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In this paper is described an easily synthesized chiral diazaborolidine that is inexpensive, stable, and provides excellent stereoselection across a number of reaction classes. These versatile compounds possess utility in four different classes of cycloaddition reactions, offering good yield and stereoselectivity. X-ray structure analysis provides insight about the origin of stereocontrol.
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Fourteen new monoterpene-flavonoid conjugates including four monoterpene-conjugated chalcones (glabratins A-D, 1-4), seven monoterpene-conjugated dihydrochalcones (glabratins E-K, 5-11), and three monoterpene-conjugated flavanones (glabratins L-N, 12-14), together with four known analogues (15-18) were isolated from the aerial parts of Sarcandra glabra. The structures and the absolute configurations of these compounds were elucidated by the spectroscopic data, single-crystal X-ray diffraction, and electronic circular dichroism (ECD) calculations. Compounds 1, 4-6, 9-14, and 18 showed obvious cell autophagy-inducing activities at 25 µM in HEK293 cells. Furthermore, the bioassay results also showed that 18 induced cell autophagy in a dose dependent manner. Our findings revealed a rare class of monoterpene-flavonoid conjugates in nature and firstly reported their autophagy-inducing activities.
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Autofagia/efectos de los fármacos , Flavonoides/farmacología , Magnoliopsida/química , Monoterpenos/farmacología , Extractos Vegetales/farmacología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Flavonoides/química , Flavonoides/aislamiento & purificación , Células HEK293 , Humanos , Estructura Molecular , Monoterpenos/química , Monoterpenos/aislamiento & purificación , Método de Montecarlo , Componentes Aéreos de las Plantas/química , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Relación Estructura-ActividadRESUMEN
A hybrid transition-metal/radical process is described that results in the addition of organozinc reagents and alkyl halides across alkenyl boron reagents in an enantioselective catalytic fashion. The reaction can be accomplished both intermolecularly and intramolecularly, providing useful product yields and high enantioselectivities in both manifolds.
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Alcoholes/síntesis química , Compuestos de Boro/química , Hidrocarburos Yodados/química , Compuestos Organometálicos/química , Zinc/química , Alcoholes/química , Radicales Libres/química , Estructura Molecular , EstereoisomerismoRESUMEN
To explore the possible roles of metallothioneins (MTs) played in cadmium (Cd) accumulation of water spinach, three IaMT genes, IaMT1, IaMT2 and IaMT3 in a high-shoot-Cd (T308) and a low-shoot-Cd accumulation cultivar (QLQ) were cloned, characterized, and quantitated. Gene expression analysis suggested that the expression of the IaMTs was differentially regulated by Cd stress in different cultivars, and T308 showed higher MTs expression overall. Furthermore, only shoot IaMT3 expression was cultivar dependent among the three IaMTs. Antioxidant analysis showed that the high production of IaMTs in T308 should be associated with its high oxidation resistance. The role of IaMTs in protecting against Cd toxicity was demonstrated in vitro via recombinant E. coli strains. The results showed that IaMT1 correlated with neither Cd tolerance nor Cd accumulation of E. coli, while IaMT2 conferred Cd tolerance in E. coli, IaMT2 and IaMT3 increased Cd accumulation in E. coli. These findings help to clarify the roles of IaMTs in Cd accumulation, and increase our understanding of the cultivar-dependent Cd accumulation in water spinach.
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Cadmio/metabolismo , Contaminantes Ambientales/metabolismo , Ipomoea/metabolismo , Metalotioneína/metabolismo , Spinacia oleracea/metabolismo , Escherichia coli/metabolismo , Peróxido de Hidrógeno/metabolismo , Malondialdehído/metabolismo , Metalotioneína/genética , Raíces de Plantas/metabolismo , Brotes de la Planta/metabolismoRESUMEN
The borylation of C-H bonds catalyzed by transition metals has been investigated extensively in the past two decades, but no iridium-catalyzed enantioselective borylation of C-H bonds has been reported. We report a set of iridium-catalyzed enantioselective borylations of aromatic C-H bonds. This reaction relies on a set of newly developed chiral quinolyl oxazoline ligands. This process proceeds under mild conditions with good to excellent enantioselectivity, and the borylated products can be converted to enantioenriched derivatives containing new C-O, C-C, C-Cl, or C-Br bonds.
