RESUMEN
MYH9-related disease (MYH9-RD) is an autosomal dominant disorder caused by mutations in the MYH9 gene. It is characterized by a triad of giant platelets, thrombocytopenia, and characteristic Döhle body-like granulocyte inclusions. In this study we report 10 unrelated patients with MYH9-RD in whom the following seven MYH9 gene mutations were found: W33R, p.Q1443_K1445dup, R702H, D1424N, E1841K, R1933X, and E1945X (the first two were novel mutations). The region of the MYH9 mutation determines in some regards the phenotype, but clinical expression can vary between individuals with the same mutation. The neutrophil inclusion bodies of two patients were too small to be detected, but could be found with immunofluorescence staining. Immunoblotting analysis revealed that the calculated NMMHC-IIA/ß-actin ratio for MYH9-RD neutrophils was 39% of normal controls. Kidney biopsy showed segmental glomerulosclerosis and NMMHC-IIA expression was decreased in podocytes. This disease is not as rare as originally thought. In any individual with persistent macrothrombocytopenia and no response to corticosteroids and immunosuppressive agents, even if neutrophil inclusions were inconspicuous in routine staining, MYH9-RD should be suspected.
Asunto(s)
Infarto Cerebral/genética , Trastornos de los Cromosomas/genética , Pérdida Auditiva Sensorineural/genética , Proteínas Motoras Moleculares/genética , Cadenas Pesadas de Miosina/genética , Nefritis Hereditaria/genética , Trombocitopenia/genética , Adolescente , Adulto , Plaquetas/ultraestructura , Niño , Preescolar , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Riñón/patología , Riñón/ultraestructura , Masculino , Microscopía Electrónica , Persona de Mediana Edad , Mutación , Fenotipo , Polimorfismo Genético , Síndrome , Trombocitopenia/patologíaRESUMEN
BACKGROUND: Endothelial cell protein C receptor (EPCR) is a cellular receptor for protein C and activated protein C (APC). In view of convincing evidence, it seems that EPCR, beyond its effects on coagulation and inflammation, could interfere with carcinogenesis. METHODS: In the present study, we investigated EPCR expression in 60 lung carcinoma tissues and 37 para-carcinoma tissues, and analyzed the relationship between EPCR expression and histopathological parameters, clinical parameters, and vascular density. RESULTS: In vitro, culturing lung cancer cell lines constitutively expressed EPCR at the level of mRNA and protein. The pathologic results clearly demonstrated that EPCR expression, including membranous and cytoplasmic staining, was significantly higher in carcinoma than that in the para-carcinoma tissues. The EPCR expression was therefore related to tumor size, lymph node metastasis as well as TNM stage. The expression of EPCR was also significantly correlated with microvessel density (MVD). CONCLUSIONS: These observations provide evidence that EPCR may be involved in the carcinogenesis of lung cancer. It is suggested that EPCR may be a useful biomarker for evaluating the clinical status of lung cancer.
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Antígenos CD/metabolismo , Neoplasias Pulmonares/metabolismo , Receptores de Superficie Celular/metabolismo , Anciano , Antígenos CD/genética , Línea Celular Tumoral , Receptor de Proteína C Endotelial , Femenino , Citometría de Flujo , Humanos , Inmunohistoquímica , Neoplasias Pulmonares/patología , Metástasis Linfática , Masculino , Persona de Mediana Edad , ARN Mensajero/genética , Receptores de Superficie Celular/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
