雾霾空气中细菌特征及对健康的潜在影响

论文从群落结构、浓度变化及粒径分布的角度论述了雾霾天气对空气细菌特征的影响,并结合了雾霾天气相关疾病的发病率,综合评价了雾霾天气空气病原菌导致的人群潜在健康风险的变化,最后提出了以前研究存在的不足及未来的发展趋势。综合研究结果,雾霾天气对空气细菌群落结构、浓度变化及粒径分布的影响,研究人员在不同城市得出的研究结果不同,可能由样点的时空环境、气象因素、雾霾程度及采集、检验、鉴定空气细菌方法等多种因素的差异引起。雾霾空气中已发现病原细菌均为条件致病菌,在空气中含量很低,但雾霾天气下部分病原菌的相对丰度增加,致病力会显著增强。此外,高浓度的细颗粒物和化学污染物可损伤皮肤黏膜屏障,打破呼吸道和皮肤的微生态平衡,为病原菌侵入创造较好的机会。两者的协同作用,显著增加了雾霾天气空气中病原菌的健康风险。     

受染巨噬细胞释放的鼠伤寒沙门菌可灭活抗蛋白酶

鼠伤寒沙门菌经口进入消化道，穿过小肠上皮屏障侵入宿主。其重要特征是能在黏膜下层的巨噬细胞和树突细胞中繁殖，随之从胃肠道向肝、脾等网状内皮组织扩散。细菌迁移时，丝氨酸纤维蛋白酶在细胞外基质（ECM）降解过程中起重要作用。此酶由丝氨酸蛋白酶抑制剂α2AP负调控。沙门菌的表面蛋白酶PgtE可通过蛋白水解作用使α2AP失活，     

原代培养脊髓神经元线状溶酶体与细胞骨架蛋白-纽蛋白关系的研究

目的研究原代培养脊髓神经元线状溶酶体(nematolysosome)的形成与分布及其与细胞骨架蛋白-纽蛋白(vinculin)的关系.方法用细胞松弛素D(cytochalasin D,CD)及佛波醇酯(phorbol myristate acetate,PMA)处理原代培养脊髓神经元,用免疫荧光双标记纽蛋白及组织蛋白酶D(cathepsin D)、酸性磷酸酶(ACPase)、电镜细胞化学及共焦激光扫描显微镜方法研究线状溶酶体与纽蛋白的关系.结果在正常对照组神经元,组织蛋白酶D(标记溶酶体)与纽蛋白分布于胞质及突起内;在CD及PMA处理神经元,纽蛋白及组织蛋白酶D的分布呈向心性移动,但集聚的部位不同;电镜酶细胞化学方法显示CD组及PMA组神经元内线状溶酶体均增多.结论组织蛋白酶D及纽蛋白在培养脊髓神经元内协同分布,CD及PMA均可引起二者分布的变化,提示纽蛋白可通过增强细胞内吞体/溶酶体系统活动而使线状溶酶体增加,也可通过促进丝状肌动蛋白聚合而影响线状溶酶体的形成及运动.     

植物蛋白酶抑制剂在植物抗虫与抗病中的作用

综述了植物蛋白酶抑制剂抗虫与抗病作用的研究进展.蛋白酶抑制剂广泛存在于植物体内,与植物抗虫抗病密切相关.植物蛋白酶抑制剂能抑制昆虫肠道蛋白酶,使昆虫生长发育缓慢,甚至死亡.但取食蛋白酶抑制剂后,昆虫能迅速分泌对抑制剂不敏感的蛋白酶,而使蛋白酶抑制剂无效.食物蛋白的含量和质量也影响植物蛋白酶抑制剂的抗虫效果.病原菌的感染能诱导植物产生蛋白酶抑制剂,诱导产生的蛋白酶抑制剂能抑制病原菌的生长.     

