细胞质雄性不育水稻幼穗和花粉发育期的蛋白酶活性变化

水稻细胞质雄性不育系珍汕97A,在花粉母细胞形成期以后可溶性蛋白含量迅速降低。单核期仅为花粉母细胞形成期的56％,二核期和三核期分别为花粉母细胞形成期的36．4％和30．3％。保持系珍汕97B虽在花粉母细胞形成期后可溶性蛋白含量降低,但在二核期则有所增高,表现为鞍形变化过程。在花粉母细胞形成期后,珍汕97A的酪蛋白水解酶活性增高,单核期为前一时期的1倍,而珍汕97B的酶活性增幅较小。第一次枝梗分化期珍汕97A的内肽酶活性较珍汕97B高,而雌雄蕊形成期两者的酶活皆降低。整个发育期中珍汕97A的内肽酶活性较珍汕97B高15％至55．5％。珍汕97A在幼穗第一次枝梗分化期时氨肽酶活性较珍汕97B高。第二次枝梗分化期后,不育系和保持系的氨肽酶活性均下降。但花粉母细胞形成期后,珍汕97A的氨肽酶活性迅速增高,三核期达最高水平,这可能意味着此时大量蛋白质被水解。蛋白酶类抑制剂的实验表明,在雌雄蕊形成期后不育系和保持系含有半胱氨酸型氨肽酶和约10％含金属型氨肽酶;而在三核期,保持系的氨肽酶则主要为半胱氨酸型。不育系三核期含有的BAPA内肽酶包括半胱氨酸型、丝氨酸型,以及部分含金属酶类;而保持系三核期的内肽酶仅为丝氨酸型。不育系肽酶类型的变化,可能反映酶基因编码的去抑制。     

脱落酸和抑制剂对萌发花生子叶肽链内切酶和花生球蛋白降解的影响

脱落酸（Ａｂｓｃｉｓｉｃａｃｉｄ,ＡＢＡ）抑制花生种子萌发的作用与核酸和蛋白质合成抑制剂的作用不同．ＡＢＡ（１００μｍｏｌ／Ｌ）在萌发零时施用,明显抑制肽链内切酶活性和同工酶表现以及花生球蛋白降解,萌发４８ｈ施用ＡＢＡ（１００μｍｏｌ／Ｌ）只降低肽链内切酶活性．ＡＢＡ的抑制作用不依赖于核酸和蛋白质合成．核酸合成抑制剂（３＇－脱氧腺苷,放线菌素Ｄ,５－氟尿嘧啶）和蛋白质合成抑制剂（亚胺环己酮）只能部分降低肽链内切酶活性,对肽链内切酶同工酶表现和花生球蛋白降解无明显影响．实验结果表明花生子叶肽链内酶不是在种子萌发过程中重新（ｄｅｎｏｖｏ）合成,文中讨论了肽链内切酶活性调节和花生贮藏蛋白降解的起始模式．     

中性肽链内切酶(CD10)生理功能研究进展

中性肽内切酶(Neutral Endopeptidase,NEP),属于M13锌金属蛋白酶家族的一种Ⅱ型整合细胞膜糖蛋白,在许多组织中有广泛的表达。同时,NEP作为一种神经肽的降解酶,已经被发现广泛地存在于中枢神经系统以及外周各种组织中。NEP通过水解疏水性氨基酸侧链,使神经肽失活。而这些神经肽参与生命调节的多重代谢活动,对生命系统的正常有序运转有重要的作用。其中P物质为一种神经肽,是造血系统网络的成员之一,同时也可以诱发炎症反应,从而,NEP可以间接参与造血系统和呼吸系统的代谢活动。而心钠素是调节血压维护心肌功能的重要调节因子,NEP通过调节心钠素的浓度来调节血压,最终参与心血管系统的代谢。由于NEP具有广泛的组织特异性,决定了NEP在多个系统的调节中都具有重要意义。因此就NEP在造血系统,心血管系统和呼吸系统的功能做一综和评述。     

