Enzyme Inhibition Rate Method for Rapid Detection of Organophosphorus and Carbamate Pesticides in Cowpea

[Objectives ] The paper was to explore enzyme inhibition rate method for rapid detection of organophosphorus and carbamate pesticides in cowpea. [ Methods ] Acetylcholinesterase （ACHE） was added to cowpea extract, to determine the inhibition rate of extract against enzyme. The influences of different sampiing methods and sampling parts on detection results were compared. [ Results] The positive rate of standard sampling was 18.18% higher than that of non-stand- ard sampling, and the positive rate of samples collected from cowpea tail was 16.67% higher than that collected from other parts. [ Condmions] Enzyme inhibi- tion rate method is suitable for rapid detection of organophosphorus and carbamate pesticides in cowpea.

In vitro and in vivo cytochrome P450 3A enzyme inhibition by Aframomum melengueta and Dennettia tripetala extracts

Objective: To evaluate the in vitro and in vivo inhibitory effects of two commonly used herbs, Aframomum melengueta(A. melengueta) and Dennettia tripetala(D. tripetala) on CYP 3A enzymes. Methods: In vitro inhibition of the enzymes were assessed with microsomes extracted from female albino rats using erythromycin-N-demethylation assay(EMND) method while their in vivo effects were measured by estimating simvastatin plasma concentrations in rats. Pharmacokinetic parameters were determined using non-compartmental anaysis as implemented in Win Nonlin pharmacokinetic program. Results: EMND assay with intestinal microsomes indicated that aqueous extracts of D. tripetala and A. melengueta significantly(P < 0.05) inhibited intestinal CYP 3A activity at both 50 μg and 100 μg concentrations. Petroleum ether extract of D. tripetala and ethanol extracts of A. melengueta inhibited intestinal CYP3 A activity at 100 μg but not at 50 μg concentrations. All the extracts showed an in vitrodose dependent CYP 3A inhibition with liver microsomes. In vivo analysis showed that pretreatment with the extracts enhanced systemic absorption of simvastatin with reductions in metabolizing enzymes activity as indicated in significant increases in maximal concentration, area under curve, area under moment curve and mean resident time of simvastatin(P < 0.05). Conclusions: Herbal preparations containing these plants' extracts should be used with caution especially in patients on CYP450 3A substrate medications.

Metabolic enzyme inhibitory abilities, in vivo hypoglycemic ability of palmleaf raspberry fruits extracts and identification of hypoglycemic compounds

The unripe palmleaf raspberry,namely Fupenzi(FPZ),is an important medicinal and edible food.This study aims to evaluate the potential of FPZ extracts prepared with different approaches in attenuating hyperglycemia,gout,Alzheimer’s disease,and pigmentation,to obtain the enriching fraction and to identify the major active compounds.Results indicated that FPZ extracts showed weak activity against acetylcholinesterase,considerable ability against tyrosinase and xanthine oxidase,but excellent inhibition onα-glucosidase.Ultrasound-assisted 40%ethanol extract(40EUS)gave the highest phenolics content,and the bestα-glucosidase inhibition(IC_(50)=0.08μg/mL),which is 877-fold higher than that of positive control acarbose.The 40%ethanol eluting fraction of 40EUS showed the strongestα-glucosidase inhibition with the IC_(50) value of 37.79 ng/mL,it could also effectively attenuate the fasting blood glucose level and oral glucose tolerance of C57BL/6 mice.Twenty-six compounds were identified from 40%ethanol fraction by using HPLC-QTOF-MS/MS,hydrolysable tannins(including 11 ellagitannins and 4 gallotannins)were the major compounds,phenolic acids came to the second.Above results could provide important technical supporting for the further application and research of FPZ in health foods and drugs against diabetes.