柞蚕卵组织蛋白酶B纯化及性质

昆虫卵内蛋白酶在胚胎发育中水解卵黄蛋白,为胚胎发育提供氨基酸。昆虫中已报道过几类卵蛋白酶,如家蚕中半胱氨酸蛋白酶和丝氨酸蛋白等。但是,目前尚不清楚这些蛋白酶是否存在于其他鳞翅目昆虫。了解这些蛋白酶的作用机理可以为我们提供害虫防治的新方法,并且,由于蛋白水解在许多生理过程中具有重要作用。如蛋白质的成熟和转运、受精、萌芽、肿瘤转移和其他形态发生等。因此,阐明这些蛋白酶的生物功能具有重要意义。由于Oi蚕卵粒大,产卵量也很大,因此被选作研究鳞翅目昆虫卵蛋白酶的材料,我们希望通过对数种昆虫卵内蛋白酶的研究,找出卵黄蛋白水解的一般规律。在我们前一篇文章中报道了oi蚕组织蛋白酶B的鉴定。该蛋白酶属于半胱酸蛋白酶类的组织蛋白,最适pH为3．5,可被E－64抑制。本文报道蛋白酶的纯化和蛋白质。经过5步纯化过程,从oi蚕卵母细胞中纯化出组织蛋白酶B,用SDS聚丙烯酰胺凝胶电泳测得蛋白酶的亚基分子量在47kDa左右。纯化的蛋白酶活性可被E－64和Leupeptin抑制。因此,该蛋白酶属于半胱氨酸蛋白酶。天冬氨酸蛋白酶特异性抑制Pepstatin不抑制其活性。其活性可被DFP和PMSF部分抑制制。这两种抑制剂通常抑制丝氨酸蛋白酶活性,但在家蚕中有报道,半氨酸蛋白也可被这两种抑制剂抑制。推测该知性中心除含有半胱氨酸残基外,可能还含有丝氨酸残基。由牛血红蛋白测得蛋白酶的最适pH为3．5。在pH3．5条件下对胚胎发育中蛋白酶活性变化进行了研究,并用纯化的蛋白酶制备了抗血清,采用单向免疫扩散对胚胎发育中组织蛋白酶B的含量进行了测定,结果表明这种蛋白酶在胚胎发育中含量较高,是胚肥发育中蛋白酶活性来源之一。     

MBL调控MASP激活补体系统

甘露聚糖结合凝集素(MBL;或甘露聚糖结合蛋白,MBP)是由相同的多肽链组成的寡聚物,它通过结合细胞表面的碳水化合物能够有效地识别侵入体内的多种致病微生物,并激活补体来杀灭病原微生物。MBL能与血清中其相关蛋白酶(MASP)结合,MASP包含3个丝氨酸蛋白酶MASP-1、MASP-2、MASP-3和非酶蛋白MAp19。研究显示,MBL通过2种机理调控MASP-2的活性,在先天性免疫中具有重要的作用。本文简要综述MBL调控MASP激活补体的作用机理。     

哈维氏弧菌胞外蛋白酶基因的克隆表达及免疫原性

根据实验室分离的大黄鱼弧菌病主要病原菌哈维氏弧菌(Vib rio harveyi)GYC1108-1株的胞外蛋白酶基因序列,设计胞外蛋白酶基因(ΔProA)特异性引物,扩增获得1552bp的ΔProA基因,克隆于pUC57-T载体;将ΔProA基因亚克隆到原核表达载体pET-28a进行融合表达,SDS-PAGE电泳检测发现,ΔProA融合蛋白经IPTG诱导后在大肠杆菌中以包涵体形式表达,分子量大小约55kD,诱导5h的表达蛋白产量约占细菌总蛋白的21%。Western blot分析,表达的55kD蛋白具有较高免疫原性。用纯化的ΔProA融合蛋白对大黄鱼进行免疫试验,结果免疫保护率达到75%。       

皮肤的保健

维护皮肤健康、发挥皮肤的正常生理功能,是预防皮肤病的关键,是保证人体健美不可缺少的一个重要环节。这里只从皮肤的保护、清洁、锻炼三个方面来谈其保健。皮肤的保护皮肤是保护机体内各种器官和组织免受外界环境中机械的、物理的、化学的和生物的有害因素侵入的屏障。保护皮肤的完整性就能阻止有害物质的侵入。比如在正常情况下,皮肤表面有很多细菌,有人统计每平方厘米约有6-8万多个细菌,当皮肤有轻微的损伤,化脓菌就会侵入,引起毛囊炎、疖肿、蜂窝组     

