Synthesis of Alkylating 1-Glycosyl-5-substituted 1,2,4-Triazoles1

Abstract

A series of alkylating derivatives of 5-subs-tituted 1-glycosyl-1,2,4-triazole having cytostatic activity has been prepared. The compounds synthesized include the 5-hydroxymethyl, 5-halomethyl, 5-(1-aziridino) methyl, and 5-bis (2-chloroethyl) aminomethyl derivatives.     

超氧化物歧化酶和乳酸脱氢酶在凝胶板上同时显色的方法

本文报道一种能使乳酸脱氢酶(LDH)和超氧化物歧化酶(SOD)同工酶在同一块电泳凝胶板上经同一种染色液一次处理达到同时显色的方法。显色结果,LDH为有色区带,SOD为无色透明区带,两者一目了然。而且前者泳动慢,后者泳动快,两种区带互不重迭,不发生干扰,结果可靠。此法不需光照,显色效率高,有利于同一样品同时进行此两种同工酶的电泳分析,达到一举两得的效果。     

Detection of conformationally changed MBP using intramolecular FRET

The principal objective of this study was to explore protein conformational changes using fluorescence resonance energy transfer (FRET) technology. Maltose binding protein (MBP) was adopted as a target model, due to its well-characterized structure and ligand specificity. To the best of our knowledge, this is the first report to provide information regarding the biological distance between the two lobes of MBP upon maltose binding. For the FRET pair, ECFP and EYFP were used as the donor and the acceptor, and were linked genetically to the C-terminal and N-terminal regions of MBP (ECFP:MBP:EYFP), respectively. After the FRET reaction, maltose-treated MBP was shown to exhibit a considerable energy transfer (FRET efficiency (E) = ∼0.11, Distance (D) = ∼6.93 nm) at the ensemble level, which was regarded as reflective of the increase in donor quenching and the upshift in acceptor emission intensity, thereby suggesting that the donor and the acceptor had been brought close together as the result of structural alterations in MBP. However, upon glucose treatment, no FRET phenomenon was detected, thereby implying the specificity of interaction between MBP and maltose. The in vitro FRET results were also confirmed via the acceptor photobleaching method. Therefore, our data showed that maltose-stimulated conformational changes of MBP could be measured by FRET, thereby providing biological information, including the FRET efficiency and the intramolecular distance.     

Evaluation of alpha-cyanoesters as fluorescent substrates for examining interindividual variation in general and pyrethroid-selective esterases in human liver microsomes

Carboxylesterases hydrolyze many pharmaceuticals and agrochemicals and have broad substrate selectivity, requiring a suite of substrates to measure hydrolytic profiles. To develop new esterase substrates, a series of alpha-cyanoesters that yield fluorescent products upon hydrolysis was evaluated for use in carboxylesterase assays. The use of these substrates as surrogates for Type II pyrethroid hydrolysis was tested. The results suggest that these novel analogs are appropriate for the development of high-throughput assays for pyrethroid hydrolase activity. A set of human liver microsomes was then used to determine the ability of these substrates to report esterase activity across a small population. Results were compared against standard esterase substrates. A number of the esterase substrates showed correlations, demonstrating the broad substrate selectivity of these enzymes. However, for several of the substrates, no correlations in hydrolysis rates were observed, suggesting that multiple carboxylesterase isozymes are responsible for the array of substrate hydrolytic activity. These new substrates were then compared against alpha-naphthyl acetate and 4-methylumbelliferyl acetate for their ability to detect hydrolytic activity in both one- and two-dimensional native electrophoresis gels. Cyano-2-naphthylmethyl butanoate was found to visualize more activity than either commercial substrate. These applications demonstrate the utility of these new substrates as both general and pyrethroid-selective reporters of esterase activity.     

Protein changes in abalone foot muscle from three geographical populations of Haliotis diversicolor based on proteomic approach

Using two‐dimensional gel electrophoresis, the foot muscle proteome of three geographical populations of Haliotis diversicolor were examined, with a total of 922 ± 21 protein spots detected in the Japanese population (JJ), 904 ± 25.6 in the Taiwanese population (TT), and 936 ± 16.2 in the Vietnamese population (VV). Of these, 254 spots showed differential expression and 85 protein spots percentage volumes varied more than twofold. Both “genotype” and “spot” analysis of variance approaches significantly showed differences among the three populations. Hierarchical clustering analysis showed that TT and VV clustered together followed by clustering with JJ, which is consistent with their geographical location. Following matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry, 30 differentially expressed proteins involved in major biological processes including energy production and storage and stress response were identified. Of these proteins, proteins pertaining to muscle contraction and muscle protein regulation showed highest expression levels in VV samples. Proteins involved in energy production and storage, including ATP synthase beta subunit, fructose‐1,6‐bisphosphate aldolase, arginine kinase, enolase, triosephosphate isomerase, and tauropine dehydrogenase, showed diverse expression patterns among the three populations. For stress‐responsive proteins, the expression of heat shock protein 70 was JJ > VV > TT. The expression pattern of Cu/Zn‐superoxide dismutase was JJ > VV > TT. Overall, these results may aid in the detection of new differentially expressed proteins within three different abalone populations.     