Methanol extracts of Brachystegia eurycoma and Detarium microcarpum seeds flours inhibit some key enzymes linked to the pathology and complications of type 2 diabetes in vitro

The inhibitory effect of methanol extracts of Brachystegia eurycoma and Detarium microcarpum seeds flours on some key enzymes[α-amylase,α-glucosidase and aldose reductase(AR)]linked to the pathology and complications of type 2 diabetes(T2D);and their antioxidant properties were evaluated.The antioxidant properties evaluated were DPPH•and ABTS•^+scavenging abilities,reducing power,and antioxidant phytochemicals(total phenolics,tannins,total flavonoids and total saponins).Extracts of both flours inhibitedα-amylase,α-glucosidase and AR in a dose-dependent manner.The half-maximal inhibitory concentrations(IC50)of B.eurycoma onα-amylase,α-glucosidase,AR and lipid peroxidation were lower than those of D.microcarpum,indicating that it had stronger inhibitory potency than D.microcarpum.B.eurycoma also had significantly(P<0.05)higher DPPH•and ABTS•^+scavenging abilities,and reducing power than D.microcarpum.The antioxidant phytochemicals(total phenolics,tannins,total flavonoids and total saponins)were also significantly(P<0.05)higher in B.eurycoma than D.microcarpum.The inhibitory effect of B.eurycoma and D.microcarpum extracts onα-amylase,α-glucosidase and AR activities may be attributed to the combined action of their polyphenols and total saponins,and this may be a possible mechanism of action providing support for their use in managing hyperglycemia and the complications of T2D.

Enzymes inhibitory property,antioxidant activity and phenolics profile of raw and roasted red sorghum grains in vitro

Whole grain cereals are important dietary sources for management of metabolic diseases due to the bioactive components they contain.Hence,this study investigated enzymes(pancreatic lipase,-amylase,-glucosidase,xanthine oxidase and angiotensin 1-converting enzyme)inhibitory property,antioxidant activity and phenolics profile of raw and roasted red sorghum(Sorghum bicolor)grains in vitro.Extracts of flours of raw and roasted(150◦C and 180◦C,for 20 min)grains were assayed for enzymes inhibitory and antioxidant activities using spectrophotometric methods;while their phenolic constituents were characterized using HPLC-DAD.The raw grains exhibited strong enzymes inhibitory and antioxidant activities,and contained phenolic acids(gallic,chlorogenic,caffeic,ellagic and p-coumaric acids)and flavonoids(quercetin,luteolin and apigenin).However,whereas the enzymes inhibitory activity and levels of the phenolic compounds in the grains decreased significantly(p<0.05)with increasing roasting temperature,the antioxidant activity increased.Hence,roasting at high temperature may not be recommended for the optimum retention of the enzymes inhibitory property and phenolic compounds of red sorghum grains.

Effects of genistein and equol on human and rat testicular 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase 3 activities

The objective of the present study was to investigate the effects of genistein and equol on 3β-hydroxysteroid de- hydrogenase （3β-HSD） and 17β-hydroxysteroid dehydrogenase 3 （17β-HSD3） in human and rat testis microsomes. These enzymes （3β-HSD and 17β-HSD3）, along with two others （cytochrome P450 side-chain cleavage enzyme and cytochrome P450 17α-hydroxylase/17-20 lyase）, catalyze the reactions that convert the steroid cholesterol into the sex hormone testosterone. Genistein inhibited 3β-HSD activity （0.2 μmol L^-1 pregnenolone） with half-maximal inhibition or a half-maximal inhibitory concentration （IC50） of 87 ± 15 （human） and 636 ± 155 nmol L^-1 （rat）. Genistein＇s mode of action on 3β-HSD activity was competitive for the substrate pregnenolonrge and noncompetitive for the cofactor NAD＋. There was no difference in genistein＇s potency of 3β-HSD inhibition between intact rat Leydig cells and testis microsomes. In contrast to its potent inhibition of 3β-HSD, genistein had lesser effects on human and rat 17β-HSD3 （0.1 μmol L^-1 androstenedione）, with an IC50 〉 100μmol L^-1. On the other hand, equol only inhibited human 3β-HSD by 42%, and had no effect on 3β-HSD and 17β-HSD3 in rat tissues. These observations imply that the ability of soy isoflavones to regulate androgen biosynthesis in Leydig cells is due in part to action on Leydig cell 3β- HSD activity. Given the increasing intake of soy-based food products and their potential effect on blood androgen levels, these findings are greatly relevant to public health.

