家蚕丝氨酸蛋白酶BmHP14基因的克隆与表达模式分析

家蚕受到外源微生物侵染或损伤时,前酚氧化酶原级联反应中的起始丝氨酸蛋白酶会激活下游信号通路,最终产生黑色素。采用RACE技术,获得了家蚕前酚氧化酶原级联反应起始丝氨酸蛋白酶——血淋巴蛋白酶编码基因的全长cDNA序列,命名为BmHP14(GenBank登录号:JQ954757)。BmHP14 cDNA全长2 508 bp,开放阅读框为2 013 bp,编码670个氨基酸,预测蛋白质分子质量71 kD,等电点5.09,N端17个氨基酸预测为信号肽序列。多重序列比对显示BmHP14与烟草天蛾HP14的相似度很高,达到57%;分子进化树中二者也聚为一支。RT-PCR分析表明,BmHP14在家蚕5龄第3天幼虫脂肪体、马氏管、精巢、卵巢、表皮、血细胞、头部均有表达,其中以脂肪体中的表达水平最高。以黑胸败血芽孢杆菌、大肠杆菌、球孢白僵菌注射侵染家蚕5龄第3天幼虫,Real-time PCR检测显示在受到病菌侵染后,幼虫脂肪体中的BmHP14表达上调。Westernblotting检测结果显示,BmHP14在家蚕体液中以前体和成熟体的形式共同存在。研究结果提示,BmHP14是家蚕前酚氧化酶原级联反应信号通路中的关键酶。     

油茶籽油的蛋白酶水解法提取工艺研究

采用物理破碎油茶籽细胞和酶降解相结合的方法提取油茶籽油,对油茶籽原材料的预处理时间和温度,酶解方法进行了研究,探索蛋白酶在水性条件下酶解油茶籽的提油工艺及其影响因素,结果表明:油茶籽原材料预处理的适宜加热温度为90℃、加热时间为2 h,适宜的酶解温度为38℃、pH值为8、水解酶用量为0.25%(占油料的质量比重)、酶解时间为4 h。     

Protease Inhibitors from Marine Venomous Animals and Their Counterparts in Terrestrial Venomous Animals

The Kunitz-type protease inhibitors are the best-characterized family of serine protease inhibitors, probably due to their abundance in several organisms. These inhibitors consist of a chain of ~60 amino acid residues stabilized by three disulfide bridges, and was first observed in the bovine pancreatic trypsin inhibitor (BPTI)-like protease inhibitors, which strongly inhibit trypsin and chymotrypsin. In this review we present the protease inhibitors (PIs) described to date from marine venomous animals, such as from sea anemone extracts and Conus venom, as well as their counterparts in terrestrial venomous animals, such as snakes, scorpions, spiders, Anurans, and Hymenopterans. More emphasis was given to the Kunitz-type inhibitors, once they are found in all these organisms. Their biological sources, specificity against different proteases, and other molecular blanks (being also K⁺ channel blockers) are presented, followed by their molecular diversity. Whereas sea anemone, snakes and other venomous animals present mainly Kunitz-type inhibitors, PIs from Anurans present the major variety in structure length and number of Cys residues, with at least six distinguishable classes. A representative alignment of PIs from these venomous animals shows that, despite eventual differences in Cys assignment, the key-residues for the protease inhibitory activity in all of them occupy similar positions in primary sequence. The key-residues for the K⁺ channel blocking activity was also compared.     

Efficient Screening of Marine Extracts for Protease Inhibitors by Combining FRET Based Activity Assays and Surface Plasmon Resonance Spectroscopy Based Binding Assays

The screening of extracts from marine organisms is a widely used strategy to discover new drug leads. A common problem in the screening process is the generation of false positive hits through unspecific effects from the complex chemical composition of the crude extracts. In this study, we explored a combination of a fluorescence resonance energy transfer (FRET) based activity assay and a surface plasmon resonance (SPR) based binding assay to avoid this problem. An aqueous extract was prepared from rest raw material of the Norwegian spring spawning herring, and further fractionated by methanol solubility and solid phase extraction. FRET based activity assays were used to determine the influence of each extract on the activity of different proteases. Several extracts showed more than 50% inhibition. The inhibition mechanisms were elucidated by SPR based competition experiments with known inhibitors. For the secreted aspartic proteases 1, 2, 3 and HIV-1 protease, the results indicated that some extracts contain inhibitors interacting specifically with the active site of the enzymes. The study shows that a combination of an activity assay and an SPR based binding assay is a powerful tool to identify potent inhibitors in marine extracts. Furthermore, the study shows that marine vertebrates offer an interesting source for new bioactive compounds, although they have rarely been explored for this purpose.     

Cytotoxic,Cytostatic and HIV-1 PR Inhibitory Activities of the Soft Coral Litophyton arboreum