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Compuestos de Boro/química , Nitrógeno/química , Catálisis , Hidroquinonas/química , Iridio/química , Ligandos , Estructura Molecular , Oxazoles/química , EstereoisomerismoRESUMEN
Iridium catalysts containing dative nitrogen ligands are highly active for the borylation and silylation of C-H bonds, but chiral analogs of these catalysts for enantioselective silylation reactions have not been developed. We report a new chiral pyridinyloxazoline ligand for enantioselective, intramolecular silylation of symmetrical diarylmethoxy diethylsilanes. Regioselective and enantioselective silylation of unsymmetrical substrates was also achieved in the presence of this newly developed system. Preliminary mechanistic studies imply that C-H bond cleavage is irreversible, but not the rate-determining step.
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Iridio/química , Nitrógeno/química , Oxazoles/química , Silanos/síntesis química , Catálisis , Ligandos , Estructura Molecular , Silanos/química , EstereoisomerismoRESUMEN
In this study, two short-cut sulfur autotrophic denitrification (SSADN) reactors were initiated using different reduced sulfur forms as electron donors and their effects on the start-up speed of the SSADN process, NO2--N accumulation characteristics, and microbial community were investigated. Results revealed that during the same period, due to the relatively slow S0 dissolution rate, the NO2--N production rate realized by microorganisms in S0-SSADN (NO2--N production rate (NPR), 174 mg/(L·d)) was significantly slower than S2--SSADN (NPR, 679 mg/(L·d)). The NO2--N accumulation efficiency (NAE) was maintained > 80%, which was significantly higher than S2--SSADN. In the SSADN system using different reduced sulfur forms, the microbial community structure and abundance considerably differed. The main sulfur-oxidizing bacteria (SOB) in S0-SSADN were Sulfurimonas (6.5%) and Thiobacillus (5.3%). The main SOB species in S2--SSADN was Thiomonas (13.6%). Thermomonas played an important role in the two reactors as an important NO3--N denitrifying bacteria species.
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Reactores Biológicos , Desnitrificación , Procesos Autotróficos , Bacterias , Electrones , Nitratos , Nitrógeno/química , Dióxido de Nitrógeno , Azufre/químicaRESUMEN
Objective: The present study aims to examine whether secondhand smoke exposure (SHSE) in university students can affect three indices of self-satisfaction related to depression as indiced by appearance, weight, and freedom of life choice. Participants: We collected data from 740 nonsmoking students in the summer of 2018, of which 57.84% were exposed to secondhand smoke. Methods: Depressive symptoms, SHSE, smoking status, weight satisfaction, appearance satisfaction, and freedom of life choice were self-reported via a questionnaire. Results: The generalized linear analyses revealed that SHSE was linked to lower scores of perceived freedom of life choice but not significantly associated with weight nor appearance satisfaction. The mediation analyses indicated that perceived freedom of life choice fully mediated the association between SHSE and depressive symptoms. Conclusions: These findings shed light on the importance of SHSE and its effects on mental health in university students. Preventive strategies should therefore locally target university campuses.
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Thrombopoietin (TPO) and its receptor (Mpl) have long been associated with megakaryocyte proliferation, differentiation, and platelet formation. However, studies have also shown that the extracellular domain of Mpl (Mpl-EC) interacts with human (h) NUDC, a protein previously characterized as a human homolog of a fungal nuclear migration protein. This study was undertaken to further delineate the putative binding domain on the Mpl receptor. Using the yeast two-hybrid system assay and co-immunoprecipitation, we identified that within the Mpl-EC domain 1 (Mpl-EC-D1), amino acids 102-251 were strongly involved in ligand binding. We subsequently expressed five subdomains within this region with T7 phage display. Enzyme-linked immunosorbent binding assays identified a short stretch of peptide located between residues 206 and 251 as the minimum binding domain for both TPO and hNUDC. A series of sequential Ala replacement mutations in the region were subsequently used to identify the specific residues most involved in ligand binding. Our results point to two hydrophobic residues, Leu(228) and Leu(230), as having substantial effects on hNUDC binding. For TPO binding, mutations in residues Asp(235) and Leu(239) had the largest effect on binding efficacy. In addition, deletion of the conservative motif WGSWS reduced binding capacity for hNUDC but not for TPO. These separate binding sites on the Mpl receptor for TPO and hNUDC raise interesting implications for the cytokine-receptor interactions.