OBJECTIVE: To identify and characterize a missence mutation Ser250 Phe underlying coagulation factor â ¦ (Fâ ¦) deficiency in a Chinese patient and his family. METHODS: The Fâ ¦ gene (F7) was analyzed by DNA sequencing, and the Fâ ¦ levels (including antigen and activity) in patient's plasma were determined with enzyme-linked immunoabsorbent assay (ELISA) and one stage prothrombin time based method. In addition, a Fâ ¦-250 Phe mutant corresponding to the identified mutation was expressed in HEK293 cells, and a subcellular localization experiment in CHO cells was performed to clarify the molecular mechanism of Fâ ¦ deficiency caused by the Fâ ¦-250 Phe mutation. RESULTS: The patient had a prolonged prothrombin time (PT: 36.5 s) and low levels of both Fâ ¦ antigen and activity (130.2 ng/mL and 4.0%, respectively). Two heterozygous mutations were identified in the F7 gene (NG-009262.1), which included a g.15975 G>A mutation at the splice receptor site of intron 6 (IVS6-1G>A) and a novel g.16750 C>T mutation in exon 8, which resulted in replacement of Ser (TCC) 250 with Phe (TTC)250 in the vicinity of a charge-stablizing system. By gene expression experiments, the antigen and activity levels of Fâ ¦-250 Ser and Fâ ¦-250 Phe in the culture medium were (37.77 ± 2.30) ng/mL and (4.02 ± 0.52) ng/mL, respectively. ELISA and Western blotting analyses indicated that expression of the mutant Fâ ¦-250 Phe and wild type Fâ ¦-250 Ser was (130.51 ± 2.32) ng/mL and (172.45 ± 2.25) ng/mL, respectively. Fâ ¦-250 Phe was found in endoplasmic reticulum and Golgi apparatus, suggesting that the mutant Fâ ¦-250 Phe could be normally synthesized in the cells but was inefficiently secreted. CONCLUSION: Compound heterozygous mutations in F7 gene (g.15975G>A and g.16750C>T) may be responsible for the Fâ ¦ deficiency in this patient. The novel Fâ ¦ 250 Phe can be transported from endoplasmic reticulum to Golgi apparatus, but may be degraded or inefficient.
Asunto(s)
Deficiencia del Factor VII/genética , Factor VII/genética , Mutación Missense , Adulto , Animales , Células CHO , Cricetinae , Cricetulus , Ensayo de Inmunoadsorción Enzimática , Factor VII/fisiología , Células HEK293 , Humanos , MasculinoRESUMEN
OBJECTIVE: To apply Bionano Saphyr visual full-length DNA optical mapping technology to the precise genetic diagnosis of hemophilia A carriers. METHODS: For 2 suspected F8 gene deficiency female carriers who could not be diagnosed by conventional next-generation sequencing technology, the full-length DNA optical mapping technology was used to detect and scan the sample X chromosome full-length visual haplotype characteristic map, which was compared with the normal haplotype. The gene structure variation information of the samples was obtained by compare with DNA atlas library. RESULTS: The average fluorescent marker length of the X chromosome DNA molecular where the F8 gene was located in the two samples was greater than 2.5 Mbp, and the average copy number was greater than 20×. After comparative analysis, one of the samples was a proximal inversion of intron 22 of the F8 gene, and another was an inversion of intron 22 accompanied by multiple deletions of large fragments. CONCLUSIONS: Bionano technology has a good detection rate for gene defects with large length and complex variation. In the absence of a proband or accurate genetic diagnosis results of the proband, the application of this technology to detect the heterozygous complex variant of the F8 gene is of great significance for the prenatal diagnosis and pre-pregnancy diagnosis of hemophilia carriers.
RESUMEN
Increasing evidence shows that beyond its role in coagulation, thrombosis interferes with carcinogenesis. Endothelial cell protein C receptor (EPCR) is a cellular receptor for protein C and activated protein C (APC). Such EPCR-induced signal transduction promotes cancer cell migration, invasion, and angiogenesis and inhibits cancer cell apoptosis. However, its role in lung carcinoma biology is yet to be demonstrated. Here, the recombinant EPCR siRNA plasmids were constructed to investigate the effects of inhibition of EPCR on human lung cancer H1299. EPCR siRNA led to inhibition of endogenous EPCR mRNA and protein expression as determined by RT-PCR and Western blotting analysis. EPCR siRNA significantly inhibited cell growth, blocked entry into the S phase of the cell cycle, and reduced the migration of H1299 cells. EPCR siRNA also decreases MMP-2 and cyclin E expression in H1299 cells. In addition, siRNA targeting of EPCR inhibited the growth of H1299 cells and decreased MVD in SCID mice tumor models. Taken together, EPCR was involved in regulating progression of human lung cancer cells. Manipulation of EPCR expression may be a potential therapeutic strategy for lung cancer.