高效可溶性重组蛋白表达载体的构建

本研究构建了两种高效表达可溶性重组蛋白的原核表达载体。一种载体由HisSUMO序列与pET30a(+)载体连接而成(命名为HisSUMO Express),表达的融合蛋白用Ni-NTA纯化,用SUMO蛋白酶I切割后可获得不留任何残基的重组蛋白。SUMO-蛋白酶I价格较贵,为减少表达蛋白的成本,第二种载体即在His-SUMO和目的序列之间加入羟胺切割位点(命名为HisSUMO Economic)。在HisSUMO Economic中表达的融合蛋白用Ni-NTA纯化,羟胺液切割后可获得仅留一个甘氨酸残基的重组蛋白。以在常规表达载体中难以表达的鼠源成纤维细胞生长因子-21(mFGF-21)为例,经葡萄糖消耗实验检测其活性,验证两种表达载体的效果。结果表明mFGF-21在两种载体中均获得了高效表达,融合蛋白占菌体总蛋白的40%以上,Ni-NTA纯化后的融合蛋白分别利用羟胺切割液和SUMO蛋白酶I切割,纯化的mFGF-21成熟蛋白回收量约为54mg/L,回收率约为6%。经两种载体表达后的mFGF-21蛋白均具有生物学活性,可促进脂肪细胞消耗葡萄糖,为进一步研究提供了基础。     

钙蛋白酶对兔脑皮质中细胞骨架蛋白tau的降解作用

钙蛋白酶 (calpain)是钙依赖性中性蛋白酶 ,根据其对钙敏感性的不同 ,可分为m 和 μ 钙蛋白酶两型。分别用不同浓度CaCl2 溶液温育Wistar大鼠脑皮质匀浆液 ,并用Western印迹和定量图像分析技术检测不同亚型钙蛋白酶对tau蛋白的降解作用。发现 :在 37℃用 1mmol LCa2 温育底物 15min ,即出现大量分子量为 2 9kD的tau蛋白降解片段 ;当Ca2 浓度为 5mmol L时 ,tau蛋白几乎全部被降解 ;这种tau蛋白降解可被特异性的钙蛋白酶抑制剂完全逆转。进一步的研究发现 ,分别用 μ 钙蛋白酶抑制剂 ( 0 .0 5 μmol Lcalpastatin) ,m 钙蛋白酶抑制剂 ( 10 0 μmol LcalpaininhibitorIV)或总钙蛋白酶抑制剂 ( 5 5 2 μmol Lcalpeptin)与 1mmol LCa2 共同温育Wistar大鼠脑皮质匀浆液 ,1mmol LCa2 激活的tau蛋白降解分别被抑制 8.6 %、92 .5 %和 97.8%。该研究结果表明 ,一定浓度的Ca2 可同时激活 μ 钙蛋白酶和m 钙蛋白酶 ,这两种亚型均参与降解tau蛋白 ,但m 钙蛋白酶的作用比 μ 钙蛋白酶更强     

角蛋白酶及其研究进展

角蛋白在一般情况下很难被降解,工业上用高温、高压的办法降解角蛋白。然而,角蛋白酶能在温和的条件下降解角蛋白。本文对角蛋白的理化性质、发酵条件和分子生物学进行了综述。     

角蛋白酶研究进展

角蛋白化学结构稳定,不易被一般的蛋白酶降解;但作为角蛋白酶的专一性底物,角蛋白可被角蛋白酶降解.本就角蛋白酶的生产菌、降解角蛋白的生化机制、角蛋白酶的生化及分子生物学特性等方面的最新研究进展作一综述.     

New Feather-Degrading Filamentous Fungi

Among 106 filamentous fungi isolated from poultry farm waste, 13 species belonging to seven genera (Aspergillus, Acremonium, Alternaria, Beauvaria, Curvularia, Paecilomyces, and Penicillium) were able to grow and produce keratinase in stationary cultures using poultry feather powder as the only substrate. The four most efficient keratinase producers were selected for a comparative study of keratinase production in submerged and stationary conditions. The highest keratinolytic activities were produced after 4-6 days of cultivation in submerged conditions: 53.8 +/- 6.1 U/mL (Alternaria tenuissima), 51.2 +/- 5.4 U/mL (Acremonium hyalinulum), 55.4 +/- 5.2 U/mL (Curvularia brachyspora), and 62.8 +/- 4.8 U/mL (Beauveria bassiana). These novel nondermatophytic keratinolytic fungi have potential use in biotechnological processes involving keratin hydrolysis. The results of this work contribute to show that keratinolytic activity is relatively widespread among common filamentous fungi and may have an important rule in feather decomposition in natural settings.     

