Validated spectrofluorimetric method for the determination of clonazepam in pharmaceutical preparations

A simple, highly sensitive and validated spectrofluorimetric method was applied in the determination of clonazepam (CLZ). The method is based on reduction of the nitro group of clonazepam with zinc/CaCl₂, and the product is then reacted with 2‐cyanoacetamide (2‐CNA) in the presence of ammonia (25%) yielding a highly fluorescent product. The produced fluorophore exhibits strong fluorescence intensity at ?_em = 383 nm after excitation at ?_ex = 333 nm. The method was rectilinear over a concentration range of 0.1–0.5 ng/mL with a limit of detection (LOD) of 0.0057 ng/mL and a limit of quantification (LOQ) of 0.017 ng/mL. The method was fully validated and successfully applied to the determination of CLZ in its tablets with a mean percentage recovery of 100.10 ± 0.75%. Method validation according to ICH Guidelines was evaluated. Statistical analysis of the results obtained using the proposed method was successfully compared with those obtained using a reference method, and there was no significance difference between the two methods in terms of accuracy and precision. Copyright © 2015 John Wiley & Sons, Ltd.     

Gel filtration chromatography as a method for removing bacterial endotoxin from antibody preparations

The removal of bacterial endotoxins from biological samples is critical to avoid the potentially fatal pyrogenic response possible when introduced to mammalian systems. Endotoxins have a variety of specific characteristics that can be exploited to target their isolation and subsequent removal, but one that has not been extensively characterized is their difference in size from that of monoclonal antibodies. Here, we present a study which utilizes gel filtration chromatography as a method for endotoxin removal from both aggregated and nonaggregated antibody preparations, outlining a mechanistically simple method for removal of this impurity. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1497–1501, 2014     

转cry1Ab/cry2Aj和G10evo-epsps基因玉米中Bt蛋白的时空表达及抗性评价

【目的】探讨转cry1Ab/cry2Aj和G10evo-epsps基因玉米双抗505-12-5中外源Bt蛋白时空表达规律,对3种主要鳞翅目害虫亚洲玉米螟、黏虫和棉铃虫的抗性进行鉴定,为转基因玉米双抗505-12-5的商业化推广提供科学的数据支撑。【方法】Bt蛋白时空表达规律采用酶联免疫法(ELISA),田间抗虫性和室内抗虫性鉴定分别采用田间人工接虫和离体组织生测方法。【结果】在玉米6~8叶期,Bt含量表现为根心叶茎,分别为517.3、453.8和312.8 ng·g~(-1);大喇叭口期,Bt含量表现为心叶根茎,分别为353.3、281.3和232.9 ng·g~(-1);吐丝期,Bt含量表现为心叶根茎,分别为188.9、114.1和53.6 ng·g~(-1);乳熟期,根、茎和心叶含量相当,分别为178.0、160.3和185.4 ng·g~(-1);繁殖器官中Bt蛋白含量表现为籽粒花丝花粉雄穗,分别为181.3、100.1、95.0和79.8 ng·g~(-1)。室内抗虫性鉴定表明,转基因玉米双抗505-12-5心叶饲喂黏虫24 h,幼虫死亡率低,但48 h后达98.21%;双抗505-12-5心叶、花丝和籽粒饲喂玉米螟,24 h幼虫死亡率分别为87.37%、100%、100%;双抗505-12-5花丝饲喂棉铃虫,24 h幼虫死亡率达80.18%,48 h死亡率为92.45%。田间鉴定结果显示,转基因玉米双抗505-12-5在心叶期和雌穗期对玉米螟、心叶期对黏虫、雌穗期对棉铃虫的抗性均达高抗水平。【结论】转基因玉米双抗505-12-5各器官在不同生育期中均能表达Bt蛋白,尤其在鳞翅目害虫为害的主要时期6~8叶期和吐丝期及乳熟期,易受害器官中Bt蛋白表达量较高。转基因玉米双抗505-12-5田间及室内对3种鳞翅目害虫均表现了显著的抗性效果,具有推广应用的潜力。     

Characterization of a root-specific β-thioglucoside glucohydrolase gene in Carica papaya and its recombinant protein expressed in Pichia pastoris