Asunto(s)
Antígenos CD/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Receptores de Superficie Celular/genética , Animales , Antígenos CD/metabolismo , Apoptosis/genética , Movimiento Celular/genética , Proliferación Celular , Ciclina E/metabolismo , Receptor de Proteína C Endotelial , Humanos , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones , Ratones SCID , Invasividad Neoplásica , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , ARN Interferente Pequeño/genética , Receptores de Superficie Celular/metabolismo , Transducción de SeñalRESUMEN
To improve wine taste and flavor stability, a novel indigenous strain of Saccharomyces cerevisiae with enhanced glycerol and glutathione (GSH) production for winemaking was constructed. ALD6 encoding an aldehyde dehydrogenases of the indigenous yeast was replaced by a GPD1 and CUP1 gene cassette, which are responsible for NAD-dependent glycerol-3-phosphatase dehydrogenase and copper resistance, respectively. Furthermore, the α-acetohydroxyacid synthase gene ILV2 of the indigenous yeast was disrupted by integration of the GSH1 gene which encodes γ-glutamylcysteine synthetase and the CUP1 gene cassette. The fermentation capacity of the recombinant was similar to that of the wild-type strain, with an increase of 21 and 19 % in glycerol and GSH production. No heterologous DNA was harbored in the recombinant in this study.
Asunto(s)
Glutatión/metabolismo , Glicerol/metabolismo , Ingeniería Metabólica/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Vino/microbiología , Acetolactato Sintasa/genética , Acetolactato Sintasa/metabolismo , Aldehído Oxidorreductasas/genética , Aldehído Oxidorreductasas/metabolismo , Fermentación , Técnicas de Inactivación de Genes , Glutamato-Cisteína Ligasa/genética , Glutamato-Cisteína Ligasa/metabolismo , Glicerol-3-Fosfato Deshidrogenasa (NAD+)/genética , Glicerol-3-Fosfato Deshidrogenasa (NAD+)/metabolismo , Metalotioneína/genética , Metalotioneína/metabolismo , Recombinación Genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Genetic engineering is widely used to meliorate biological characteristics of industrial brewing yeast. But how to solve multiple problems at one time has become the bottle neck in the genetic modifications of industrial yeast strains. In a newly constructed strain TYRL21, dextranase gene was expressed in addition of α-amylase to make up α-amylase's shortcoming which can only hydrolyze α-1,4-glycosidic bond. Meanwhile, 18s rDNA repeated sequence was used as the homologous sequence for an effective and stable expression of LSD1 gene. As a result, TYRL21 consumed about twice much starch than the host strain. Moreover TYRL21 speeded up the fermentation which achieved the maximum cell number only within 3 days during EBC tube fermentation. Besides, flavor evaluation comparing TYRL21 and wild type brewing strain Y31 also confirmed TYRL21's better performances regarding its better saccharides utilization (83% less in residual saccharides), less off-flavor compounds (57% less in diacetyl, 39% less in acetaldehyde, 67% less in pentanedione), and improved stability index (increased by 49%) which correlated with sensory evaluation of final beer product.
Asunto(s)
Dextranasa/genética , Glutatión/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , alfa-Amilasas/genética , Secuencia de Bases , Cerveza/microbiología , Carbono/metabolismo , ADN de Hongos/genética , Dextranasa/metabolismo , Fermentación , Expresión Génica , Genes Fúngicos , Ingeniería Genética , Inestabilidad Genómica , Glutatión/metabolismo , Microbiología Industrial , Cinética , Mutación , Plásmidos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , alfa-Amilasas/metabolismoRESUMEN
Copper is an essential trace element for living organisms. Copper enriched by yeast of Saccharomyces cerevisiae is regarded as the biologically available organic copper supplement with great potentiality for application. However, the lower uptake ratio of copper ions makes the production of copper enriched by yeast uneconomically and environmentally unfriendly. In this study, S. cerevisiae Cu-5 with higher copper tolerance and intracellular copper accumulation was obtained by screening of our yeast strains collection. To increase the uptake ratio of copper ions, the medium composition and cultivation conditions for strain Cu-5 were optimized systematically. A medium comprised of glucose, yeast extract, (NH4)2SO4, and inorganic salts was determined, then a novel cultivation strategy including pH control at 5.5 and increasing amounts of yeast extract for a higher concentration of copper ion in the medium was developed. The uptake ratios of copper ions were more than 90% after combining 50 to 100 mg/L copper ions with 3.5 to 5.0 g/L yeast extract, which is the highest until now and is conducive to the cost-effective and environmentally friendly production of bioactive copper in yeast-enriched form.
