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Background: Cinnamomum camphora is a commercially important tree species in China, and it's also a common native tree in the forests of southern China. However, literature on the impact of Cinnamomum camphora essential oil (CCEO) on human psychophysiological activity is scarce. Hence, the primary objective of this study was to examine the effect of exposure to CCEO on the functioning of the human autonomic nervous system, electroencephalographic (EEG) activity, and emotional state. Methods: Forty-three healthy university students participated. The data collected included heart rate (HR), blood pressure (BP), pulse rate, blood oxygen saturation (SpO2), electroencephalographic (EEG) activity, and the results of the Profile of Mood States (POMS) test. Results: A drop in diastolic pressure (DBP) and pulse rate was also noticed after participants inhaled CCEO. Furthermore, EEG studies have demonstrated notable reductions in absolute beta (AB), absolute gamma (AG), absolute high beta (AHB), and relative gamma (RG) power spectra during exposure to CCEO. Conversely, the relative theta (RT) and power spectra values showed a significant increase. Additionally, the finding from POMS indicated that the fragrance evoked positive emotions and suppressed negative feelings. Conclusion: The results suggest that exposure to CCEO may promote mental and physical relaxation, facilitate cognitive processes such as memory and attention, and enhance mood states.
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BACKGROUND: Pichia pastoris (Komagataella phaffii) is a promising production host, but the usage of methanol limits its application in the medicine and food industries. RESULTS: To improve the constitutive expression of heterologous proteins in P. pastoris, four new potential transcription regulators (Loc1p, Msn2p, Gsm1p, Hot1p) of the glyceraldehyde triphosphate dehydrogenase promoter (pGAP) were revealed in this study by using cellulase E4 as reporter gene. On this basis, a series of P. pastoris strains with knockout or overexpression of transcription factors were constructed and the deletion of transcription factor binding sites on pGAP was confirmed. The results showed that Loc1p and Msn2p can inhibit the activity of pGAP, while Gsm1p and Hot1p can enhance the activity of pGAP; Loc1p, Gsm1p and Hot1p can bind directly to pGAP, while Msn2p must be treated to expose the C-terminal domain to bind to pGAP. Moreover, manipulating a single transcription factor led to a 0.96-fold to 2.43-fold increase in xylanase expression. In another model protein, aflatoxin oxidase, knocking out Loc1 based on AFO-∆Msn2 strain resulted in a 0.63-fold to 1.4-fold increase in expression. It can be demonstrated that the combined use of transcription factors can further improve the expression of exogenous proteins in P. pastoris. CONCLUSION: These findings will contribute to the construction of pGAP-based P. pastoris systems towards high expression of heterologous proteins, hence improving the application potential of yeast.
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Regulación Fúngica de la Expresión Génica , Regiones Promotoras Genéticas , Factores de Transcripción , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Pichia/genética , Pichia/metabolismoRESUMEN
BACKGROUND: α-Glucosidase (AG) is a bifunctional enzyme, it has a capacity to synthesize 2-O-α-d-glucopyranosyl-l-ascorbic acid (AA-2G) from l-ascorbic acid (L-AA) and low-cost maltose under mild conditions, but it can also hydrolyze AA-2G, which leads to low synthesis efficiency of AA-2G. MAIN METHODS AND MAJOR RESULTS: This study introduces a rational molecular design strategy to regulate enzymatic reactions based on inhibiting the formation of ground state of enzyme-substrate complex. Y215 was analyzed as the key amino acid site affecting the affinity of AG to AA-2G and L-AA. For the purpose of reducing the hydrolysis efficiency of AA-2G, the mutant Y215W was obtained by analyzing the molecular docking binding energy and hydrogen bond formation between AG and the substrates. Compared with the wild-type, isothermal titration calorimetry (ITC) results showed that the equilibrium dissociation constant (KD ) of the mutant for AA-2G was doubled; the Michaelis constant (Km ) for AA-2G was reduced by 1.15 times; and the yield of synthetic AA-2G was increased by 39%. CONCLUSIONS AND IMPLICATIONS: Our work also provides a new reference strategy for the molecular modification of multifunctional enzymes and other enzymes in cascade reactions system.
