ISAMn-MOF treatment facilitates the infiltration of stimulatory immune cells in tumors and lymphoid organs. This study provides insight into the design of bioactive MOFs for improved cancer metalloimmunotherapy.


Asunto(s)
Estructuras Metalorgánicas , Neoplasias , Ratones , Animales , Estructuras Metalorgánicas/farmacología , Manganeso/farmacología , Proteínas de la Membrana/metabolismo , Nucleotidiltransferasas/metabolismo , Neoplasias/tratamiento farmacológico
14.
ACS Nano ; 17(10): 8966-8979, 2023 05 23.
Artículo en Inglés | MEDLINE | ID: mdl-37133900

RESUMEN

Enabling macrophages to phagocytose tumor cells holds great potential for cancer therapy but suffers from tremendous challenges because the tumor cells upregulate antiphagocytosis molecules (such as CD47) on their surface. The blockade of CD47 alone is insufficient to stimulate tumor cell phagocytosis in solid tumors due to the lack of "eat me" signals. Herein, a degradable mesoporous silica nanoparticle (MSN) is reported to simultaneously deliver anti-CD47 antibodies (aCD47) and doxorubicin (DOX) for cancer chemo-immunotherapy. The codelivery nanocarrier aCD47-DMSN was constructed by accommodating DOX within the mesoporous cavity, while adsorbing aCD47 on the surface of MSN. aCD47 blocks the CD47-SIRPα axis to disable the "don't eat me" signal, while DOX induces immunogenic tumor cell death (ICD) for calreticulin exposure as an "eat me" signal. This design facilitated the phagocytosis of tumor cells by macrophages, which enhanced antigen cross-presentation and elicited efficient T cell-mediated immune response. In 4T1 and B16F10 murine tumor models, aCD47-DMSN generated a strong antitumor effect after intravenous injection by increasing tumor-infiltration of CD8+ T cells. Taken together, this study offers a nanoplatform to modulate the phagocytosis of macrophages for efficacious cancer chemo-immunotherapy.


Asunto(s)
Nanopartículas , Neoplasias , Ratones , Animales , Calreticulina , Linfocitos T CD8-positivos , Fagocitosis , Neoplasias/metabolismo , Inmunoterapia , Antígeno CD47/metabolismo
15.
Appl Microbiol Biotechnol ; 96(6): 1539-50, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-22797600

RESUMEN

To develop a high efficiency Candida antarctica lipase B (CALB) yeast display system, we linked two CALB genes fused with Sacchromyces cerevisiae cell wall protein genes, the Sed1 and the 3'-terminal half of Sag1, separately by a 2A peptide of foot-and-mouth disease virus (FMDV) in a single open reading frame. The CALB copy number of recombinant strain KCSe2ACSa that harbored the ORF was identified, and the quantity of CALB displayed on the cell surface and the enzyme activity of the strain were measured. The results showed that the fusion of multiple genes linked by 2A peptide was translated into two independent proteins displayed on the cell surface of stain KCSe2ACSa. Judging from the data of immunolabeling assay, stain KCSe2ACSa displayed 94 % CALB-Sed1p compared with stain KCSe1 that harbored a single copy CALB-Sed1 and 64 % CALB-Sag1p compared with stain KCSa that harbored a single copy CALB-Sag1 on its surface. Besides, strain KCSe2ACSa possessed 170 % hydrolytic activity and 155 % synthetic activity compared with strain KCSe1 as well as 144 % hydrolytic activity and 121 % synthetic activity compared with strain KCSa. Strain KCSe2ACSa even owned 124 % hydrolytic activity compared with strain KCSe2 that harbored two copies CALB-Sed1. The heterogeneous glycosylphosphatidylinositol-anchored proteins co-displaying yeast system mediated by FMDV 2A peptide was shown to be an effective method for improving the efficiency of enzyme-displaying yeast biocatalysts.


Asunto(s)
Virus de la Fiebre Aftosa/genética , Proteínas Fúngicas/genética , Lipasa/genética , Péptidos/genética , Pichia/genética , Ingeniería de Proteínas/métodos , Candida/enzimología , Candida/genética , Virus de la Fiebre Aftosa/metabolismo , Proteínas Fúngicas/metabolismo , Expresión Génica , Lipasa/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Péptidos/metabolismo , Pichia/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
16.
Biotechnol Lett ; 33(12): 2431-8, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21809089

RESUMEN

To increase the activity of Rhizomucor miehei lipase (RML) in organic solvent, multiple sequence alignments and rational site-directed mutagenesis were used to create RML variants. The obtained proteins were surface-displayed on Pichia pastoris by fusion to Flo1p as an anchor protein. The synthetic activity of four variants showed from 1.1- to 5-fold the activity of native lipase in an esterification reaction in heptane with alcohol and caproic acid as substrates. The increase in esterification activity may be attributed to the four mutations changing the flexibility of RML or facilitating the reaction. In conclusion, this method demonstrated that multiple sequence alignments and rational site-directed mutagenesis combined with yeast display technology is a faster and more effective means of obtaining high-efficiency esterification lipase variants compared with previous similar methods.


Asunto(s)
Mejoramiento Genético/métodos , Lipasa/química , Mutagénesis Sitio-Dirigida/métodos , Compuestos Orgánicos/química , Rhizomucor/enzimología , Rhizomucor/genética , Solventes/química , Activación Enzimática , Estabilidad de Enzimas , Esterificación , Lipasa/metabolismo , Ingeniería de Proteínas/métodos
17.
Radiol Case Rep ; 16(8): 2256-2260, 2021 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-34178200

RESUMEN

Granulomatous inflammation is rare in the musculoskeletal system and difficult to diagnose. Here we describe a case of a 62-year-old woman with a history of being stabbed by a fishbone presented with a soreness, swelling, and limitation of movement of her right palm and wrist for 4 months. Surgery was done and the histopathology of specimens demonstrated granulomatous lesion, which was negative for acid-fast bacilli. This case demonstrates the diagnosis of granulomatous tenosynovitis on MRI, ultrasound, and surgical examination under anesthesia.