Characterization of Carboxylesterase Associated with Malathion Insensitivity in the Field Population of the Oriental Migratory Locust

Carboxylesterases （CarEs） from two field populations of the oriental migratory locust, Locusta migratoria manilensis （Meyen）, were examined to try to understand their contribution to malathion insensitivity. The CarEs activities in Wudi population （WD） were 1.75- and 1.50-fold significantly higher than those in Huangliu population （HL） when a-naphthyl acetate （a-NA） and [3-naphthyl acetate were used as substrates, respectively. Such elevated CarEs activities presented in the WD could be because of an increased staining intensity of the a-NA-hydrolyzing CarEs as shown on the nondenaturing polyacrylamide gel electrophoresis. Inhibition studies of CarEs using paraoxon and malaoxon indicated that CarE activities in the HL were more strongly inhibited than those in the WD. Furthermore, a 449-bp DNA fragment of CarE was obtained from L. migratoria manilensis. Hemiquantity reverse transcription-polymerase chain reaction analysis showed that CarE gene expression level in the WD was higher than that in the HL. The higher CarE activities and the increased CarE mRNA level in the WD appeared to be associated with decreased susceptibility to malathion in the WD due to the application of organophosphorus insecticides.

Comparative Study of Malathion Toxicity and General Esterases in Larvae and Adults from a Field Population of Oxya chinensis (Thunberg) (Orthoptera: Acridoidea)

The susceptibility of Oxya chinensis to malathion was compared in larvae and adults from a field population, collected from Jinyuan outskirt, Shanxi Province. The results showed that Oxya chinensis was more susceptible to malathion in the adult stage than in the larval stage. The LD50 values for malathion susceptibility of Oxya chinensis were 4.94 and 2.44 mg g-1 body weight in the larvae and adults respectively. The results indicated that the larvae were 2.02-fold less susceptible to malathion than the adults. The general esterases and the kinetics were characterized and compared between the two life stages and between females and males. Larval preparations of Oxya chinensis were more active than adult preparations in females and males. The larvae showed 1.18-, 1.49-, and 1.17- fold higher specific activities than the adults in females with α-NA, α-NB and β-NA respectively. In males, the ratios were 1.34-, 1.70-, and 1.06-fold. Female preparations were more active than those of males in the adults. The reverse results were observed in the larvae where male preparations were more active than female preparations. Kinetic studies showed that Km values of general esterases hydrolyzing α-NA, α-NB, and β-NA in the adult stage were 1.36-, 1.32- and 1.39-fold respectively, higher than those in the larval stage in females. In males, the ratios were 1.24-, 2.14-, and 1.20-fold. The esterase from male insects had a higher affinity (lower Km value) to the substrate than those from females. The results also showed that the Vmax values of general esterase hydrolyzing α-NA, α-NB, and β-NA in the two stages were similar. From the results of bioassays and biochemical analyses, it has been inferred that a higher level of resistance to malathion in larvae than in adults would appear to result from differences in the expression of resistance mechanisms in these two life stages. Enhanced esterase activities appeared to play a major role in resistance to malathion in both larvae and adults. From the analysis of inhibition in vitro, the esterases in the two life stages were B-type, and carboxylesterases were predominant enzymes in the composition of the esterases in the two stages.