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Ácido Ascórbico , alfa-Glucosidasas , alfa-Glucosidasas/genética , alfa-Glucosidasas/metabolismo , Simulación del Acoplamiento Molecular , Ácido Ascórbico/química , Ácido Ascórbico/metabolismo , Ácido Ascórbico/farmacología , HidrólisisRESUMEN
Poly(ADP-ribosyl)lation (PARylation) is a posttranslational modification that plays an important role in a variety of biological processes in both animals and plants. Identification of PARylated substrates is the key to elucidating the regulatory mechanism of PARylation. Several approaches have been developed to identify PARylated substrates over the past decade; however, a reliable and efficient method is needed to demonstrate PARylated proteins. Here, we report a simple and sensitive assay of PARylated proteins using a clickable 6-alkyne-NAD+ analog. The 6-alkyne-NAD+ is incorporated into substrate proteins in the in vitro PARylation assay. The labeled proteins are covalently captured by disulfide azide agarose beads through copper-catalyzed azide-alkyne cycloaddition (CuAAC), cleaved under reducing conditions, and analyzed by immunoblotting. The covalent bonds between the PARylated proteins and azide beads allow high stringent washing to eliminate nonspecific binding. Furthermore, the disulfide linker permits efficient cleavage and recovery of highly enriched PARylated proteins. Therefore, this approach can detect proteins that undergo PARylation at very low levels.
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Azidas , NAD , Animales , NAD/metabolismo , Proteínas/química , Alquinos , Disulfuros , Adenosina DifosfatoRESUMEN
As a valid tumor marker, vascular endothelial growth factor 165 (VEGF165) is an effective therapeutic target for anticancer treatments. Aptamers hold great promise for the development of anti-VEGF strategies. In this study, anti-VEGF165 ssDNA aptamers were screened using a semirational design and a multilevel screening strategy. Recombinant human VEGF165 protein was used as a target for the construction of an ssDNA virtual aptamer library with ssDNA that had one sole secondary structure. After silicon-assisted prescreening, circular dichroism and isothermal titration calorimetry were used to further screen for candidates. Three aptamers (nos. 524, 529, and 64) with one sole secondary and tertiary structure, showing a high affinity for VEGF165, were identified. The KD values obtained using surface plasmon resonance analysis were 36.3, 288, and 79.3 nM for aptamers 524, 529, and 64, respectively. Cytological tests revealed that the three aptamers inhibit rhVEGF165-induced proliferation of HUVECs. Specifically, aptamer 529 had the strongest inhibitory effect (nearly 100% inhibition). The screening strategy used in our study showed improved screening efficiency relative to other methods and resulted in aptamers with one sole conformation. The aptamers had an advantage in ensuring the uniqueness of aptamer targeting. This semirational design and multilevel screening strategy provide a reference for the screening of other aptamers.
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Aptámeros de Nucleótidos , Técnica SELEX de Producción de Aptámeros , Humanos , Técnica SELEX de Producción de Aptámeros/métodos , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Aptámeros de Nucleótidos/química , Silicio , Factores de Crecimiento Endotelial Vascular , Biomarcadores de Tumor/metabolismoRESUMEN
BACKGROUND: Acute kidney injury is a clinical syndrome with both high morbidity and mortality. However, the underlying molecular mechanism of AKI is still largely unknown. The role of SENP1 in AKI is unclear, while one of its substrates, HIF-1α possesses nephroprotective effect in AKI. Herein, this study aimed to reveal the role of SENP1/HIF-1α axis in AKI by using both cell and animal models. METHODS: We investigated the effects of AKI on SENP1 expression using clinical samples, and cisplatin-induced AKI model based on mice or HK-2 cells. The influence of SENP1 knockdown or over-expression on cisplatin-induced AKI was studied in vitro and in vivo. Following the exploration of the change in HIF-1α expression brought by AKI, the synergistic effects of SENP1 knockdown and HIF-1α over-expression on AKI were examined. RESULTS: The results showed the up-regulation of SENP1 in clinical specimens, as well as cell and animal models. The knockdown or over-expression of SENP1 in HK-2 cells could promote or inhibit AKI through regulating cell apoptosis, respectively. Moreover, SENP1+/- mice suffered from much more serious AKI compared with mice in wild type group. Furthermore, we found that HIF-1α over-expression could attenuate the promoted cell apoptosis as well as AKI induced by SENP1 knockdown. CONCLUSIONS: we showed that SENP1 provided protection for kidney in AKI via regulating cell apoptosis and through the regulation of HIF-1α. This study could benefit for the understanding of the pathogenesis of AKI and provide potential therapeutic target for AKI treatment.
