细粒棘球绦虫水通道蛋白基因家族成员密码子偏好性分析

水通道蛋白(aquaporin, AQP)是一种主要内在蛋白家族成员,具有通透水及其他小分子溶质的功能,参与虫体渗透稳态、物质转运等过程。本研究运用CodonW、SPSS等生物信息学软件分析细粒棘球绦虫(Echinococcus granulosus)水通道蛋白基因家族成员(EgAQPs)的密码子使用偏好性,并与非洲爪蟾(Xenopus laevis)卵母细胞、酵母(Saccharomyces cerevisiae)进行密码子偏好性比较,以期找到EgAQPs最合适的外源表达系统。结果表明EgAQPs密码子选用偏好性普遍较弱,偏好以C/G作为结尾。在密码子使用频率上,EgAQPs与非洲爪蟾卵母细胞的差异高于酵母,表明酵母表达系统可能更适合于EgAQPs表达。若要进一步提高EgAQPs在非洲爪蟾卵母细胞或酵母中的表达水平,尚需对其密码子进行优化。     

非洲爪蟾孵化酶分子对卵黄膜作用方式的研究

非洲爪蟾的孵化液对卵黄膜和二甲基酷蛋白具有降妥活性。用非洲爪蟾孵化酶的特异性抗ＧＳＴ－ＵＶ．２抗体进行Ｗｅｓｔｅｒｎ杂交的结果表明,孵化液中出现一种分子量为６０ｋＤ的大组分,有时也会出现一种分子量为４０ｋＤ的小组分。     

四膜虫(Tetrahymena shanghaiensis)大核核仁与去膜大核能诱导非细胞体系核重建

外源DNA或染色质在非洲爪蟾卵提取物中可以诱导细胞核样结构的重建。重建核除不具有核仁样结构外,在其它形态结构上与真核细胞核十分相似。前人的工作表明在重建核中具有核仁前体结构。但可能是由于缺少活性核仁组织者的缘故,这些核仁前体不能相互融合形成新生核仁。那么活性核仁组织者在重建核中是否能发挥其功能呢?为了研究这一问题,我们提取纯化了四膜虫的大核与大核的周边核仁。进一步去除大核的核被膜,并将去除核被膜的大核与大核核仁分别加入非洲爪赡卵非细胞体系中。通过电镜超薄切片观察,我们发现无论是与大核染色质相连的周边核仁还是分离纯化的核仁结构在非洲爪赡卵非细胞体系中都不能保持其原有结构特征,而是发生了典型核重建变化,并且在诱导形成的重建核中也看不到核仁样结构。这些结果说明具有活性的核仁组织者在加入非洲爪蟾卵提取物后既不能继续保持其原有的RNA转录功能也不能诱导新的核仁的出现。     

爪蟾(Xenopus laevis)的四倍体及其子一代

在爪蟾受精卵第一次有丝分裂中期进行静液压处理,抑制细胞质分裂从而得到四倍体。实验中获得三只存活了三年以上的四倍体雌性爪蟾。其中一只雌性与二倍体雄性爪蟾作人工催青得到受精卵。现已产卵三批,发育的胚胎400余,经检查子代染色体为三倍体。     

推荐一种理想的实验动物——非洲爪蟾

非洲爪蟾(Xenopus)属于两栖纲,无尾目,是Xenopinae亚科三个属中的一种。虽然它是两栖动物,却纯属水栖,饲养方便。 在国外,非洲爪蟾最初应用于临床,供作妊娠诊断用。由于在实验室条件下经促性腺激素     

非洲爪蟾中一种长型肽聚糖识别蛋白的克隆与表达分析(英文)

肽聚糖识别蛋白(peptidoglycan recognition proteins,PGRPs)是固有免疫系统中一类重要的模式识别受体。该文首次从两栖类模式生物-非洲爪蟾(Xenopus tropicalis)中克隆得到了一个长型PGRP(XtPGRP-L)基因。XtPGRP-L具有5个外显子和4个内含子的基因组结构,该结构在进化的过程中比较保守。序列比对与系统进化分析显示XtPGRP-L具有保守的酰胺酶活性位点。蛋白质建模显示XtPGRP-L拥有保守的3-D结构。实时定量PCR检测显示,XtPGRP-L在非洲爪蟾胚胎早期不表达,到72h蝌蚪期开始表达。在成体的肝脏、肺、肠和胃高表达。同时,在LPS刺激后,XtPGRP-L在肝脏、肠和胃中呈明显上调表达。结果表明,XtPGRP-L在非洲爪蟾固有免疫系统中可能具有重要的作用。     