Plant β-thioglucoside glucohydrolases (TGGs or myrosinases) are a young class of enzymes in the glycosyl hydrolase family 1 and have a narrow distribution. TGG genes have mainly been cloned from crucifers, while TGGs in other species have received little attention. The TGG gene CpTGG2 and its recombinant protein from papaya were characterized in this paper. This is the first plant TGG gene without unusual intron splicing borders, as present in all other available TGG genes. Phylogenetic analysis indicated that plant myrosinases are divided into two major lineages. CpTGG2 is located in the lineage constituted by AtTGG4–6 from Arabidopsis thaliana, while the rest of myrosinases (including MA, MB and MC subfamilies) are grouped into another lineage. RT-PCR analysis indicated that CpTGG2 was specifically expressed in the root. The recombinant CpTGG2 expressed in yeast had a subunit mass of 70 kDa, and had low basal TGG activity without addition of ascorbate. Low concentrations of ascorbate stimulated CpTGG2 activity, while high concentrations were inhibitory. CpTGG2 was active in broad pH and temperature ranges, similar to AtTGG4 and AtTGG5. The apparent K_m and V_max were 2.24 mM and 24.3 μmol min⁻¹ mg⁻¹ when sinigrin was the substrate. The calculated k_cat/K_m value was 1.3 × 10⁴ S⁻¹ M⁻¹_. Our results reshaped and expanded the myrosinase family structure and provided clues to the evolution of myrosinase genes.     

The Saccharomyces cerevisiae genes ( CMP1 and CMP2 ) encoding calmodulin-binding proteins homologous to the catalytic subunit of mammalian protein phosphatase 2B

Summary Saccharomyces cerevisiae genomic clones that encode calmodulin-binding proteins were isolated by screening a λgt11 expression library using¹²⁵I-labeled calmodulin as probe. Among the cloned yeast genes, we found two closely related genes (CMP1 andCMP2) that encode proteins homologous to the catalytic subunit of phosphoprotein phosphatase. The presumed CMP1 protein (62999 Da) and CMP2 protein (68496 Da) contain a 23 amino acid sequence very similar to those identified as calmodulin-binding sites in many calmodulin-regulated proteins. The yeast genes encode proteins especially homologous to the catalytic subunit of mammalian phosphoprotein phosphatase type 213 (calcineurin). The products of theCMP1 andCMP2 genes were identified by immunoblot analysis of cell extracts as proteins of 62000 and 64000 Da, respectively. Gene disruption experiments demonstrated that elimination of either or both of these genes had no effect on cell viability, indicating that these genes are not essential for normal cell growth.     

Measurements of lipopolysaccharide (endotoxin) in meningococcal protein and polysaccharide preparations for vaccine usage

Lipopolysaccharide (LPS, i.e. endotoxin) present in meningococcal outer-membrane protein and polysaccharide preparations made for vaccine use was quantitated by a silver-stain method following SDS-PAGE. The reactivities of LPS in the preparations were also measured by rabbit pyrogenicity and Limulus amoebocyte lysate (LAL) assay. Although rabbit pyrogenicity and LAL assay are more sensitive than the silver stain method, the latter provided an actual amount of LPS present in the protein or in the polysaccharide. For a meningococcal protein preparation, rabbit pyrogenicity showed about one-tenth, and even less by LAL assay, of the actual amount of LPS. This is because protein-bound LPS in meningococcal protein preparations is about 10-fold less active in causing fever in rabbits, and 20- to 40-fold less active in the gelation of LAL than the same amount of a purified free LPS which is generally used as a reference in quantitating LPS in these two assays. As for the small amount of LPS present in a meningococcal polysaccharide preparation, similar LPS content was obtained when measured by the three methods suggesting that the LPS is not bound to the polysaccharide in contrast to that in the proteins mentioned above. The purified meningococcal LPS was pyrogenic in rabbits at 1 ng/kg.     

Cry2Ab蛋白对龟纹瓢虫的安全性研究

【目的】转基因作物对非靶标昆虫的影响是转基因作物环境安全评价的重要内容,研究Cry2Ab蛋白对龟纹瓢虫的影响,对转基因作物的环境安全评价具有重要意义。【方法】采用实验动物学、分子生物学等方法,研究Cry2Ab蛋白对龟纹瓢虫发育历期、成虫体重、雌雄比例及体内氨基酸种类和含量的影响。【结果】与蔗糖对照组相比,Cry2Ab蛋白对龟纹瓢虫不同龄期的发育历期、成虫体重和雌雄比例均无明显差异,对体内氨基酸种类和含量也没有显著差异。【结论】Cry2Ab蛋白对龟纹瓢虫的生长发育及代谢无显著影响。     