Comparisons of Properties of Acetylcholinesterase from Two Field-Collected Populations of Oxya chinensis Thunberg(Orthoptera:Acrididae)and the Role of Acetylcholinesterase in the Susceptibility to Malathion

In this study, acetylcholinesterase (AChE) was extracted from two field-collected populations of Oxya chinensis (XinxiangCity, Henan Province and Changzhi City, Shanxi Province). AChE activities were decreased when concentrations of ATCincreased, showing a characteristic phenomenon of substrate inhibition at high concentration in both populations. Suchinhibition occurred at relatively low concentration for AChE from Xinxiang population but relatively high for AChE fromChangzhi population. The kinetic study showed that there were no significant differences between the two populations inthe Km values. The Km value in Changzhi population was only 1.09-fold higher than that in Xinxiang population. However,significant differences were observed between the two populations in Vmax values. The Vmax value in Changzhi populationwas 1.32-fold higher than that in Xinxiang population. The inhibition study in vitro showed that the AChE from bothpopulations exhibited similar rank order in sensitivity to inhibition by three OPs, as determined by comparison of theirbimolecular rate constants (ki), from the most potent inhibition to the least was chlopyrifos-oxon > paraoxon >demeton-s-methyl for AChE from the two populations and that the ki values in Xinxiang population were lower than those in Changzhipopulation. The I50 values of AChE from Xinxiang population were 4.84-, 2.66-, and 1.92-fold less sensitive to inhibition byparaoxon, chlopyrifos-oxon, and demeton-s-methyl. These results were consistent with the results in bioassay. It isinferred that AChE insensitivity to OP insecticides plays an important role in the differences of insusceptibility of Oxyachinensis to malathion between the two populations.

Effects of Yanhusuo(Rhizoma Corydalis),Baizhi(Radix Angelicae Dahuricae)and Their Combination Extracts on Cytochrome P450 Activities in Rats

Background:Yuanhu Zhitong Prescription(元胡止痛方,YZP),a well-known herbal prescription for an analgesic effect,is recorded in the China Pharmacopoeia,consisting of Yanhusuo(Rhizoma Corydalis)and Baizhi(Radix Angelicae Dahuricae).Objective:To explore the influence of 70%EtOH extracts from Yanhusuo(Rhizoma Corydalis),Baizhi(Radix Angelicae Dahuricae)and YZP on the CYP450s,especially the differences between the single drug and prescription.Materials and methods:Cocktail probe drugs method was used to evaluate Cytochrome P450 activities in rat liver microsomes,including CYP1A2,CYP2D1,CYP2C11,CYP2C6 and CYP3A1,after rats repeatedly administrated with the extracts of Yanhusuo(Rhizoma Corydalis),Baizhi(Radix Angelicae Dahuricae)and YZP for 7 days.Results:Yanhusuo(Rhizoma Corydalis)extracts significantly increased the activities of CYP1A2,2C6 and 3A1,and inhibited that of 2D1.Baizhi(Radix Angelicae Dahuricae)extracts significantly increased the activities of CYP1A2 and inhibited that of 2D1 and 2C11.YZP extracts exhibited the same effect with single drugs.Conclusion:These results might partly interpret the TCM compatibility.Moreover,co-administration of prescriptions containing Yanhusuo(Rhizoma Corydalis),Baizhi(Radix Angelicae Dahuricae)or YZP should consider a potential herb(drug)-drug interaction medicated by the induction of CYP1A2,2C6 and 3A1 and inhibition of CYP2D1 and 2C11 enzymes.

Rhus longipes (Engl.) infusions improve glucose metabolism and mitigate oxidative biomarkers in ferrous sulfate-induced renal injury

