Construction of ATMT System for Clonostachys rosea and Its Conditional Optimization of Colonization into Glycyrrhiza uralensis Seeds / 中国实验方剂学杂志

ObjectiveAgrobacterium tumefaciens-mediated transformation （ATMT） of Clonostachys rosea， an endophytic fungus of Glycyrrhiza uralensis seeds， was established and optimized， and orthogonal test was designed to optimize the colonization conditions of C. rosea for G. uralensis seeds， so as to lay foundation for the development of biofertilizer and the breeding of high-quality G. uralensis. MethodThe conditions of ATMT were optimized from three aspects， including the concentration of acetosyringone， co-culture time and the concentration of conidia of recipient fungi. Then， high-quality transformants were selected. Orthogonal test was used to optimize the colonization conditions by taking co-culture temperature， co-culture time and spore concentration as factors and colonization rate as index. ResultWhen spore concentration was 1×107 cfu·mL-1， acetosyringone concentration was 150 μmol·L-1 and the co-culture time was 60 h， the transformation efficiency of C. rosea was the highest， which was 135 transformants per 1×107 recipient fungal spores. The accuracy and stability of the transformations were tested by cloning the marker gene green fluorescent protein （GFP） and β-glucuronidase （GUS） staining. When co-culture temperature was 25 ℃， co-culture time was 36 h and the spore concentration was 1×106 cfu·mL-1， the colonizing rate for C. rosea back dyeing into G. uralensis seeds by seed soaking method was the highest， which was 71.11%. ConclusionThis study successfully establishes stable and efficient technical systems not only of ATMT in C. rosea， but also of colonization of the transformants into G. uralensis seeds， which can lay a foundation for the development of biofertilizer of G. uralensis.

Research progress on the interactions between gut bacterial β-glucuronidases and Chinese herbal medicines / 药学学报

In traditional oral practice, the presystemic interactions with gut microbiota is an important mechanism underlying the holistic health benefits of Chinese herbal medicines (CHMs), making the study of CHMs distinct from the research of Western medicines of which the systemic exposure (level in blood) is the starting point and the core. Gut microbial metabolism complements host metabolism in maintaining metabolic homeostasis of many biologically important endogenous molecules and the disposition of numerous exogenous compounds. Among them, the widely distributed gut bacterial β-glucuronidases (BGUSs) coordinate with host UDP-glucuronosyltransferases (UGTs) to play a role in the occurrence and intervention of diseases by affecting the glucuronidation homeostasis and altering the intestinal local and/or systemic exposure of endogenous compounds and xenobiotics. On one hand, many ingredients of CHMs undergo enterohepatic circulation; On the other hand, CHMs can act on BGUSs directly or indirectly change the distribution and function of BGUSs through reprogramming gut microbiome. The multiple interactions between BGUSs and CHMs may play an important role in the overall therapeutic benefits of CHMs. This work firstly summarizes the latest research progress on BGUSs; then the physiological, pathological and pharmacological significance of BGUSs are exemplified with representative endogenous and exogenous compounds from the aspects of nutrient utilization, metabolic homeostasis, and therapeutic response based on the varied substrate spectra of BGUSs; finally, the scattered data in literature were integrated to summarize the multiple interactions between BGUSs and CHMs, highlighting the important role of BGUSs in the holistic actions of CHMs.

Identification of Escherichia coli β-glucuronidase inhibitors from Polygonum cuspidatum Siebold & Zucc

Abstract Gut bacterial β-glucuronidase (GUS) can reactivate xenobiotics that exert enterohepatic circulation- triggered gastrointestinal tract toxicity. GUS inhibitors can alleviate drug-induced enteropathy and improve treatment outcomes. We evaluated the inhibitory effect of Polygonum cuspidatum Siebold & Zucc. and its major constituents against Escherichia coli GUS (EcGUS), and characterized the inhibitory mechanism of each of the components. Trans-resveratrol 4'-O-β-D-glucopyranoside (HZ-1) and (-)-epicatechin gallate (HZ-2) isolated from P. cuspidatum were identified as the key components and potent inhibitors. These two components displayed strong to moderate inhibitory effects on EcGUS, with Ki values of 9.95 and 1.95 μM, respectively. Results from molecular docking indicated that HZ-1 and HZ-2 could interact with the key residues Asp163, Ser360, Ile 363, Glu413, Glu504, and Lys 568 of EcGUS via hydrogen bonding. Our findings demonstrate the inhibitory effect of P. cuspidatum and its two components on EcGUS, which supported the further evaluation and development of P. cuspidatum and its two active components as novel candidates for alleviating drug-induced damage in the mammalian gut.

