中美高中生物实验比较研究——以影响酶活性的实验为例

通过翻译美国出版的高中生物实验教材有关温度对酶促反应速率的影响实验,并将其与我国人民教育出版社出版的高中生物教材（必修一）中相关实验进行比较分析,为我国生物教材修订提供参考,同时为生物教师该实验的教学准备提供素材。

探究实验中常见生成性问题及解决建议——影响酶活性的条件

学生自行设计实验方案可能出现很多生成性问题，引导学生通过适当的方法解决这些问题，不仅能加深学生对现有知识的理解，拓宽学生的思路，还能培养学生的创新和实践能力。

精准分工指导教学方法在中职影响酶活性因素实验中的应用

生物化学实验是培养实验技能的重要手段,精准分工指导教学方法能有效提高中职学生实验技能,为后续实验的开展打下坚实基础。

小肠结肠炎耶尔森菌尿素酶基因多态性及尿素酶活性分析

目的了解小肠结肠炎耶尔森菌尿素酶基因的多态性以及不同致病力菌株尿素酶活性之间的关联性。方法选取43株不同致病能力的小肠结肠炎耶尔森菌用PCR扩增尿素酶基因并进行序列测定。连同4株NCBI上尿素酶基因参考序列一起进行比对和构建聚类树。同时选取8株不同致病力的小肠结肠炎耶尔森菌进行尿素酶活性实验。结果在低致病性小肠结肠炎耶尔森菌中,全部的18株O∶3血清型菌株的尿素酶基因7个开放读码框(ureABCEFGD)的基因序列完全一致;在全部17株O∶9血清型菌株中,尿素酶基因7个开放读码框(ureABCEFGD)的基因序列完全一致的有16株,仅1株2/O∶9(NX1997-IE419)与其他血清型菌株的尿素酶基因序列不同;高致病性的O∶8血清型小肠结肠炎耶尔森菌以及非致病菌的尿素酶基因序列与低致病性O∶3、O∶9血清型菌株的尿素酶基因序列不同且各菌株之间基因序列差异也较大。尿素酶活性实验显示不同致病能力的小肠结肠炎耶尔森菌尿素酶活性存在多样性,无明显的规律发现。结论低致病性O∶3、O∶9血清型的小肠结肠炎耶尔森菌的尿素酶基因分别表现出高度的保守性,NX1997-IE419的尿素酶基因可能是由O∶3血清型小肠结肠炎耶尔森菌的尿素酶基因演变而来。高致病性的1B/O∶8小肠结肠炎耶尔森菌及非致病性小肠结肠炎的尿素酶基因相对不保守。尿素酶活性试验提示我们小肠结肠炎耶尔森菌的尿素酶活性大小与其致病性的强弱可能无关联。

挖掘教材实验背后的科学思维--以酶的实验设计的教学为例

围绕酶的实验设计中反应类型和检测方法的选择及其本质与原理的剖析,充分运用了归纳演绎、评价比较、综合分析、批判反思等科学思维,建构概念,提炼观念,落实了生物学学科核心素养的培养。

用淀粉探究pH对酶活性的影响

影响酶活性的实验由于受到鉴定试剂、底物和生成物等因素的影响，常难以得出理想鉴定结果。在探究pH对酶活性影响的实验中，一般不采用淀粉作为实验材料。通过中和滴定对淀粉酶进行预处理，尝试用淀粉作为实验材料探究pH对酶活性影响。

5E教学模式在高中生物教学中的应用——以“降低化学反应活化能的酶——酶的作用和本质”为例

本文以"降低化学反应活化能的酶——酶的作用和本质"为例,阐述5E教学模式在高中生物教学中的应用,在这一节课中,先以广告导入酶的学习,再引导学生设计"影响酶活性的条件"实验方案,经过探究、总结酶作用的机理,培养学生的探究精神,然后反思实验中存在的问题,为教学提供参考。