Bioassay-guided fractionation using different chromatographic and spectroscopic techniques in the analysis of the Red Sea soft coral Litophyton arboreum led to the isolation of nine compounds; sarcophytol M (1), alismol (2), 24-methylcholesta-5,24(28)-diene-3β-ol (3), 10-O-methyl alismoxide (4), alismoxide (5), (S)-chimyl alcohol (6), 7β-acetoxy-24-methylcholesta-5-24(28)-diene-3,19-diol (7), erythro-N-dodecanoyl-docosasphinga-(4E,8E)-dienine (8), and 24-methylcholesta-5,24(28)-diene-3β,7β,19-triol (9). Some of the isolated compounds demonstrated potent cytotoxic- and/or cytostatic activity against HeLa and U937 cancer cell lines and inhibitory activity against HIV-1 protease (PR). Compound 7 was strongly cytotoxic against HeLa cells (CC₅₀ 4.3 ± 0.75 µM), with selectivity index of SI 8.1, which was confirmed by real time cell electronic sensing (RT-CES). Compounds 2, 7, and 8 showed strong inhibitory activity against HIV-1 PR at IC₅₀s of 7.20 ± 0.7, 4.85 ± 0.18, and 4.80 ± 0.92 µM respectively. In silico docking of most compounds presented comparable scores to that of acetyl pepstatin, a known HIV-1 PR inhibitor. Interestingly, compound 8 showed potent HIV-1 PR inhibitory activity in the absence of cytotoxicity against the cell lines used. In addition, compounds 2 and 5 demonstrated cytostatic action in HeLa cells, revealing potential use in virostatic cocktails. Taken together, data presented here suggest Litophyton arboreum to contain promising compounds for further investigation against the diseases mentioned.     

海水养殖废水浇灌下不同盐生经济植物生理特征及其比较

采用大田试验,研究了无施肥条件下海水浇灌(对照)、1∶1海水养殖废水(1∶1养殖废水处理)、全海水养殖废水(全养殖废水处理)处理以及施肥条件下海水浇灌(施肥处理)对海蓬子和碱蓬植株生长与生理特性的影响。结果表明:1∶1养殖废水处理的植株地上部干重(DW)显著高于对照,但全养殖废水处理植株地上部DW低于对照;施肥处理植株地上部DW明显高于所有对照和其他处理,随着生育期的延长,上述效应愈加明显,尤其体现在碱蓬植株上。与施肥处理相比,两配比养殖废水灌溉抑制了2种植物生长,降低了植株地上部氮磷(NP)含量、叶绿素含量(CHL)含量和可溶性蛋白(SP)含量,并随养殖废水配比上升而降低越显著;相反,2种植物细胞相对电导率(REC)、丙二醛(MDA)含量却随着养殖废水配比的上升而升高,碱蓬的上升比海蓬子更为明显。与对照相比,1∶1养殖废水不同程度提高了2种植物DW、CHL含量、植株地上部NP含量、内肽酶、羧肽酶、超氧化物歧化酶(SOD)及过氧化物酶(POD)活性以及SP和游离氨基酸(FAA)含量;并且,随着养殖废水配比的上升,2种植物以上各指标却不同程度地下降。综上所述,1∶1养殖废水处理可有效促进2种植物叶绿素合成,提高SOD、POD活性,降低MDA积累量,减少细胞膜受损,进而加快植株生长,增加NP吸收。2种植物相比,碱蓬对海水养殖废水浓度的生理响应较海蓬子敏感。1∶1养殖废水处理可很大程度上替代肥料的施用,从而降低环境污染。     

The serine protease inhibitors and SERPINE1/2 disrupt prostaglandin E2 production and hyaluronic acid retention in FSH‐stimulated pig cumulus–oocyte complexes

The serine proteases, tissue‐ and urokinase‐type plasminogen activators (PLAT and PLAU) and their inhibitors SERPINE1/2 are regulators of plasminogen to plasmin conversion. They are widely expressed in ovarian tissues, including granulosa and cumulus cells, and their expression is regulated by gonadotropins. The aim of this work was to assess the effect of serine protease inhibitors (aprotinin and AEBSF) and SERPINE1/2 on FSH‐induced cumulus cell expansion, the production of prostaglandin E2 (PGE2) and retention of hyaluronic acid (HA) in expanding cumulus. The serine protease activity proved to be essential for the production of PGE2 and also for the retention of HA; the inhibition of plasminogen activators by SERPINE1/2 had the same effect. Collectively, these data indicate that plasmin is required for proper function of expanding cumulus cells in vitro and presumably also in vivo in the pre‐ovulatory follicles.     

Intracellular and extracellular enzyme activity in soil with reference to elemental cycling

Enzyme activities play an important role for the transformation of elements and compounds in soil and, thus, were extensively analyzed for more than 4 decades. The activity of any enzyme in soil may not only be controlled by active organisms. Substantial parts of ‘extracellular’ enzymes may be stabilized by abiotic soil components maintaining their activity. Methods to discriminate the source of enzyme activity were summarized with emphasis on the approach plotting enzyme activity versus a feature integrating the microbial biomass after the addition of glucose and nitrate. Considering the quotient between enzyme activity and microbial biomass content, protease activity will be discussed with reference to nitrogen transformation in soils.     

碱性蛋白酶水解猪血细胞最优反应条件的研究

研究旨在确立碱性蛋白酶水解猪血细胞的最优反应条件。采用二次旋转正交回归设计,建立了ｐＨ值、温度（Ｔ）、酶—底物浓度比（〔Ｅ〕／〔Ｓ〕）三个因素对水解蛋白收率影响的回归模型。通过分析,确定最优水解条件为：ｐＨ＝８２１,〔Ｅ〕／〔Ｓ〕＝４％,最后,在此最优水解条件下,确定出最佳水解时间ｔ＝９０ｍｉｎ。     

蚕蛹蛋白双酶水解工艺条件研究

蚕蛹脱脂后,用微生物酶进行双酶水解,确定其最佳工艺条件为：３９４２中性蛋白酶反应温度为４０℃,ｐＨ为７０,固液比为２０∶１００,加酶量为２０％,反应时间为２ｈ;１３９８中性蛋白酶反应温度为５０℃,ｐＨ为７５,固液比为２０∶１００,加酶量为２０％,反应时间为１ｈ．在该条件下水解,水解率高达２３７５％,水解液中的１８种氨基酸质量浓度为１５６４ｇ·Ｌ－１．