中性肽链内切酶（CD10）生理功能研究进展

中性肽内切酶（Neutral Endopeptidase,NEF）,属于M13锌金属蛋白酶家族的一种Ⅱ型整合细胞膜糖蛋白,在许多组织中有广泛的表达。同时,NEP作为一种神经肽的降解酶,已经被发现广泛地存在于中枢神经系统以及外周各种组织中。NEP通过水解疏水性氨基酸侧链,使神经肽失活。而这些神经肽参与生命调节的多重代谢活动,对生命系统的正常有序运转有重要的作用。其中P物质为一种神经肽,是造血系统网络的成员之一,同时也可以诱发炎症反应,从而,NEP可以间接参与造血系统和呼吸系统的代谢活动。而心钠素是调节血压维护心肌功能的重要调节因子,NEP通过调节心钠素的浓度来调节血压,最终参与心血管系统的代谢。由于NEP具有广泛的组织特异性,决定了NEP在多个系统的调节中都具有重要意义。因此就NEP在造血系统,心血管系统和呼吸系统的功能做一综和评述。     

血管紧张素1-7改构体对A549细胞生长的抑制作用

目的:以D型氨基酸替代的方式和C端及N端改构的方式构建2种能够抵抗蛋白酶降解的血管紧张素Ang1-7异构体小肽血管紧张素改构体1和2,对其抗肿瘤细胞系A549活性进行初步研究,以期为长效型Ang1-7改构类抗肿瘤药的应用提供理论依据。方法:HPLC法检测2种改构体抗酶降解的能力;用带荧光标记的Ang1-7对A549细胞进行药物亲和实验,并用无标记的药物拮抗这种配体亲和;用MTT法检测Ang1-7及2种改构体对A549细胞增殖的影响。结果:2种改构体均能抗血管紧张素转化酶、中性内肽酶、亮氨酸内肽酶的降解;带荧光标记的Ang1-7能够和A549细胞结合,且这种结合可以被无标记的2种改构体竞争性拮抗;Ang1-7和2种改构体能抑制A549细胞增殖。结论:构建了能够抵抗酶降解,在体外能结合于A549细胞表面并抑制A549细胞增殖的2种Ang1-7改构体小肽,为该小肽进一步的体内抗癌研究及应用奠定了基础。     

基于选择性抑制mPGES-1的表达筛选抗炎中药

通过建立的选择性膜型前列腺素E2合酶-1(mPGES-1)表达抑制剂的体外细胞筛选模型,对50味中药共236个样品进行筛选。初筛中,筛选中药不同极性提取物对mPGES-1表达的抑制作用,抑制率50%的样品进入复筛;复筛中,检测初筛活性样品对环氧化酶(COX)-1和COX-2表达的抑制作用,抑制率均20%的样品认为是阳性物。结果表明,吲哚美辛对3种酶的抑制活性与文献报道相近;筛选发现泽泻组分1、蒲黄组分2对mPGES-1的表达有可重复的选择性抑制作用,对泽泻组分1、蒲黄组分2进行进一步研究,有望确定其抗炎的有效部位或化合物。     

小麦叶片暗诱导衰老期间内肽酶的特性

研究了小麦(Triticum aestivum L.cv.Yangmai 158)叶片暗诱导衰老期间内肽酶同工酶的变化及其部分生化特性,发现叶片衰老期间,内肽酶活性升高,同时出现5种新的内肽酶同工酶(EP1、EP2、EP4、EP5、EP6).6-苄氨基嘌呤(6-BA)处理能延缓这些同工酶的出现,而脱落酸(ABA)处理则加速它们的表达.衰老期间小麦叶片内的6种内肽酶同工酶(EP1-EP6)中的EP1、EP2、EP4、EP5、EP6呈现活性的pH及温度范围较窄,而EP3有活性的pH范围和温度范围均较宽,且EP3在嫩叶、老叶中均有活性.另外,EP3、EP5、EP6对热不太敏感.蛋白酶抑制剂实验表明,EP1、EP2是需金属离子的半胱氨酸型内肽酶,EP4是丝氨酸型内肽酶.     

