To study the molecular mechanism of salt stress response of peanut small GTP binding protein gene AhRabG3f, a 1 914 bp promoter fragment upstream of the start codon of AhRabG3f gene (3f-P) from peanut was cloned. Subsequently, five truncated fragments (3f-P1-3f-P5) with lengths of 1 729, 1 379, 666, 510 and 179 bp were obtained through deletion at the 5' end, respectively. Plant expression vectors where these six promoter fragments were fused with the gus gene were constructed and transformed into tobacco by Agrobacterium-mediated method, respectively. GUS expression in transgenic tobacco and activity analysis were conducted. The gus gene expression can be detected in the transgenic tobacco harboring each promoter segment, among which the driving activity of the full-length promoter 3f-P was the weakest, while the driving activity of the promoter segment 3f-P3 was the strongest. Upon exposure of the transgenic tobacco to salt stress, the GUS activity driven by 3f-P, 3f-P1, 3f-P2 and 3f-P3 was 3.3, 1.2, 1.9 and 1.2 times compared to that of the transgenic plants without salt treatment. This suggests that the AhRabG3f promoter was salt-inducible and there might be positive regulatory elements between 3f-P and 3f-P3 in response to salt stress. The results of GUS activity driven by promoter fragments after salt treatment showed that elements included MYB and GT1 between 1 930 bp and 1 745 bp. Moreover, a TC-rich repeat between 682 bp and 526 bp might be positive cis-elements responsible for salt stress, and an MYC element between 1 395 bp and 682 bp might be a negative cis-element responsible for salt stress. This study may facilitate using the induced promoter to regulate the salt resistance of peanut.
Arachis/genetics* ; Fabaceae/genetics* ; GTP-Binding Proteins/metabolism* ; Gene Expression Regulation, Plant ; Glucuronidase/metabolism* ; Plant Proteins/metabolism* ; Plants, Genetically Modified/genetics* ; Salt Stress ; Stress, Physiological/genetics* ; Tobacco/genetics*

OBJECTIVE: To study the characteristics of gut microbiota and its association with the activity of β-glucuronidase (β-GD) in neonates with hyperbilirubinemia. METHODS: A total of 50 neonates with hyperbilirubinemia who were admitted in January to December, 2018, were enrolled as the hyperbilirubinemia group, and 30 neonates without hyperbilirubinemia were enrolled as the control group. The 16S rRNA high-throughput sequencing method was used to compare gut microbiota between the two groups. The phenolphthalein-glucuronic acid substrate method was used to measure the activity of β-GD in the intestinal tract of neonates with hyperbilirubinemia before and after treatment. RESULTS: The comparison of the distribution of gut microbiota at the genus level showed a significant difference in the abundance of 52 bacteria between the hyperbilirubinemia and control groups before treatment ( CONCLUSIONS There are differences in gut microbiota between the neonates with hyperbilirubinemia and those without hyperbilirubinemia. The activity of β-GD in feces is positively correlated with the abundance of
Feces ; Gastrointestinal Microbiome ; Glucuronidase ; Humans ; Hyperbilirubinemia, Neonatal ; Infant, Newborn ; RNA, Ribosomal, 16S

OBJECTIVE: To explore the effect of heparanase (HPSE) on apoptosis of microvascular endothelial cells (MVECs) and trans-endothelial migration of hepatocellular carcinoma (HCC) cells. METHODS: A HCC cell line with high HPSE expression was selected by real-time quantitative PCR (qRT-PCR) and Western blotting and transefected with a lentiviral vector containing an interfering RNA sequence of HPSE. Transwell migration assay was performed to detect the trans-endothelial migration (TEM) rate of the transfected HCC cells across human umbilical vein endothelial cells (HUVECs). In a Transwell indirect co-culture system, the effect of HPSE silencing in the HCC cells was determined on apoptosis of HUVECs . A nude mouse model of HCC was used to verify the effect of HPSE on apoptosis of MVECs and liver metastasis of the tumor. RESULTS: HCCLM3 cell line highly expressing HPSE was selected for the experiment. Transfection of the HCC cells with the lentiviral vector for HPSE interference the HCC cells resulted in significantly lowered TEM rate as compared with the cells transfected with the control vector ( < 0.01). In the indirect co-culture system, the survival rate of HUVECs co-cultured with HCCLM3 cells with HPSE interference was significantly higher and their apoptotic index was significantly lower than those in the control group ( < 0.05). Ultrastructural observation showed no obvious apoptosis of HUVECs co-cultured with HCCLM3 cells with HPSE interference but revealed obvious apoptotic changes in the control group. In the animal experiment, the tumor formation rate in the liver was 100% (6/6) in the control group, significantly higher than that in RNAi group (33.3%, 2/6) ( < 0.05). Under optical microscope, necrosis and apoptosis of the MVECs was detected in the liver of the control mice, while the endothelial cells remained almost intact in RNAi group. CONCLUSIONS HPSE promotes the metastasis of HCC cells by inducing apoptosis of MVECs.
Animals ; Apoptosis ; Carcinoma, Hepatocellular ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Endothelial Cells ; Gene Expression Regulation, Neoplastic ; Glucuronidase ; Humans ; Liver Neoplasms ; Mice

