基于酶抑法的农产品农药残留快速检测系统及影响因素分析

在酶抑法检测系统中,常用的酶包括乙酰胆碱酯酶和丁酰胆碱酯酶等,这些酶能够与农药发生反应,导致酶活性受到抑制。检测人员为了提高酶抑法检测的准确性,需要选择合适的农药类型和酶活性,保证提取液的质量,控制适当的温度和时间,建立标准曲线并进行质量控制,通过优化提高酶抑法在农产品农药残留检测中的应用效果。检测过程中,将酶与农产品提取液混合,一段时间后观察酶活性的变化,通过与标准曲线对比,可以确定农产品中农药残留的浓度,酶抑法是一种用于检测农产品中农药残留的快速检测方法,具有操作简便、灵敏度高、检测时间短等优点,该方法主要基于酶与农药的相互作用,通过观察酶活性的变化来检测农药残留。

基于酶抑法的农产品农药残留快速检测系统及影响因素分析

为快速检测农产品农药残留,开发了基于酶抑制法的农产品农药残留快速检测系统.该检测系统由固体光源、样品池、单片机测量电路板及微机等组成,通过测量经酶抑制反应后样品液的吸光度值实现农产品中农药残留的快速检测.研究了吸光度随反应时间的变化关系、温度对酶活性的影响及农药残留浓度与酶抑制率的关系.结果表明吸光度随反应时间逐渐增加,反应前期增速快,线性度好,适合测量;酶在20～40℃时具有最佳的活性,灵敏度高;农药残留对酶的抑制作用明显,当抑制率高于70%时可判定农药残留超标.

基于酶抑法的农产品农药残留快速检测系统及影响因素分析

当前农产品农药残留问题关注程度较高,其对人身健康影响较大,因此需要农药残留快速检测系统进行短时间、高精度测量。本文首先阐述了检测系统测量原理及结构组成,然后对相关影响因素进行分析,从而考察基于酶抑法的农产品农药残留快速检测系统的检测效果。

分析酶抑法的农产品农药残留快速检测系统及影响因素

酶抑法检测系统往往由单片机测量电路、传感器、光源及微型计算机组成,通过使用酶抑制反应的计算手段对样品液的吸光度进行测量,能快速检测农产品农药残留情况.相关试验结果显示:随着反应时间的增加,吸光度增强,尤其是在反应前期,增速快,线性度好,适合测量;20~40℃酶活性最高,灵敏度强,农药残留对酶有明显的抑制,抑制率超过70%可判定农药残留过量.

有机相中测定酶PSL活性研究

[目的]为实现在有机相中用酶抑法分析有机磷农药残留。[方法]以2,6-二氯靛酚钠为底物和显色剂,制作标准曲线,在4ml的丙酮中分析微生物酯酶PSL的催化动力学和催化活性。[结果]标准曲线相关系数R2=0.9806。酶的用量为2.5mg,1IU=75.7mg/(min.μm);酶活力为0.033IU;比活力为0.0132IU/mg。[结论]建立了有机相中测定酶活性的分析方法,为有机相中酶抑法分析农药残留提供理论依据。

Effect of Enzymes in Buccal Mucous Membrane on Buccal Absorption of Insulin

To evaluate the effect of proteolytic enzymes on the absorption of insulin in the buccal mucosa, the trichloroacetic acid (TCA) method was used to estimate the degradation of insulin under different conditions in the buccal mucosal homogenates. In vivo experiments estimating the enhancement of hypoglycaemic effect by enzyme inhibitors were also conducted. The results showed that proteolytic enzymes in the buccal mucosa were less active than in the intestine. Bacitracin, aprotinin and sodium deoxycholate could inhibit the degradation of insulin in the buccal mucosal homogenates. The degradation of insulin in buccal mucosal homogenates of normal hamsters was smaller than that of diabetic hamsters. In vivo experiments of hypoglycaemia supported the in vitro results. When given buccally, bacitracin, aprotinin and sodium deoxycholate could increase the relative pharmacological bioavailability of insulin. When co-administered with aprotinin(0.1%), bacitracin(0.5%) and sodium deoxycholate(5%), the relative pharmacological bioavailabilities of insulin were 4.84%, 6.60% and 14.95% respectively. The in vitro and in vivo results suggest that proteolytic enzymes are present in the buccal mucosa, which limit absorption of insulin. Co-administration with some enzyme inhibitors can improve the bioavailability of insulin via buccal delivery and sodium deoxycholte is more efficient than some enzyme inhibitors used for improving buccal absorption.