棉铃虫和甜菜夜蛾中肠丝氨酸蛋白酶活性测定以及活化、降解CrylAc毒素分析

昆虫中肠对Bt原毒素活化与对活化毒素降解的变化被认为是害虫对Bt产生的机制之一,研究比较棉铃虫Helicoverpa armigern（Hǔbner）与甜菜夜蛾Spodoptera exigm（Hǔbner）的中肠液、BBMV蛋白酶的活性,通过SDS-PAGE分析2种昆虫对原毒素的活化速度与对活化毒素的降解速度。2种昆虫的中肠液蛋白酶活性均显著高于BBMV蛋白酶活性,中肠液与BBMV均能迅速活化原毒素并继续降解活化后的毒素,与中肠液相比,BBMV对原毒素的活化与对活化毒素的降解均慢于中肠液,甜菜夜蛾对毒素的活化与降解又慢于棉铃虫。另外,还测定抑制剂对中肠液蛋白酶活性的抑制作用,结果表明,各抑制剂对棉铃虫和甜菜夜蛾相应酶活性的抑制表现出相同的趋势,TLCK对丝氨酶蛋白酶具较好的抑制作用,而PMSF对胰蛋白酶的抑制作用次之,TPCK对胰凝乳蛋白酶的抑制作用较弱。     

棉铃虫和甜菜夜蛾中肠丝氨酸蛋白酶活性测定以及活化、降解Cry1Ac毒素分析

昆虫中肠对Bt原毒素活化与对活化毒素降解的变化被认为是害虫对Bt产生的机制之一,研究比较棉铃虫Helicoverpa armigera(Hübner)与甜菜夜蛾Spodoptera exigua(Hübner)的中肠液、BBMV蛋白酶的活性,通过SDS-PAGE分析2种昆虫对原毒素的活化速度与对活化毒素的降解速度。2种昆虫的中肠液蛋白酶活性均显著高于BBMV蛋白酶活性,中肠液与BBMV均能迅速活化原毒素并继续降解活化后的毒素,与中肠液相比,BBMV对原毒素的活化与对活化毒素的降解均慢于中肠液,甜菜夜蛾对毒素的活化与降解又慢于棉铃虫。另外,还测定抑制剂对中肠液蛋白酶活性的抑制作用,结果表明,各抑制剂对棉铃虫和甜菜夜蛾相应酶活性的抑制表现出相同的趋势,TLCK对丝氨酶蛋白酶具较好的抑制作用,而PMSF对胰蛋白酶的抑制作用次之,TPCK对胰凝乳蛋白酶的抑制作用较弱。     

人类疾病研究的新焦点:中性肽链内切酶(NEP)——NEP生理功能及其潜在调控因子研究之进展

 Neprilysin（ NEP）是M13锌金属蛋白酶家族的一种Ⅱ型整合细胞膜糖蛋白.自从1974年首次被定义以来,NEP作为一种神经肽的降解酶,已经被发现广泛地存在于中枢神经系统以及外周各种组织中,并具有众多组织特异性功能.其中, NEP具有两大重要生理功能：一是NEP通过抑制细胞迁移增殖并诱导细胞程序性死亡具有抗肿瘤作用,这依赖它的酶学特性以及与涉及细胞信号转导通路的关键分子直接的蛋白-蛋白间相互作用;另外, NEP具有神经保护作用,这主要通过阻止神经毒物——淀粉样蛋白Aβ在大脑中的聚集来实现.通过研究各种人类疾病的进展,目前发现这些疾病经常伴随有NEP表达的削弱和活性下降.基于这些发现,调控NEP在病理细胞中的不同水平正在逐渐被认可为具有治疗应用前景的一种策略,从而达到恢复正常的细胞功能并缓解病症的目的.现阶段的研究重点,主要在于揭示涉及NEP的表达以及活性的一些调控机制,并且获得了大量鼓舞人心的研究成果.例如,男性荷尔蒙已知是前列腺中NEP的转录调节因子.除它之外,最新研究结果还发现,女性雌激素,柳栎的水合萃取物,绿茶的一些组分,各种神经肽比如韩蛙皮素和生长激素抑制素以及淀粉样前体蛋白的胞内区,也都对NEP的表达和活性有刺激作用.     

