Proteinases and the instability of isocitrate lyase in extracts of developing flax seedlings

In extracts of flax seedlings 4 days after imbibition, isocitrate lyase activity is unstable in comparison to that in extracts from 2.5-day seedlings or to malate syntheses analysed at several stages of development. This instability in extracts of 4-day seedlings is especially pronounced when a large number of seedlings is homogenized per unit volume of Tris-Mg²⁺-EDTA-dithioerythritol buffer. However, isocitrate lyase can be stabilized when the resultant homogenate is diluted soon after seedling breakage. The pronounced instability in more concentrated extracts is not due to inadequate buffering by the homogenization medium, nor can it be due to polyphenols because added polyvinylpyrrolidone has no effect. Mixing of a heated supernatant from concentrated extract with dilute unheated extract yields the units of stable isocitrate lyase expected in the dilute extract, ruling out stoichiometric inactivation by a heat-stable component. The pronounced instability is attributed to the action of proteinases. A theoretical model assuming a decay process that is first order in isocitrate lyase and first-order in one or more proteinases is in reasonable agreement with the results. Malate synthase and NADP⁺-isocitrate dehydrogenase are much more stable in concentrated extracts prepared from 4-day flax seedlings. Isocitrate lyase is stable in concentrated extracts of 5-day watermelon seedlings, which is a developmental stage analogous to that for 4-day flax seedlings.     

The formation and biotransformation of cysteine conjugates of halogenated ethylenes by rabbit renal tubules

The nephrotoxicity of chlorotrifluoroethylene (CTFE) was examined using isolated rabbit renal tubules suspensions. Exposure of the tubules to CTFE resulted in consumption of CTFE, formation of a glutathione conjugate and inhibition of active organic acid transport. Synthetic cysteine, N-acetylcysteine or glutathione conjugates of CTFE inhibited transport indicating S-conjugation as a possible toxic pathway. 1,2-dichlorovinyl glutathione (DCVG), a model synthetic glutathione conjugate, was used to examine the degradation and toxicity of these conjugates. DCVG inhibited rabbit renal tubule transport in vivo and in vitro. The DCVG was found to be degraded with the evolution of glutamine and glycine to produce the ultimate nephrotoxicant, dichlorovinyl cysteine. Dichlorovinyl cysteine is then bioactivated with the release of ammonia. This sequential degradation explains the latency of DCVG-induced renal transport inhibition relative to dichlorovinyl cysteine. It is now evident that certain halogenated ethylenes are capable of being biotransformed to glutathione conjugates in the kidney with their subsequent hydrolysis to nephrotoxic cysteine conjugates.     

Human Dickkopf-1 (huDKK1) protein: characterization of glycosylation and determination of disulfide linkages in the two cysteine-rich domains

Human Dickkopf‐1 (huDKK1), an inhibitor of the canonical Wnt‐signaling pathway that has been implicated in bone metabolism and other diseases, was expressed in engineered Chinese hamster ovary cells and purified. HuDKK1 is biologically active in a TCF/lef‐luciferase reporter gene assay and is able to bind LRP6 coreceptor. In SDS‐PAGE, huDKK1 exhibits molecular weights of 27–28 K and 30 K at ～ 1:9 ratio. By MALDI‐MS analysis, the observed molecular weights of 27.4K and 29.5K indicate that the low molecular weight form may contain O‐linked glycans while the high molecular weight form contains both N‐ and O‐linked glycans. LC‐MS/MS peptide mapping indicates that ～ 92% of huDKK1 is glycosylated at Asn²²⁵ with three N‐linked glycans composed of two biantennary forms with 1 and 2 sialic acid (23% and 60%, respectively), and one triantennary structure with 2 sialic acids (9%). HuDKK1 contains two O‐linked glycans, GalNAc (sialic acid)‐Gal‐sialic acid (65%) and GalNAc‐Gal[sialic acid] (30%), attached at Ser 30 as confirmed by β‐elimination and targeted LC‐MS/MS. The 10 intramolecular disulfide bonds at the N‐ and C‐terminal cysteine‐rich domains were elucidated by analyses including multiple proteolytic digestions, isolation and characterization of disulfide‐containing peptides, and secondary digestion and characterization of selected disulfide‐containing peptides. The five disulfide bonds within the huDKK1 N‐terminal domain are unique to the DKK family proteins; there are no exact matches in disulfide positioning when compared to other known disulfide clusters. The five disulfide bonds assigned in the C‐terminal domain show the expected homology with those found in colipase and other reported disulfide clusters.     