Determination of mercury and nickel by amperometric biosensor prepared with thermostable lactate dehydrogenase

Response of biosensor prepared with the thermostable bacterial LDH enzyme was analyzed in the presence of mercury and nickel.For electrode preparation,the enzyme was purified and immobilized on a gold sheet coated by PGA-pyrrole polymeric material.The working electrode was tested at increasing concentration of lactate in the presence of two different concentrations of mercury and nickel.Current response of biosensor decreased from 0.32 μA to 0.09 μA and 4.13 μA to 2.63 μA when 25×10-7 mmol/L mercury and 17×10-5 mmol/L nickel were included in the working solution,respectively.Sensitivity of the electrode decreased from 0.010 2 μA/（mmol·L-1） to 0.0043 μA/（mmol·L-1） in the presence of 25×10-7 mmol/L mercury.On the other hand,the presence of nickel did not result in a decrease in electrode sensitivity.The results pointed out that the prepared biosensor is useful to detect mercury in a sample containing both mercury and nickel together.

Effect of L158,809 and Cilazapril on the Expression of TGF-β_1 and Secretion of Extracellular Matrix Proteins in Cultured Human Mesangial Cells

Objective: To explore the effect of L158, 809 (angiatensin Ⅱ receptorMockers, ARBs) and Cilazapril (Angiotensin converting enzyme inhibitor, ACEI) on the expression oftransforming growth factor-β_1 (TGF-β_1) and secretion of fibronectin, laminin and type Ⅳcollagen from the cultured human mesangial cells . Methods: Human mesangial cells were cultured indifferent glucose (5.6 mmol/L and 30 mmol/L) and agents (1, 10, 100 and 500 μmol/L) concentrations. The proliferation of mesangial cells were detected at 24, 48 and 72 h . Then the mesangial cellsare divided into four groups, low glucose (5.6 mmol/L) control group, high glucose (30 mmol/L)control group , L158, 809 (10 μmol/L) group and cilazapril (10 μmol/L) group. Forty- eight hourslater, the expression of TGF-β_1 were detected by RT-PCR. Concentrations of TGF-β_1 ,fibronection, laminin and type Ⅳ collagen in the su-pematants of the, mesangial cells weredetermined by EUSA and radioimmunoassay methods. Results: Compared with low glucose control group,the mesangial cells under high glucose medium show excessive proliferation and more TGF-β_1,fibronectin, laminin and type Ⅳ collagen in the supernatant. The expression of TGF-β_1 mRNA wasalso significantly increased under high glucose. The levels of TGF-β_1 and ECM (extracellularmatrix) proteins in the L158, 809 group and cilazapril group are obviously lower than that of thehigh glucose control group. The expression of TGF-β_1 mRNA was markedly decreased in the L158, 809group and cilazapril group compared with that of high glucose control group . Conclusion: Highglucose stimulated the cultured human mesangial cells to excessively proliferate, express TGF-β_1and secrete ECM proteins, and the high glucose-indeced changes were suppressed by either L158, 809and cilazapril.

Molecular mechanism underlying the functional loss of cyclindependent kinase inhibitors p16 and p27 in hepatocellular carcinoma

Hepatocellular carcinoma （HCC） is one of the most common human cancers, and its incidence is still increasing in many countries. The prognosis of HCC patients remains poor, and identification of useful molecular prognostic markers is required. Many recent studies have shown that functional alterations of cellcycle regulators can be observed in HCC. Among the various types of cell-cycle regulators, p16 and p27 are frequently inactivated in HCC and are considered to be potent tumor suppressors, p16, a G1-specific cell-cycle inhibitor that prevents the association of cyclindependent kinase （CDK） 4 and CDK6 with cyclin DI, is frequently inactivated in HCC via CpG methylation of its promoter region, p16 may be involved in the early steps of hepatocarcinogenesis, since p16 gene methylation has been detected in subsets of pre-neoplastic liver cirrhosis patients, p27, a negative regulator of the G1-S phase transition through inhibition of the kinase activities of Cdk2/cyclin A and Cdk2/cyclin E complexes, is now considered to be an adverse prognostic factor in HCC. In some cases of HCC with increased cell proliferation, p27 is overexpressed but inactivated by sequestration into cyclin D1-CDK4-containing complexes. Since loss of p16 is closely related to functional inactivation of p27 in HCC, investigating both p16 and p27 may be useful for precise prognostic predictions in individuals with HCC.