Asunto(s)
Cobre , Saccharomyces cerevisiae , Transporte Biológico , Medios de Cultivo , IonesRESUMEN
OBJECTIVE: To construct a mouse model of Glanzmann's thrombasthenia (GT) with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation by CRISPR/Cas9 technology, and then further explore the expression and function of glycoprotein αIIbß3 on the surface of platelet membrane. METHODS: The donor oligonucleotide and gRNA vector were designed and synthesized according to the ITGA2B gene sequence. The gRNA and Cas9 mRNA were injected into fertilized eggs with donor oligonucleotide and then sent back to the oviduct of surrogate mouse. Positive F0 mice were confirmed by PCR genotyping and sequence analysis after birth. The F1 generation of heterozygous GT mice were obtained by PCR and sequencing from F0 bred with WT mice, and then homozygous GT mice and WT mice were obtained by mating with each other. The phenotype of the model was then further verified by detecting tail hemorrhage time, saphenous vein bleeding time, platelet aggregation, expression and function of αIIbß3 on the surface of platelet. RESULTS: The bleeding time of GT mice was significantly longer than that of WT mice (P<0.01). Induced by collagen, thrombin, and adenosine diphosphate (ADP), platelet aggregation in GT mice was significantly inhibited (P<0.01, P<0.01, P<0.05). Flow cytometry analysis showed that the expression of αIIbß3 on the platelet surface of GT mice decreased significantly compared with WT mice (P<0.01), and binding amounts of activated platelets to fibrinogen were significantly reduced after thrombin stimulation (P<0.01). The spreading area of platelet on fibrinogen in GT mice was significantly smaller than that in WT mice (P<0.05). CONCLUSION: A GT mouse model with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation has been established successfully by CRISPR/Cas9 technology. The aggregation function of platelet in this model is defective, which is consistent with GT performance.
Asunto(s)
Codón sin Sentido , Integrina alfa2 , Trombastenia , Animales , Sistemas CRISPR-Cas , Modelos Animales de Enfermedad , Fibrinógeno/genética , Humanos , Integrina alfa2/genética , Ratones , Oligonucleótidos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , ARN Guía de Kinetoplastida , Trombastenia/diagnóstico , Trombastenia/genética , Trombina/genéticaRESUMEN
OBJECTIVE: To provide genetic consulting and prenatal diagnosis for two families with congenital factor V deficiency based on the known mutations of factor V gene (G16088C and G69969T). METHODS: Chorionic DNA was obtained at 12 weeks of gestation and analyzed to exclude maternal cell contamination through microsatellite DNA analysis. It was then amplified with PCR and sequenced to determine the presence of mutations in exons 3 and 23. Factor V activity of the blood was assayed at 22 weeks of gestation and 6 months after birth. RESULTS: The fetus in case 1 was found to be a heterozygous carrier of the G16088C mutation, for whom factor V activity of the cord blood and peripheral blood were 15% and 53%, respectively. Fetus 2 did not carry the familiar G69969T mutation, for whom the factor V activity of cord blood and peripheral blood has measured 32% and 93%, respectively. Follow-up studies demonstrated that the two infants were both in good health without a tendency for bleeding. CONCLUSION: In both cases, the genotypes were consistent with the phenotypes. This is the first report of prenatal diagnosis of congenital factor V deficiency.
Asunto(s)
Deficiencia del Factor V/diagnóstico , Factor V/genética , Diagnóstico Prenatal , Adulto , Secuencia de Bases , Niño , Factor V/metabolismo , Deficiencia del Factor V/genética , Femenino , Humanos , Masculino , Repeticiones de Microsatélite , Mutación , EmbarazoRESUMEN
Glutathione in beer works as the main antioxidant compounds which correlates with beer flavor stability. High residual sugars in beer contribute to major non-volatile components which correlate to high caloric content. In this work, Saccharomyces cerevisiae GSH1 gene encoding glutamylcysteine synthetase and Scharomycopsis fibuligera ALP1 gene encoding alpha-amylase were co-expressed in industrial brewing yeast strain Y31 targeting at alpha-acetolactate synthase (AHAS) gene (ILV2) and alcohol dehydrogenase gene (ADH2), and new recombinant strain TY3 was constructed. The glutathione content from the fermentation broth of TY3 increased to 43.83 mg/l compared to 33.34 mg/l from Y31. The recombinant strain showed high alpha-amylase activity and utilized more than 46% of starch after 5 days growing on starch as sole carbon source. European Brewery Convention tube fermentation tests comparing the fermentation broth of TY3 and Y31 showed that the flavor stability index increased to 1.3 fold and residual sugar concentration were reduced by 76.8%, respectively. Due to the interruption of ILV2 gene and ADH2 gene, the amounts of off-flavor compounds diacetyl and acetaldehyde were reduced by 56.93% and 31.25%, comparing with the amounts of these from Y31 fermentation broth. In addition, as no drug-resistance genes were introduced to new recombinant strain, consequently, it should be more suitable for use in beer industry because of its better flavor stability and other beneficial characteristics.