Hepatotoxicity of Main Components of Polygonum multiflorum and Its Effects on Drug Metabolism Enzyme / 中国药房

OBJECTIVE：To study the hepatotoxicity of main components of Polygonum multiflorum ，and investigate its toxic mechanism based on metabolic enzymes. METHODS ：ADMETlab 2.0 platform was used to forecast the toxic or carcinogenic effects of emodin ，physcion，rhein，stilbene glycoside and gallic acid on liver ，skin and heart. The effects of those components on cytochrome P 450 enzyme system （CYP1A2，CYP2C9，CYP2C19，CYP2D6，CYP3A4）were evaluated. The effects of different concentrations of emodin ，rhein，stilbene glycoside and gallic acid （10，20，40，80 μmol/L）on the survival rate of normal hepatocyte L 02 were detected. The effects of major components of P. multiflorum on the activity of UGT 1A1 enzyme were studied by in vitro reaction system ，using bilirubin as substrate. RESULTS ：Main components of P. multiflorum ，ie. emodin ，physcion， rhein and gallic acid ，showed strong toxic effects on the liver ，while stilbene glycosides possessed weak toxic effects on the liver. Emodin and physcion had strong inhibitory effects on CYP 1A2 and medium inhibitory effects on CYP 2C9，CYP2D6 and CYP3A4；rhein showed medium inhibitory effects on CYP 1A2 and CYP 2C9，while stilbene glycoside and gallic acid possessed weak inhibitory effects on the above enzymes. Emodin （40，80 μmol/L）and gallic acid （40，80 μmol/L）could significantly reduce the survival rate of L 02 cells（P＜0.01）. The inhibition rate of 5，10，20，40，80 μmol/L emodin and gallic acid（except for 5 μmol/L emodin）on UGT 1A1 enzyme increased significantly （P＜0.01），and the inhibition effect of emodin on UGT 1A1 enzyme was reversible competitive inhibition. CONCLUSIONS ：The main components of P. multiflorum ，ie. emodin ，rhein and physcion ， are hepatotoxic ；the mechanism of it may be associated with inhibiting the activity of CYP 1A2 and CYP 2C9 and competitively blocking rate-limiting enzyme UGT 1A1 in the process of bilirubin metabolism.

Presence of benznidazole conjugated metabolites in urine identified by β-glucuronidase treatment

Chagas disease is a serious public health problem in Latin America and, due to migration, in other non-endemic regions. Benznidazole (BNZ) is first choice drug in pediatric therapeutics. However, little is known regarding its metabolism in humans. The aim of the study was to isolate and identify products of human BZN metabolism in urine samples obtained from a pediatric Chagas patient and a healthy adult volunteer both treated with BZN. Urine samples were collected after dose of BNZ. Urine was treated with β-glucuronidase followed by an extraction procedure under two different pH conditions and a HPLC/UV and MS/MS identification of BZN and its metabolites. BZN (m/z 260.09847) was identified in all urine extracts. Peaks from each extracted chromatograms were selected for MS and MS/MS identification. Three compounds structurally related to BZN were identified: BZN-Na+ (m/z 283.08009), N-amine-BZN (m/z 230.12307) and N-hydroxi-amine-BZN (m/z 246.11702). BNZ-Na+ was identified in all extracts, but N-amine-BZN and N-hydroxi-amine-BZN were only observed in those extracts treated with β-glucuronidase. This is the first experimental report showing elimination of BZN N-reduced metabolites in urine. As they were released after treatment with β-glucuronidase it can be suggested that glucuronization plays a role in BNZ metabolism and renal elimination.

Development and optimization of an intergeneric conjugation system and analysis of promoter activity in Streptomyces rimosus M527 / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

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 (3-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.