Objective:To explore the antioxidant and antidiabetic activities of Rhus longipes(R.longipes)leaf and stem bark aqueous infusions.Methods:R.longipes leaf and stem bark infusions were characterized via gas-chromatography mass-spectroscopy(GC-MS)analysis.In vitro antioxidant and carbohydrate and lipid digestive enzyme inhibitory activities of R.longipes infusions were determined.Additionally,the modulatory effects of R.longipes infusions on intestinal glucose absorption,muscle glucose uptake,and biomarkers of renal oxidative injury were evaluated.Molecular docking was performed to determine the binding affinities of the identified compounds from the leaf and stem bark infusions on carbohydrate and lipid digestive enzymes.Results:GC-MS analysis revealed the presence of several phytocompounds,including palmitoleic acid,octadecanamide,24,25-dihydroxyvitamin D and L-ascorbic acid.The bark infusion had significantly higher total phenolic contents compared with the leaf infusion,with better DPPH scavenging[IC_(50):(10.50±1.03)μg/mL]and ferric reducing[IC_(50):(9.85±0.32)μg/mL]activities(P<0.05).Both R.longipes infusions at their highest concentrations significantly increased glucose uptake in yeast suspension and rat psoas muscle with marked suppression of glucose absorption in the rat jejunum(P<0.05).With no cytotoxicity on Vero cells,the infusions lowered lipid peroxidation,increased cellular reduced glutathione concentration,and the activities of superoxide dismutase and catalase in renal homogenate treated with FeSO_(4).Conclusions:R.longipes shows antioxidant and antidiabetic activities and could be a potential therapeutic candidate for diabetes.

Novel semicarbazide-sensitive amine oxidase inhibitor screening from anti-obesity drugs using HPLC-MS based technique

Semicarbazide-sensitive the metabolism of glucose and fat, amine oxidase （SSAO） has been considered to be associated with and elevated SSAO activity was observed in obese patients. In the present study, an in vitro SSAO activity-based method was developed to screen inhibitors from 15 an- ti-obesity drugs. Among the fifteen anti-obesity drugs, four drugs including caffeine, fenfluramine, bumetanide and amfebutamone inhibited SSAO activity, and caffeine was the most effective one. When the concentration of caffeine was 1.4 mmol/L, the inhibition ratio was 31.9% and 18.8% in rabbit serum and rat adipose tissue, respectively. Inhibition of SSAO activity by caffeine was also confirmed in the in vivo study, showing the inhibition ratio of 15.6% on serum SSAO. Caffeine pro- vides a natural source of inhibition of SSAO activity and may be a promising inhibitor for the study of SSAO.

Crystal Structures of Urokinase-type Plasminogen Activator in Complex with 4-(Aminomethyl) Benzoic Acid and 4-(Aminomethyl-phenyl)-methanol

Urokinase-type plasminogen activator （uPA） is a trypsin-like serine protease and plays a key role in several biological processes, including tissue remodeling, cell migration, and matrix degradation. The inhibitors of uPA have been shown to prevent the spread of metastasis and tumor growth, and accordingly uPA is widely recognized as a target for the treatment of cancer. In this work, we report the crystal structures of the complexes of uPA with its inhibitors： 4- （aminomethyl）-benzoic acid （AMBA） and 4-（aminomethyl-phenyl）-methanol （AMPM）, both at a resolution of 2.35 А. The inhibitory constants of these two inhibitors were measured by a chromogenic competitive assay, and it was found that AMBA is a better inhibitor for uPA （Ki = 2.68 mM） than AMPM （Ki = 13.99 mM）. The structural study shows that the binding mode of inhibitor AMBA on uPA is similar to that of AMPM on uPA, both docked into the active site S1 pocket of uPA. Structural details of these complexes are provided to explain the difference of inhibitory constants.

Six New 3,5-Dimethylcoumarins from Chelonopsis praecox,Chelonopsis odontochila and Chelonopsis pseudobracteata

Ten 3,5-dimethylcoumarins(1-6 and 8‒11)involving six new ones(1-6),together with a known 3-methylcoumarin(7),were isolated from the aerial parts of three Chelonopsis plants,C.praecox,C.odontochila,and C.pseudobracteata.The struc-tures of the new compounds were determined by extensive HRESIMS,1D and 2D NMR spectroscopic analyses.According to the substitution at C-5,these coumarins were classified into 5-methyl,5-hydroxymethyl,5-formyl,and 5-nor types.All the isolates were assayed for their inhibition onα-glucosidase,protein tyrosine phosphatase 1B,and T-cell protein tyrosine phosphatase in vitro.

