利用伴刀豆球蛋白检测O-甘露糖基化

目的:基于伴刀豆球蛋白A(ConA)特异性识别并结合甘露糖的特性,建立一种检测O-甘露糖基化的方法,为酵母等宿主表达蛋白的O-糖基化提供一种高效筛选和分析的方法。方法:利用糖苷酶F(PNGF)切除检测蛋白的N-糖链,排除N-糖基化的干扰;通过Q阴离子交换柱和ConA Sepharose 4B柱纯化Western印迹膜封闭蛋白牛血清白蛋白(BSA),除去BSA中甘露糖修饰的蛋白的干扰,优化膜封闭条件;利用辣根过氧化物酶标记的ConA检测具有低甘露糖型N-糖基化修饰能力的毕赤酵母GJK01-HL(Δoch1)表达的抗Her-2抗体是否存在O-甘露糖基化现象。结果:通过PNGF酶切处理,可以完全去除糖蛋白的N-糖链的干扰;BSA经过Q阴离子交换柱和ConASepharose 4B柱纯化后,除去了大部分甘露糖蛋白,可作为封闭蛋白;用建立的方法检测,发现毕赤酵母工程菌GJK01-HL(Δoch1)表达的抗Her-2抗体存在O-甘露糖基化现象。结论:本方法是研究糖蛋白是否发生O-甘露糖基化的有效检测手段,可用于酵母等表达蛋白的O-糖基化的高效筛选和分析。     

重组人MASP2蛋白在大肠杆菌中的表达和纯化

目的:获得酶原形式的重组人甘露聚糖结合凝集素相关丝氨酸蛋白酶2(MASP2)。方法:在大肠杆菌中诱导表达重组人MASP2全长蛋白,包涵体裂解后,经复性、透析、浓缩、考马斯亮蓝染色、SDS-PAGE及Western印迹,鉴定纯化结果及酶活性。结果:复性后的MASP2蛋白经考马斯亮蓝染色未见杂带。自激活实验表明,当MASP2浓度在1μmool/L以下时,无论在4℃还是37℃,都能较稳定地保持酶原形式;蛋白浓度为3.5μmool/L时只能在4℃保持稳定,37℃发生自激活;蛋白浓度达到12μmool/L后,在4℃时已不能稳定存在。结论:获得了较纯的重组人MASP2蛋白,且具有自激活活性。     

The influence of variable and constant magnetic fields on biota and biological activity of ordinary chernozem soils

In model experiments on influence variable magnetic fields of industrial frequency (50 Hz) an induction of 1500 and of 6000 mkTl and the constant magnetic field an induction of 6000 mkTl and of 15000 mkTl during 5 days of exposure on biological properties of chernozem ordinary is shown, that the soil microflora is more sensitive to magnetic fields, than enzymes activity. Bacteria are more sensitive, than microscopic mushrooms. Dehydrogenase it is steady against influence of all variants. Constant magnetic field by the induction of 15000 mkTl rendered practically identical authentic overwhelming influence on catalase and saccharase activity - on 51 and 47% accordingly.     

Within‐plant distribution of 1,4‐benzoxazin‐3‐ones contributes to herbivore niche differentiation in maize

Plant defences vary in space and time, which may translate into specific herbivore‐foraging patterns and feeding niche differentiation. To date, little is known about the effect of secondary metabolite patterning on within‐plant herbivore foraging. We investigated how variation in the major maize secondary metabolites, 1,4‐benzoxazin‐3‐one derivatives (BXDs), affects the foraging behaviour of two leaf‐chewing herbivores. BXD levels varied substantially within plants. Older leaves had higher levels of constitutive BXDs while younger leaves were consistently more inducible. These differences were observed independently of plant age, even though the concentrations of most BXDs declined markedly in older plants. Larvae of the well‐adapted maize pest Spodoptera frugiperda preferred and grew better on young inducible leaves irrespective of plant age, while larvae of the generalist Spodoptera littoralis preferred and tended to grow better on old leaves. In BXD‐free mutants, the differences in herbivore weight gain between old and young leaves were absent for both species, and leaf preferences of S. frugiperda were attenuated. In contrast, S. littoralis foraging patterns were not affected. In summary, our study shows that plant secondary metabolites differentially affect performance and foraging of adapted and non‐adapted herbivores and thereby likely contribute to feeding niche differentiation.     