18.
Biomater Sci ; 9(7): 2508-2518, 2021 Apr 07.
Artículo en Inglés | MEDLINE | ID: mdl-33459733

RESUMEN

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. Sorafenib (sfb) is widely used in clinics for advanced HCC therapy. However, the therapeutic efficacy of sfb is suboptimal due to its poor water solubility, low bioavailability, and side effects. Here, we employed a clinically safe polymer poly(ethylene glycol)-b-poly(lactic acid) (PEG-b-PLA) to prepare a nanoparticle (NP)-based sfb formulation (NP-sfb) and tested its antitumor effect in multiple HCC models. NP-sfb could achieve effective drug loading and remain stable under physiological conditions. NP-sfb could be taken up by HepG2, Hepa1-6, and H22 cells and could efficiently inhibit cell proliferation and/or promote cell apoptosis. In vivo studies indicated that NP-sfb showed significantly improved therapeutic efficacy compared with free-sfb at the same dose or even higher doses. Mechanistic studies demonstrated that NP-sfb not only inhibited tumor proliferation and angiogenesis but also stimulated the tumor microenvironment by reducing the infiltration of immunosuppressive myeloid cells and increasing the ratio of cytotoxic T cells. This study demonstrates that the NP-based formulation is a promising strategy to improve the clinical application of sfb.


Asunto(s)
Antineoplásicos , Carcinoma Hepatocelular , Neoplasias Hepáticas , Nanopartículas , Antineoplásicos/uso terapéutico , Disponibilidad Biológica , Carcinoma Hepatocelular/tratamiento farmacológico , Línea Celular Tumoral , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Polímeros/uso terapéutico , Sorafenib , Microambiente Tumoral
19.
Appl Microbiol Biotechnol ; 86(5): 1493-501, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-20033404

RESUMEN

Two alternative cell-surface display systems were developed in Pichia pastoris using the alpha-agglutinin and Flo1p (FS) anchor systems, respectively. Both the anchor cell wall proteins were obtained originally from Saccharomyces cerevisiae. Candida antarctica lipase B (CALB) was displayed functionally on the cell surface of P. pastoris using the anchor proteins alpha-agglutinin and FS. The activity of CALB displayed on P. pastoris was tenfold higher than that of S. cerevisiae. The hydrolytic and synthetic activities of CALB fused with alpha-agglutinin and FS anchored on P. pastoris were investigated. The hydrolytic activities of both lipases displayed on yeast cells surface were more than 200 U/g dry cell after 120 h of culture (200 and 270 U/g dry cell, respectively). However, the synthetic activity of CALB fused with alpha-agglutinin on P. pastoris was threefold higher than that of the FS fusion protein when applied to the synthesis of ethyl caproate. Similarly, the CALB displayed on P. pastoris using alpha-agglutinin had a higher catalytic efficiency with respect to the synthesis of other short-chain flavor esters than that displayed using the FS anchor. Interestingly, for some short-chain esters, the synthetic activity of displaying CALB fused with alpha-agglutinin on P. pastoris was even higher than that of the commercial CALB Novozyme 435.


Asunto(s)
Biotecnología/métodos , Candida/enzimología , Aromatizantes/metabolismo , Lipasa/genética , Pichia/metabolismo , Catálisis , Clonación Molecular , Ésteres/metabolismo , Proteínas Fúngicas , Lipasa/metabolismo , Lectinas de Unión a Manosa/genética , Lectinas de Unión a Manosa/metabolismo , Factor de Apareamiento , Péptidos/genética , Péptidos/metabolismo , Pichia/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
20.
Front Physiol ; 11: 579233, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33041875

RESUMEN

The mirid bug, Cyrtorhinus lividipennis Reuter, is an important predator of rice planthoppers in Asia. In a previous study, C. lividipennis fed on gramineous weeds with brown planthopper (BPH) eggs had reduced development compared to those fed on rice with BPH eggs. In the current study, the concentrations of selected amino acids (AAs) were higher in rice than five gramineous species, which might explain the enhanced growth of C. lividipennis on rice. When C. lividipennis was fed on AA-deprived artificial diets, the Wnt/ß-catenin pathway was inhibited. Furthermore, C. lividipennis females silenced for expression of Frizzled 2 (Fz2) showed a significant reduction in the Wnt/ß-catenin and target of rapamycin (TOR) pathways. Silencing Fz2 led to decreased expression of the vitellogenin gene (Vg), lower Vg accumulation in oocytes, reduced soluble protein in ovaries and fat bodies, reduced titers of juvenile hormone, prolonged preoviposition periods, and lower predation capacity, body weight, and egg numbers as controlled to controls. Fz2 silencing resulted in undeveloped ovaries and the inhibition of oocyte growth in the ovarioles, resulting in decreased numbers of offspring and reduced hatching rates. The silencing of Fz2 also resulted in aberrant embryos with undeveloped eyespots and organs, suggesting that Fz2 is an essential gene for embryonic development, oogenesis, and egg maturation. In summary, this study established a potential link between Wnt and TOR pathways, which interact synergistically to regulate C. lividipennis reproduction in response to AA signals. These results provide valuable new information that can be applied to large-scale rearing of C. lividipennis predators, which is important for reducing planthopper damage in rice fields.

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