Factores que afectan la expresión transitoria del gen GUS en yuca (Manihot esculenta Crantz) / Factors affecting the transient expression of gene GUS in cassava (Manihot esculenta Crantz)

RESUMEN La expresión transitoria es una métodología ampliamente utilizada para el estudio de genes. Sin embargo, hasta la fecha no existe un reporte en donde se utilice esta técnica en hojas de yuca de plantas adultas. Por esta razón este trabajo se centró en la determinación de algunos parámetros críticos para la expresión transitoria del gen GUS en yuca como son: la metodología para introducir [a bacteria, [a cepa de Agrobacterium, el tiempo post-inoculación, la introducción del gen VirG y la expresión del gen GUS en algunas variedades de yuca. Los resultados indicaron niveles más altos de expresión del gen GUS entre 5-7 días postinoculación (dpi), agroinfiltrando con la cepa GV3101 y un incremento en la virulencia de esta cepa mediante la introducción del gen VirG. Por último se observaron diferentes niveles de expresión del gen GUS entre [as variedades de yuca evaluadas, [o que indica que el factor genético es clave en la eficiencia de la agroinfiltración en este cultivo.

ABSTRACT Transient expression is a common technique used for the co-expression of proteins for a wide range of experiments in molecular biology. This technique has been frequently used for gene study in plant pathogen interaction and has proved to be versatile and effective. However, there are still few reports and sparse data for transient expression in cassava leaves from adult/mature plants. For this reason, in this study we evaluated some regards of transient expression for the gene GUS in cassava: bacteria introduction methods, type of Agrobacterium strains, time post-inoculation, introduction of gene VirG and GUS and their expression into different cassava varieties. The results show that GUS expression is more efficient between 5-7 days post-inoculation with the GV3101 strain, moreover the virulence of this strain increases with the VirG gene. Finally, we observed a range of GUS expression pattern between different cassava varieties, altogether showing that the genetic factor is important for an accurate and effective agroinfiltration in cassava.

Effect of Klotho protein on calcium and phosphorus metabolism in chronic kidney disease / 中国医师杂志

The incidence of chronic kidney disease (CKD) gradually increased.With the decline of renal function,CKD patients gradually appear intractable hypertension,anemia,malnutrition,calcium and phosphorus metabolic disorders,micro-inflammatory State and other complications which can seriously affect the survival and prognosis.Among them,calcium and phosphorus metabolism disorders are more common complications,and can lead to high incidence of cardiovascular disease and increased mortality.Recent studies have shown that Klotho protein has anti-inflammatory,anti-oxidative stress,anti-apoptosis and antifibrosis effects.It can inhibit renal fibrosis and regulate calcium and phosphorus metabolism disorders in patients with CKD.This article reviews the regulatory role of Klotho protein in calcium and phosphorus metabolism.

Metabolic profile and β-glucuronidase inhibitory property of three species of Swertia

ABSTRACT β-Glucuronidase inhibitors are suggested as potential hepatoprotective agents. Swertia chirayita (Roxb.) Buch.-Ham. ex C.B. Clarke, Gentianaceae, is known for its hepatoprotective and anti-hepatotoxic activity in Ayurvedic system of medicine for ages. This plant is substituted by other species like S. decussata Nimmo ex C.B. Clarke and S. bimaculata (Siebold & Zucc.) Hook. f. & Thomson ex C.B. Clarke. The aim of the study was to compare metabolite profile and β-glucuronidase inhibitory activity of these three important species of Swertia and to identify the active constituents. S. chirayita (IC50 210.97 µg/ml) and S. decussata (IC50 269.7 µg/ml) showed β-glucuronidase inhibitory activity significantly higher than that of silymarin, the known inhibitor of the enzyme. The activity of S. bimaculata was low. The metabolites present in the three species were analyzed by HPLC and GC-MS based metabolomics approach. Five amino acids, twenty one organic acids, one inorganic acid, eight fatty acids, twenty one phenols including xanthones, eight sugars, seven sugar alcohols, five terpenoids and amarogentin were identified. Activities of the xanthones mangiferin (IC50 16.06 µg/ml), swerchirin (IC50 162.84 µg/ml), decussatin (IC50 195.11 µg/ml), 1-hydroxy-3,5,8-trimethoxy xanthone (IC50 245.97 µg/ml), bellidifolin (IC50 390.26 µg/ml) were significantly higher than that of silymarin (IC50 794.62 µg/ml). Quinic acid (IC50 2.91 mg/ml), O-acetylsalicylic acid (IC50 48.4 mg/ml), citric acid (IC50 1.77 mg/ml), D-malic acid (IC50 14.82 mg/ml) and succinic acid (IC50 38.86 mg/ml) also inhibited the enzyme β-glucuronidase. The findings suggest that constituents, in addition to the xanthones, probably also contribute to the bioactivity of different Swertia species by synergistic effect. Further in vivo study is required to support the claim.