Enkephalin-degrading enzyme inhibitors. Crucial role of the C-terminal residue on the inhibitory potencies of retro-hydroxamate dipeptides

The retro-inversion of the amide bond in kelatorphan and analogs, the first series of complete inhibitors of enkephalin metabolism, led to compounds highly efficient only against the neutral endopeptidase 24-11 (NEP). In order to increase the recognition of the aminopeptidase N (APN) and dipeptidylaminopeptidase (DAP), without loss of affinity for NEP, the malonyl group of these retro-inhibitors was replaced by diversely substituted succinyl moieties. All the molecules synthesized are highly efficient NEP inhibitors with Ki's in the 0.2-1 nM range, indicating that NEP possesses a relatively large and not very selective S'2 subsite. In contrast, inhibition of DAP activity is crucially dependent on the size and the position of the substituent in the succinyl moiety. Inhibitory potencies in the nanomolar range are obtained with compounds containing a benzyl group in the alpha-position related to the retro amide bond. Finally, a relatively modest inhibition of APN was observed with Ki's in the 0.5-1 microM range for compounds with benzyl or cyclohexyl group in P'2 position. However, these data demonstrate that efficient and complete inhibition of enkephalin degradation can be obtained with hydroxamate dipeptides containing a retro amide bond. The analgesic potency of the most active inhibitors was measured using the hot plate test in mice. Significant antinociceptive responses were obtained but these effects were rather weaker than those expected from the in vitro inhibitory potencies of these compounds on the three enkephalin-degrading enzymes.     

Inhibitors of the enkephalin degrading enzymes. Modulation of activity of hydroxamate containing compounds by modifications of the C-terminal residue

To further characterize the S'2 subsite of both the neutral endopeptidase (EC 3.4.24.11, NEP) and aminopeptidase N (EC 3.4.11.2, APN), two enzymes physiologically involved in enkephalin metabolism, a new series of hydroxamate inhibitors containing a cyclic amino acid as the P'2 component were synthesized. These amino acids differ by the size of the cycle, the relative position of the functional groups, and their absolute configuration. Highly efficient inhibitors of NEP were obtained whatever the modification on the P'2 component, while for APN inhibition, a cyclic beta-amino acid was preferred. The most active inhibitors contained a trans cyclopentyl beta-amino acid and a cis or a trans cyclohexyl beta-amino acid. When injected intracerebroventricularly in mice, these two latter compounds elicited potent antinociceptive responses on both the jump latency and the fore paw lick times.     

Enzymes and inhibitors in leu-enkephalin in metabolism in human plasma

The enzymes degrading leucine enkephalin in human plasma and the inhibitors active on these enzymes were studied by kinetic and chromatographic techniques. Data obtained evidence the existence of complex kinetics of leu-enkephalin hydrolysis and of formation of its hydrolysis byproducts. These appear to originate from the combined effect of further hydrolysis of the enkephalin's fragments after their release and of competition between the different enzymes present in plasma. Chromatographic separation of plasma proteolysis inhibitors indicates the existence of several pools of substances acting on all three enzyme groups that degrade leu-enkephalin. The partial specificity of these substances induces competition effects: consequently, the actual protection over leu-enkephalin is considerably lower that the total inhibitory activity. That notwithstanding, plasma inhibitors control enkephalin hydrolysis to a relevant extent, while they modify only slightly the ratio of hydrolysis between the different enzymes. This latter parameter—and specifically the large prevalence of aminopeptidases over dipeptidylaminopeptidases and dipeptidylcarboxypeptidases—appears controlled mainly by kinetic factors.     

Biological Insights and NMR Metabolic Profiling of Different Extracts of Spermacoce verticillata (L.) G. Mey.