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Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Receptores de Trombopoyetina/metabolismo , Trombopoyetina/metabolismo , Aminoácidos , Sitios de Unión , Secuencia Conservada , Humanos , Unión Proteica , Estructura Terciaria de Proteína , Receptores de Trombopoyetina/química , Trombopoyetina/químicaRESUMEN
Intramolecular amination of organoboronates occurs with a 1,2-metalate shift of an aminoboron "ate" complex to form azetidines, pyrrolidines, and piperidines. Bis(boronates) undergo site-selective amination to form boronate-containing azacycles. Enantiomerically enriched azacycles are formed with high stereospecificity.
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Recently our laboratory reported evidence showing that hNUDC acts as an additional cytokine for thrombopoietin receptor (Mpl). Previously known as the human homolog of a fungal nuclear migration protein, hNUDC plays a critical role in megakaryocyte differentiation and maturation. Here we sought to further clarify the hNUDC-Mpl ligand-receptor relationship by utilizing interference RNA (RNAi) to knockdown Mpl expression in a megakaryocyte cell line. We created U6 promoter driven constructs to express short hairpin RNAs (shRNA) with affinity for different sites on Mpl mRNA. By including Mpl-EGFP fusion protein in these constructs, we were able to effectively screen the shRNA that was most efficient in inhibiting Mpl mRNA expression. This shRNA was subsequently transferred into a lentivirus vector and transduced into Dami cells, a cell line which constitutively expresses endogenous Mpl. This lentiviral vector was also designed to simultaneously express EGFP to monitor transfection efficiency. Our results show that lentivirus can be used to effectively deliver shRNAs into Dami cells and cause specific inhibition of Mpl protein expression after transduction. Furthermore, we show the functional effects of shRNA-mediated Mpl silencing by demonstrating reduced hNUDC stimulated megakaryocyte proliferation and differentiation. Thus, the use of a RNAi knockdown strategy has allowed us to pinpoint the connection of hNUDC with Mpl in the regulation of megakaryocyte maturation.
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Proteínas de Ciclo Celular/fisiología , Diferenciación Celular/fisiología , Megacariocitos/citología , Proteínas Nucleares/fisiología , Interferencia de ARN , Receptores de Trombopoyetina/fisiología , Proteínas de Ciclo Celular/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Expresión Génica/genética , Vectores Genéticos/genética , Humanos , Lentivirus/genética , Megacariocitos/efectos de los fármacos , Megacariocitos/metabolismo , Proteínas Nucleares/farmacología , Glicoproteína IIb de Membrana Plaquetaria/metabolismo , Poliploidía , ARN sin Sentido/genética , Receptores de Trombopoyetina/deficiencia , Receptores de Trombopoyetina/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
The weather is arguably one of the most common topics in daily conversation. However, it is also a well-known factor in psychiatric problems worldwide such as depression, where particularly elders and females are prone to the condition. Previous studies have mostly focused on the association between long-term climate/season change and depressive symptoms to test the belief that people feel depressed when the weather is bad. Yet, little is known about the relationship between intraday weather conditions and depressive symptoms. This study aimed to decompose this association and explore age and gender differences in depressive symptoms' report under different intraday weather conditions. We analyzed data with a total sample size of 20,987 individuals aged 15-96 years obtained via the China Labor-force Dynamic Survey (CLDS) project in 2016. The surveyors filled in the intraday weather conditions (i.e., sunny, cloudy, overcast, light rain, moderate rain, and heavy rain) in the questionnaire with depression being self-reported using the Center for Epidemiologic Studies Depression (CES-D) Scale. In the survey, 17.3% [95% confidence interval (CI) = 16.8%, 17.8%] individuals were assessed to have depressive symptoms (CES-D scores >15). Overcast day was the peak weather condition for depressive symptoms, while gender and age differences of depressive symptoms were biggest in the weather condition of moderate rain. Multivariate logistic regression revealed that female and middle-old age individuals were more likely to report having depressive symptoms, and people under overcast day were more likely to report having depressive symptoms compared to under sunny day [odds ratio (OR) = 1.25, 95%CI = 1.11, 1.39]. This intraday weather conditions-depression link was significant in males and middle-old age individuals but not in females and youngsters.