非洲爪蟾BAFF及其信号通路相关基因的比较生物信息学分析

目的:对非洲爪蟾BAFF和BAFF信号通路相关基因进行了分析.方法:采用生物信息学方法对两栖类重要的模式生物-非洲爪蟾的基因组和EST数据库进行分析.结果:非洲爪蟾BAFF cDNA全长为557 bp,编码218个氨基酸.与人BAFF序列相似性为37.5％.该文一共得到了14个BAFF信号通路相关基因.通过与人BAFF信号通路进行比较,对非洲爪蟾这14个BAFF信号通路相关基因进行了分析.结论:BAFF和BAFF信号通路在进化过程中较为保守,这为进一步研究低等脊椎动物BAFF功能和信号通路具有重要的指导作用.     

敬钊毒素-V对Kv4.3通道的抑制作用

K+通道亚型Kv4.3在调节心肌细胞动作电位的幅度与时程方面具有重要作用,是治疗心律失常的有效作用靶点,但目前世界上该通道的特异性抑制剂非常缺乏。敬钊毒素-V(Jingzhaotoxin-V,JZTX-V)是从敬钊缨毛蜘蛛粗毒中纯化到的一种新型肽类神经毒素,能够部分抑制大鼠背根神经节细胞上的瞬时外向K+电流,其半数有效抑制浓度(IC50值)为52.3nmol/L。为了研究JZTX-V对Kv4.3通道的作用,本实验通过多肽固相化学合成的方法得到JZTX-V,并用双电极杆电压钳技术检测JZTX-V对表达在非洲爪蟾卵母细胞上的Kv4.3通道电流的作用。结果显示,JZTX-V能够完全抑制Kv4.3通道电流,并且这种抑制作用具有浓度依赖性和时间依赖性,其IC50值为425.1nmol/L,JZTX-V还能够使通道的电流-电压关系曲线和稳态失活曲线分别向去极化方向漂移大约29mV和10mV,改变Kv4.3通道的动力学特征,因此我们推测JZTX-V是一种Kv4.3通道门控调制毒素。以上研究结果对于开发心肌Kv4.3通道的分子探针及以Kv4.3通道为靶点的药物设计具有借鉴作用。     

四膜虫（Tetrahymena shanghaiensis）大核核仁与去膜大核能诱导非…

外源ＤＮＡ或染色质在非洲爪蟾卵提取物中可以诱导细胞核样结构的重建。重建核除不具有核仁样结构外，在其它形态结构上与真核细胞核十分相似。前人的工作 重建中具有核仁前体结构。但可以是由于缺洗涤戌一核仁组织者的缘故，这些核仁前体不能相互融合形成新生核仁。那么活性核仁组织 重建核中是否能发挥其功能呢？为了研究这一问题，我们提取纯化了四膜虫的大核与大核的周边核仁。进一步去除大核与大核核仁分别加入非洲爪蟾卵非细     

基于定点突变技术对苦荞麦胰蛋白酶抑制剂活性位点的研究

目的:为了研究胰蛋白酶抑制剂的活性位点,揭示Ft TI结构与功能的关系。将Ft TI和突变体aFtTI-R65L,aFtTI-D67V和aFtTI-R65L/D67V经IPTG诱导培养5h,收集菌液经过超声波破碎得到粗产物,经过纯化后的胰蛋白酶抑制剂对胰蛋白酶的摩尔抑制比分别为1∶1,1∶1.15,1∶1.3,1∶1.2;抑制常数Ki分别为1.62n M,1.69 n M,1.9 n M,1.8 n M(BAp NA作为底物)。结果:SDSPAGE分析表明突变前和突变后表达产物胰蛋白酶抑制剂的大小一致,均为9.5 k Da。对突变体aFtTI-R65L,aFtTI-D67V和aFtTI-R65L/D67V抑制反应温度研究表明,其最适反应温度均为40℃。在10~80℃保温30 min后,突变体对胰蛋白酶的抑制活性仍保留80%以上;在90℃保温30min,突变体的抑制活性开始显著下降,只保留其39%。具有较高的耐热性。将aFtTI在pH 3.0~10.0的不同缓冲溶液中放置30 min后,其抑制活性可保留90%左右,在pH 2.0条件下,aFtTI抑制活性丧失约31%;在pH 11.0条件下,aFtTI抑制活性丧失约43%。结论:对苦荞麦蛋白酶抑制剂Ft TI的定点突变并不会改变它是一种偏碱性的胰蛋白酶抑制剂的性质,突变前后均保持了耐碱性的特点。     