Spermacoce verticillata (L.) G. Mey. is commonly used in the folk medicine by various cultures to manage common diseases. Herein, the chemical and biological profiles of S. verticillata were studied in order to provide a comprehensive characterization of bioactive compounds and also to highlight the therapeutic properties. The in vitro antioxidant activity using free-radical scavenging, phosphomolybdenum, ferrous-ion chelating and reducing power assays, and the inhibitory activity against key enzymes such as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), tyrosinase, α-amylase and α-glucosidase of S. verticillata extracts (dichloromethane, ethyl acetate, methanol and water) were investigated. The highest total phenolic and flavonoid content were observed in the methanolic and aqueous extracts. Exhaustive 2DNMR investigation has revealed the presence of rutin, ursolic and oleanoic acids. The methanolic extract, followed by aqueous extract have showed remarkable free radical quenching and reducing ability, while the dichloromethane extract was the best source of metal chelators. The tested extracts showed notable inhibitory activity against cholinesterases (AChE: 1.63–4.99 mg GALAE/g extract and BChE: 12.40–15.48 mg GALAE/g extract) and tyrosinase (60.85–159.64 mg KAE/g extract). No inhibitory activity was displayed by ethyl acetate and aqueous extracts against BChE and tyrosinase, respectively. All the tested extracts showed modest α-amylase inhibitory activity, while only the ethyl acetate and aqueous extracts were potent against α-glycosidase. This study further validates the use of S. verticillata in the traditional medicine, while advocating for further investigation for phytomedicine development.     

黄顶菊茎叶浸提液对白菜和水稻幼苗化感作用的初步研究

研究了不同浓度的黄顶菊茎叶浸提液对白菜和水稻幼苗的化感作用.结果表明:黄顶菊茎叶浸提液显著地抑制了白菜和水稻幼苗的根长和苗高,降低了白菜和水稻的根系活力,提高了丙二醛含量,并且增大了受体细胞膜透性,使细胞内含物外渗,导致电导率值增高;同时,黄顶菊茎叶浸提液对白菜和水稻的过氧化氢酶(CAT)、过氧化物酶(POD)和超氧化物歧化酶(SOD)的活性均有显著的抑制作用,导致受体对外界不良环境的抵抗力降低.由此可见黄顶菊通过影响植物体内的一系列生理过程,降低植物对环境的抗胁迫能力,使受体植物的生长发育受到抑制,从而表现出强烈的化感作用.综合各项指标可以看出,白菜较水稻对黄顶菊化感作用较敏感.     

蟹爪叶盾蕨石油醚提取物的成分分析及抗菌活性研究

为了研究蟹爪叶盾蕨(Neolepisorus ovatus(Bedd.)Ching f. doryopteris(Christ)Ching)石油醚提取物的化学组成及抗菌活性,采用气相色谱-质谱法分析蟹爪叶盾蕨石油醚提取物的成分,采用琼脂平板打孔法检测提取物对大肠杆菌、金黄色葡萄球菌、藤黄八叠球菌的抑制活性。从蟹爪叶盾蕨石油醚提取物中鉴定出38种化学成分,占出峰总数的92. 76%,其中豆甾-4-烯-3-酮(19. 83%)、何帕烯(16. 40%)、β-香树脂酮(14. 93%)等甾体和萜类化合物含量丰富。抑菌活性结果显示,蟹爪叶盾蕨石油醚提取物对藤黄八叠球菌的抑菌圈为(7. 78±0. 55)cm,对大肠杆菌、金黄色葡萄球菌、白色念珠菌没有抑制作用。所鉴定的化合物均首次从蟹爪叶盾蕨检测到,蟹爪叶盾蕨石油醚提取物对藤黄八叠球菌有抑制作用。此研究结果为该植物的开发利用提供了科学依据。     

Different inhibition of enalaprilat and imidaprilat on bradykinin metabolizing enzymes

Effects of angiotensin-converting enzyme (ACE) inhibitors, enalaprilat and imidaprilat, on bradykinin (BK) metabolizing enzymes, aminopeptidase P (APP), neutral endopeptidase (NEP) and carboxypeptidase N (CPN), were examined. APP activity in the mouse lung was inhibited by enalaprilat in a concentration-dependent manner while imidaprilat did not influence the enzyme activity. The inhibitory effects of these ACE inhibitors on the NEP activity in the mouse lung and the CPN activity in the mouse serum were negligible. These data suggested that the influence of enalaprilat on the APP activity and subsequent BK metabolism are different from those of imidaprilat.     