BACKGROUND: Gut microbiota is closely associated with development and exacerbation of inflammatory bowel diseases (IBD). The aim of this study was to investigate differences in gut microbiota depending on sex and changes of gut microbiota during IBD developments. METHODS: 16s rRNA metagenomic sequencing was performed for fecal materials from 8-week-old wild type (WT) and interleukin 10 (IL-10) knockout (KO) C57BL/6 mice of both sexes. Diversity indices, relative abundance of microbiota, and linear discriminant analysis effect size were examined to compare microbial communities between groups. Clustering of groups was performed by principal coordinates analysis (PCoA) and unweighted pair group method with arithmetic mean (UPGMA). Functional capabilities of microbiota were estimated using phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) based on Kyoto Encyclopedia of Genes and Genomes database. RESULTS: PCoA and UPGMA tree analysis of beta-diversity demonstrated significant differences in gut microbiota between male and female groups of WT mice, but not of IL-10 KO mice. Firmicutes to Bacteroides ratio was higher in male group than that in female group in both WT mice and IL-10 KO mice. Phylum Proteobacteria significantly increased in female IL-10 KO mice than that in female WT mice. At species level, Lactobacillus murinus, Bacteroides acidifaciens, and Helicobacter hepaticus significantly increased in IL-10 KO mice than in WT mice. The relative abundance of beta-glucuronidase (K01195) was higher in female IL-10 KO mice than that in female WT mice by PICRUSt. CONCLUSIONS: Our results suggest that microbiota-host interactions might differ between sexes during development of IBD.
Animals ; Bacteroides ; Female ; Firmicutes ; Gastrointestinal Microbiome ; Genome ; Glucuronidase ; Helicobacter hepaticus ; Humans ; Inflammatory Bowel Diseases ; Interleukin-10 ; Lactobacillus ; Male ; Metagenomics ; Methods ; Mice ; Microbiota ; Proteobacteria ; Sequence Analysis ; Sex Characteristics ; Trees

β-Glucosidase activity assays constitute an important indicator for the early diagnosis of neonatal necrotizing enterocolitis and qualitative changes in medicinal plants. The drawbacks of the existing methods are high consumption of both time and reagents, complexity in operation, and requirement of expensive instruments and highly trained personnel. The present study provides a simplified, highly selective, and miniaturized glucometer-based strategy for the detection of β-glucosidase activity. Single-factor experiments showed that optimum β-glucosidase activity was exhibited at 50 °C and pH 5.0 in a citric acid-sodium citrate buffer when reacting with 0.03 g/mL salicin for 30 min. The procedure for detection was simplified without the need of a chromogenic reaction. Validation of the analytical method demonstrated that the accuracy, precision, repeatability, stability, and durability were good. The linear ranges of β-glucosidase in a buffer solution and rat serum were 0.0873-1.5498 U/mL and 0.4076-2.9019 U/mL, respectively. The proposed method was free from interference from β-dextranase, snailase, β-galactosidase, hemicellulase, and glucuronic acid released by baicalin. This demonstrated that the proposed assay had a higher selectivity than the conventional dinitrosalicylic acid (DNS) assay because of the specificity for salicin and unique recognition of glucose by a personal glucose meter. Miniaturization of the method resulted in a microassay for β-glucosidase activity. The easy-to-operate method was successfully used to detect a series of β-glucosidases extracted from bitter almonds and cultured by Aspergillus niger. In addition, the simplified and miniaturized glucometer-based assay has potential application in the point-of-care testing of β-glucosidase in many fields, including medical diagnostics, food safety, and environmental monitoring.
Animals ; Aspergillus niger ; Calibration ; Cellulase/analysis* ; Chemistry, Clinical/methods* ; Dextranase/analysis* ; Enterocolitis, Necrotizing/diagnosis* ; Equipment Design ; Flavonoids/analysis* ; Glucose/analysis* ; Glucuronic Acid/analysis* ; Glucuronidase/analysis* ; Glycoside Hydrolases/analysis* ; Hydrogen-Ion Concentration ; Linear Models ; Multienzyme Complexes/analysis* ; Plants, Medicinal ; Polygalacturonase/analysis* ; Rats ; Reproducibility of Results ; beta-Galactosidase/analysis* ; beta-Glucosidase/analysis*