DNA ploidy and pS2 protein expression in breast cancer

The DNA content of ductal breast carcinomas of varying histological grade was measured using static image cytometry and correlated with pS₂ expression in the tumour cells. Our study was performed on imprint of surgical biopsies of 60 women with ductal breast cancer. A statistically significant difference was observed between pS₂⁺ expression and grade of malignancy ( P <0.001). The percentage of euploid tumours significantly decreased from grade I to grade II to grade III ( P =0.01). The percentage of aneuploid tumours increased from pS₂⁺ to pS₂⁻ breast tumours ( P <0.001). These findings may be indicative of pS₂ and DNA ploidy alterations and tumour aggressiveness.     

A novel approach for assessing protein synthesis in channel catfish, Ictalurus punctatus

A comprehensive understanding of animal growth requires adequate knowledge of protein synthesis (PS), which in fish, has traditionally been determined by the flooding dose method. However, this procedure is limited to short-term assessments and may not accurately describe fish growth over extended periods of time. Since deuterium oxide (²H₂O) has been used to non-invasively quantify PS in mammals over short- and long-term periods, we aimed at determining if ²H₂O could also be used to measure PS in channel catfish. Fish were stocked in a 40-L aquarium with ~ 4% ²H₂O and sampled at 4, 8 and 24 h (n = 6 at each time period) to determine ²H-labeling of body water (plasma), as well as protein-free and protein-bound ²H-labeled alanine. The labeling of body water reflected that of aquarium water and the labeling of protein-free alanine remained constant over 24 h and was ~ 3.8 times greater than that of body water. By measuring ²H-labeled alanine incorporation after 24 h of ²H₂O exposure we were able to calculate a rate of PS: 0.04 ± 0.01% h^− 1. These results demonstrate that PS in fish can be effectively measured using ²H₂O and, because this method yields integrative measures of PS, is relatively inexpensive and accounts for perturbations such as feeding, it is a novel and practical assessment option.     

The effect of DTT in protein preparations for proteomic analysis: Removal of a highly abundant plant enzyme, ribulose bisphosphate carboxylase/oxygenase

Rubisco is a major photosynthetic plant enzyme in the chloroplasts, catalyzing a photosynthetic reaction through carboxylation and oxygenation in the leaves. Despite its biological importance, its high abundance causes difficulties in the proper separation of protein mixtures during 2-dimensional gel electrophoresis (2-DE). Here, we resolved those plant soluble proteins by efficiently removing Rubisco. This resulted in a high quality and resolution of 2-DE gels. Rubisco removal was achieved through aggregation in the presence of a high DTT concentration, which subsequently increased the visualization of less abundant proteins and reduced horizontal streaking. This simple method may provide a means for finding more biologically important protein targets via plant proteomics.     

Expression of the pyranose 2-oxidase from Trametes pubescens in Escherichia coli and characterization of the recombinant enzyme

cDNA-encoding pyranose 2-oxidase (P2O) from Trametes pubescens was sequenced and cloned into Escherichia coli strain BL21/DE3 on a multicopy plasmid under the control of trc promoter. The synthesis of P2O was studied in a batch culture in M9-based mineral medium: the enzyme was synthesized constitutively at 28 °C in amount corresponding to 8% of the cell soluble protein (0.6 U mg⁻¹). Only small portion of P2O (11%) was in the form of non-active inclusion bodies. Purified recombinant enzyme has similar physico-chemical and kinetic parameters with other P2Os. When compared to the expression of p2o of Trametes ochracea, a ratio of the mature enzyme to inclusion bodies found in the same E. coli host at 28 °C is as much as nine times higher. The finding makes the enzyme from T. pubescens preferable for the large-scale production by recombinant bacteria. The difference in amino acid sequences of the P2O from T. ochracea and T. pubescens may explain the favourable trait of the latter enzyme regarding protein folding.     