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Lesión Renal Aguda , Cisplatino , Animales , Apoptosis , Cisteína Endopeptidasas , Subunidad alfa del Factor 1 Inducible por Hipoxia , Riñón , RatonesRESUMEN
L-ascorbic acid 2-glucoside (AA-2G) is a derivative of L-ascorbic acid (L-AA). Compared with L-AA, it has good stability and is easily decomposed by enzyme in the human body. α-Glucosidase (AG) was the first enzyme found capable of producing AA-2G. However, researches on this enzyme is still in infancy. We took AG derived from Aspergillus niger (AAG), Japanese rice (JrAG) and Rattus rattus (RAG), and compared their specific enzymatic activity and transglycosidation rate, with the aim to improve the synthesis of AA-2G by the transglycosidation of AG. The genes encoding these three different AG were cloned and expressed in engineered yeast. The conditions for the transglycosidation reaction of these three enzymes were optimized and the transglycosidation efficiency and yield of AA-2G under the optimized conditions were compared. The specific activity of AAG reached 1.0 U/mg, while the yield of AA-2G reached 153.1 mg/L with a transglycosidation rate of 0.5%. The specific activity of RAG reached 0.4 U/mg, while the yield of AA-2G reached 861.0 mg/L with a transglycosidation rate of 2.5%. JrAG showed the highest specific activity and transglycosidation rate. The enzyme specific activity of JrAG reached 1.9 U/mg, while the yield of AA-2G reached 2 577.2 mg/L with a transglycosidation rate of 7.6%, much higher than that of the other two glucosidases. JrAG may thus have potential to improve the synthesis of AA-2G.
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Ácido Ascórbico , alfa-Glucosidasas , Animales , Ácido Ascórbico/análogos & derivados , Aspergillus niger/genética , Humanos , Ratas , alfa-Glucosidasas/genéticaRESUMEN
Renal ischemia-reperfusion (I/R) injury may lead to acute kidney injury, which is characterized by high morbidity and mortality rates. Resveratrol (RSV) can be extracted from Chinese herbs, and multiple animal experiments have demonstrated its potential for renal protection. This systematic review evaluates the protective effect of RSV against renal I/R injury in animal models. The PubMed, Embase, Web of Science, and Science Direct databases were searched for animal experiments related to RSV in renal I/R injury from their establishment to June 2022. In total, 19 studies were included with 249 animals (129 treated with RSV and 120 as controls). The pooled analysis revealed that RSV administration significantly decreased serum creatinine (SCr) levels (16 studies, n = 243, WMD = -58.13, 95% CI = -79.26 to -37.00, p < 0.00001) and blood urea nitrogen (BUN) levels (12 studies, n = 163, WMD = -34.37, 95% CI = -46.70 to -22.03, p < 0.00001) in the renal I/R injury model. The level of malondialdehyde (MDA), an oxidative stress index, was alleviated [7 studies, n = 106, standardized mean difference (SMD) = -6.05, 95% CI = -8.90 to -3.21, p < 0.0001] and antioxidant enzymes such as glutathione (GSH) (7 studies, n = 115, SMD = 9.25, 95% CI = 5.51-13.00, p < 0.00001) and catalase (CAT) (4 studies, n = 59, SMD = 8.69, 95% CI = 4.35-13.03, p < 0.0001) were increased after treatment of RSV. The subgroup analysis suggested that 5-10 mg/kg of RSV optimally protects against renal I/R injury as both the BUN and SCr levels were significantly decreased at this dosage. The protective effects of RSV against renal I/R injury might be attributed to multiple mechanisms, such as inhibiting oxidative stress, apoptosis, inflammation, fibrillation, and promoting autophagy. For a deeper understanding of the protective effects of RSV, experimental studies on animal models and large randomized controlled trials in humans are needed.