An efficient genetic transformation system and suitable promoters are essential prerequisites for gene expression studies and genetic engineering in streptomycetes. In this study, firstly, a genetic transformation system based on intergeneric conjugation was developed in Streptomyces rimosus M527, a bacterial strain which exhibits strong antagonistic activity against a broad range of plant-pathogenic fungi. Some experimental parameters involved in this procedure were optimized, including the conjugative media, ratio of donor to recipient, heat shock temperature, and incubation time of mixed culture. Under the optimal conditions, a maximal conjugation frequency of 3.05×10^-5 per recipient was obtained. Subsequently, based on the above developed and optimized transformation system, the synthetic promoters SPL-21 and SPL-57, a native promoter potrB, and a constitutive promoter permE^* commonly used for gene expression in streptomycetes were selected and their activity was analyzed using gusA as a reporter gene in S. rimosus M527. Among the four tested promoters, SPL-21 exhibited the strongest expression activity and gave rise to a 2.2-fold increase in β-glucuronidase (GUS) activity compared with the control promoter permE^*. Promoter SPL-57 showed activity comparable to that of permE^*. Promoter potrB, which showed the lowest activity, showed a 50% decrease in GUS activity compared with the control permE^*. The transformation system developed in this study and the tested promotors provide a basis for the further modification of S. rimosus M527.
Conjugation, Genetic ; Glucuronidase/genetics* ; Promoter Regions, Genetic ; Streptomyces rimosus/genetics*

OBJECTIVE: To observe the effect of Ronghuang granule on serum fibroblast growth factor 23 (FGF23), fibroblast growth factor receptor (FGFRs) and Klotho protein levels in non-dialysis patients with chronic kidney disease-mineral and bone disorder (CKD-MBD) and kidney deficiency and damp heat syndrome. METHODS: Seventy non-dialysis CKD-MBD patients with kidney deficiency and dampness-heat syndrome were randomized into control group (=35) and treatment group (=35). All the patients were given routine treatment combined with traditional Chinese medicine retention enema, and the patients in the treatment group received additional Ronghuang granule treatment (3 times a day). After the 12-week treatments, the patients were examined for changes of TCM syndromes. Serum levels of Ca, P, parathyroid hormone (iPTH), FGF23, FGFRs and Klotho proteins were detected before and after treatment. These parameters were also examined in 20 healthy volunteers. RESULTS: Sixty-five patients completed the study, including 33 in the control group and 32 in the treatment group. The patients in the treatment group showed significantly better treatment responses than those in the control group ( < 0.05 or 0.01). At 4, 8, and 12 weeks of treatment, the patients in the treatment group had significantly lowered scores of TCM syndromes compared with the score before treatment ( < 0.05 or 0.01), while in the control group, significant reduction of the scores occurred only at 12 weeks ( < 0.05); at each of the time points, the treatment group had significantly greater reductions in the score than the control group ( < 0.01). Significant improvements in serum Ca, P and iPTH levels were observed at 4, 8, and 12 weeks in the treatment group ( < 0.05) but only at 12 weeks in the control group ( < 0.05). The patients in the control and treatment groups all showed elevated serum levels of FGF23, FGFRs and Klotho protein compared with the normal subjects ( < 0.01); FGF23, FGFRs and Klotho levels were significantly reduced in the treatment group ( < 0.05) but remained unchanged in the control group (>0.05), showing significant differences between the two groups. CONCLUSIONS Ronghuang granule improves the clinical symptoms of non-dialysis CKD-MBD patients with kidney deficiency and dampness heat syndrome by reducing serum levels of FGF23, FGFRs and Klotho, improving calcium and phosphorus metabolism disorder, and inhibiting secondary hyperparathyroidism.
Calcium ; blood ; Chronic Kidney Disease-Mineral and Bone Disorder ; blood ; therapy ; Drugs, Chinese Herbal ; pharmacology ; Enema ; Fibroblast Growth Factors ; blood ; Glucuronidase ; blood ; Humans ; Parathyroid Hormone ; blood ; Phosphorus ; blood ; Receptors, Fibroblast Growth Factor ; blood ; Renal Insufficiency, Chronic ; blood ; therapy ; Sweating Sickness ; blood ; therapy ; Syndrome