Asunto(s)
Cerveza/análisis , Glutatión/metabolismo , Microbiología Industrial , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Cerveza/microbiología , Fermentación , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ingeniería Genética , Glutamato-Cisteína Ligasa/genética , Glutamato-Cisteína Ligasa/metabolismo , Humanos , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycopsis/enzimología , Saccharomycopsis/genética , Saccharomycopsis/metabolismo , Gusto , alfa-Amilasas/genética , alfa-Amilasas/metabolismoRESUMEN
In this study, the problems of high caloric content, increased maturation time and off-flavors in commercial beer manufacture arising from residual sugar, diacetyl, and acetaldehyde levels were addressed. A recombinant industrial brewing yeast strain (TQ1) was generated from T1 [Lipomyces starkeyi dextranase gene (LSD1) introduced, alpha-acetohydroxyacid synthase gene (ILV2) disrupted] by introducing Saccharomyces cerevisiae glucoamylase (SGA1) and a strong promoter PGK1 while disrupting the genes coding alcohol dehydrogenase (ADH2). The highest glucoamylase activity for TQ1 was 93.26 U/ml compared with host strain T1 (12.36 U/ml) and wild-type industrial yeast strain YSF5 (10.39 U/ml), respectively. European Brewery Convention (EBC) tube fermentation tests comparing the fermentation broths of TQ1 with T1 and YSF5 showed that the real extract were reduced by 15.79% and 22.47%; the main residual maltotriose concentration were reduced by 13.75% and 18.82%; the caloric content were reduced by 27.18 and 35.39 calories per 12 oz. Due to the disruption of ADH2 gene in TQ1, the off-flavor acetaldehyde concentration in the fermentation broth were 9.43% and 13.28% respectively lower than that of T1 and YSF5. No heterologous DNA sequences or drug-resistance genes were introduced into TQ1. So, the gene manipulations in this work properly solved the addressed problems in commercial beer manufacture.
Asunto(s)
Alcohol Deshidrogenasa/metabolismo , Cerveza/microbiología , Ingeniería Genética/métodos , Glucano 1,4-alfa-Glucosidasa/metabolismo , Microbiología Industrial/métodos , Recombinación Genética/genética , Saccharomyces cerevisiae/metabolismo , Alcohol Deshidrogenasa/genética , ADN de Hongos/química , ADN de Hongos/genética , Glucano 1,4-alfa-Glucosidasa/genética , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Saccharomyces cerevisiae/genética , GustoRESUMEN
OBJECTIVE: To investigate whether miRNA-30a is involved in the pathogenesis of ITP by affecting the differentiation of Th17 cells, and to explore its possible mechanism of miRNA-30a involved in the pathogenesis of ITP through the verification of the target gene SOCS3 for the prediction of miRNA-30a. METHODS: Firstly, a chronic ITP mouse model was established. The expression of miRNA-30a and RORγt in the spleen mononuclear cells were detected and their correlation were analyzed. Secondly, the luciferase vector containing 3'UTR of the target gene and green fluorescent vector containing miRNA were constructed. Luciferase fluorescence detection, real-time fluorescent quantitative PCR (qPCR) and Western blot were used to verify whether SOCS3 is the target gene of miRNA-30a. RESULTS: The platelet count of mice in experimental group decreased to below 20% of normal ones after 48 hours of injection of anti-mouse platelet serum (APS), which was maintained for 14 days at least; the expression of miRNA-30a and RORγt in the spleen mononuclear cells in experimental group were higher than those in the control group(Pï¼0.05), moreover, there was a positive correlation between them (r=0.54); the activity of luciferase in PMDH-GFP-miRNA-30a and pMIR-report-UTR was significantly lower than that in PMDH-GFP empty plasmid and pMIR-report-UTR(Pï¼0.05); The expression of SOCS3 at mRNA and protein level was not different from that in the control group. CONCLUSION: Chronic ITP mouse model has been established successfully; miRNA-30a expression in spleen mononuclear cells of ITP mouse increase, and positively correlated with the expression of RORγt, which contribute to the pathogenesis of ITP by affecting the differentiation of Th17 cells; SOCS3 is able to bind to the target site of miRNA-30a, but might not be its functional target gene.