Characterization of Drosophila nitric oxide synthase: a biochemical study

The heme and flavin-binding domains of Drosophila nitric oxide synthase (DNOS) were expressed in Escherichia coli using the expression vector pCW. The denatured molecular mass of the expressed protein was 152kDa along with a proteolytically cleaved product of 121kDa. The DNOS heme protein exhibited very low Ca(2+)/calmodulin-dependent NO synthase activity. The trypsin digestion patterns were different from nNOS. The full-length DNOS protein had high degree of stability against trypsin. The activity assay of trypsin-digested protein confirmed the same result. Urea dissociation profile of DNOS full-length protein showed that the reductase domain activity was much more susceptible towards urea than the oxygenase domain activity. Urea gradient gel of DNOS full-length protein established distinct transition of dissociation and unfolding in the range 3-4M urea. Reductase domain activity of full-length DNOS protein against external electron acceptors like cytochrome c indicated slow electron transfer from FMN. The bacterial expression of DNOS full-length protein represents an important development in structure-function studies of this enzyme and comparison with other mammalian NOS enzymes which is evolutionary significant.     

Amyloid β binds procaspase-9 to inhibit assembly of Apaf-1 apoptosome and intrinsic apoptosis pathway

Apoptosis is essential in the death process induced by Amyloid-β (Aβ), a major constituent of diffuse plaques found in Alzheimer's disease patients. However, we have found that caspase activation and cell death induced by staurosporine, employed to induce the intrinsic mitochondria-dependent apoptotic pathway, were significantly reduced by 42 amino-acid Aβ42, implying that the peptide also has a negative effect on the apoptotic process. The inhibitory effect of Aβ42 on the apoptotic pathway is associated with its interaction with procaspase-9 and consequent inhibition of Apaf-1 apoptosome assembly. We detected the inhibitory effect in the early stage (< 8 h) of apoptosis, but later caspase activation becomes obvious. Thus we inferred that the inhibitory process on apoptosis begins at an early stage, and the later robust activation surpasses it. We propose that the apoptotic manifestation in Aβ-treated cells is a combined consequence of those anti- and pro-apoptotic processes.     

Specific labelling of microsomal proteins by reactive intermediates generated from 2-acetylaminofluorene in vitro

Incubation of 2-[9-¹⁴C] acetylaminofluorene (2-[9-¹⁴C]AAF) in vitro with rat liver microsomes, leads to covalent binding of label to microsomal proteins. The binding is NADPH-dependent, increases linearly with time, and is inhibited by SKF-525A and 7,8-benzoflavone (7,8-BF). Binding is increased more than 8-fold in microsomes from 3-methylcholanthrene(MC)-pretreated rats, but only less than 2-fold in those from phenobarbital(PB)-pretreated rats. In the presence of cytosolic proteins, there is slight enhancement of the labelling of microsomes and some labelling of the cytosolic proteins. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and 2-dimensional gel electrophoresis indicate that covalent labelling by 2-AAF derivatives is concentrated in specific proteins. The pattern of labelling varies between microsomes from animals pretreated with PB, MC and 2-AAF. Factors which may contribute to the specificity of labelling are discussed.     

茶树叶片蛋白双向电泳体系建立与应用

为开展茶树Camellia sinensis 低温和干旱胁迫下差异蛋白的分离和鉴定,以抗逆性较强的茶树品种‘迎霜’为试材,通过对提取方法、IPG 胶条pH 范围、上样量、分离胶浓度、染色方法的比较,筛选适用于茶树叶片的蛋白质双向电泳体系。结果表明,采用TCA-丙酮法或Tris-HCl 法提取叶片总蛋白,选用17 cm pH 4~7IPG 胶条用于等电聚焦,选择1.6~2.2 mg 上样量、13.5%聚丙烯酰胺凝胶进行分离,随后通过高敏考马斯亮蓝R-250 法染色;最终,叶片各分子量的蛋白充分分离,获得的双向电泳图谱分辨率高、背景清晰、重复性好,适用于‘迎霜’低温和干旱胁迫下叶片差异蛋白分析。     

A strategy for the comparative analysis of serum proteomes for the discovery of biomarkers for hepatocellular carcinoma

Many of the emerging technologies for the global evaluation of gene expression, at both the RNA and protein level, are being applied to the problem of finding biomarkers for human disease progression. These analyses can be made difficult, however, by variation between samples that arises from both technical and nondisease related physiological or genetic causes. In an effort to identify serum polypeptides whose presence or absence correlates with the clinical status of patients at high risk for hepatocellular carcinoma (HCC), we have developed a strategy that helps to focus the analysis on meaningful changes in protein levels above the background of variation. For the current study we divided the patient population into four clinically defined diagnostic groups that represent a generally increasing risk for HCC. Chronic infection with hepatitis B virus (HBV) is a major risk factor for HCC and our groups included patients with no indication of liver disease (healthy), those with inactive chronic HBV, those with active chronic HBV, and patients with a diagnosis of HCC and history of chronic HBV infection. Serum polypeptides from these patients were first analyzed in two-dimensional gels by combining the serum from patients in each of the four groups to generate composite gel profiles. Analysis of these composite gels allowed us to identify two relatively abundant features that were reduced in the HCC group as compared to the healthy group. Tryptic fragment mass fingerprinting identified the features as a carboxy terminal fragment of complement C3 and an isoform of apolipoprotein A1. These two features were examined by two-dimensional gel electrophoresis of serum from each individual in the four groups in order to verify that the inter-group differences seen in composite gels reported changes in abundance for most members of the group, rather than extreme changes for a small fraction of the group. These preliminary studies suggest that a proteomic methodology can be used for the identification of serum biomarkers for HCC and other liver disease.