Klotho is highly expressed in the kidney, while soluble Klotho is detectable in the blood, urine, and cerebrospinal fluid, and has multiple hormone-like functions. The role of Klotho in kidney injury has attracted more and more attentions from researchers. Emerging evidence revealed that the transient deficiency of Klotho is an early event of acute kidney injury (AKI), whereas, in chronic kidney disease, this deficiency is sustained not only in the kidney, but also in other organ systems. Therefore, Klotho could be a potential biomarker for early diagnosis of AKI, as well as for its progression to chronic kidney disease. Moreover, Klotho might have therapeutic value to renal injury. Nevertheless, there are only few studies on the involvement of Klotho in post AKI repair. This review focused on the role of Klotho in not only kidney injury, but also its repair, in particular the relationship between Klotho and cell fate (autophagy/apoptosis/necrosis), repair/regeneration, Wnt/β-catenin and erythropoietin receptor, one of the Klotho effectors.
Acute Kidney Injury ; metabolism ; Biomarkers ; Disease Progression ; Glucuronidase ; physiology ; Humans ; Kidney ; metabolism ; pathology ; Signal Transduction

The increasing demand of Chinese materia medica could not be supplied by wild resource, and the cultivated medicinal materials become popular, which led to decreased quality of many medicinal materials due to the difference of the circumstance between the wild and the cultivated. How to improve quality becomes key points of Chinese medicine resource. The leaves of Scutellaria baicalensis were sprayed with H₂O₂, the activities of superoxide dismutase (SOD) and catalase (CAT) changed little, but there had been a marked decrease of peroxidase (POD) and ascorbic oxidase (APX), which showed that the antioxidase system declined. Meanwhile, H₂O₂, as enhanced the expression of phenylalnine ammonialyase (PAL) and β-glucuronidase (GUS) as well as activity of PAL, promoted the biosynthesis and biotransformation of flavonoids. At the day 2 after treated, H₂O₂ of 0.004 μmol·L⁻¹ the contents of the baicalin and the wogonoside decreased slightly, but the contents of the baicalein and the wogonin increased significantly, the baicalein from 0.094% to 0.324%, the wogonin from 0.060% to 0.110%, i. e. increased 246% and 83.3%, respectively.
Ascorbate Oxidase ; metabolism ; Catalase ; metabolism ; Drugs, Chinese Herbal ; chemistry ; Flavanones ; analysis ; Flavonoids ; analysis ; Glucosides ; analysis ; Glucuronidase ; metabolism ; Hydrogen Peroxide ; Peroxidase ; metabolism ; Phenylalanine Ammonia-Lyase ; metabolism ; Scutellaria baicalensis ; metabolism ; Secondary Metabolism ; Superoxide Dismutase ; metabolism

To improve bond selectivity of recombinant β-glucuronidase in Escherichia coli (PGUS-E), based on the PGUS-E structure guidance, three key points R329, T369 and N467 were identified to be responsible for the bond selectivity of PGUS-E, and further saturation mutagenesis was conducted. Two positive mutants R329K and T369V were obtained by a combined selection technique of thin-layer chromatography and high performance liquid chromatography. Compared to PGUS-E, the bond selectivity of mutants R329K and T369V increased by 26.9% and 34.3%, respectively; whereas the biochemical properties such as pH and temperature profile were unchanged. Nevertheless, the activity was decreased compared to PGUS-E. These results further confirmed that sites R329 and T369 played important roles for the bond selectivity and activity. In summary, this study significantly increased the bond selectivity of PGUS-E by structure guided saturation mutagenesis, providing experimental support for elucidating the relationship between the structure and function of PGUS-E.
Chromatography, High Pressure Liquid ; Chromatography, Thin Layer ; Escherichia coli ; metabolism ; Glucuronidase ; chemistry ; Industrial Microbiology ; Mutagenesis ; Recombinant Proteins ; chemistry ; Temperature