Asunto(s)
Púrpura Trombocitopénica Idiopática , Animales , Diferenciación Celular , Ratones , MicroARNs , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares , Células Th17RESUMEN
BACKGROUND: Immune imbalance of regulatory T cells (Treg)/T helper 17 cells (Th17) contributes to the development of immune thrombocytopenic purpura (ITP). The dysregulation of miRNAs is important in the development of ITP. However, the role of miR-106b-5p in Treg/Th17 imbalance remains unknown in ITP. MATERIALS AND METHODS: Peripheral blood was collected from patients with ITP and healthy controls, and CD4 + T cells were further isolated. miR-106b-5p, nuclear receptor subfamily 4 group A member 3 (NR4A3), forkhead box protein 3 (Foxp3), IL-17A, and TGF-ß expressions were detected by qRT-PCR, western blot, or ELISA. The effect of miR-106b-5p on NR4A3 was detected by dual-luciferase reporter gene assay. RESULTS: Compared with healthy controls, miR-106b-5p was elevated in peripheral blood of patients with ITP, and NR4A3 expression was decreased. sh-NR4A3 significantly decreased Foxp3 and TGF-ß expressions, indicating that NR4A3 may regulate Treg differentiation via Foxp3. Additionally, NR4A3 was identified to be a target of miR-106b-5p, and miR-106b-5p was able to negatively modulate NR4A3 expression. Moreover, we found miR-106b-5p induced immune imbalance of Treg/Th17 through NR4A3. In vivo experiments revealed that silencing miR-106b-5p promoted Treg differentiation and increased the number of platelets, suggesting the relief of ITP. CONCLUSION: miR-106b-5p regulated immune imbalance of Treg/Th17 in ITP through the NR4A3/Foxp3 pathway.
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Proteínas de Unión al ADN/metabolismo , Factores de Transcripción Forkhead/metabolismo , MicroARNs/metabolismo , Púrpura Trombocitopénica Idiopática/genética , Púrpura Trombocitopénica Idiopática/inmunología , Receptores de Esteroides/metabolismo , Receptores de Hormona Tiroidea/metabolismo , Transducción de Señal , Linfocitos T Reguladores/inmunología , Células Th17/inmunología , Animales , Secuencia de Bases , Diferenciación Celular , Niño , Preescolar , Femenino , Silenciador del Gen , Humanos , Masculino , Ratones Endogámicos BALB C , MicroARNs/sangre , MicroARNs/genética , Recuento de Plaquetas , Púrpura Trombocitopénica Idiopática/sangreRESUMEN
A self-cloning module for gene knock-out and knock-in in industrial brewing yeast strain was constructed that contains copper resistance and gamma-glutamylcysteine synthetase gene cassette, flanked by alcohol dehydrogenase II gene (ADH2) of Saccharomyces cerevisiae. The module was used to obtain recombined strains RY1 and RY2 by targeting the ADH2 locus of host Y1. RY1 and RY2 were genetically stable. PCR and enzyme activity analysis of RY1 and RY2 cells showed that one copy of ADH2 was deleted by GSH1+CUP1 insertion, and an additional copy of wild type was still present. The fermentation ability of the recombinants was not changed after genetic modification, and a high level of glutathione (GSH) was secreted, resulting from GSH1 overexpression, which codes for gamma-glutamylcysteine synthetase. A pilot-scale brewing test for RY1 and RY2 indicated that acetaldehyde content in fermenting liquor decreased by 21-22%, GSH content increased by 20-22% compared with the host, the antioxidizability of the recombinants was improved, and the sensorial evaluation was also better than that of the host. No heterologous DNA was harbored in the recombinants; therefore, they could be applied in the beer industry in terms of their biosafety.