浅析高中生物实验教学中的几点想法

在准备实验和实施高中生物学的实验过程中,遇到了一些具体问题,包括实验器具材料、实验时间等,尝试对一些实验步骤和实验材料进行改进,取得了良好的实验结果。

Hsa-miR-98-5p/DKK3信号轴对乳腺癌细胞生物学行为的影响

背景与目的:乳腺癌威胁着全世界女性的健康。虽然已发现大量微小RNA(microRNA,miRNA)在乳腺癌中异常表达,但仍需要构建一个完整的miRNA-信使RNA(messenger RNA,mRNA)网络。方法:利用癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库下载乳腺癌相关数据集,分析肿瘤组织与正常组织之间差异表达的miRNA。利用miRDB、miRTarBase和StarBase数据库分析差异miRNA靶向的基因。使用R语言中的ClusterProfiler包对靶基因进行富集分析。使用String数据库联合Cytoscape 3.6.2软件进行蛋白质-蛋白质相互作用(protein-protein interaction,PPI)网络分析及Hub基因的筛选;构建miRNA-Hub mRNA调控网络确定研究信号轴,然后通过细胞实验进行验证。结果:采用TCGA数据集识别出两个差异miRNAs。3个数据库取交集预测得到278个靶基因。共鉴定出10个Hub基因,从构建的miRNA-Hub基因网络图中发现,hsa-miR-98-5p/DKK3轴可能在乳腺癌的进展中起关键作用。细胞功能学实验证实hsa-miR-98-5p可抑制细胞凋亡,促进细胞的增殖、迁移和侵袭。双荧光素酶报告基因实验进一步验证了hsa-miR-98-5p与DKK3的结合作用。结论:本研究首先通过生物信息学分析鉴定出一个与乳腺癌进展相关的hsa-miR-98-5p/DKK3轴,初步证实hsa-miR-98-5p可通过靶向DKK3抑制乳腺癌细胞凋亡,促进细胞的增殖、迁移和侵袭。

Effect of synbiotics on intestinal microflora and digestive enzyme activities in rats

AIM: To investigate the effect of synbiotics, i.e. probiotics and prebiotics mixture, on the gut microbial ecology and digestive enzyme activities in rats. METHODS: Forty-eight SD rats weighing about 280 g were used in this study. Rats were divided into three groups according to the contents of probiotics and prebiotics mixture in the feed as control, low and high dose groups. The duration of the experiment was 8 wk. RESULTS: Compared with the control group, thefecal Lactobacillus and Bifidobacterium counts were significantly increased and the fecal Coliform organism counts were markedly reduced in the low and high dose groups. Concerning the digestive enzyme activity of jejunum, only lactase activity increased in low dose group. However, significant increase of lipase, lactase, sucrase, and isomaltase activities were observed in high dose group.CONCLUSION: Intake of low and high dosages of probiotics and prebiotics mixture significantly improved the ecosystem of the intestinal tract by increasing the probiotics population and digestive enzyme activities in rats.

Interplay between nitric oxide and VIP in CCK-8-induced phasic contractile activity in the rabbit sphincter of Oddi

AIM: The sphincter of Oddi (SO) plays an important role in delivery of bile into the duodenum. To establish whether vasoactive intestinal polypeptide (VIP) and nitric oxide (NO) were involved in phasic contractile activity of the rabbit SO stimulated by cholecystokinin-octapeptide (CCK-8). METHODS: Isolated SO muscle rings were cleaned of fat and mounted horizontally on two small L-shaped hooks one of which was connected to a force transducer for the measurement of isometric tension.The experiments were carried out in a thermostatically controlled (37±0.2℃) organ bath (5 mL) containing Krebs solution.The organ fluid was gassed with 95% O2 and 50 mL/L CO2 to keep the pH at 7.40±0.05. Contractile responses to CCK-8 (1 μmol/L) were evaluated in the presence and absence of NG-nitro-L-arginine (LNNA), an inhibitor of NO synthase (100 μmol/L), and (p-chloro-D-Phe6-Leu17)-VIP (VlPa, 30 μmol/L), a VIP receptor antagonist. RESULTS: CCK-8 stimulated the phasic activity of the SO. NO synthase inhibition increased the frequency and amplitude of contractions with a slight increase in developed tension. Pre-incubation with VlPa also attenuated this CCK-8 effect. The combined application of LNNA and VlPa abolished the phasic activity of the muscle rings with a marked increase in tension in response to CCK-8. CONCLUSION: VIP and NO together contribute to an increase in phasic activity of SO.