Phosphorescent Inner Filter Effect-based Sensing System for Determination of β-Glucuronidase Using Manganese-doped Zinc Sulfide Quantum Dots / 分析化学

The key to maximize the sensitivity of inner filter effect ( IFE)-based assay is to enlarge the overlap between the absorption spectra of the absorber and the excitation or emission spectra of the fluorophore. In this work, Mn-doped ZnS quantum dots ( QDs) were chosen for IFE-based detection of β-glucuronidase ( GUS) , since the excitation of QDs was perfectly overlapped with the absorption of the substrate of GUS, namely 4-nitrophenyl-β-D-glucuronide ( PNPG) . In addition, the phosphorescence emission from Mn-doped ZnS QDs could eliminate the fluorescence background from biological samples. Therefore, a simple turn-on phosphorescent GUS assay was developed, with the linear range of 10-300 U/L and limit of detection of 7 U/L (S/N=3).

Research Progress in β-Glucuronidase / 中国药师

Objective:To summarize the structure, properties and biological functions ofβ-glucuronidase. Methods:By referring to the relevant literatures at home and abroad onβ-glucuronidase in recent years, this paper induced, analyzed and drew conclusions. Results:The crystal structure of humanβ-glucuronidase is comprised of three distinct structural domains. The most common mutation of β-glucuronidase is missense mutation, which results in the change of enzyme activity, and then induces a series of pathological chan-ges like MPSⅦ. The researches on β-glucuronidase used in antibody-directed enzyme prodrug drug therapy and enzyme replacement therapy are becoming deeper and deeper. Conclusion:β-Glucuronidase as an important acid hydrolytic enzyme in human has a variety of genetic mutations, and plays an important role in the fields of medicine and disease diagnosis, which has become one of research hotspots.

The role of endogenous β-glucuronidase expression in the formation of hepatolithiasis / 中华肝胆外科杂志

Objective To study the mechanisms involved in the regulation of endogenous β-glucuronidase expression and to explore the more effective methods to prevent recurrence of hepatolithiasis formation.Methods The expression levels of c-myc and endogenous β-glucuronidase in the liver specimens of hepatolithiasis were examined by immunohistochemical staining.The expressions of c-myc and endogenous β-glucuronidase in the human intrahepatic biliary epithelial cell line (HiBEpiC),and normal liver cell line (L02) treated with different concentrations of lipopolysaccharide (LPS) were studied using western blot.The c-myc siRNA transfection was utilized to detect the role of c-myc in the regulation of the expression of endogenous β-glucuronidase.Results Compared with normal liver samples,the expressions of endogenous β-glucuronidase and c-myc in the liver specimens of hepatolithiasis were significantly increased,and they were positively correlated with each other.LPS induced increased expressions of endogenous β-glucuronidase and c-myc in a dose-dependent manner.C-myc siRNA transfection effectively inhibited the increased expression of endogenous β-glucuronidase as induced by LPS.Conclusion LPS played a crucial role in the formation of hepatolithiasis by stimulating the endogenous expression of β-glucuronidase in liver and biliary epithelial cells via c-myc.

Association of serum soluble Klotho with episode of nonfatal cardiovascular disease and mortality in maintenance hemodialysis patients / 中华肾脏病杂志