Mechanisms of cholecystokinin-induced calcium mobilization in gastric antral interstitial cells of Cajal

AIM:To investigate the effect of sulfated cholecystokinin-8(CCK-8S) on calcium mobilization in cultured murine gastric antral interstitial cells of Cajal(ICC) and its possible mechanisms.METHODS:ICC were isolated from the gastric antrum of mice and cultured.Immunofluorescence staining with a monoclonal antibody for c-Kit was used to identify ICC.The responsiveness of ICC to CCK-8S was measured using Fluo-3/AM based digital microfluorimetric measurement of intracellular Ca2+ concentration([Ca2+]i).A confocal laser scanning microscope was used to monitor [Ca2+]i changes.The selective CCK1 receptor antagonist lorglumide,the intracellular Ca2+-ATPase inhibitor thapsigargin,the type Ⅲ inositol 1,4,5-triphosphate(InsP3) receptor blocker xestospongin C and the L-type voltage-operated Ca2+ channel inhibitor nifedipine were used to examine the mecha-nisms of [Ca2+]i elevation caused by CCK-8S.Immunoprecipitation and Western blotting were used to determine the regulatory effect of PKC on phosphorylation of type Ⅲ InsP3 receptor(InsP3R3) in ICC.Protein kinase C(PKC) activator phorbol 12-myristate 13-acetate(PMA) and inhibitor chelerythrine were used to assess the role of PKC in the CCK-8S-evoked [Ca2+]i increment of ICC.RESULTS:ICC were successfully isolated from the gastric antrum of mice and cultured.Cultured ICC were identified by immunofluorescence staining.When given 80 nmol/L or more than 80 nmol/L CCK-8S,the [Ca2+]i in ICC increased and 100 nmol/L CCK-8S significantly increased the mean [Ca2+]i by 59.30% ± 4.85%(P < 0.01).Pretreatment of ICC with 5 μmol/L lorglumide inhibited 100 nmol/L CCK-8S-induced [Ca2+]i increment from 59.30% ± 4.85% to 14.97% ± 9.05%(P < 0.01),suggesting a CCK1R-mediated event.Emptying of intracellular calcium stores by thapsigargin(5 μmol/L) prevented CCK-8S(100 nmol/L) from inducing a [Ca2+]i increase.Moreover,pretreatment with xestospongin C(1 μmol/L) could also abolish the CCK-8S-induced effect,indicating that Ca2+ release from InsP3R-operated stores appeared to be a major mechanism responsible for CCK-8S-induced calcium mobilization in ICC.On the other hand,by removing extracellular calcium or blocking the L-type voltage-operated calcium channel with nifedipine,a smaller but significant rise in the [Ca2+]i could be still elicited by CCK-8S.These data suggest that the [Ca2+]i release is not stimulated or activated by the influx of extracellular Ca2+ in ICC,but the influx of extracellular Ca2+ can facilitate the [Ca2+]i increase evoked by CCK-8S.CCK-8S increased the phosphorylation of InsP3R3,which could be prevented by chelerythrine.Pretreatment with lorglumide(5 μmol/L) could significantly reduce the CCK-8S intensified phosphorylation of InsP3R3.In the positive control group,treatment of cells with PMA also resulted in an enhanced phosphorylation of InsP3R3.Pretreatment with various concentrations of PMA(10 nmol/L-10 μmol/L) apparently inhibited the effect of CCK-8S and the effect of100 nmol/L PMA was most obvious.Likewise,the effect of CCK-8S was augmented by the pretreatment with chelerythrine(10 nmol/L-10 μmol/L) and 100 nmol/L chelerythrine exhibited the maximum effect.CONCLUSION:CCK-8S increases [Ca2+]i in ICC via the CCK1 receptor.This effect depends on the release of InsP3R-operated Ca2+ stores,which is negatively regulated by PKC-mediated phosphorylation of InsP3R3.