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Alcohol Deshidrogenasa/genética , Bebidas Alcohólicas/microbiología , Técnicas de Sustitución del Gen/métodos , Técnicas de Inactivación de Genes/métodos , Glutamato-Cisteína Ligasa/genética , Metalotioneína/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimología , Acetaldehído/metabolismo , Alcohol Deshidrogenasa/metabolismo , Glutamato-Cisteína Ligasa/metabolismo , Glutatión/metabolismo , Metalotioneína/metabolismo , Recombinación Genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
New industrial brewing yeast strains, free of vector sequences and drug-resistance genes, were constructed by disrupting alpha-acetohydroxyacid synthase (AHAS) gene (ILV2) and introducing Lipomyces starkeyi dextranase (DEX) gene (LSD1) as a selective marker. The resulting recombinant strains can survive on YNB minimal medium plate with dextran T-70 as sole carbon source and showed lower AHAS activity. Fermentation test with recombinant strains in 500 ml conical flask confirmed DEX activity and lower AHAS activity compared with their host strain. Moreover, the fermentative performance of recombinant strains T1 and Q9 was better than their host, and the residual sugar content was reduced by 20-25% in fermented wort with recombinant strains compared to their host, too.
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Acetolactato Sintasa/genética , Cerveza/microbiología , Dextranasa/genética , Microbiología Industrial , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , Medios de Cultivo/química , Dextranasa/química , Dextranasa/metabolismo , Fermentación , Industria de Alimentos , Regulación Fúngica de la Expresión Génica , Datos de Secuencia Molecular , Plásmidos/genética , Proteínas Recombinantes , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/enzimología , Saccharomycetales/genética , Selección Genética , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: von Willebrand factor (vWF) mediates the initial capture of platelets to vascular subendothelium and is essential for platelet aggregation under high fluid shear stress as in arterial stenosis. On release from endothelial cells, vWF is rapidly cleaved by ADAMTS13/vWF-cleaving protease (vWF-CP). We investigated the clinical significance of changes in plasma vWF and vWF-CP activities in chronic renal disease. METHODS: Plasma vWF and vWF-CP activities were measured using enzyme-linked immunosorbent assay (ELISA) and residual collagen binding assay respectively in patients with lupus nephritis (n = 31), primary nephritic syndrome (n = 25), diabetic nephropathy (n = 45), chronic glomerulonephritis (n = 38) and 40 normal controls. The relation of their levels with pathological and renal status was analyzed. RESULTS: In all diseased patients the levels of vWF were significantly higher and vWF-CP activity significantly lower than the controls (both P < 0.01). vWF in the four subgroups did not correlate with the stage of disease but correlated negatively with vWF-CP activity. vWF-CP activity was not changed two weeks after renal transplantation. Renal biopsy demonstrated that the vWF level in stage IV was higher than in stages II and III while vWF-CP activity was lower in patients with lupus nephritis. After eight-week treatment, the vWF level significantly decreased and the vWF-CP activity significantly increased in systemic lupus erythema, disease activity index < 9, but not with index = 9. Even though the vWF-CP activity was significantly lower in membranous nephropathy than in minimal change disease, mesangial proliferative glomerulonephritis or IgA glomerulonephritis, the vWF level was not significantly different. CONCLUSIONS: The alterations of plasma vWF and vWF-CP activities were associated with different renal pathologies. Injury to endothelial cells and autoantibodies against vWF-CP activity may result in higher vWF level and lower vWF-CP activity in chronic renal disease and thus a mechanism for worsening of chronic renal disease and thrombosis.
Asunto(s)
Proteínas ADAM/sangre , Enfermedades Renales/sangre , Factor de von Willebrand/análisis , Proteína ADAMTS13 , Adolescente , Adulto , Anciano , Enfermedad Crónica , Femenino , Humanos , Trasplante de Riñón , Nefritis Lúpica/sangre , Masculino , Persona de Mediana EdadRESUMEN
Hereditary platelet disorders are a heterogeneous group of disorders characterized by abnormal number or function of platelets, sometimes even involving other systems apart from blood abnormalities. The great clinical and genetic heterogeneity makes the diagnosis and treatment of hereditary platelet disorders as a huge challenge for clinicians. At present, only a small number of patients have received a clear molecular diagnosis of hereditary platelet diseases, and a lot of pathogenic genetic variations still remain unknown. The popularity of next generation sequencing (NGS) promotes the development of individualized gene sequencing. Researchers have made great progress in the field of hemostasis and thrombosis using whole genome sequencing (WGS), whole exone sequencing (WES) and target gene sequencing (TGS). The development of NGS has not only promoted the individualized molecular diagnosis of hereditary platelet diseases, but also laid a solid foundation for gene therapy in the future. In this review, the new progress of the diagnosis of platelet-related diseases by using next generation sequencing techniques, is summarized.