L‐Cysteine capped CdTe–CdS core–shell quantum dots: preparation,characterization and immuno‐labeling of HeLa cells

Functionalized CdTe–CdS core–shell quantum dots (QDs) were synthesized in aqueous solution via water‐bathing combined hydrothermal method using L‐cysteine (L‐Cys) as a stabilizer. This method possesses both the advantages of water‐bathing and hydrothermal methods for preparing high‐quality QDs with markedly reduced synthesis time, and better stability than a lone hydrothermal method. The QDs were characterized by transmission electronic microscopy and powder X‐ray diffraction and X‐ray photoelectron spectroscopy. The CdTe–CdS QDs with core–shell structure showed both enhanced fluorescence and better photo stability than nude CdTe QDs. After conjugating with antibody rabbit anti‐CEACAM8 (CD67), the as‐prepared l ‐Cys capped CdTe–CdS QDs were successfully used as fluorescent probes for the direct immuno‐labeling and imaging of HeLa cells. It was indicated that this kind of QD would have application potential in bio‐labeling and cell imaging. Copyright © 2009 John Wiley & Sons, Ltd.     

Alleviation of chromium toxicity in rice seedlings by applying exogenous glutathione

The effect of exogenous reduced glutathione (GSH) on alleviation of hexavalent chromium (Cr⁶⁺) toxicity to rice seedlings and its physiological mechanisms were comprehensively investigated in a series of experiments. Our results showed that growth and nutrient uptake of rice seedlings were dramatically reduced under 100 μM Cr⁶⁺ stress, and the reduction was significantly alleviated by exogenous GSH. Cr⁶⁺ stress also reduced cell viability in root tips and damaged ultrastructure of both chloroplasts and root cells, while the addition of GSH alleviates those negative effects. Cr-induced toxicity and GSH-caused Cr alleviation differed significantly between Cr-tolerant Line 117 (L117) and Cr-sensitive Line 41 (L41). Under Cr⁶⁺ stress, cystine content was increased and GSH content was decreased in rice plants, exogenous GSH, however, mitigated the Cr-toxicity by reversing the Cr-induced changes of the two compounds. The types of Cr-induced secretion of organic acids varied between the genotypes, where reduction in the contents of acetic and lactic acids and tartaric and malic acids were observed in L117 and L41, respectively. The addition of GSH alleviated the reduction of secretion of these organic acids. Exogenous GSH also altered the forms of Cr ions in the rhizosphere and the fraction of distribution at subcellular level in both shoots and roots. It may be concluded that the alleviation of Cr⁶⁺ toxicity by exogenous GSH is directly attributed to its regulation on forms of Cr ions in rhizosphere and their distribution at subcellular levels.     

Cationic Inhibitors of Serine Proteinases from Buckwheat Seeds: Study of Their Interaction with Exogenous Proteinases

The inhibition of exogenous serine proteinases of different origin by cationic protease inhibitors BWI-1c, -2c, -3c, and -4c from buckwheat (Fagopyrum esculentum Moench) seeds has been studied. High efficiency of the inhibitors in binding bovine trypsin and chymotrypsin as well as their broad antiprotease effect, including inhibition of proteinases secreted by fungi and bacteria, has been demonstrated. According to the data obtained, it is proposed that cationic inhibitors from buckwheat seeds may participate in the defense of plants against fungal and bacterial infection.     