Objective To explore the association of serum soluble Klotho (sKlotho) with nonfatal cardiovascular disease (CVD) and all-cause/CVD mortality in maintenance hemodialysis (MHD) patients.Methods A total of 132 MHD patients admitted during October 2011 were enrolled.Serum sKlotho was measured by ELISA.Demographic data,including age,gender and comorbid conditions,were obtained from their medical histories,and parameters including calcium,phosphorus and albumin were assessed.The occurrence time of nonfatal CVD and all-cause mortality were recorded during the 60 months follow-up.MHD patients were categorized into four groups according to the quartiles of sKlotho:group Ⅰ (sKlotho ＜ 361.34 ng/L),group Ⅱ (361.34 ng/L≤sKlotho＜ 398.81 ng/L),group Ⅲ (398.81 ng/L≤sKlotho＜445.99 ng/L) and group Ⅳ (sKlotho≥445.99 ng/L).Spearman correlation analysis and binary Logistic regression analysis were used to test the association between sKlotho and nonfatal CVD events.The impacts of sKlotho on all-cause mortality and CVD mortality were assessed by Kaplan-Meier method with log-rank test.Cox regression model was applied to evaluate the effect of sKlotho on MHD patients outcomes.Results All 132 MHD patients had sKlotho ranging from 304.02 ng/L to 550.62 ng/L.And 87 patients suffered from nonfatal CVD,with 192 episodes of nonfatal CVD during the follow-up period.The sKlotho had negative correlations with coronary artery disease (r=-0.286,P=0.001),congestive heart failure (r=--0.190,P=0.029),cerebrovascular accident (r=-0.240,P=0.006) and peripheral arterial occlusion (r=-0.243,P=0.005).The sKlotho were risk factors of coronary artery disease (OR=0.989,P=0.023) and peripheral artery occlusion (OR=0.988,P=0.046).35 patients died in the follow-up period,including 27 death from CVD.The all-cause mortality and CYD mortality rates were significantly different among four groups (P=0.036,P=0.047).Survival rates of all-cause death and CVD death varied among four groups (x2=8.076,P=0.044;X2=7.866,P=0.049).Patients in group Ⅳ had higher survival rates of allcause death and CVD death than those in group Ⅰ and group Ⅱ (all P ＜ 0.05).Multivariate Cox regression analyses revealed diabetes and age were independent risk factors for all-cause mortality and CVD mortality (all P ＜ 0.05),but sKlotho was not associated with the poor prognosis (HR=0.996,P=0.256;HR=0.996,P=0.287).Conclusions Patients with lower sKlotho have worse nonfatal CVD ratio,especially coronary artery disease and peripheral arterial occlusion.Reduced serum sKlotho is associated with all-cause and CVD mortality,but sKlotho is still not a predictive indicator of prognosis of MHD patients.

Research Progress in β-Glucuronidase / 中国药师

Objective:To summarize the structure, properties and biological functions ofβ-glucuronidase. Methods:By referring to the relevant literatures at home and abroad onβ-glucuronidase in recent years, this paper induced, analyzed and drew conclusions. Results:The crystal structure of humanβ-glucuronidase is comprised of three distinct structural domains. The most common mutation of β-glucuronidase is missense mutation, which results in the change of enzyme activity, and then induces a series of pathological chan-ges like MPSⅦ. The researches on β-glucuronidase used in antibody-directed enzyme prodrug drug therapy and enzyme replacement therapy are becoming deeper and deeper. Conclusion:β-Glucuronidase as an important acid hydrolytic enzyme in human has a variety of genetic mutations, and plays an important role in the fields of medicine and disease diagnosis, which has become one of research hotspots.

Expression of mRNA and protein of Klotho gene in placental tissue of macrosomia and its relationship with birth weight of neonates / 中华妇产科杂志

Objective To explore the the expression of Klotho mRNA and protein in placenta of macrosomia and its relationship with the birth weight of neonates. Methods The cases were from November 2014 to March 2015 in Shengjing Hospital of China Medical University, divided into 4 groups:the gestational diabetes with macrosomia group (GM), the gestational diabetes with normal birth weight group (GN), the normal pregnancy with macrosomia group (NM) and the normal pregnancy with normal birth weight group (NN). Klotho mRNA and protein expression in the placenta were detected by immunohistochemistry SP method, real-time fluorescent quantitative PCR and western blot, respectively, and were compared among the 4 groups. Results (1) Immunohistochemical detection showed the positive rate of Klotho protein was significantly higher in the placenta of GM (93%,28/30) than in the GN (73%,22/30;P0.05). Conclusions The up-regulation of Klotho gene may be associated with macrosomia. The relationship is not affected by the complication of gestational diabetes.

Research progress of the relationship between klotho protein and chronic kidney disease / 中国医师杂志

Klotho gene is closely related to the aging,which plays an important role in modulation of mineral metabolism,anti-apoptosis,anti-oxidation and antisenescence,etc.Klotho deficiency leads to a syndrome resembling aging,such as shortened life span,atherosclerosis,skin atrophy,hyperphosphatemia,etc.This performance is similar to that of the patients with chronic kidney disease (CKD).The levels of klotho expression and transcription are down-regulated in CKD resulted from various causes.Thus,CKD may be viewed as a state of accelerated aging and/or an age-related disease associated with Klotho deficiency.This article reviewed the relationship between Klotho and CKD.