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Depresión , Tiempo (Meteorología) , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , China , Depresión/epidemiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Autoinforme , Factores Sexuales , Adulto JovenRESUMEN
Large number of data showed that allele variants in certain genes are markers for hepatocellular carcinoma (HCC). GRP78 is a stress-associated protein which is a central regulator of endoplasmic reticulum homeostasis due to its multiple functional roles in the folding, maturation and transport of proteins. A case-control study was conducted on 576 HCC patients, and 539 age- and gender-matched healthy subjects to examine whether rs430397 polymorphism in the fifth intron of GRP78 gene is associated with the development and prognosis of HCC. Polymorphism in rs430397 was analyzed by resequencing and TaqMan real-time PCR. Allele A, genotype AA and combined genotypes (AG+AA) displayed significantly increased risk for HCC (OR = 1.48, 95%CI = 1.07-1.79, p = 0.010; OR = 2.25, 95%CI = 1.08-3.38, p = 0.019; and OR = 1.50, 95%CI = 1.09-1.85, p = 0.012, respectively). Genotypes AA and AG were mainly associated with HBV-related HCC (85.8%; p < 0.00001 versus HBV noncarriers with HCC) and cirrhosis-related HCC (90%; p = 0.011 versus noncirrhosis HCC). Patients carrying the AA genotype had a shorter survival time (median 23.0 months in all cases; median 21.0 months in the cases carrying HBsAg). Like HBV and cirrhosis, the rs430397 is an independent prognostic factor influencing the survival of HCC. In conclusion, allele A and genotypes AA and AG of rs430397 may represent high risk and poor prognosis for HCC.
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Pueblo Asiatico/genética , Carcinoma Hepatocelular/genética , Proteínas de Choque Térmico/genética , Intrones/genética , Neoplasias Hepáticas/genética , Polimorfismo de Nucleótido Simple/genética , Adulto , Anciano , Carcinoma Hepatocelular/complicaciones , Carcinoma Hepatocelular/virología , Estudios de Casos y Controles , Chaperón BiP del Retículo Endoplásmico , Femenino , Genotipo , Hepatitis B/complicaciones , Hepatitis B/genética , Virus de la Hepatitis B/fisiología , Humanos , Neoplasias Hepáticas/complicaciones , Neoplasias Hepáticas/virología , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Factores de Riesgo , Tasa de SupervivenciaRESUMEN
Drosomycin (Drs) encoding an inducible 44-residue antifungal peptide is clustered with six additional genes, Dro1, Dro2, Dro3, Dro4, Dro5, and Dro6, forming a multigene family on the 3L chromosome arm in Drosophila melanogaster. To get further insight into the regulation of each member of the drosomycin gene family, here we investigated gene expression patterns of this family by either microbe-free injury or microbial challenges using real time RT-PCR. The results indicated that among the seven drosomycin genes, Drs, Dro2, Dro3, Dro4, and Dro5 showed constitutive expressions. Three out of five, Dro2, Dro3, and Dro5, were able to be upregulated by simple injury. Interestingly, Drs is an only gene strongly upregulated when Drosophila was infected with microbes. In contrast to these five genes, Dro1 and Dro6 were not transcribed at all in either noninfected or infected flies. Furthermore, by 5' rapid amplification of cDNA ends, two transcription start sites were identified in Drs and Dro2, and one in Dro3, Dro4, and Dro5. In addition, NF-kappaB binding sites were found in promoter regions of Drs, Dro2, Dro3, and Dro5, indicating the importance of NF-kappaB binding sites for the inducibility of drosomycin genes. Based on the analyses of flanking sequences of each gene in D. melanogaster and phylogenetic relationship of drosomycins in D. melanogaster species-group, we concluded that gene duplications were involved in the formation of the drosomycin gene family. The possible evolutionary fates of drosomycin genes were discussed according to the combining analysis of gene expression pattern, gene structure, and functional divergence of these genes.