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Background and aims: Pancreatic adenocarcinoma (PAAD) is highly aggressive and characterized by a poor prognosis. Oxidative stress has great impacts on the occurrence and development of tumors. However, the predictive role of oxidative stress related genes on PAAD patients' prognosis remains unclear. In this study, we aimed to construct a prognostic model for PAAD based on oxidative stress genes and to evaluate its predictive value. Methods: The Cancer Genome Atlas (TCGA) and three Gene Expression Omnibus (GEO) datasets were used to identify differentially expressed oxidative stress genes. Univariate Cox regression, Kaplan-Meier and multivariate Cox regression analysis were used to select genes and to construct a prognosis model. According to the median value of the model's risk score, patients were divided into high and low risk groups, and gene set enrichment analysis (GSEA), immune infiltration and immunotherapy effect, drug resistance and the expression of immune checkpoint related genes and synthetic driver genes of T cell proliferation were analyzed. Finally, the mRNA and protein levels of four genes in PAAD were verified by the clinical proteomic tumor analysis consortium (CPTAC) database and the immunostaining of patients' tissue. Results: 55 differentially expressed oxidative stress genes were identified, and four genes including MET, FYN, CTTN and CDK1 were selected to construct a prognosis model. GESA indicated that immune related pathways, metabolic pathways and DNA repair pathways were significantly enriched in the high risk group as compared to the low risk group. The frequency of genetic mutations was also significantly higher in high risk groups than that in low risk groups. Moreover, the infiltration level of 23 immune cells as well as the expression of immune checkpoint related and synthetic driver genes of T cell proliferation were significantly altered, with the better immunotherapy effect occurring in low risk group. In patient PAAD tissues, the mRNA and protein levels of these four genes were up-regulated. Conclusion: We have successfully constructed a four oxidative stress gene prognostic model that has important predictive value for PAAD patients, and this model might be a promising guidance for prognostic prediction and efficacy monitoring in clinical individualized therapy.
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Vascular endothelial growth factor (VEGF165) is a highly specific vascular endothelial growth factor that can be used to treat many cardiovascular diseases. The development of anti-tumor drugs and disease detection reagents requires highly pure VEGF165 (at least 95% purity). To date, the methods for heterologous expression and purification of VEGF165 require multiple purification steps, but the product purity remains to be low. In this study, we optimized the codons of the human VEGF165 gene (vegf165) according to the yeast codon preference. Based on the Pichia pastoris BBPB vector, we used the Biobrick method to construct a five-copy rhVEGF165 recombinant expression vector using Pgap as the promoter. In addition, a histidine tag was added to the vector. Facilitated by the His tag and the heparin-binding domain of VEGF165, we were able to obtain highly pure rhVEGF165 (purity > 98%) protein using two-step affinity chromatography. The purified rhVEGF165 was biologically active, and reached a concentration of 0.45 mg/mL. The new design of the expression vector enables production of active and highly pure rhVEGF165 ) in a simplified purification process, the purity of the biologically active natural VEGF165 reached the highest reported to date.