Kinetic method for enzymatic analysis by predicting background with uricase reaction as model

Objective：To investigate the reliability for kinetic assay of substance with background predicted by the integrated method using uricase reaction as model. Methods： Absorbance before uricase action （Δ0） was estimated by extrapolation with given lag time of steady-state reaction. With Km fixed at 12.5μmol/L, background absorbance （Δb） was predicted by nonlinearly fitting integrated Michaelis-Menten equation to Candida utilis uricase reaction curve. Uric acid in reaction solution was determined by the difference （ΔA） between Δ0 and Δb. Results .Ab usually showed deviation 〈3% from direct assay with residual substrate done fifth of initial substrate for analysis. ΔA showed CV 〈5% with resistance to common interferences except xanthine, and it linearly responded to uric acid with slope consistent to the absorptivity of uric acid. The lower limit was 2.0 μmol/L and upper limit reached 30 μmol/L in reaction solution with data monitored within 8 min reaction at 0. 015 U/ml uricase. Preliminary application to serum and urine gave better precision than the direct equilibrium method without the removal of proteins before analysis. Conclusion .This kinetic method with background predicted by the integrated method was reliable for enzymatic analysis, and it showed resistance to common interferences and enhanced efficiency at much lower cost.

Effects of long-term ethanol consumption on jejunal lipase and disaccharidase activities in male and female rats

AIM: To study the effect of long-term ethanol consumption on jejunal lipase and disaccharidase (sucrase, maltase,and lactase) activities in rats and its gender difference. METHODS: Age-matched male and female Wistar rats were fed control or ethanol-containing liquid diets for 12 wk following the Lieber-DeCarli model. According to both theplasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, 40 rats were divided into four groups as follows: male control group (MC), male ethanol group (ME), female control group (FC), and female ethanol group (FE).RESULTS: After ethanol feeding for 12 wk, the results revealed that plasma AST and ALT activities of group MEwere significantly increased by 58% and 92%, respectively,than those of group MC (P＜0.05). Similarly, plasma AST and ALT activities of group FE were also significantly increased by 61% and 188%, respectively, than those of group FC (P＜0.05). Fat accumulation was observed in both ethanol treated groups, while fatty changes were more severe in group FE than those in group ME. The induction of hepatic microsomal cytochrome P450 2E1 (CYP2E1) was obviously seen in group ME and group FE, but was not detected in group MC and group FC. Jejunal lipase activity of group ME was significantly increased by 1.25-fold than that of group MC (P＜0.05). In contrast to, sucrase, maltase, and lactase activities of group ME were significantly decreased by 63%, 62% and 67%, respectively, than those of group MC (P＜0.05). Similarly, activities of these three enzymes of group FE were also significantly decreased by 43%, 46% and 52%, respectively, than those of group FC (P＜0.05).There were no significant epithelial changes of the duodenal mucosa in any group.CONCLUSION: Long-term ethanol consumption significantly can increase jejunal lipase and decrease jejunal disaccharidase activities in both male and female rats.

Effect of endothelin-1 receptor antagonists on histological and ultrastructural changes in the pancreas and trypsinogen activation in the early course of caerulein-induced acute pancreatitis in rats