Frog albumin is expressed in skin and characterized as a novel potent trypsin inhibitor

A novel potent trypsin inhibitor was purified and characterized from frog Bombina maxima skin. A full-length cDNA encoding the protein was obtained from a cDNA library constructed from the skin. Sequence analysis established that the protein actually comprises three conserved albumin domains. B.maxima serum albumin was subsequently purified, and its coding cDNA was further obtained by PCR-based cloning from the frog liver. Only two amino acid variations were found in the albumin sequences from the skin and the serum. However, the skin protein is distinct from the serum protein by binding of a haem b (0.95 mol/mol protein). Different from bovine serum albumin, B. maxima albumin potently inhibited trypsin. It bound tightly with trypsin in a 1:1 molar ratio. The equilibrium dissociation constants (KD) obtained for the skin and the serum proteins were 1.92 x 10(-9) M and 1.55 x 10(-9) M, respectively. B. maxima albumin formed a noncovalent complex with trypsin through an exposed loop formed by a disulfide bond (Cys53-Cys62), which comprises the scissile bond Arg58(P1)-His59(P1'). No inhibitory effects on thrombin, chymotrypsin, elastase, and subtilisin were observed under the assay conditions. Immunohistochemical study showed that B. maxima albumin is widely distributed around the membranes of epithelial layer cells and within the stratum spongiosum of dermis in the skin, suggesting that it plays important roles in skin physiological functions, such as water economy, metabolite exchange, and osmoregulation.     

Purification and Characterization of Albumin from Frog Skin of <Emphasis Type="Italic">Duttaphrynus melanostictus</Emphasis>

Following determination of trypsin inhibitory activity, a serine protease inhibitor was purified and characterized from frog Duttaphrynus melanostictus serum. It was identified as serum albumin, with molecular weight of 67 kDa (DmA-serum). Different from bovine serum albumin, DmA-serum potently inhibited trypsin with similar K _i values around 1.6 × 10⁻⁷ M. No inhibitory effect on thrombin, chymotrypsin, elastase and subtilisin was observed under the assay conditions. The N-terminal amino acid is EAEPHSRI. Subsequently, a protein with same N-terminal amino acid was purified from skin, termed as DmA-skin. However, DmA-skin is distinct from DmA-serum by binding of a haem b (0.5 mol/mol protein), and with low trypsin inhibitory activity. Frog albumin is distributed in frog skin and exhibited trypsin inhibitory activity, suggesting that it plays important roles in skin physiological functions, like water economy, metabolite exchange and osmoregulation, etc.     

Effect of Bovine Serum Albumin Incubation on Lettuce Chloroplasts Digested by Trypsin

1. Bovine serum albumin stimulates the DCIP photoreduction activity of lettuce chloroplasts which has been treated with trypsin. When these chloroplast preparations were washed by tricine buffer such "reversible action" can still be obtained. It is possible that bovine serum albumin may be incorporated into trypsin destroyed site of the membrane. 2. Trypsin-induced CCCP inhibitory effect on DCIP photoreduction activity is reversed by bovine serum albumin. 3. Bovine serum albumin partially reverses the trypsin-induced unstacking of lettuce chloroplast membranes. 4. After trypsin digestion, there are absorbance decreases around 500–640 nm. Bovine serum albumin has no effect on these absorbance decreases. It is concluded that the membrane-bound proteins responsible for different functions of chloroplast are heterogeneous. The results also show that there are gate and channel near the position of PSⅡ on chloroplast membrane.     

Are the proteinase inhibitory activities in lenticular tissues real?

