一种新型免疫层析技术在临床乳腺癌Her-2蛋白定量检测中的应用

建立一种靶点蛋白质快速定量检测方法。在原有侧向流动免疫层析技术的基础上,通过优化层析材料和纳米微球的均一性、改进检测区的检测方法,经逐点扫描技术,建立标准浓度曲线,以达到对临床靶点蛋白质的定量检测。以乳腺癌组织中的Her2表达为例,通过对已知浓度样品的检测,验证本技术方法的准确度大于96%。另外,以蛋白质免疫印迹作为组织中特定蛋白质检测金标准,分析临床肿瘤组织中Her2蛋白的含量,其准确率也达到95.5%,而免疫组织化学方法检测准确率仅为69.58%。新型免疫层析法检测结果与靶向治疗患者的愈后密切相关(P<0.01)。改进后的新型免疫层析方法能够准确地对临床靶点蛋白质进行定量检测,而且结合侧向流动技术的简单、快速和易用性,这种新型检测方法可以广泛应用于临床组织标本、血液标本和体液标本中靶点蛋白质的临场定量检测,在一定程度上可以替代免疫组化技术。     

De novo organogenesis and plant regeneration in <Emphasis Type="Italic">Pongamia pinnata</Emphasis>, oil producing tree legume

Role of Thidiazuron (TDZ) in inducing adventitious organogenesis in Pongamia was studied. TDZ at different concentrations (0, 0.45, 2.27, 4.54, 6.71, 9.08, 11.35, 13.12 and 22.71 μM) were used for induction of caulogenic bud formation in deembryonated cotyledon explants. Each cotyledon was cut into three segments and identified as proximal, middle and distal. Duration of TDZ exposure, influence of the segment and orientation of the explant were studied. TDZ at 11.35 μM concentration was optimum for the induction of shoots and rapid elongation. Shoots induced at higher concentration elongated after several passages in growth regulator free medium, thereby extending the period of differentiation. Exposure of the explant for 20 days yielded more number of buds than 10 days. Proximal segment of the cotyledon was more responsive. Contact of abaxial surface in the medium was more effective and generated more buds than the adaxial side. Buds differentiated and elongated on transfer to MS basal medium for 8–12 passages of 15 days each. Rooting and elongation of shoots was achieved in charcoal supplemented half-strength MS medium. Rooted plantlets survived on transfer to sand soil mixture. The plants were hardened and transferred to green house. This is the first report on in vitro regeneration of Pongamia pinnata via adventitious organogenesis using TDZ. This protocol may find application in studies in genetic transformation, isolation of somaclonal variants and in induction of mutants. It also provides a system to study the inhibitory role of TDZ on shoot differentiation.     

多西紫杉醇修饰的人工晶体与眼组织相容性分析

目的：探讨经多西紫杉醇修饰的人工晶体对眼组织相容性的影响。方法：按照随机数字表法将32 只日本大耳兔分为两组： 实验组通过手术植入表面经多西紫杉醇修饰处理后的疏水性人工晶体，对照组植入疏水性人工晶体。比较两组人工晶体亲水角、 术后24 小时光耀斑块计数以及人工晶体周围组织炎症浸润数。结果：实验组的亲水角小于对照组，差异有统计学意义（P<0.05）。 实验组光耀斑块计数低于对照组，差异有统计学意义（P<0.05）。实验组家兔人工晶体周围组织炎症浸润计数低于对照组，差异有 统计学意义（P<0.05）。结论：人工晶体表面经多西紫杉醇修饰后，其亲水性、与眼组织的组织相容性增加，且可缓解光耀斑炎症感 染和降低并发症的发生，有重要的临床参考价值。     

Nitrate induced iron deficiency chlorosis in Juncus acutiflorus

Chlorosis caused by iron deficiency is commonly associated with high bicarbonate levels in the soil. However, in rare cases such chlorosis has been observed in soils with high nitrate levels. In a dutch rich-fen, chlorosis has been noted in stands of Juncus acutiflorus at locations where groundwater containing high levels of nitrate reached the surface. Experiments revealed that the chlorosis could be attributed to iron deficiency although iron levels in the shoots were well above the known physiological threshold values for iron deficiency. It is postulated that increased nitrate assimilation leads to an increased apoplastic pH and to a concomitant immobilisation of iron and/or lower iron (III) reduction. Moreover free amino acid levels were markedly higher in the iron deficient plants in the field. It was found, however, that the percentage of nitrogen present as free amino acids was not influenced directly by low iron levels but mainly by the C/N ratios in the shoots. Nowadays, nitrate concentrations in ground water as high 1000 µM are no longer an exception in the Netherlands. We propose that strongly increased nitrate inputs may cause iron stress in natural vegetations, especially in wet habitats.     

Direct somatic embryogenesis from axes of mature peanut embryos

Summary Plant regeneration via somatic embryogenesis was obtained in peanut (Arachis hypogaea L.) from axes of mature zygotic embryos. The area of greatest embryogenic activity was a 2-mm region adjacent to and encircling the epicotyl. Somatic embryogenesis was evaluated on Murashige and Skoog media supplemented with a variety of auxin treatments. Maximum production occurred on medium supplemented with 3 mg · liter⁻¹ 4-amino-3,5,6-trichloropicolinic acid. Explant cultures were transferred to half-strength medium supplemented with 1 mg · liter⁻¹ gibberellic acid for somatic embryo germination and early plantlet growth. Plantlets, transferred to soil, were placed in a greenhouse and grown to maturity.     