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Pichia , Factor A de Crecimiento Endotelial Vascular , Codón/genética , Humanos , Pichia/genética , Proteínas Recombinantes/genética , Saccharomycetales , Factor A de Crecimiento Endotelial Vascular/genética , Factores de Crecimiento Endotelial VascularRESUMEN
Recently, cadmium (Cd) contamination in paddy soils has become a highly concerning pollution problem. Endophytic microbes in rice not only affect the plant growth but also contribute to ion absorption by the roots. Therefore, they are a promising, ecologically sound means of reducing the Cd transport from soils to shoots and grains of the plant. In this study, a Cd-resistant endophytic bacterium, named 181-22, with high Cd absorption capacity (90.8%) was isolated from the roots of rice planting in heavily Cd-contaminated paddy soils and was identified as Bacillus koreensis CGMCC 19,468. The strain significantly increased fresh weight of roots and shoots (44.4% and 42.7%) and dry weight of roots and shoots (71.3% and 39.9%) and decreased Cd content in the rice roots (12.8%), shoots (34.3%), and grains (39.1%) under Cd stress compared to uninoculated plant by colonizing rice roots via seed inoculation. Moreover, colonization of 181-22 reprogrammed rice physiology to alleviate Cd stress by increasing pigment and total protein content, regulating Cd-induced oxidative stress enzymes such as superoxide dismutase and catalase and reducing malondialdehyde. Thus, B. koreensis 181-22 has the potential to protect rice against Cd stress and can be used as a biofertilizer to bioremediate paddy soils contaminated with Cd. KEY POINTS: ⢠Bacillus koreensis 181-22 colonized the inside of rice roots at high numbers via seed inoculation. ⢠B. koreensis 181-22 promoted rice growth and decreased Cd accumulation in grains. ⢠B. koreensis 181-22 regulated the physiological response to alleviated Cd stress in rice.
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Bacillus , Oryza , Contaminantes del Suelo , Cadmio/análisis , Cadmio/toxicidad , Raíces de Plantas/química , Suelo , Contaminantes del Suelo/análisis , Contaminantes del Suelo/toxicidadRESUMEN
Poly(ADP-ribosyl)ation (PARylation) is a posttranslational modification reversibly catalyzed by poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribose) glycohydrolases (PARGs) and plays a key role in multiple cellular processes. The molecular mechanisms by which PARylation regulates innate immunity remain largely unknown in eukaryotes. Here we show that Arabidopsis UBC13A and UBC13B, the major drivers of lysine 63 (K63)-linked polyubiquitination, directly interact with PARPs/PARGs. Activation of pathogen-associated molecular pattern (PAMP)-triggered immunity promotes these interactions and enhances PARylation of UBC13. Both parp1 parp2 and ubc13a ubc13b mutants are compromised in immune responses with increased accumulation of total pathogenesis-related (PR) proteins but decreased accumulation of secreted PR proteins. Protein disulfide-isomerases (PDIs), essential components of endoplasmic reticulum quality control (ERQC) that ensure proper folding and maturation of proteins destined for secretion, complex with PARPs/PARGs and are PARylated upon PAMP perception. Significantly, PARylation of UBC13 regulates K63-linked ubiquitination of PDIs, which may further promote their disulfide isomerase activities for correct protein folding and subsequent secretion. Taken together, these results indicate that plant immunity is coordinately regulated by PARylation and K63-linked ubiquitination.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Inmunidad de la Planta , Poli ADP Ribosilación , Enzimas Ubiquitina-Conjugadoras/metabolismo , Arabidopsis/metabolismo , Glicósido Hidrolasas/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , UbiquitinaciónRESUMEN
Emodin, a major component of Chinese herbal rhubarb, delays the progression of chronic renal failure. However, the effect and working mechanisms of Emodin on renal tubulointerstitial fibrosis remains elusive. We hypothesized that emodin inhibits renal tubulointerstitial fibrosis through EZH2, a histone methyltransferase. Our in vivo and in vitro studies demonstrate that emodin reduced extracellular collagen deposition and inhibited Smad3 and CTGF pro-fibrotic signaling pathways, which were correlated with the down-regulation of EZH2 and reduced trimethylation of histone H3 on lysine 27 (H3k27me3) in NRK-49F fibrotic cells and UUO kidneys. Inhibition of EZH2 by 3-DZNeP blocked or attenuated the anti-fibrotic effect of emodin in UUO kidneys and NRK-49F cells. These data indicate that emodin inhibits renal tubulointerstitial fibrosis in obstructed kidneys and this effect is mediated through EZH2.