The possibility of proteinase inhibitory activities in lenses measured with synthetic substrates being spurious, due to the effective competition of lens proteins as substrates for the target enzymes, was investigated. Goat, sheep and human cataractous lens proteins were found to be poor substrates for trypsin, elastase and papain compared to casein or bovine serum albumin. Further, the inhibition of elastase catalyzed hydrolysis of succinyl trialanyl p-nitroanilide by casein (500 μg, 53%) and albumin (500 μg, 49%) and of trypsin-catalyzed hydrolysis of benzoyl argininep-nitroanilide by albumin (1 mg, 24%) were significant only at high protein concentrations. These data indicated that the relatively high antielastase and antitryptic activities observed in human cataractous lenses were real. On the other hand, coincident lens protein hydrolysis elevating the true antitryptic and antielastase activities in goat and sheep lenses (that have low activities) could not be ruled out The lesser papain inhibitory activities observed in lenses when albumin was used as substrate compared to activities with benzoyl arginine p-nitroanilide as substrate, appeared to be partly due to lens protein hydrolysis masking the actual inhibition in the former method. Preincubation of goat, sheep and human lens extracts with trypsin for 1 h resulted in complete loss of antitryptic and antielastase activity except in the case of human lens antielastase activity which underwent 50% loss. Papain inhibitory activity was fully stable. Similar papain treatment caused loss of 80–100% of antielastase activity and 45–55% loss of antitryptic activity.     

Characterization of a serum factor that decreases albumin mRNA in cultured hepatocytes

Summary When primary cultures of hepatocytes are exposed to media containing fetal bovine serum (FBS) there is a rapid decrease in levels of tissue-specific mRNAs such as albumin mRNA. We used Northern blot analysis to examine mRNA levels in cultured hepatocytes, and characterized the factor in FBS that significantly reduces the steady state albumin mRNA level. Neonatal bovine serum or serum derived from platelet-poor calf plasma proved as potent as did FBS, but commercial bovine serum albumin did not exhibit this inhibitory activity. Inhibitory activity of FBS was not removed by moderate heat treatment, dialysis, or extraction with organic solvents. However, incubation of FBS with a highly anionic detergent such as 0.1% sodium dodecyl sulfate orN-lauroyl sarcosine, followed by extensive dialysis, resulted in sera that did not inhibit expression of albumin mRNA. These sera supported cell attachment and seemed non-toxic toward the cells. Ammonium sulfate fractionation of FBS showed the activity was present in the 45 to 70% fraction, and trypsin digestion destroyed the inhibitory activity. Gel exclusion chromatography gave a molecular weight 60 000 to 70 000. Fractionation of serum proteins by DEAE-Sephacel or Cibacron blue-agarose showed enrichment for albumin in the most active fractions. Interestingly, metabolic labeling of secreted and cellular proteins with³⁵S-methionine and cysteine showed no significant difference between hepatocytes maintained for 2 days beforehand in serum-free or serum-supplemented media, and no difference between detergent-treated FBS and control FBS. Therefore, FBS contains a factor that causes a significant decrease in steady state levels of mRNA for albumin and other mRNAs of tissue specific function, but under these conditions albumin mRNA levels are not paralleled by synthesis of albumin or other proteins.     

Identification and molecular cloning of a novel amphibian Bowman Birk-type trypsin inhibitor from the skin of the Hejiang Odorous Frog; Odorrana hejiangensis

In this study, an amphibian (Odorrana hejiangensis) skin extract was fractionated by reverse phase HPLC and fractions were screened for trypsin inhibitory activity. Using this initial approach, a novel trypsin inhibitory peptide was detected with an apparent protonated molecular mass of 1804.83 Da, as determined by MALDI-TOF mass spectrometry. It was named Hejiang trypsin inhibitor (HJTI) in accordance. The primary structure of the biosynthetic precursor of HJTI was deduced from a cDNA sequence cloned from a skin-derived cDNA library. The primary structure of the encoded predicted mature active peptide was established as: GAPKGCWTKSYPPQPCS (non-protonated monoisotopic molecular mass--1802.81Da). On the basis of this unequivocal amino acid sequence, a synthetic replicate was synthesized by solid phase Fmoc chemistry. This replicate displayed a moderately potent trypsin inhibition with a K(i) of 388 nM. Bioinformatic analysis of the primary structure of this peptide indicated that it was a member of the Bowman-Birk family of protease inhibitors. The substitutions of Gln-14 and Ser-17 by Lys, resulted in an increase in cationicity and a small increase in potency to a K(i) value of 218nM. Neither HJTI nor its synthetic analog, possessed any significant antimicrobial activity.     