Effects of aqueous extract of rice plants (Oryza sativa L.) on seed germination and radicle elongation of Monochoria vaginalis var. plantaginea

Laboratory experiments were conducted to examine the allelopathic potentials of rice plants (Oryza sativa L.) on seed germination and radicle elongation of Monochoria vaginalis (Burm. f.) Presl var. plantaginea (Roxb.) Solms-Laubat. To compare the relative allelopathic potentials of rice plants at different growth stages, aqueous extracts of dried tissues were obtained from the seeds, husks, and seedlings harvested 10, 20, 40 and 120 days after seeding. When M. vaginalis seeds were incubated in solutions containing aqueous extracts, significant increases in the germination rate were observed for all the aqueous extracts except for the extract of 10 d-old shoots at higher dosages. When partitioned with ethyl acetate, the germination-promoter(s) in the aqueous extracts remained in the aqueous phase while some inhibitors were extracted with ethyl acetate. In contrast, aqueous shoot extracts added to the incubation media at the same dosage for promotion of seed germination inhibited radicle elongation of M. vaginalis, indicating that the sensitivity of the weed varies between these two growth stages.     

Amelioration of intracerebroventricular streptozotocin induced cognitive impairment by Evolvulus alsinoides in rats: In vitro and in vivo evidence

Evolvulus alsinoides, also known as Shankpushapi, is a commonly used traditional medicine for enhancing memory. We evaluated the in vitro free radical scavenging and enzymes [acetylcholinesterase, butyrylcholinestrase, glycogen synthase kinase-3-β (GSK-3-β), rho kinase (ROCK II), prolyl endopeptidase (PEP), catechol-O-methyl transferase (COMT) and lipoxygenase (LOX)] inhibitory activities of aqueous and hydro-alcoholic extracts of E. alsinoides. Hydro-alcoholic extract of E. alsinoides demonstrated more free radical scavenging activity as compared to aqueous extract. Hydro-alcoholic extract also showed higher cholinesterase, GSK-3-β, ROCK II, PEP, COMT and LOX enzyme inhibitory activities as compared to aqueous extract. Phytochemical analysis revealed more flavanoids in hydro-alcoholic extract as compared to aqueous extract but no significant difference in phenolic content of the two extracts was observed. Based on in vitro data, hydro-alcoholic extract (100, 300 and 500 mg/kg, p.o.) was selected for in vivo study in intracerebroventricularly injected streptozotocin (STZ) induced cognitive impairment in male Wistar rats. Elevated plus maze, passive avoidance and Morris water maze were used for assessment of cognitive function on 14th, 21st and 28th day after STZ injection. Oxidative stress parameters (malondialdehyde, reduced glutathione, nitric oxide levels and superoxide dismutase activity), cholinergic dysfunction and rho kinase (ROCK II) expression were studied in cerebral cortex and hippocampus of rat brain at the end of the study. Hydro-alcoholic extract of E. alsinoides dose dependently prevented STZ induced cognitive impairment by reducing the oxidative stress, improving cholinergic function and preventing the increase in rho kinase expression. The results suggest an anti-Alzheimer potential of hydro-alcoholic extract of E. alsinoides.     

Nonpeptidic endothelin-converting enzyme inhibitors and their potential therapeutic applications

Endothelins (ETs) are potent vasoconstrictors, promitogens, and inflammatory mediators. They have been implicated in the pathogenesis of various cardiovascular, renal, pulmonary, and central nervous system diseases. Since the final step of the biosynthesis of ETs is catalyzed by a family of endothelin-converting enzymes (ECEs), inhibitors of these enzymes may represent novel therapeutic agents. Currently, seven isoforms of these metalloproteases have been identified; they all share a significant amino acid sequence identity with neutral endopeptidase 24.11 (NEP), another metalloprotease. Therefore, it is not surprising that the majority of ECE inhibitors also possess potent NEP inhibitory activity. To date, three classes of ECE inhibitors have been synthesized: dual ECE/NEP inhibitors, triple ECE/NEP/ACE inhibitors, and selective ECE inhibitors. Potential clinical applications of these compounds in hypertension, chronic heart failure, restenosis, renal failure, and cerebral vasospasm deduced from studies with relevant animal models are reviewed.