Serological and molecular properties of isolates of Bean common mosaic virus-BCMV from Khorasan Razavi province

During growing season, 2011–2012, selected bean plant samples with symptoms of mosaic, vein clearing, leaf rolling, were collected from different part of Khorasan Razavi province of north-eastern Iran. To identify the virus, leaf samples were tested serologically by DAS-ELISA and tissue blot, using specific BCMV polyclonal antibody. Elisa positive samples were rechecked by RT-PCR and IC-RT-PCR using set of primers directed to the coat protein gene which were designed to detect and characterise the viral species. Similarly, the BCMV primers amplified product of approximately 373?bp in 1% agarose gel electrophoresis. In phylogenic analysis, A and B groups were formed for 12 Iranian isolate comparing with 18 BCMV isolates from GenBank. Iranian isolates were classified into three clusters indicating to have more homology 99.3% with Mexican Isolates. There are also indications of some diversities among Iranian BCMV isolates.     

Proteomic identification of camel seminal plasma: Purification of β-nerve growth factor

The camel seminal plasma contains a diverse array of components including lipids, carbohydrates, peptides, ions and proteins. These are essential for maintaining normal physiology of spermatozoa and are secreted mainly from the prostrate, epidydimis and bulbo-urethral glands of reproductive system. The protein profiles of camel seminal plasma were resolved by two-dimensional gel electrophoresis (2D-PAGE). The majority of the protein was found in acidic regions below pI 7.0 and the 19 brightly stained proteins were identified by MALDI-TOF/MS analysis. On the basis of proteomic profiles, β-nerve growth factor (β-NGF) was purified by ion-exchange and gel filtration chromatography and identified by SDS-PAGE and MALDI-TOF/MS analysis. It was further confirmed by western blotting experiments using rabbit anti-β-NGF primary antibody.     

Glyceraldehyde-3-phosphate dehydrogenase is a reliable internal control in Western blot analysis of leukocyte subpopulations from children

To study differences in the development of immunity, leukocytes from cord blood are often compared with those from adult peripheral blood. Western blot analysis is a common method for detecting proteins. In this study, we investigated the reliability of using different housekeeping proteins (β-actin, β-tubulin, and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) as internal controls for different leukocyte subpopulations from infants, children, and adults. Our results showed that the expression levels of β-actin and β-tubulin were much lower in cord blood leukocytes than in adult leukocytes, and this expression pattern persisted in children up to 3 years old. Further study revealed that the β-actin expression level in newborns was especially lower in CD14-positive monocytes. However, cord blood and adult peripheral blood monocytes had similar expression levels of β-actin messenger RNA (mRNA). Further experiments showed that posttranslational regulation was responsible for the low β-actin expression level in neonatal monocytes. Thus, researchers should carefully assess the appropriate use of housekeeping gene-encoded proteins as internal standards to normalize samples for comparisons of different leukocyte populations from subjects of different ages. In this study, we determined that GAPDH was a more reliable internal control than others in Western blot analysis for comparing the development of immunity among infants, children, and adults.     

Schistosoma mansoni: molecular characterization of Alkaline Phosphatase and expression patterns across life cycle stages

Here we describe the cloning and characterization of the Schistosoma mansoni Alkaline Phosphatase (SmAP), previously identified in the tegument of adult worms. SmAP encodes a complete sequence composed of 536 amino acids containing an N-terminal signal peptide, five N-glycosylation sites, and a GPI anchor signal, similar to that described for mammalian orthologs. Real-time RT-PCR and Western blot experiments suggest a rapid translation as soon as cercariae are transformed into schistosomula. Immunolocalization analysis shows that the protein is widely distributed in the worm tissues, with increased concentration in the vitelline glands of female parasites. Furthermore, the surface localization of this enzyme was quantitatively supported by its enzymatic activity in live ex vivo or cultured parasites throughout the life cycle stages. The fact that cercariae accumulate large amounts of SmAP mRNA, which rapidly translates into protein upon schistosomula transformation, indicates it may have an important role in host invasion.     

The effect of prefeeding on fixed-ratio pausing is jointly determined by past and upcoming reinforcer magnitudes

Pausing within multiple fixed-ratio schedules differing in reinforcer magnitude is jointly controlled by both past and upcoming conditions of reinforcement. Abrupt shifts from a just-received large reinforcer to a signaled upcoming small reinforcer (i.e., a negative incentive shift) produce marked disruptions in responding, as indexed by extended pausing. The purpose of this experiment was to determine if reducing the level of food deprivation via prefeeding enhanced these disruptive effects. Five Long Evans rats lever-pressed according to a fixed-ratio schedule. Half of the components ended in a relatively large reinforcer (three 45-mg food pellets) and half ended in a relatively small reinforcer (one pellet). Components alternated irregularly, yielding four transitions between reinforcers: small-small, small-large, large-small (the negative incentive shift), and large-large. During five, 1-session prefeeding probes, rats were given 12 g of food in their home cages 1 h prior to the start of the session. Under steady-state conditions, negative incentive shifts engendered the longest pausing. Prefeeding produced large absolute and relative increases in pausing during negative incentive shifts, and small increases in pausing in the other transitions. The results are interpreted within a resistance to change framework.     