Analysis of heparanase isoforms and cathepsin B in the plasma of patients with gastrointestinal carcinomas: analytical cross-sectional study / Análises das isoformas de heparanase e da catepsina B em plasma de pacientes com carcinomas gastrointestinais: estudo transversal analítico

CONTEXT AND OBJECTIVE: Heparanase-1 degrades heparan sulfate and has been correlated with tumor progression. Although the isoform heparanase-2 has no catalytic activity, it seems to be important for modulating heparanase-1 activity. Cathepsin B is a proteinase involved in tumor metastasis. The aim of this study was to analyze heparanase isoform expression and cathepsin B activity in plasma samples from patients with gastrointestinal carcinomas, compared with healthy individuals (control group). DESIGN AND SETTING: This was an analytical cross-sectional study. Peripheral blood samples were collected at a Brazilian public hospital, from 21 patients with histopathological diagnoses of gastrointestinal carcinomas and from 43 healthy individuals. The analyses were performed in two Brazilian medical schools. METHODS: Heparanase isoforms were identified and quantified in plasma samples by means of Western blot. The enzymatic activities of heparanase-1 and cathepsin B were also measured. RESULTS: The results demonstrated that the expression of both heparanase isoforms was significantly greater in plasma samples from gastrointestinal carcinoma patients, compared with the control group. Logistic regression analysis showed that increased heparanase-1 and heparanase-2 expression was exclusively dependent on the ...

CONTEXTO E OBJETIVO: A heparanase-1 degrada heparam sulfato e está relacionada à progressão de tumor. Apesar de a isoforma heparanase-2 não possuir atividade catalítica, parece ser importante para modular a atividade da heparanase-1. A catepsina B é uma proteinase envolvida na metástase de tumores. O objetivo deste estudo foi analisar a expressão das isoformas de heparanase e atividade da catepsina B em amostras de plasma de pacientes com carcinomas gastrointestinais, comparando-se com indivíduos saudáveis (grupo controle). TIPO DE ESTUDO E LOCAL: Este é um estudo transversal analítico. Foram coletadas amostras de sangue periférico, em hospital público brasileiro, de 21 pacientes com diagnóstico histopatológico de carcinoma gastrointestinal e 43 indivíduos saudáveis. As análises foram realizadas em duas faculdades de medicina brasileiras. MÉTODOS: As isoformas da heparanase foram identificadas e quantificadas em amostras de plasma por Western blot. As atividades enzimáticas de heparanase-1 e catepsina B foram também mensuradas. RESULTADOS: Os resultados demonstraram que as expressões das isoformas de heparanase foram significativamente maiores nas amostras de plasma de pacientes com carcinoma gastrointestinal em comparação com ...

(1H-Pyrrolo [2,3-b]pyridine)7-Azaindole Derivatives and Their Antiurease, Phosphodiesterase and ẞ-glucuronidase Activity.

A variety of 7-azaindole analogs 1-14 with variable substituents on phenyl ring of phenacyl moiety were synthesized and evaluate for their urease, phosphodiesterase and -glucuronidase Inhibitory potential. Compound 9 (IC50 = 2.19±0.37μM) showed potent urease inhibitory potential than standard thiourea (IC50 = 21.00±0.01μM). However, while compounds 10 (IC50 = 255.11±6.62μM) and 8 (IC50 = 133.3±0.46μM), exhibited moderate urease potential. Moreover, compound 2 (IC50 = 20.83± 0.234μM) showed potent phosphodiesterase inhibitory potential than standard EDTA (IC50 = 274.00+0.007μM). Compound 8 (IC50 192.6±3.53μM) was found to be moderate ẞ-glucuronidase inhibitor, as compare to standard 1,4, lactone D saccharic acid (IC50 = 48.41±1.24μM). Nevertheless, compounds 13 (36.81% inhibition) and 14 (47.11% inhibition) showed less than 50% ẞ-glucuronidase inhibition, therefore they were not further evaluated for their IC50 values. The size of the substituent, electron donating or withdrawing affect of substituents as well as the position of substituent on phenyl affects the activity.