高粱高亲和硝酸盐转运蛋白NRT2/3基因家族鉴定、表达与DNA变异分析

硝态氮是作物吸收无机氮素的主要形态,硝酸盐转运蛋白2(nitrate transporter 2,NRT2)作为高亲和性的转运蛋白,以硝酸盐作为特异性底物,在可利用的硝酸盐受限时,高亲和性转运系统被激活,在硝酸盐吸收、转运过程中发挥着重要作用。大多数NRT2不能单独转运硝酸盐,需在硝酸盐同化相关蛋白2(nitrate assimilation related protein 2,NAR2)的协助下才能完成硝酸盐的吸收或转运。作物氮利用效率受环境条件影响,品种间存在差异,因此培育高氮素利用效率品种有重大意义。高粱(Sorghum bicolor)具有耐贫瘠特性,对土壤中的氮素吸收和利用效率较高。本研究结合高粱基因组数据库对NRT2/3基因家族成员基因结构、染色体定位、理化性质、二级结构与跨膜结构域、信号肽与亚细胞定位、启动子区顺式作用元件、系统进化、单核苷酸多态性(single nucleotide polymorphism,SNP)的识别与注释及选择压力进行了全面分析。通过生物信息学分析,筛选出5个NRT2s(命名为SbNRT2-1a、2-1b、SbNRT2-2–4)基因和2个NAR2s(SbNRT3-1–2)基因,较谷子略少。分布在3条染色体上,分为4个亚家族,同一亚族中基因结构高度相似;高粱NRT2/3亲水性平均值均为正值,表明均为疏水性蛋白;α-螺旋和无规则卷曲占二级结构总量的比例大于70%;亚细胞定位均在质膜上,其中NRT2s蛋白不含信号肽,NRT3s蛋白含信号肽;进一步对其跨膜结构域进行分析,发现NRT2s家族成员跨膜结构域个数均大于10个,而NRT3s家族成员跨膜结构域个数为2个;高粱与玉米(Zea mays)NRT2/3s的共线性较好;蛋白结构域显示存在MFS_1和NAR2蛋白结构域,可执行高亲和力硝酸盐转运;系统进化树分析可知,高粱与玉米和谷子的NRT2/3基因亲缘关系更近;基因启动子顺式作用元件分析发现,SbNRT2/3基因的启动子区均具有数个植物激素和逆境应答元件,可以响应高粱生长和环境变化;基因表达热图显示低氮条件下在根诱导表达的是SbNRT2-1a、SbNRT2-1b和SbNRT3-1,推测可在高粱根部表达并调控对硝酸盐的吸收或转运过程。在SbNRT2-4和SbNRT2-1a等发现多个非同义SNP变异;选择压力分析表明,高粱NRT2/3基因家族在进化过程中受纯化选择作用。SbNRT2/3基因表达及蚜虫侵染影响与基因在不同组织中的表达分析结果一致,SbNRT2-1b和SbNRT3-1在感染蚜虫品系5-27sug根部表达显著,高粱蚜虫侵染叶片显著降低了SbNRT2-3、SbNRT2-4和SbNRT3-2的表达水平。本研究初步对高粱全基因组NRT2/3基因家族进行鉴定、表达与DNA变异分析,为高粱氮高效研究提供了基础。     

Evidence for Ca-dependent protein phosphorylation in vitro in guard cells from Vicia faba L.

Protein phosphorylation in vitro was investigated in guard cells from Vicia faba. A number of proteins with apparent molecular masses of 72, 67, 57, 52, 49, 44, 37, and 26 kDa were phosphorylated when guard-cell extract was incubated with [γ-³²P]ATP under Ca²⁺-free conditions. In the presence of Ca²⁺ at 1 μM, several proteins with apparent molecular masses of 125, 83, 41, 31, and 25 kDa were newly phosphorylated. These Ca²⁺-dependent protein phosphorylations were suppressed by (8R^*,9S^*,11S^*)-(−)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a- triazadibenzo[a,g]cycloocta[cde]trinden-1-one (K-252a), a wide-range inhibitor of protein kinases, suggesting that the protein phosphorylations were mediated by protein kinases. Several proteins were phosphorylated in vitro in mesophyll extract from Vicia. In contrast to guard cells, there was no detectable Ca²⁺-dependent protein phosphorylation in mesophyll cells. 1-(5-Indonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-7), an inhibitor of myosin light chain kinase (MLCK), and an antagonist of calmodulin (CaM), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), inhibited Ca²⁺-dependent phosphorylation of 41- and 25-kDa proteins in guard cells. Fractionation experiments revealed that the Ca²⁺-dependent phosphorylated proteins with molecular masses of 41 and 25 kDa were present in the mitochondria, and the 125- and 31-kDa proteins in the cytosol. These results suggest that Ca²⁺-dependent protein phosphorylation occurs markedly in guard cells, and that Ca²⁺-dependent phosphorylation of 41- and 25-kDa proteins may be catalyzed by MLCK or MLCK-like protein kinase in guard cells.     