Statins role in cancer prevention and development-recent meta-analyses

The therapeutic indications of 3-hydroxy-3-methylgl-utaryl-coenzyme A （HMG-CoA） reductase inhibitors （statins） include hypercholesterolaemia and the pre-vention of cardiovascular events. Statins are well toler-ated and beyond their unambiguous positive cardio-vascular effects there are a steadily increasing number of pleiotropic actions emerging. In this regard, growth inhibition, apoptosis, anti-infammatory and immuno-modulatory actions have been attributed to statins. The anti-proliferative effects have been the basis for massive preclinical investigations to elucidate a func-tional role for statins in carcinogenesis and tumor cell growth. However, preclinical and clinical studies are conflicting, although there is accumulating evidence that statins are capable to suppress and decrease the incidence and recurrence of some human cancers. Giv-en the fact that statins are well tolerated they might also have some impact in combinations with conven-tional and targeted chemotherapy. While synergism has been shown for many combinations in vitro this does not hold true yet in the clinics. Here we review the rational behind usage of statins in oncological set-tings. Positive effects have been observed in patients with melanoma and cancers from the breast, colon, prostate, lung, liver and hematologic tissues. However, substantial evidence from clinical studies is still weak and confounded by several factors, which are inherent in the study design. The majority of the studies are ob-servational or of retrospective nature. Defnitely, there is substantial need for larger, prospective randomized, placebo-controlled trials. Finally, we conclude that statins at the current status of evidence should not be recommended in the prevention or during progression of any cancers, however, individual statins may have benefcial effects in specifc tumor subgroups.

A Mew Technique to Rapidly Test Agrochemicai Residues In Fruits and Vegetables

Extraction of AchE, relationship between substrate and enzyme concentration, and inhibition effects of the agrochemicals to AchE are discussed in this paper. Through the re-search, the proper AchE concentration for hydrolysis of 1 ml 1mmol/L substrate and I50 val-ues of the agrochemicals to AchE are decided. It is proved that Asch-DTNB method is a rapid test tool for agrochemical residues in fruits and vegetables.A rapid test card has been developed with sensitivity of 0.05mg/L.

Study of the Correlation of EGFR and K-RAS Gene Mutations with Its Protein Expression in Non-small Cell Lung Cancer

OBJECTIVE To investigate gene mutations of epidermal growth factor receptor （EGFR） and K-RAS （Kirsten rat sarcoma viral oncogene） in Chinese patients with non-small cell lung cancer （NSCLC）, and study the correlation with its protein expression and its clinical significance on gefitinib.METHODS Detect the EGFR and K-RAS gene mutations status by gene sequencing and use the method of immunohistochemistry to detect EGFR and K-RAS protein expression.RESULTS The frequency of EGFR mutations was 33%, mainly located in exon 19 and exon 21. The frequency of K-RAS mutations was 5.5%, mainly located in codon 12. There was no case which both had EGFR and K-RAS mutations, suggesting a mutually exclusive relationship between the two. EGFR mutations are more common in adenocarcinomas （particularly those with bronchioloalveolar features）, nonsmokers and females. 16% were detected EGFR positive expression and had no correlation with EGFR mutation （P 〉 0.05）, but had significant correlation with mutation in exon 19 （P 〈 0.05）. The frequency of K-RAS positive expression was 52.5% and had no correlation with K-RAS mutation （P 〉 0.05）. Twelve （8 cases were protein-negative） out of 15 gefitinib-treated NSCLC patients with disease control carry EGFR mutations.CONCLUSION EGFR protein expression has some correlation with exon 19 mutations. Combined detection of EGFR and K-RAS gene mutations can help clinicians to choose patients who may benefit from EGFR tyrosine kinase inhibitor （EGFR-TKI） and to predict the response and prognosis of gefitinib.

Epidemiological and antibiotic resistant study on extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae in Zhejiang Province

To investigate the epidemiological status of extended spectrum β lactamase (ESBL) producing Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) and the drug resistance profiles of such organisms Methods A total of 282 clinical isolates of E coli and 180 of K pneumoniae were collected from different districts of Zhejiang Province Inhibitor potentiated broth dilution tests were performed for detecting extended spectrum β lactamases Etests were performed to detect the drug resistance of these strains against nine commonly used antibiotics Results The prevalence of extended spectrum β lactamases in E coli and K pneumoniae was 34 0% and 38 3%, respectively The average prevalence of extended spectrum β lactamases in E coli and K pneumoniae was 35 7% The resistance prevalence of extended spectrum β lactamase producing strains to ceftazidime and cefotaxime was 40% and 26% respectively, so were those to cefepime, cefoxitin, piperacillin tazobactam, cefoperazone sulbactam, amikacin and ciprofloxacin All these strains were sensitive to imipenem Conclusion The results in this study showed that the prevalence of extended spectrum β lactamases was high, while extended spectrum β lactamase producing strains were resistant to most antimicrobial agents except imipenem