Identification and Biological Activities of the Phenolic Compounds in <i>Eisenia arborea</i>

Four polyphenols were isolated and purified from a brown alga Eisenia arborea. These phlorotannin compounds showed strong radical scavenging and some enzyme inhibitory activities. All of the compounds showed strong antioxidative, acetylcholinesterase and butyrylcholinesterase inhibitory, and tyrosinase inhbibitory activities at 100 μg/mL. Dieckol and PFF inhibited butyrylcholinesterase, a new target for the treatment of Alzheimer’s disease, very strongly even at 10 μg/mL, more strongly than AChE. These two compounds also effectively inhibited tyrosinase. These results support the potential of developing natural antioxidants and antidementia agents from the brown alga.

Biochemical characterization of chitin synthase activity and inhibition in the African malaria mosquito, Anopheles gambiae

Chitin synthase （CHS） is an important enzyme catalyzing the formation of chitin polymers in all chitin containing organisms and a potential target site for insect pest control. However, our understanding of biochemical properties of insect CHSs has been very limited. We here report enzymatic and inhibitory properties of CHS prepared from the African malaria mosquito, Anopheles gambiae. Our study, which represents the first time to use a nonradioactive method to assay CHS activity in an insect species, determined the optimal conditions for measuring the enzyme activity, including pH, temperature, and concentrations of the substrate uridine diphosphate N-acetyl-D-glucosamine （UDP- GlcNAc） and Mg＋＋. The optimal pH was about 6.5-7.0, and the highest activity was detected at temperatures between 37℃ and 44℃. Dithithreitol is required to prevent melanization of the enzyme extract. CHS activity was enhanced at low concentration of GIcNAc, but inhibited at high concentrations. Proteolytic activation of the activity is significant both in the 500×g supernatant and the 40 000×g pellet. Our study revealed only slight in vitro inhibition ofA. gambiae CHS activity by diflubenzuron and nikkomycin Z at the highest concentration （2.5μmol/L） examined. There was no in vitro inhibition by polyoxin D at any concentration examined. Furthermore, we did not observe any in vivo inhibition of CHS activity by any of these chemicals at any concentration examined. Our results suggest that the inhibition of chitin synthesis by these chemicals is not due to direct inhibition of CHS in A. gambiae.

Broken electron transfer pathway in enzyme:Gold clusters inhibiting TrxR1/Trx via cell studies and theory simulations

Thioredoxin reductase 1(TrxR1)is over activity in tumor cell to maintain their redox balance.Although gold clusters have great potential in antitumor drug as they could well inhibit TrxR1,the molecular mechanism has not been disclosed yet.In this work,we revealed gold clusters can well inhibit the activity of TrxR1 in lung tumor cells and further disclosed the inhibition mechanism by using computational simulation methods.We firstly inferred the binding sites of gold in the hydrophobic cavities on TrxR1.The simulation results show that the gold ion(released from Au cluster)interact with–SH of Cys189 in TrxR1,this greatly increase the distance between the C-terminal redox center of TrxR1 and the Trx redox center,thereby destroy the electron transfer pathway between them.Our electron transfer destroying mechanism is different from the previous hypothesis that gold binds to the Sec498 of TrxR1 which has never been proved by experimental and theory studies.This work provides a new understanding of the gold clusters to inhibit TrxR1 activity.

Biotransformation of quinoa phenolic compounds with Monascus anka to enhance the antioxidant capacity and digestive enzyme inhibitory activity