Secretion of keratinolytic enzymes and keratinolysis by Scopulariopsis brevicaulis and Trichophyton mentagrophytes: regression analysis

A survey on keratinophilic fungi from poultry-farm soils at Namakkal and from feather dumping soils at Chennai, India, revealed the existence of 34 species of fungi. Most of the fungi exhibited variable efficiency in producing extracellular keratinase when grown in plates with chicken feathers as the sole carbon and nitrogen source. The fungi Aspergillus flavus, Aspergillus niger, Aspergillus versicolor, Chrysosporium state of Arthroderma tuberculatum, Paecilomyces carneus, Scopulariopsis brevicaulis, Trichoderma viride, and Trichophyton mentagrophytes were efficient candidates to degrade the feathers. However, when cultivating the strains in submerged conditions in a medium containing chicken feathers as the sole nutrients source, Aspergillus glaucus, Chrysosporium keratinophilum, Curvularia lunata, Fusarium solani, and Penicillium citrinum also proved to be potent. Among all species, S. brevicaulis and Trichophyton mentagrophytes produced higher amounts of keratinase in both methods. Conditions for keratinase production were optimized by statistical design and surface plots. The highest keratinase activity was estimated by S. brevicaulis (3.2 KU/mL) and Trichophyton mentagrophytes (2.7 KU/mL) in the culture medium with chicken feathers and shows (79% and 72.2% of degrading ability, respectively).     

Synthesis and regulation of extracellular keratinase in three fungi isolated from the grounds of a gelatin factory,Jabalpur, India

Chrysosporium queenslandicum, Graphium penicilloideus andScopulariopsis brevicaulis were grown on various supplemented basal salts media to compare keratinase induction, activity and repression. All three fungi can utilize keratin as a sole source of carbon, nitrogen and sulfur. Total keratinase activity inC. queenslandicum andS. brevicaulis, was not repressed by supplementation of keratin-containing medium with glucose, ammonium or sulfate. The production of keratinase activity was not derepressible in keratin-free media. Keratin utilization commenced before the detection of significant extracellular keratinase activity which was always associated with mycelial growth.     

Similarities and specificities of fungal keratinolytic proteases: comparison of keratinases of Paecilomyces marquandii and Doratomyces microsporus to some known proteases

Based on previous screening for keratinolytic nonpathogenic fungi, Paecilomyces marquandii and Doratomyces microsporus were selected for production of potent keratinases. The enzymes were purified and their main biochemical characteristics were determined (molecular masses, optimal temperature and pH for keratinolytic activity, N-terminal amino acid sequences). Studies of substrate specificity revealed that skin constituents, such as the stratum corneum, and appendages such as nail but not hair, feather, and wool were efficiently hydrolyzed by the P. marquandii keratinase and about 40% less by the D. microsporus keratinase. Hydrolysis of keratin could be increased by the presence of reducing agents. The catalytic properties of the keratinases were studied and compared to those of some known commercial proteases. The profile of the oxidized insulin B-chain digestion revealed that both keratinases, like proteinase K but not subtilisin, trypsin, or elastase, possess broad cleavage specificity with a preference for aromatic and nonpolar amino acid residues at the P-1 position. Kinetic studies were performed on a synthetic substrate, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The keratinase of P. marquandii exhibited the lowest Km among microbial keratinases reported in the literature, and its catalytic efficiency was high in comparison to that of D. microsporus keratinase and proteinase K. All three keratinolytic enzymes, the keratinases of P. marquandii and D. microsporus as well as proteinase K, were significantly more active on keratin than subtilisin, trypsin, elastase, chymotrypsin, or collagenase.     

Evaluation of keratinolytic potential of some fungal isolates from gelatin factory campus.