Effect of 1,3-O,N spiroheterocyclic inhibitors of heparanase on the growth of HeLa cells / 中华妇产科杂志

Objective To provide the theoretical supportting for targeted heparanase (HPA) inhibition of cervical cancer through observing the anti-proliferative effect of the HPA inhibitor on HeLa cell line of cervical cancer. Methods The two series of 13 kinds of novel HPA inhibitors were synthesized and optimized. Heparan degrading enzyme assay kit was used to test the effect of the inhibitors on the inhibition of HPA enzyme activity. Methyl thiazolyl tetrazolium (MTT) method and scratch test were used to observe the anti-proliferative and the migration effect of the inhibitors on HeLa cells. Flow cytometry was performed to determine the cell cycles and apoptosis. The expression of HPA was evaluated by reverse transcription (RT)-PCR, western blot and immunocytochemistry. Results All tested inhibitors could inhibit the activity of HPA enzyme [the range of 50% inhibiting concentration (IC50) values from 4.47 to 47.19 μmol/L] and the growth of HeLa cells (the range of IC50 values from 48.16 to 96.64μmol/L). Among them, No.16 compound exhibits the strongest inhibition against the growth of HeLa, which could arrest the cell into G0/G1 and G2/M phases. The rate of cell apoptosis in the group treated with 50μmol/L No.16 for 48 hours [(11.9±1.2)%] was significantly higher than that [(6.6 ± 1.8)%] in untreated group (P=0.013). Real time PCR and western blot showed that expression levels of HPA mRNA (1.23±0.46) and protein (0.46±0.31) significantly decreased in the treated group as compared with the levels of HPA mRNA (3.43 ± 0.45) and protein (1.30 ± 0.58) in the untreated group (both P<0.05). Immunocytochemistry also showed that the treatment of No.16 significantly reduced the average optical density (0.39 ± 0.04) of HPA immuostaining signal compared with that in the control group (0.50 ± 0.09; P=0.026). Conclusion Novel 1,3-O,N spiroheterocyclic HPA inhibitors could inhibit the proliferation of HeLa cells,inhibit the HPA enzyme activity in different degree, and down-regulate the expression of HPA protein.

Transient GUS gene expression in cassava (Manihot esculenta Crantz) using Agrobacterium tumefaciens leaf infiltration / Expresión transitoria del gen GUS en yuca (Manihot esculenta Crantz) utilizando infiltración con Agrobacterium tumefaciens

Objective. Assess transient gene expression of GUS in cassava (Manihot esculenta Crantz) leaves using Agrobacterium tumefaciens infiltration. Materials and methods. A. tumefaciens strains GV3101 and AGL1 containing pCAMBIA1305.2 were used to evaluate transient gene expression of β-glucuronidase (GUS). A. tumefaciens infiltration (agroinfiltration) was made using both leaves from in vitro and 1 month old greenhouse plants. Leaves were incubated in X-GLUC buffer, stained and photographed to detect GUS activity. Results. Agroinfiltration assays showed GUS transient expression in leaves of cassava varieties widely cultivated in the north coast and eastern savannah, MCOL2215 (Venezuelan) and CM6438-14 (Vergara), respectively. A. tumefaciens agressive strain AGL1 showed high efficiency inducing GUS expression in cassava leaves. Conclusions. We recommend using A. tumefaciens agressive strain AGL1 for agroinfiltration to assess transient expression in cassava leaves.

Objetivo. Evaluar la expresión transitoria del gen GUS en hojas de yuca (Manihot esculenta Crantz) por medio de infiltración con Agrobacterium tumefaciens. Materiales y métodos. Se utilizaron las cepas GV3101 y AGL1 de A. tumefaciens conteniendo el plásmido pCAMBIA1305.2, para evaluar la expresión transitoria del gen GUS. La infiltración de A. tumefaciens (agroinfiltración) se realizó tanto en hojas de plantas "in Vitro" como de plantas adultas de 1 mes. Las hojas se incubaron en tampón X-GLUC, se destiñeron y se fotografiaron para detectar la actividad de la enzima β-glucuronidasa (GUS). Resultados. Los ensayos de agroinfiltración en hoja muestraron la expresión transitoria del gen GUS en variedades cultivadas en la costa norte y en los llanos orientales, MCOL2215 (Venezolana) y CM6438-14 (Vergara) respectivamente, tanto en plantas "in Vitro" como en plantas adultas. La cepa hipervirulenta de A. tumefaciens AGL1 mostró una mayor eficiencia para la expresión transitoria en hojas de yuca. Conclusiones. Se recomienda utilizar la cepa AGL1 para evaluar la expresión transitoria de genes de interés por agroinfiltración en hojas de yuca.