AIM: To assess the effect of non-selective ETA/B (LU 302872)and selective ETA (LU 302146) antagonist on pancreatic histology and ultrastructure of acinar cells in connection with trypsinogen activation in early caerulein-induced AP.METHODS: Male Wistar rats with caerulein-induced AP,lasting 4 h, were treated i.p. with 10 and 20 mg/kg b.w.of each antagonist. Edema, inflammatory infiltration,necrosis and vacuolization of acinar cells in the pancreas were scored at 0-3 scale. Free active trypsin (FAT), total potential trypsin (TPT) after activation with enterokinase,and index of trypsinogen activation (%FAT/TPT) were assayed in pancreatic homogenates.RESULTS: In untreated AP, the edema, inflammatory infiltration, necrosis and vacuolization increased as compared to control healthy rats (P＜0.01). None of the treatment exerted any meaningful effect on the edema and inflammatory infiltration. The selective antagonist increased slightly the necrosis score to 0.82±0.06 at higher dose (P＜0.05) vs 0.58±0.06 in untreated AP. The nonselective antagonist increased slightly the vacuolization score to 2.41±0.07 at higher dose (P＜0.01) vs 1.88±0.08in untreated AP. The decrease in the number of zymogen granules, disorganization of endoplasmic reticulum,autophagosomes and cytoplasmic vacuoles were more prominent in treated AP than in untreated AP groups.%FAT/TPT in untreated AP increased about four times (18.4±3.8 vs4.8±1.3 in control group without AP, P＜0.001).Treatment of AP with both antagonists did not affect significantly augmented trypsinogen activation.CONCLUSION: The treatment with endothelin-1 receptors (non-selective ETA/B and selective ETA) antagonists has essential effect neither on the edema and inflammatory infiltration nor on trypsinogen activation observed in the early course of caerulein-induced AP. Nevertheless a slight increase of the necrosis and vacuolization score and some of the ultrastructural data could suggest the possibility of their undesired effects in caerulein-induced AP at investigated doses.

Measurement of serum paraoxonase-1 activity in the evaluation of liver function

Paraoxonase-1 (PON1) is an esterase and lactonase synthesized by the liver and found in the circulation associated with high-density lipoproteins. The physiological function of PON1 seems to be to degrade specific oxidized cholesteryl esters and oxidized phospholipids in lipoproteins and cell membranes. PON1 is, therefore, an antioxidant enzyme. Alterations in circulating PON1 levels have been reported in a variety of diseases involving oxidative stress including chronic liver diseases. Measurement of serum PON1 activity has been proposed as a potential test for the evaluation of liver function. However, this measurement is still restricted to research and has not been extensively applied in routine clinical chemistry laboratories. The reason for this restriction is due to the problem that the substrate commonly used for PON1 measurement, paraoxon, is toxic and unstable. The recent development of new assays with non-toxic substrates makes this proposal closer to a practical development. The present editorial summarizes PON1 biochemistry and function, its involvement with chronic liver impairment, and some aspects related to the measurement of PON1 activity in circulation.

An experimental research of Weining granule in treating gastric precancerous lesions

Objective： To investigate potential therapeutic effects and mechanism of Weining granule in the treatment of gastric precancerous lesions. Methods： Sixty rats were randomly assigned to a blank group or a model group or to receive retinoic acid or high-, medium- or low- dose of Weining granule. General conditions of the animals were observed before and after treatment. Changes in gastric mucosal pathohistology, telomerase activity, proliferation index （PI） and apoptosis index （AI） were measured. Results： General conditions, including activity and eating, were improved in all Weining-granule-treated groups with the numbers of rats having intestinal metaplasia （IM）, atypical hyperplasia （ATP） or positive telomerase activity being significantly lower than those in the model group （P 〈 0.05 or P 〈 0.01）. Compared with the model group, all doses of Weining granule significantly decreased PI （P 〈 0.01） and increased AI （P 〈 0.05）. Conclusion： Weining granule may provide a therapeutic benefit for the treatment of gastric precancerous lesions by inhibiting telomerase activity and proliferation of gastric cancer cells and by accelerating their apoptosis.