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Emodina/farmacología , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Insuficiencia Renal Crónica/tratamiento farmacológico , Animales , Factor de Crecimiento del Tejido Conjuntivo/antagonistas & inhibidores , Modelos Animales de Enfermedad , Medicamentos Herbarios Chinos/uso terapéutico , Inhibidores Enzimáticos/farmacología , Fibrosis , Técnicas In Vitro , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Masculino , Ratas , Ratas Sprague-Dawley , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patología , Transducción de Señal/efectos de los fármacos , Proteínas Smad/antagonistas & inhibidores , Obstrucción Ureteral/tratamiento farmacológico , Obstrucción Ureteral/metabolismo , Obstrucción Ureteral/patologíaRESUMEN
Aflatoxins (AFs) are potent carcinogens present in numerous crops. Access to accurate methods for evaluating contamination is a critical factor in aflatoxin risk assessment. Versicolorin A (Ver A), a precursor of aflatoxin B1 (AFB1), can be used as an indicator for the presence of AFB1, even when the AF is not yet detectable. Currently employed Ver A detection methods are expensive, time consuming, and difficult to apply to numerous samples. Herein, Ver A was detected via near-infrared spectroscopy. Both quantitative and two-grade sorting methods were set-up using the extreme gradient boosting algorithm coupled with a support vector machine. This two-tiered method obtained a root-mean-square error of prediction value of 3.57 µg/kg for the quantitative model, and an accuracy rate of 90.32% for the sorting approach. This novel method is rapid, accurate, solvent free, requires no sample pretreatment, and detects Ver A in maize, making it convenient for practical use.
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Aflatoxina B1/análisis , Antraquinonas/análisis , Espectroscopía Infrarroja Corta/métodos , Zea mays/química , Contaminación de Alimentos/análisis , HumanosRESUMEN
Hepatocyte nuclear factor 4 alpha (HNF4α) regulates the expression of essential genes involved in very-low-density lipoprotein (VLDL) homeostasis and gluconeogenesis.ã18ß-glycyrrhetinic acid (GA) is an active ingredient of Glycyrrhiza uralensis an herbal medicine used for treating liver aliments. In this study, we established that GA functions as a partial antagonist of HNF4α through HNF4α-driven reporter luciferase assay and co-immunoprecipitation experiments with co-activator PGC1α. By virtual docking and site-directed mutagenesis analysis, we confirmed that serine 190 and arginine 235 of HNF4α are both essential for GA to exert its antagonistic action on HNF4α. Importantly, GA suppressed the expression of HNF4α target genes such as apolipoprotein B (ApoB), microsomal triglyceride transfer protein (MTP) and phospholipase A2 G12B (PLA2G12B) modulating hepatic VLDL secretion in mice fed on a high fat diet. In addition, GA also suppressed gluconeogenesis and ameliorated glucose intolerance via down-regulating the expression of HNF4α target genes glucose-6-phosphatase (G6pc) and phosphoenolpyruvate carboxykinase (Pepck). Furthermore, GA significantly lowered blood glucose and improved insulin resistance in db/db mice. In all, we established that GA acts as a partial HNF4α antagonist modulating lipid and carbohydrate metabolism.