This research aims to change the phenolic fractions and the bioactivities of quinoa by solid-state fermentation(SSF)with the edible fungus Monascus anka(M.anka).The contents of protein and fat increased in the fermented product,while the carbohydrate decreased.After 6-day fermentation with M.anka,the amount of phenolic compound reached the highest level.The majority of phenolic forms in fermented quinoa were phenolic acids,mainly ferulic acid,protocatechuic acid and p-hydroxybenzoic acid.Ultrahigh-performance liquid chromatography-mass spectrometry(UHPLC-MS)results showed that SSF was an effective method to transform quinoa phenolic compounds.Free fractions could be rapidly absorbed in the small intestine,suggesting that SSF with M.anka was a useful method to enhance bioavailable antioxidants.Antioxidant ability in vitro results showed that phenolic fractions from fermented quinoa were greater than the unfermented quinoa,and a significant cellular antioxidant activity(CAA)increment of 135%was obtained in the free phenolic fraction of fermented quinoa.Moreover,α-amylase andα-glucosidase inhibition activities were enhanced with fermentation.Correlation matrix analysis revealed that most of the free phenolic compounds showed strong positive correlations with antioxidant activities and digestive enzyme activities.Consequently,fermentation with M.anka was a particularly promising method to enhance the bioactivity of quinoa.

Assembly of IMPDH2-Based,CTPS-Based,and Mixed Rod/Ring Structures Is Dependent on Cell Type and Conditions of Induction

Inhibition of guanosine triphosphate（GTP） and cytidine triphosphate（CTP） biosynthetic pathways induces cells to assemble rod/ring（RR） structures,also named cytoophidia,which consist of the enzymes cytidine triphosphate synthase（CTPS） and inosine-50-monophosphate dehydrogenase 2（IMPDH2）.We aim to explore the interaction of CTPS and IMPDH2 in the generation of RR structures.He La and COS-7 cells were cultured in normal conditions or in the presence of 6-diazo-5-oxo-L-norleucine（DON）,ribavirin,or mycophenolic acid（MPA）.Over 90% of DON-treated cells presented RR structures.In He La cells,35% of the RR structures were positive for IMPDH2 alone,26% were CTPS alone,and 31% were IMPDH2/CTPS mixed,while in COS-7 cells,42% of RR were IMPDH2 alone,41% were CTPS alone,and 10% were IMPDH2/CTPS mixed.Ribavirin and MPA treatments induced only IMPDH2-based RR.Cells were also transfected with an N-terminal hemagglutinin（NHA）-tagged CTPS1 construct.Over 95% of NHA-CTPS1 transfected cells with DON treatment presented IMPDH2-based RR and almost 100% presented CTPS1-based RR;when treated with ribavirin,over 94% of transfected cells presented IMPDH2-based RR and 37% presented CTPS1-based RR,whereas 2% of untreated transfected cells presented IMPDH2-based RR and 28% presented CTPS1-based RR.These results may help in understanding the relationship between CTP and GTP biosynthetic pathways,especially concerning the formation of filamentous RR structures.

Synergistic control of sex hormones by 17β-HSD type 7: a novel target for estrogen-dependent breast cancer

17β-hydroxysteroid dehydrogenase(17β-HSD)type 1 is known as a critical target to block the final step of estrogen production in estrogen-dependent breast cancer.Recent confirmation of the role of dyhydroxytestosterone(DHT)in counteracting estrogeninduced cell growth prompted us to study the reductive 17β-HSD type 7(17β-HSD7),which activates estrone while markedly inactivatingDHT.The role ofDHTin breast cancer cell proliferation isdemonstratedby its independent suppression of cell growthin the presence of a physiological concentration of estradiol(E2).Moreover,an integral analysis of a large number of clinical samples in Oncomine datasets demonstrated the overexpression of 17β-HSD7 in breast carcinoma.Inhibition of 17β-HSD7 in breast cancer cells resulted in a lower level of E2 and a higher level of DHT,successively induced regulation of cyclinD1,p21,Bcl-2,and Bik,consequently arrested cell cycle in the G0/G1 phase,and triggered apoptosis and auto-downregulation feedback of the enzyme.Such inhibition led to significant shrinkage of xenograft tumors with decreased cancer cell density and reduced 17β-HSD7 expression.Decreased plasma E2 and elevated plasma DHT levels were also found.Thus,the dual functional 17β-HSD7 is proposed as a novel target for estrogen-dependent breast cancer by regulating the balance of E2 andDHT.Thisdemonstrates aconceptual advance on the general belief that the major role of this enzyme is in cholesterol metabolism.