Epigenetic silencing of TIMP4 in heart failure

Tissue inhibitor of matrix metalloprotease 4 (TIMP4) is endogenously one of the key modulators of matrix metalloprotease 9 (MMP9) and we have reported earlier that cardiac specific TIMP4 instigates contractility and helps in differentiation of cardiac progenitor cells. Although studies show that the expression of TIMP4 goes down in heart failure but the mechanism is unknown. This study aims to determine the mechanism of silencing of TIMP4 in heart failure progression created by aorta‐vena cava (AV) fistula. We hypothesize that there is epigenetic silencing of TIMP4 in heart failure. To validate this hypothesis, we created heart failure model by creating AV fistula in C57BL/6 mice and looked into the promoter methylation (methylation specific PCR, high resolution melting, methylation sensitive restriction enzyme and Na bisulphite treatment followed by sequencing), histone modification (ChIP assay) and microRNAs that regulate TIMP4 (mir122a) and MMP9 (mir29b and mir455‐5p). The physiological parameters in terms of cardiac function after AV fistula were assessed by echocardiography. We observed that there are 7 CpG islands in the TIMP4 promoter which get methylated during the progression of heart failure which leads to its epigenetic silencing. In addition, the up‐regulated levels of mir122a in part, contribute to regulation of TIMP4. Consequently, MMP9 gets up‐regulated and leads to cardiac remodeling. This is a novel report to explain the epigenetic silencing of TIMP4 in heart failure.     

Study of thiol proteases of normal human skin fibroblasts

The protease activity of cultured normal human skin fibroblasts was studied using the synthetic fluorigenic peptides, the modified protein 4-methylumbelliferyl-casein, the thiol inhibitors and the affinity for concanavalin A-Sepharose. The majority of the activity to N-benzyloxycarbonyl-L-phenylalanyl-L-arginyl-7-amido-4-methyl-coumarin and N-a-benzyloxycarbonyl-L-arginyl-arginyl-7-amido-4-methylcoumarin had a pH optimum of 6.0, and was thiol-dependent and inhibited by leupeptin and antipain. The activity toward N-benzyloxycarbonyl-L-phenylalanyl-L-arginyl-7-amido-4-methylcoumarin represents both cathepsin B and cathepsin L, whereas the activity towards 4-methylumbelliferyl-casein represent only cathepsin L. Cathepsin H could not be detected when assayed with L-arginine-7-amido-4-methylcoumarin substrate. Cathepsin D was present in comparatively small amounts when assayed with 4-methylumbelliferyl-casein. Activity towards 4-methylumbelliferyl-casein had pH optima at 3 and 6 and was stimulated by dithiothreitol. A proportion of the activity at pH 6.0 was not dependent on thiols and not inhibited by leupeptin, and had the general characteristics of a carboxyl proteinase. Over 70 per cent of the activity was in the lysosomal fraction and showed structure-linked latency. All the detectable protein emerged from the immobilized concanavalin A column and the fractions eluted by alpha-methyl-D-mannoside were significantly hydrolysed the synthetic peptides. Only that fraction which bound to concanavalin A was active towards 4-methylumbelliferyl-casein. Cathepsin B had no affinity for concanavalin A-Sepharose due to the absence of glycoprotein content, unlike cathepsin L which showed a strong affinity for concanavalin A-Sepharose.     

A mannose-specific lectin from Vicia villosa seeds

A lectin specific to mannose has been purified from Vicia villosa seed by (NH₄)₂SO₄ fractionation, GalNAc-Sepharose and Man-Sepharose affinity chromatography. It was defined as VVL_M, which showed a single band on an acidic-PAGE stained with Coosmassie brilliant blue. The molecular weight of VVL_M was 50 kDa as determined by gel filtration on Biogel P-100 column. The VVL_M molecule consists of 2 distinct subunits with apparent molecular weight of 30 kDa and 22kDa determined by SDS-PAGE. VVL_M has at least four isolectins with similar haemagglutinating activity. Its extinction coefficient is calculated as A_1cm¹ = 16.4 at 280 nm. Sugars could not be detected phenol-sulfuric acid method. The circular dichroism analysis at far UV indicated that VVL_M was a β-sheet-rich protein, and gave no α-helix, 69% β-sheet, 14% β-turn by Provencher and Glockner method. The lectin was inhibited by α-methyl-d-mannose at 12.5 mM and glucose or GlcNAc at 50 mM. The carbohydrate binding specificity of VVL_M was investigated by using affinity chromatography on a VVL_M-Sepharose column. Among various Asn-linked oligosaccharides, core structure Manα1→3(Manα1→6)Manβ1→4GlcNAcβ1→4GlcNAc_OT were found to have high affinity for VVL_M-Sepharose. The antisera of VVL_M did not produce precipitin line with VVL_G in agar double diffusion plate indicating so serological relationship between VVL_M and VVL_G. However VVL_M showed similar immunodeterminants of some other lectins of mannose specificity such as Con A, PSL, LCA and VFL.