Asunto(s)
Trastornos de las Plaquetas Sanguíneas , Plaquetas , Secuenciación de Nucleótidos de Alto Rendimiento , HumanosRESUMEN
Foam stability is often influenced by proteinase A, and flavor stability is often affected by oxidation during beer storage. In this study, PEP4, the gene coding for proteinase A, was disrupted in industrial brewing yeast. In the meantime, one copy of GSH1 gene increased in the same strain. GSH1 is responsible for gamma-glutamylcysteine synthetase, a rate-limiting enzyme for synthesis of glutathione which is one kind of important antioxidant and beneficial to beer flavor stability. In order to improve the brewer's yeast, plasmid pYPEP, pPC and pPCG1 were firstly constructed, which were recombined plasmids with PEP4 gene, PEP4's disruption and PEP4's disruption+GSH1 gene respectively. These plasmids were verified to be correct by restriction enzymes' assay. By digesting pPCG1 with AatII and PstI, the DNA fragment for homologous recombination was obtained carrying PEP4 sequence in the flank and GSH1 gene internal to the fragment. Since self-cloning technique was applied in the study and the modified genes were from industrial brewing yeast itself, the improved strains, self-cloning strains, were safe to public. The genetic stability of the improved strains was 100%. The results of PCR analysis of genome DNA showed that coding sequence of PEP4 gene had been deleted and GSH1 gene had been inserted into the locus of PEP4 gene in self-cloning strains. The fermentation ability of self-cloning strain, SZ-1, was similar to that of the host. Proteinase A could not be detected in beer brewed with SZ-1, and GSH content in the beer increased 35% compared to that of the host, Z-1.
Asunto(s)
Ácido Aspártico Endopeptidasas/genética , Cerveza/microbiología , Regulación Fúngica de la Expresión Génica , Glutamato-Cisteína Ligasa/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Ácido Aspártico Endopeptidasas/metabolismo , Clonación Molecular , Fragmentación del ADN , ADN de Hongos/química , ADN de Hongos/genética , Dipéptidos/metabolismo , Fermentación , Amplificación de Genes , Glutamato-Cisteína Ligasa/metabolismo , Glutatión/metabolismo , Plásmidos , Recombinación Genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
OBJECTIVE: To evaluate the clinical significance of plasma von Willebrand factor (vWF) and von Willebrand factor-cleaving protease (vWF-CP) activity in systemic lupus erythematosus (SLE). METHODS: vWF antigen (vWF:Ag) and vWF-CP activity were respectively evaluated by using ELISA and residual-collagen binding assay (R-CBA) in 30 patients with SLE and 40 normal controls. RESULTS: The level of the vWF:Ag in SLE patients (114.6 +/- 16.3)% was significantly higher than that in the normal controls (71.3 +/- 49.5)% (P < 0.01), while the level of plasma vWF-CP activity in the SLE patients (57.7 +/- 16.3)% was significantly lower than that in the controls (86.6 +/- 1.8)% (P < 0.01). The level of vWF-CP activity was positively correlated with SLE disease activity index (SLEDAI). The alterations of these two indices were remarkable in lupus nephritis (LN) patients in comparison with those in non-LN patients. The level of vWF-CP activity in type IV LN was lowest among the four types of LN (II, III, IV, V). The level of the vWF:Ag in 30 SLE patients was reduced four weeks after therapy, on the other hand, the vWF-CP activity was significantly increased in those with SLEDAI below 9, but unchanged in whose with SLEDAI above 9. CONCLUSIONS: The injury of endothelial cells and to production autoantibodies against vWF-CP were the factors that resulted in lower vWF-CP activity in SLE patients and this lowered activity may influence the progression of SLE.