During hair degradation, majority of organic sulphur was oxidized to inorganic sulphate and thiosulphate by four fungal isolates (Cylindrocarpon lichenicola, Graphium cuneiferum, Microsporum gypseum, and M. fulvum) from gelatin factory soil. Inorganic thiosulphate, an unusual metabolite, was regularly detected in the culture filtrates of all fungi, although in less amounts. Maximum quantity (44 micrograms/ml) was released by G. cuneiferum on 50th day of incubation. All four fungi showed significant extracellular keratinase activity on human hair. Sulphydryl compounds were present in low amounts throughout the experiment. Detection of inorganic sulphate and thiosulphate with significant release of total protein and keratinase and changes in alkalinity, established the role of sulphitolysis and peptidolysis during keratin biodegradation by fungi which ultimately results in complete keratin degradation.     

微生物角蛋白酶的特性及其应用研究进展

角蛋白作为家禽加工和农业废弃物的主要成分,因其结构中富含能抵抗普通蛋白酶和化学催化剂降解的稳定交联二硫键而难以被利用,因此每年都在环境中大量积累,造成了严重的环境污染。微生物角蛋白酶可将角蛋白废弃物转化为可再次利用的产物,带来了经济的可行性及环境的可持续发展。本文主要综述了角蛋白酶的生物化学特性、角蛋白酶的基本结构及其表达特性,总结了其应用价值及角蛋白降解机制,最后展望了微生物角蛋白酶的进一步研究方向。     

Similarities and Specificities of Fungal Keratinolytic Proteases: Comparison of Keratinases of Paecilomyces marquandii and Doratomyces microsporus to Some Known Proteases

Based on previous screening for keratinolytic nonpathogenic fungi, Paecilomyces marquandii and Doratomyces microsporus were selected for production of potent keratinases. The enzymes were purified and their main biochemical characteristics were determined (molecular masses, optimal temperature and pH for keratinolytic activity, N-terminal amino acid sequences). Studies of substrate specificity revealed that skin constituents, such as the stratum corneum, and appendages such as nail but not hair, feather, and wool were efficiently hydrolyzed by the P. marquandii keratinase and about 40% less by the D. microsporus keratinase. Hydrolysis of keratin could be increased by the presence of reducing agents. The catalytic properties of the keratinases were studied and compared to those of some known commercial proteases. The profile of the oxidized insulin B-chain digestion revealed that both keratinases, like proteinase K but not subtilisin, trypsin, or elastase, possess broad cleavage specificity with a preference for aromatic and nonpolar amino acid residues at the P-1 position. Kinetic studies were performed on a synthetic substrate, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The keratinase of P. marquandii exhibited the lowest K_m among microbial keratinases reported in the literature, and its catalytic efficiency was high in comparison to that of D. microsporus keratinase and proteinase K. All three keratinolytic enzymes, the keratinases of P. marquandii and D. microsporus as well as proteinase K, were significantly more active on keratin than subtilisin, trypsin, elastase, chymotrypsin, or collagenase.     

Stability and Stabilisation of Doratomyces microsporus Keratinase

Thermal and pH stabilities of a new crude keratinase ( Doratomyces microsporus ) were investigated in the ranges of 20-40°C and pH 4-10, respectively. The stability test was followed by activity measurement on two different substrates: human stratum corneum and haemoglobin. Activity measurement lasted more than 100 h. The effect of calcium ions on enzyme stability was also studied. Crude keratinase was stabilised by crosslinking with glutaraldehyde (GA). The same characteristics were determined for Proteinase K, the commercial enzyme, for comparative purposes. Crude keratinase was most stable at pH 8 in Tris/HCl and borate buffers. The type of buffer used proved to have higher effect on crude keratinase stability than on Proteinase K. Both enzymes were most stable at 20°C. Keratinase stability rapidly decreased at 40°C while Proteinase K showed higher thermal stability. A 1 mM solution of Ca 2+ ions did not significantly influence enzyme stability, but 2.5% GA solution stabilised crude keratinase at 40°C reducing the k d value by about 50%. Crude and crosslinked crude keratinase were used for crude calf skin degradation. A mathematical model, based on Michaelis-Menten kinetics, was developed to describe the crude calf skin degradation in a batch reactor. Validation of the model showed that it could describe the process over a defined range of its conditions.