Protective role of purified cysteine proteinases against Fasciola gigantica infection in experimental animals

Fascioliasis is one of the public health problems in the world. Cysteine proteinases (CP) released by Fasciola gigantica play a key role in parasite feeding, migration through host tissues, and in immune evasion. There has been some evidence from several parasite systems that proteinases might have potential as protective antigens against parasitic infections. Cysteine proteinases were purified and tested in vaccine trials of sheep infected with the liver fluke. Multiple doses (2 mg of CP in Freund's adjuvant followed by 3 booster doses 1 mg each at 4 week intervals) were injected intramuscularly into sheep 1 week prior to infect orally with 300 F. gigantica metacercariae. All the sheep were humanely slaughtered 12 weeks after the first immunization. Changes in the worm burden, ova count, and humoral and cellular responses were evaluated. Significant reduction was observed in the worm burden (56.9%), bile egg count (70.7%), and fecel egg count (75.2%). Immunization with CP was also found to be associated with increases of total IgG, IgG(1), and IgG(2) (P<0.05). Data showed that the serum cytokine levels of pro-inflammatory cytokines, IL-12, IFN-γ, and TNF-α, revealed significant decreases (P<0.05). However, the anti-inflammatory cytokine levels, IL-10, TGF-β, and IL-6, showed significant increases (P<0.05). In conclusion, it has been found that CP released by F. gigantica are highly important candidates for a vaccine antigen because of their role in the fluke biology and host-parasite relationships.     

Partial Purification and Properties of a Cysteine Protease from Citrus Red Mite Panonychus citri

Several studies have reported that the citrus red mites Panonychus citri were an important allergen of citrus-cultivating farmers in Jeju Island. The aim of the present study was to purify and assess properties of a cysteine protease from the mites acting as a potentially pathogenic factor to citrus-cultivating farmers. A cysteine protease was purified using column chromatography of Mono Q anion exchanger and Superdex 200 HR gel filtration. It was estimated to be 46 kDa by gel filtration column chromatography and consisted of 2 polypeptides, at least. Cysteine protease inhibitors, such as trans poxy-succinyl-L-leucyl-amido (4-guanidino) butane (E-64) and iodoacetic acid (IAA) totally inhibited the enzyme activities, whereas serine or metalloprotease inhibitors did not affect the activities. In addition, the purified enzyme degraded human IgG, collagen, and fibronectin, but not egg albumin. From these results, the cysteine protease of the mites might be involved in the pathogenesis such as tissue destruction and penetration instead of nutrient digestion.     

我国大豆种子中球蛋白2S组分的分离纯化及部分性质的研究

根据大豆种子球蛋白和清蛋白溶解性不同的原理,分离出我国红丰3号大豆种子球蛋白2S粗制剂,再用SephadexG-100柱层析对其进行纯化。纯化后的球蛋白表现SDS-聚丙烯酰胺凝胶电泳非均一性。对这样纯化过的2s球蛋白进行DEAE-纤维素离子交换柱层析分离,用0.1—0.5mol/L pH7.6磷酸盐缓冲液进行线性梯度洗脱,得到四个洗脱峰,每个峰都获得了PAGE单一条带。四个组分分别命名为SⅡP_1,SⅡP_2,SⅡP_3和SⅡP_4。然后对这四种蛋白质的某些性质进行了研究,结果表明四者的分子量按以上顺序分别为22800,21500,19200和17800。所含残基数分别为191,179,163和147个。SⅡP_1,SⅡP_2和SⅡP_3三者的沉降系数(S_(20),w)分别为2.1S,1.9S和1.8S。N-末端分析表明这四种蛋白质的N-末端均为天门冬氨酸.还发现SⅡP_2具有能抑制α-胰凝乳蛋白酶的活性。本实验所提纯的这个抑制剂的一个ATEE(N-乙酰-L-酪氨酸乙酯)单位为0.4μg抑制剂蛋白(仅指对α-chymotrypsin发生作用)。α-chymotrypsin与此抑制剂相互作用时的摩尔数比初步判断为E/I=2/1。     

丙泊酚对全肝缺血再灌注大鼠脑损伤的保护作用及机制研究

目的:探究丙泊酚对全肝缺血再灌注(THIR)大鼠脑损伤的保护作用及机制.方法:选取72只健康成年雄性SD大鼠,将其按照抽签法分成假手术组、对照组以及丙泊酚组.所有大鼠予以12h禁食处理,采用3％戊巴比妥钠行腹腔注射麻醉处理,常规消毒后取上腹部正中切口进入腹腔.假手术组仅暴露肝门,不予以阻断处理.对照组与丙泊酚组则以无创...