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Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Evolución Molecular , Regulación de la Expresión Génica , Variación Genética , Familia de Multigenes/genética , Animales , Sitios de Unión , Elementos Transponibles de ADN/genética , Proteínas de Drosophila/metabolismo , Perfilación de la Expresión Génica , Genes de Insecto , Filogenia , Regiones Promotoras Genéticas/genética , Factores de Transcripción/metabolismo , Sitio de Iniciación de la Transcripción , Transcripción GenéticaRESUMEN
The Fry gene, located on chromosome 13, is an evolutionarily conserved large protein from yeast to human. Our previous study genetically linked the Fry gene with differential susceptibility to mammary carcinogenesis, but whether Fry affects mammary gland development and function, as well as the growth of breast cancer cells, is largely unknown. To define the consequences of Fry loss in the mammary glands, we have generated mice conditionally deficient of the Fry gene in the mammary glands using the Cre-loxP recombination system. We examined multiple phenotypes with male and female homozygous Fry conditional knockout mice (Mfry) and control mice (WT), including body weight, preliminary observations (health and neurological flexes), open field locomotion, sensory abilities, auditory threshold, and glucose metabolism. The loss of Fry in the mammary glands didn't cause a significant difference in these genotypes between Mfry and WT mice. However, our data showed that Fry was required during pregnancy, while it was functionally dispensable in virgin mammary gland development. Loss of Fry led to more lateral buds, and the lobuloalveoli were smaller and showed undistended morphology in mammary glands during late pregnancy. in vitro experiment, ectopic expression of FRY could alter the morphology and significantly suppress the growth and proliferation of the breast cancer cell lines, MDA-MB-231 (ER-/PR-/HER2-, Basal-like) and BT474 (ER+/PR+/HER2+, Luminal B). The following genome-wide transcriptomic analysis of these cells suggested that FRY interacted with protein kinases relevant signaling pathways and induced massive changes in gene expression, including the activation of the Hippo/Yap pathway. Together, our data suggest that the FRY is required for mammary glands developments during pregnant periods, and affects breast cancer cell growth and proliferation.
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A bottleneck in recent gene synthesis technologies is the high cost of oligonucleotide synthesis and post-synthesis sequencing. In this article, a simple and rapid method for low-cost gene synthesis technology was developed based on DNAWorks program and an improved single-step overlap extension PCR (OE-PCR). This method enables any DNA sequence to be synthesized with few errors, then any mutated sites could be corrected by site-specific mutagenesis technology or PCR amplification-assembly method, which can amplify different DNA fragments of target gene followed by assembly into an entire gene through their overlapped region. Eventually, full-length DNA sequence without error was obtained via this novel method. Our method is simple, rapid and low-cost, and also easily amenable to automation based on a DNAWorks design program and defined set of OE-PCR reaction conditions suitable for different genes. Using this method, several genes including Manganese peroxidase gene (Mnp) of Phanerochaete chrysosporium (P. chrysosporium), Laccase gene (Lac) of Trametes versicolor (T. versicolor) and Cip1 peroxidase gene (cip 1) of Coprinus cinereus (C. cinereus) with sizes ranging from 1.0 kb to 1.5 kb have been synthesized successfully.
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Clonación Molecular/métodos , Bases de Datos de Ácidos Nucleicos , Reacción en Cadena de la Polimerasa/métodos , Programas Informáticos , Genes , Modelos Biológicos , Peroxidasas/genética , Pichia/genética , Análisis de Secuencia de ADNRESUMEN
Human NudC nuclear distribution protein (hNUDC) is differentially expressed between normal and cancer cells. Based on its marked altered expression and its roles in modulating cell division, cytokineses and migration, a detailed understanding of the mechanisms regulating hNUDC expression in cancer cells is critical. In this study, we identified miR-194 as a downstream target of hNUDC and linked its expression to reduced metastatic capacity and tumorigenicity of prostate cancer (PCa) cells. Using miRNA target prediction programs, hNUDC mRNA was found to contain a potential binding site for miR-194 within its 3'UTR. A Reporter assay confirmed that post-transcriptional regulation of hNUDC was dependent on the miR-194 binding site. Forced expression of miR-194 in PCa cell lines, PC-3 and DU-145, led to a decrease in the mRNA and protein levels of hNUDC. Overexpression of miR-194 in these cells inhibited cell migration and invasion, and induced multinucleated cells. Our data showed that hNUDC knockdown by siRNA significantly reduced the migration and invasion in the PC-3 and DU-145 cells, phenocopying the results of miR-194 overexpression. Furthermore, lentivirus-mediated stable expression of miR-194 in PCa cells reduced the ability of colony formation as detected by a soft agar assay and exhibited significantly less tumorigenic ability in vivo. Our results suggest a novel role for miR-194 in effectively controlling cell metastatic processes in PCa cells via the regulation of hNUDC expression.