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Glucemia/efectos de los fármacos , Diabetes Mellitus/tratamiento farmacológico , Ácido Glicirretínico/análogos & derivados , Factor Nuclear 4 del Hepatocito/antagonistas & inhibidores , Hepatocitos/efectos de los fármacos , Hipoglucemiantes/farmacología , Hipolipemiantes/farmacología , Lípidos/sangre , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Animales , Glucemia/metabolismo , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Gluconeogénesis/efectos de los fármacos , Ácido Glicirretínico/farmacología , Células HEK293 , Factor Nuclear 4 del Hepatocito/genética , Factor Nuclear 4 del Hepatocito/metabolismo , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Resistencia a la Insulina , Masculino , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Transducción de SeñalRESUMEN
Hepatic nuclear factor 4 alpha (HNF4α) drives the expression of apolipoprotein B (ApoB), microsomal triglyceride transfer protein (MTP) and phospholipase A2 G12B (PLA2G12B), governing hepatic very-low-density lipoprotein (VLDL) production and secretion. Andrographolide (AP) is a major constituent isolated from Andrographis paniculata. We found that AP can disrupt the interaction between HNF4α and its coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α). Virtual docking and mutational analysis indicated that arginine 235 of HNF4α is essential for binding to AP. As a consequence of antagonizing the activity of HNF4α, AP suppresses the expression of ApoB, MTP and PLA2G12B and reduces the rate of hepatic VLDL secretion in vivo. AP additionally reduced gluconeogenesis via down-regulating the expression of HNF4α target genes phosphoenolpyruvate carboxykinase (Pepck) and glucose-6-phosphatase (G6pc). Collectively, our results suggest that AP affects liver function via modulating the transcriptional activity of HNF4α.
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Diterpenos/farmacología , Factor Nuclear 4 del Hepatocito/antagonistas & inhibidores , Hígado/efectos de los fármacos , Animales , Células Cultivadas , Gluconeogénesis/efectos de los fármacos , Gluconeogénesis/genética , Células HEK293 , Células Hep G2 , Factor Nuclear 4 del Hepatocito/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/antagonistas & inhibidores , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Unión Proteica/efectos de los fármacosRESUMEN
Beta-glucosidase (BGL1) is widely used in animal feed industries. However, degradation caused by digestive enzymes in the intestine hampers its application. Improving the resistance of feed enzymes against proteases is crucial in livestock farming. To improve the resistance of beta-glucosidase against pepsin and trypsin, a rational molecular design based on the inhibition of bound-state formation and secondary design was developed. The strategy includes: (1) prediction of the interaction surface of the pepsin-BGL1 complex structure, (2) prediction of key amino acids affecting the formation of the complex, (3) optimization of pepsin-resistant mutants by structural evaluation, (4) secondary molecular design based on pepsin-resistant mutants, and optimization of pepsin and trypsin-resistant mutants. Two BGL1 protein mutants (BGL1Q627C and BGL1Q627C/R543H/R646W) were constructed, and then mutated and wild-type BGL1s were expressed in Pichia pastoris. The half-life of BGL1Q627C and BGL1Q627C/R543H/R646W were 1.36 and 1.51 times that of the wild type upon pepsin exposure, respectively. For trypsin resistance, the half-life were 0.93 and 1.53 times that of the wild type, respectively. Compare to those of the wild type, most of the basic enzymatic properties of both mutants were not significantly changed except for increased Michaelis constants. The rational design method can be used as a guide for modifying other feed enzymes.
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Pepsina A/metabolismo , Trichoderma/enzimología , Tripsina/metabolismo , beta-Glucosidasa/genética , Hidrólisis , Trichoderma/genética , beta-Glucosidasa/metabolismoRESUMEN
Sequence-indexed insertional libraries are important resources for functional gene study in model plants. However, the maize (Zea mays) UniformMu library covers only 36% of the annotated maize genes. Here, we generated a new sequence-indexed maize Mutator insertional library named ChinaMu through high-throughput sequencing of enriched Mu-tagged sequences. A total of 2,581 Mu F2 lines were analyzed, and 311,924 nonredundant Mu insertion sites were obtained. Based on experimental validation, ChinaMu contains about 97,000 germinal Mu insertions, about twice as many as UniformMu. About two-thirds (66,565) of the insertions are high-quality germinal insertions (positive rate > 90%), 89.6% of which are located in genic regions. Furthermore, 45.7% (20,244) of the 44,300 annotated maize genes are effectively tagged and about two-thirds (13,425) of these genes harbor multiple insertions. We tested the utility of ChinaMu using pentatricopeptide repeat (PPR) genes. For published PPR genes with defective kernel phenotypes, 17 out of 20 were tagged, 11 of which had the previously reported mutant phenotype. For 16 unstudied PPR genes with both Mu insertions and defective kernel phenotypes, 6 contained insertions that cosegregated with the mutant phenotype. Our sequence-indexed Mu insertional library provides an important resource for functional genomics study in maize.