Calcium binding protein expression in the optic tectum of Alligator during development

The onset and distribution of the calcium binding proteins, calretinin, calbindin, and parvalbumin, were examined in the optic tectum of Alligator mississipiensis embryos between Stages 18 and 26–28. The immunoreactivity of each calcium binding protein correlated well with the results from the Western blot experiments. In terms of onset and distribution, calretinin expressison was the most widespread of the three calcium binding proteins that were examined, and was also the earliest to be visualized. Calbindin expression occurred next, whereas parvalbumin expression was the most limited and appeared last. For small calretinin (+) neurons, the pattern of immunoreactivity during development was from inside to outside, whereas for the larger cells, it was from outside to inside. For calbindin immunoreactive cells in the superficial zone, the pattern was from outside to inside. The distribution of the parvalbumin immunopositive neurons did not change significantly over the time period examined. Similar data on other amniotes is limited. However, the pattern in Alligator shares some similarities with kittens in regards to the distribution of calbindin and parvalbumin in the developing superior colliculus. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 899–910, 2013     

宫颈鳞状细胞癌中ADAM17 的表达及其临床意义

目的:探讨去整合素-金属蛋白酶17(ADAM17)在宫颈鳞状细胞癌中的表达及其临床病理意义。方法:运用免疫组织化学法分别检测正常宫颈上皮、宫颈鳞状细胞癌及宫颈上皮内瘤样变组织中ADAM17的表达,并分析其与宫颈癌临床分期及淋巴结转移的相关性。结果:ADAM17在正常宫颈上皮组织切片中无明显表达,在宫颈上皮内瘤样变组织中少部分表达,呈浅黄色,在宫颈鳞状细胞癌中癌细胞大量表达,ADAM17表达呈棕褐色,数量较多且浓染。不同临床分期的宫颈鳞状细胞癌组织中ADAM17的阳性表达率比较存在显著统计学差异(P0.05),且随临床分期的上升,ADAM17的表达逐渐升高;有淋巴结转移的宫颈鳞状细胞癌组织中ADAM17的阳性表达率显著高于无淋巴结转移的宫颈鳞状细胞癌组织,差异具有统计学意义(P0.05)。结论:ADAM17蛋白在宫颈鳞状细胞癌组织中呈异常高表达,且与宫颈鳞状细胞癌的临床分期和淋巴结转移密切相关,通过检测ADAM17蛋白的表达可能有助于宫颈鳞状细胞癌的诊断、治疗和预后预测。     

Immunoblot analysis of the expression of genes for barley rubisco activase inE. coli

Rubisco activity during photosynthesis is regulated by the rubisco activase, which facilitates the dissociation of RuBP and other inhibitory sugar phosphates from the active site of rubisco in an ATP-dependent reaction. In this paper, barleyRca genes (RcaA1,RcaA2 andRcaB) were expressed inE. coli and the activity of rubisco activase expressed was assayed biochemically by chromatography. Then the protein was identified electrophoretically by SDS-PAGE and detected immunologically by Western blot analysis using polyclonal antibodies raised against the kidney bean rubisco activase as probe. The band pattern of purified proteins on the polyacrylamide gel showed two polypeptides of 46 kD and 42 kD. Anti-rubisco activase antibodies reacted specifically with both polypeptides of 46 kD and 42 kD present in the crude extracts ofE. coli transformants. Therefore, it was found that the genes of barley rubisco activase was successfully expressed inE. coli as active forms of 46 kD and 42 kD.     

一个在肺腺癌细胞系中差异表达cDNA片段的定位及表达分析

为了研究和克隆肺癌转移相关候选基因,探讨肺癌发生及转移的分子基础,应用细胞培养,cDNA克隆,Noorthern印记杂交和生物信息学技术分析了在细胞来源相同,但转移能力不同的肺腺癌细胞系AGZY83－a和Anip973中差异表达片段OPB7－1在人不同组织中和不同人肺癌细胞系中的表达情况,并应用RH定位技术对该片段进行了基因定位。表明OPB7－1与已知基因同源性差,该基因在正常人多种组织中有表达,心肌和骨骼肌中高表达,转录本均为3.0kb左右。在不同人肺癌细胞系中存在该基因的表达差异,高转移潜能,低分化及高浸润的细胞系中呈高表达趋势,且表达的片段大小略有差别。提示OPB7－1是一个具有广泛表达为的基因,可能是与肺癌的发生发展相关的新基因。     

反相斑点杂交法对解脲脲原体分型的研究

目的研究以聚合酶链反应为基础的快速检测与鉴定解脲脲原体基因型的方法。方法选择2003年11月至2005年11月在中山大学附属第二医院门诊就诊的有外阴阴道炎症状和体征的患者601例,设为病例组,同期无自觉症状的正常体检人群306例,设为对照组,分别取宫颈分泌物待检测。将解脲脲原体不同基因型的特异探针固定在硝酸纤维素膜上,临床标本按常规方法提取解脲脲原体DNA,采用生物素标记的解脲脲原体特异通用引物PCR扩增DNA,然后分别与解脲脲原体不同基因型特异探针杂交、显色。结果病例组解脲脲原体阳性421例占70.0%,对照组解脲脲原体阳性126例占41.2%。病例组中单型别感染的U.parvum占65.4%,其中1型、3型、6型和14型分别占28.8%、43.3%、26.0%和1.9%,U.urealyticum占18.4%;对照组中单型别感染的U.parvum占79.3%,其中1型、3型、6型和14型分别占63.2%、21.1%、15.7%和0.0%,U.urealyticum占13.8%。18例阳性标本随机DNA测序鉴定,均为相应的解脲脲原体基因型。结论U.parvum群,尤其是其中的1、3、6型别是正常人群携带的可能性较大,U.urealyticum则有可能和1型起协同作用或独自导致疾病。用反相斑点杂交进行解脲脲原体基因分型,方法简单、实用,适用于临床。