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Proteínas de Ciclo Celular/metabolismo , Movimiento Celular , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Proteínas Nucleares/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Animales , Apoptosis , Western Blotting , Proteínas de Ciclo Celular/genética , Proliferación Celular , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica , Proteínas Nucleares/genética , Neoplasias de la Próstata/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Chronic arsenic exposure via drinking water has become a worldwide public health concern. In humans, inorganic arsenic (iAs) is metabolized to monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) mainly mediated by arsenic (+3 oxidation state) methyltransferase (As3MT). We reported recently that N-6 adenine-specific DNA methyltransferase 1 (N6AMT1) was involved in arsenic metabolism, and examined its interactive effect with As3MT on arsenic metabolism in vitro To further evaluate the interactive effect of N6AMT1 and As3MT on arsenic biomethylation in humans, we conducted a human population-based study including 289 subjects living in rural villages in Inner Mongolia, China, and assessed their urinary arsenic metabolites profiles in relation to genetic polymorphisms and haplotypes of N6AMT1 and As3MT Five N6AMT1 single nucleotide polymorphisms (SNPs; rs1003671, rs7282257, rs2065266, rs2738966, rs2248501) and the N6AMT1 haplotype 2_GGCCAT were significantly associated with the percentage of iAs (% iAs) in urine (e.g., for rs7282257, mean was 9.62% for TT, 6.73% for AA). Rs1003671 was also in a significant relationship with urinary MMA and DMA (the mean of %MMA was 24.95% for GA, 31.69% for GG; the mean of % DMA was 69.21% for GA, 59.82% for GG). The combined effect of N6AMT1 haplotype 2_GGCCAT and As3MT haplotype 2_GCAC showed consistence with the additive significance of each haplotype on % iAs: the mean was 5.47% and 9.36% for carriers with both and null haplotypes, respectively. Overall, we showed that N6AMT1 genetic polymorphisms were associated with arsenic biomethylation in the Chinese population, and its interaction with As3MT was observed in specific haplotype combinations.
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Arsénico/metabolismo , Variación Genética , Metiltransferasas/genética , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/genética , Adulto , China , Femenino , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido SimpleRESUMEN
A new protease gene (pro1437)was separated from an oil-polluted Mud flat metagenomic library. Pro1437 belongs to a peptidase M48 superfamily according to the results of sequence analysis, and it showed very low identities compared to other known proteases or peptidases. The error-prone PCR was used to introduce random mutations and improve the expression of pro1437. After two rounds of mutagenesis and screening, a mutant (Pro2T21) with a 6.6-fold higher activity and a 4.8-fold higher expression level than Pro1437 was obtained. Sequence analysis found three amino acid substitutions (A54V, L192H, F224L) in Pro2T21. 3D structure modelling analysis indicated A54V and L192H probably played a crucial role in the improvement of enzymatic activity and soluble expression level of Pro2T21. Furthermore, Pro2T21opti displayed a 5.8-fold higher expression level than the wild type under optimal pH 8.0 at 50°C after codon-optimization. Also, Pro2T21opti represented robust compatibility with several popular laundry detergents, and blood stains on white cloth pieces were completely washed away when endogenous protease-inactivated Tide and Pro2T21opti were used together. Therefore, Pro2T21opti has great potential for use as an additive in detergents after further study.
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Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Endopeptidasas/genética , Endopeptidasas/metabolismo , Metagenoma , Contaminación por Petróleo , Microbiología del Suelo , Sustitución de Aminoácidos , Proteínas Bacterianas/aislamiento & purificación , Manchas de Sangre , Detergentes , Evolución Molecular Dirigida , Endopeptidasas/aislamiento & purificación , Escherichia coli/enzimología , Escherichia coli/genética , Humanos , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Mutagénesis , Filogenia , Conformación Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Solubilidad , TemperaturaRESUMEN
In this study, the aroA-M12 gene encoding bacterial 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and the Btslm recombinant gene encoding Bacillus thuringiensis (Bt) toxin gene were introduced into a Brassica napus variety, Xiangyou No. 15, via Agrobacterium-mediated transformation using glyphosate as a selectable agent. PCR amplification and Southern blot analyses of T0 transgenic plants showed that the alien genes were transferred and integrated stably into the genome of Xiangyou No. 15. Western blot further confirmed that the alien genes were expressed in these plants. Transgenic plants with tolerance to both glyphosate and the tested pest were demonstrated by bioassays. Our result also clearly shows that the aroA-M12 gene can be used as an efficient selectable marker gene instead of antibiotic resistant markers in plant transformation.