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Biblioteca de Genes , Genómica , Mutagénesis Insercional/genética , Mutación/genética , Zea mays/genética , Alelos , Secuencia de Bases , Cruzamientos Genéticos , Elementos Transponibles de ADN/genética , Genes de PlantasRESUMEN
The objective of this study was to evaluate the feasibility of the predictive monitoring of aflatoxin B1 (AFB1) under granary conditions, since mycotoxin contamination of the stored grain represents an important issue. Using the storage test, we investigated the relationship between versicolorin A (Ver A, an intermediate in AFB1 biosynthesis) levels and the levels of aflatoxigenic fungi, and their relationship with aflatoxin production. All samples, except for one, were found to be contaminated with aflatoxigenic fungi using PCR analyses, while their AFB1 levels were not detectable before the storage test using an enzyme-linked immunosorbent assay (ELISA) method with an LOD of 2 µg/kg. Aflatoxigenic fungi levels were analysed, as well as Ver A levels prior to the accumulation of AFB1 (Levels were ≥5 µg/kg; the permissible levels of AFB1 in corn intended for direct consumption are <5 µg/kg (EC)). Statistical analyses demonstrated that aflatoxin levels after both actual storage and safe storage (AFB1Ë5µg/kg) times are significantly correlated with the Ver A levels and the changes in Ver A levels (ΔVer A). Both high and variable Ver A levels were indicative of the vigorous metabolic activity of aflatoxigenic fungi. In contrast, steady Ver A levels showed that aflatoxin production by the fungi was not active. Monitoring Ver A levels and their changes may allow an earlier detection of harmful aflatoxin contamination in the stored grain. Additionally, the toxicity of Ver A should be further examined. The results of our study indicate that the monitoring of Ver A levels, even when the AFB1 levels are very low, may increase the safety of grain consumption, especially considering Ver A toxicity.
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Aflatoxina B1/análisis , Antraquinonas/análisis , Contaminación de Alimentos/análisis , Almacenamiento de Alimentos , Zea mays/química , Biomarcadores/análisis , Ensayo de Inmunoadsorción EnzimáticaRESUMEN
Berberine (BBR) has previously been found to exert beneficial effects on renal injury in experimental rats. However, the mechanisms underlying these effects are not yet fully understood. Tumor necrosis factor (TNF) receptor-associated factor 5 (TRAF5) has been demonstrated to mediate the activation of nuclear factor-κB (NF-κB), which has been implicated in the pathogenesis of chronic kidney disease (CKD). The aim of this study was to investigate the effects of BBR on kidney injury and the activation of the NF-κB signaling pathway in mouse podocytes. TRAF5 was found to be overexpressed in patients with CKD and chronic renal failure (CRF) (data obtained from the dataset GSE48944, as well as from patients at Shuguang Hospital). TRAF5 overexpression significantly inhibited cell viability and induced the apoptosis of mouse podocytes. However, BBR prevented the decrease in cell viability and the apoptosis induced by TRAF5 overexpression. The NF-κB inhibitor, caffeic acid phenethyl ester (CAPE), mimicked the protective effects of BBR, as evidenced by the increased expression of nephrin and podocin, and the decreased the expression of caspase-3 and the ratio of Bax/Bcl-2. Moreover, BBR prevented the decrease in cell viability decrease and the apoptosis induced by TNF-α, interleukin (IL)-6 and lipopolysaccharide (LPS). Taken together, our data indicate that BBR exerts protective effects against CRF partly through the TRAF5-mediated activation of the NF-κB signaling pathway in mouse podocytes.