Inhibitory selectivity of canecystatin: a recombinant cysteine peptidase inhibitor from sugarcane

The cDNA of a cystein peptidase inhibitor was isolated from sugarcane and expressed in Escherichia coli. The protein, named canecystatin, has previously been shown to exert antifungal activity on the filamentous fungus Trichoderma reesei. Herein, the inhibitory specificity of canecystatin was further characterized. It inhibits the cysteine peptidases from plant source papain (Ki =3.3nM) and baupain (Ki=2.1x10(-8)M), but no inhibitory effect was observed on ficin or bromelain. Canecystatin also inhibits lysosomal cysteine peptidases such as human cathepsin B (Ki=125nM), cathepsin K (Ki=0.76nM), cathepsin L (Ki=0.6nM), and cathepsin V (Ki=1.0nM), but not the aspartyl peptidase cathepsin D. The activity of serine peptidases such as trypsin, chymotrypsin, pancreatic, and neutrophil elastases, and human plasma kallikrein is not affected by the inhibitor, nor is the activity of the metallopeptidases angiotensin converting enzyme and neutral endopeptidase. This is the first report of inhibitory activity of a sugarcane cystatin on cysteine peptidases.     

Turkey serum trypsin inhibitors: description and characterization

1. Turkey serum trypsin inhibitors were studied on whole and chromatographically fractionated normal turkey serum using both quantitative (trypsin inhibitory capacity measurement) and qualitative (antitryptic activity detection methods) determinations, coupled to electrophoretic and isoelectrophoretic studies. 2. Five proteins with trypsin inhibitory activity were described, the most important ones being alpha 2 and beta-globulins with a multibanded pattern revealed by isoelectric focusing. 3. Trypsin inhibitory capacity assays, performed on individual sera, as well as isoelectric focusing studies, failed to find any quantitative and/or qualitative deficiency of these antiproteases. 4. Evidence is given that round heart disease in turkeys is not related to serum trypsin inhibitor deficiency.     

Binding of low-density lipoprotein to heparin-Sepharose: characterization and inhibition by serum high-molecular-weight components

In this study, the interaction of human serum low-density lipoprotein (LDL) with heparin immobilized on Sepharose was reinvestigated. Binding of isolated LDL (stabilized with human serum albumin (HSA] was compared with that of LDL in full serum. (1) Binding of isolated LDL was slightly decreased by CaCl2 and was not affected by MgCl2. In contrast, with full serum LDL binding was increased by these divalent cations. (2) In both situations, binding of LDL was saturable, but the maximum degree of binding that could be reached was much higher with isolated LDL than with LDL in full serum. This could be ascribed to an inhibitory action of a factor found in the d greater than 1.24 fraction of serum. (3) The effect of this factor was diminished in the presence of CaCl2 or MgCl2, which suggests that the stimulation of LDL binding by these cations in full serum is due to suppression of the inhibitory activity of this factor. (4) The inhibitory factor in the d greater than 1.24 fraction can be partially purified by absorption to heparin-Sepharose, followed by elution with 6 M guanidine chloride. The resulting preparation had a 30- to 50-fold higher specific activity. Attempts to purify the factor further resulted in loss of activity. (5) The activity is decreased upon treatment with trypsin and also upon acetylation or reduction with dithiothreitol, indicating that free amino groups and S-S bridges are essential.     

A subclass of albumin recognized by inhibition of phosphatidylethanolamine-mediated agglutination of mouse erythrocytes

Agglutination of mouse erythrocytes by non-choline phospholipids is inhibited by a factor in mammalian sera. The inhibitor cochromatographed with albumin on dye-agarose conjugates, was retained by an anti-albumin affinity column, was neutralized by anti-albumin antibody and found in a serum fraction in which only albumin could be detected. A variety of commercial preparations of albumin (fraction V, crystalline) did not inhibit. However, they acquired potent inhibitory activity when treated with low molecular weight thiols. The inhibitory activity of serum was increased 8-fold by treatment with dithiothreitol. Other proteins were not activated in this way. Inhibitory activity increased with average free sulphydryl content of treated albumin, up to six thiol groups per molecule. Alkylation of these sulphydryl groups did not diminish inhibitory activity. Thiols also induced polymerization of albumin. Inhibitory albumin in serum was largely monomeric. We propose that the inhibitor is a type of serum albumin which is lost or inactivated during preparation of commercial albumin, and which shares a structural feature, necessary for inhibition, with thiol-reduced albumin and the ligand on mouse erythrocytes.