Enzymatic Activity of Escherichia Coil Lactate Dehydrogenase and Cytochrome c Oxidase

The catalytic activity of two common bacterial enzymes, lactate dehydrogenase （LDH） and cytochrome c oxidase （COX） from Escherichia coli was examined following bacterial exposure to microwave （MW） radiation under well-defined experimental conditions. The experiments were conducted in a specialized microwave processing apparatus, with an exposure frequency of 18 GHz, and a temperature profile that was restricted to below 40℃ to avoid thermal degradation of the bacteria. The absorbed power was calculated to be 1,500 kW/m3 and the electric field was determined to be 300 Wm. Both values were theoretically confirmed using Computer Simulation Technology （CST） Microwave Studio 3D Electromagnetic Stimulation Software. Results showed that the activity of both enzymes was increased following MW radiation compared to negative controls and thermally treated samples subjected to similar temperature profiles. It is suggested that the increase in COX and LDH enzyme activity could not be explained by conventional heating alone, but was rather a result of micro-thermal effects that incorporated ＇undetectable＇ thermal mechanisms.

Biochemical and behavioral characterization of the double transgenic mouse model (APPswe/PS1dE9) of Alzheimer’s disease

Objective The double transgenic mouse model （APPswe/PSldE9） of Alzheimer＇s disease （AD） has been widely used in experimental studies. β-Amyloid （Aβ） peptide is excessively produced in AD mouse brain, which affects synaptic function and the development of central nervous system. However, little has been reported on characterization of this model. The present study aimed to characterize this mouse AD model and its wild-type counterparts by biochemical and functional approaches. Methods Blood samples were collected from the transgenic and the wild-type mice, and radial arm water maze behavioral test was conducted at the ages of 6 and 12 months. The mice were sacrificed at 12-month age. One hemisphere of the brain was frozen-sectioned for immunohistochemistry and the other hemisphere was dissected into 7 regions. The levels ofAβ1-40, Aβ1-42 and 8-hydroxydeoxyguanosine （8-OHdG） in blood or/and brain samples were analyzed by ELISA. Secretase activities in brain regions were analyzed by in vitro assays. Results The pre-mature death rate of transgenic mice was approximately 35% before 6-month age, and high levels of Aβ1-40 and Aβ1-42 were detected in these dead mice brains with a ratio of 1 ： 1 0. The level of blood-borne Aβ at 6-month age was similar with that at 12-month age. Besides, Aβ1-40 level in the blood was significantly higher than Aβ1-42 level at the ages of 6 and 12 months （ratio 2.37：1）. In contrast, the level of Aβ1-42 in the brain （160.6 ng/mg protein） was higher than that of Aβ1-40 （74 ng/mg protein） （ratio 2.17：1）. In addition, the levels of Aβ1-40 and Aβ1-42 varied markedly among different brain regions. Aβ1-42 level was significantly higher than Aβ1-40 level in cerebellum, frontal and posterior cortex, and hippocampus. Secretase activity assays did not reveal major differences among different brain regions or between wild-type and transgenic mice, suggesting that the transgene PS1 did not lead to higher 7-secretase activity but was more efficient in producing Aβ1-42 peptides. 8-OHdG, the biomarker of DNA oxidative damage, showed a trend of increase in the blood of transgenic mice, but with no significant difference, as compared with the wild-type mice. Behavioral tests showed that transgenic mice had significant memory deficits at 6-month age compared to wild-type controls, and the deficits were exacerbated at 12-month age with more errors. Conclusion These results suggest that this mouse model mimics the early-onset human AD and may represent full-blown disease at as early as 6-month age for experimental studies.

Enzyme Activities in Perfluorooctanoic Acid (PFOA)-Polluted Soils

Perfluorooctanoic acid （PFOA） is a popular additive of the chemical industry; its effect on activities of important soil enzymes is not well understood. A laboratory incubation experiment was carried out to analyze the PFOA-induced changes in soil urease, catalase, and phosphatase activities. During the entire incubation period, the activities of the three soil enzymes generally declined with increasing PFOA concentration, following certain dose-response relationships. The values of EC10, the contaminant concentration at which the biological activity is inhibited by 10%, of PFOA for the soil enzyme activity calculated from the modeling equation of the respective dose-response curve suggested a sensitivity order of phosphatase 〉 catalase 〉 urease. The effect of PFOA on soil enzyme activities provided a basic understanding of the eco-toxicological effect of PFOA in the environment. Results of this study supported using soil ohosohatase as a convenient biomaxker for ecological risk assessment of PFOA-oolluted soils.