The nitrone spin trap PBN alters the cellular response to H(2)O(2): activation of the EGF receptor/ERK pathway

The nitrone spin trap PBN has been shown to protect neuronal cells from reactive oxygen species both in culture and in vivo. As an approach to understanding the molecular mechanisms by which PBN may function to protect cells, we examined whether PBN alters the cellular response to reactive oxygen species. H(2)O(2) stimulation of PC-12 cells results in weak activation of both the ERK and JNK signal transduction pathways. PBN pretreatment of PC-12 cells, followed by H(2)O(2) stimulation, results in strong and selective activation of the pro-survival ERK pathway. H(2)O(2) induction of ERK activity in PBN-pretreated cells was shown to be dependent on extracellular Ca(+2) influx. Further analysis of the ERK pathway showed that in PBN-pretreated cells, EGF receptor and the adapter protein SHC were phosphorylated in a Ca(+2)-dependent, ligand-independent manner following H(2)O(2) stimulation. Interestingly, H(2)O(2) stimulation of PBN-pretreated cells results in only 30% of the increase in intracellular Ca(+2) as compared to untreated cells following H(2)O(2) stimulation. These data suggest a model in which PBN attenuates H(2)O(2)-induced Ca(+2) entry, yet magnifies or alters Ca(+2) action, resulting in the activation of the EGF receptor/ERK pathway.     

Role of membrane lipids in regulation of vulnerability to oxidative stress in PC12 cells: implication for aging

Previously, we reported that PC12 cells showed increased vulnerability to oxidative stress (OS) induced by H2O2 (as assessed by decrements in calcium recovery, i.e., the ability of cells to buffer Ca(2+) after a depolarization event) when the membrane levels of cholesterol (CHL) and sphingomyelin (SPH) were modified to approximate those seen in the neuronal membranes of old animals. The present study was designed to examine whether the enrichment of the membranes with SPH-CHL and increased cellular vulnerability to OS are mediated by neutral SPH-specific phospholipase C (N-Sase) and the intracellular antioxidant GSH. The results showed a significant up-regulation of N-Sase activity by both low (5 microM) and high (300 microM) doses of H2O2. However, under high doses of H2O2 the up-regulation of N-Sase is accompanied by a significant increase in reactive oxygen species and by a decrease in intracellular GSH. The enrichment of membranes with SPH-CHL significantly potentiated the effects of high doses of H2O2, by further reducing the intracellular GSH and further up-regulating the N-Sase activity. Furthermore, repleting intracellular GSH with 20 mM N-acetylcysteine treatment was sufficient to attenuate the effect of a low dose of H2O2 on Ca(2+) recovery in SPH-CHL-treated cells. Thus, these results suggested that age-related alterations in the membrane SPH-CHL levels could be important determinants of the susceptibility of neuronal cells to OS.     

Epitope mapping of antigenic determinants of hepatitis C virus proteins by phage display

Study of individual hepatitis C (HCV) proteins could help to find a molecular structure and conformation, localization of antigenic and immunogenic determinants, to reveal of protective epitopes. It is necessary for practical medicine - development of diagnostic test-systems, vaccines and therapeutics. Linear and conformation dependent epitopes of HCV proteins was localized in this work and immunogenic properties of phage displayed peptides screened on monoclonal antibodies to HCV proteins have been investigated. Eleven epitopes of four HCV proteins have been studied. Three epitopes was found as linear, two epitopes were dependent on secondary structure of proteins and one epitope was dependent on tertiary structure of NS3 protein. Aminoacid sequences of other determinants have been determined and the distinct localization of these determinants will be continued after discovering of tertiary structure of HCV proteins. It was shown, that phage mimotope 3f4 is immunogenic and could induce specific hu- moral immune response to NS5A HCV protein. The data obtained could be useful for improving of HCV diagnostic test-systems, studying of amino acid substitutions and its influence on antigenic properties of the HCV proteins. The results could help to study an immune response in patients infected with different genotypes of HCV. Phage displayed peptides mimicking the antigenic epitopes of HCV proteins could be applied to development of HCV vaccine.     

Potentialities of new radiation technologies (ultrasonography, computed tomography, magnetic resonance imaging) in the diagnosis of endocrine diseases

In the paper providing the data available in the literature and many years' experience gained by the Department of the Moscow Regional Clinical Research Institute, the authors describe the potentialities of the currently available radiation techniques in the diagnosis of surgical endocrine diseases. The incidence of various organ dysfunctions is presented. The authors also consider the introscopic symptoms of various endocrinopathies and outline the common specific features of the ultrasound, computed tomographic, and magnetic resonance imaging pattern of individual masses (including adipose tissue cysts and tumors). Emphasis is laid on the leading role of ultrasonography (USG) in the diagnosis of thyroid and parathyroid diseases and on the priorities of computed tomography (CT) and magnetic resonance imaging (MRI) in visualIzing the abnormal thymus, mediastinally ectopic glands, and pituitary tumors. The capacities of each technique in the diagnosis of endocrine space lesions of the pancreas and adrenals are defined. The increase in the frequency of endocrine abnormalities accidentally identified at USG, CT, and MRI, which is noted in the paper, fosters the development of new trends in endocrinology and points to the need for screening surveys (particularly patients who live in poor environmental areas).     

The stability of enzyme activity and the content of microflora of different soils of the South of Russia to influence of a variable magnetic field of industrial frequency

In modelling experiments of the influence of variable magnetic field of industrial frequency (50 Hz) by induction of 1500 and 6000 mkTl during 5 days on microflora and on enzyme activity of soils the South of Russia of different genesis and properties is investigated.