High-level expression of soluble human β-defensin-2 in Escherichia coli

Human β-defensin-2 (hBD2) is a short cationic peptide with a broad antimicrobial spectrum. The coding sequence of hBD2 was cloned into pET-32a (+) to construct a fusion expression plasmid, pET32–hBD2, which was transformed into E. coli BL21 (DE3) for expression. The cultivation parameters of the expression vector harboring strain were optimized to produce the fusion protein in soluble form efficiently and to avoid the formation of insoluble inclusion bodies. The optimal conditions were determined as following: cultivation at 28 °C in MBL medium, induction at middle stage of exponential growth with 0.8 mM IPTG, and post-induction expression for 8 h. Under the above conditions, a high percentage of the target fusion protein (≥92.3%) was expressed in soluble form and the volumetric productivity of soluble fusion protein reached 1.3 g/l. The culture process was successfully scaled up in a 10 l bench-top fermentor.     

A ribosomal protein from Thermus thermophilus is homologous to a general shock protein

The gene encoding the ribosomal protein from Thermus thermophilus, TL5, which binds to the 5S rRNA, has been cloned and sequenced. The codon usage shows a clear preference for G/C rich codons that is characteristic for many genes in thermophilic bacteria. The deduced amino acid sequence consists of 206 residues. The sequence of TL5 shows a strong similarity to a general shock protein from Bacillus subtilis, named CTC. The protein CTC is homologous in its N-terminal part to the 5S rRNA binding protein, L25, from E coli. An alignment of the TL5, CTC and L25 sequences displays a number of residues that are totally conserved. No clear sequence similarity was found between TL5 and other proteins which are known to bind to 5S rRNA. The evolutionary relationship of a heat shock protein in mesophiles and a ribosomal protein in thermophilic bacteria as well as a possible role of TL5 in the ribosome are discussed.     

板栗疫病菌致病性机理的双向凝胶电泳法研究

双向凝胶电泳技术是蛋白质组学研究的基础性技术平台。如何得到一张高质量的双向凝胶电泳图谱是进行后续研究的关键。为探索适用于板栗疫病菌可溶性总蛋白的最佳提取条件,从蛋白组学角度来探索板栗疫病菌致病性机理,比较了目前在丝状真菌中常用的两种蛋白质提取方法,制备的蛋白质样品经双向凝胶电泳后,在凝胶上呈现的蛋白质斑点的丰度和分布特点。结果表明,两种方法获得的蛋白质主要集中分布在pH4~7的范围内;TCA-丙酮沉淀法得到的图谱分辨率高但是蛋白质总量很少。裂解液-TCA-丙酮沉淀法得到的蛋白质总量较大,通过cleanupkit处理后图谱分辨率可以达到差异蛋白组的要求。随机提取几个银染蛋白点用MALDI-TOFMS/MS进行分析,可以得到高质量的肽质量指纹谱。表明该样品制备方法可以满足蛋白质鉴定的要求。     

H2O2对蛇足石杉离体培养物诱变效应的研究

为了获得高产石杉碱甲(Huperzine A,Hup A)的蛇足石杉[Huperzia serrata(Thunb.)Trev.]叶状体,对H_2O_2诱变后的叶状体进行了研究。结果表明,诱变后叶状体株系的Hup A含量显著提高,并获得高产株系SH42,其相对生长率和Hup A含量分别达到4499.28%和261.17μg g~(–1) DW,比起始叶状体分别提高了2.35倍和2.43倍;且株系间可溶性蛋白质谱带和SOD同工酶谱均存在差异,经过连续9代培养,变异叶状体可以稳定遗传。因此,H_2O_2对叶状体细胞具有良好的诱变效应,可以用于筛选高产Hup A株系。     

Cloning and sequencing of the HU-2 gene of Escherichia coli

Summary The Escherichia coli HU-2 gene was cloned using a DNA fragment from the HU-1 gene as a probe. The amino acid sequence of the HU-2 protein deduced from the nucleotide sequence is in good agreement with the published sequence. The nucleotide sequence has a possible promoter and a typical ribosomal binding site upstream of the translation initiation codon (AUG) and a possible rhoindependent terminater site downstream of the termination codon (UAA) of the gene.