一种快速简易的苏芸金杆菌变种鉴别法——菌落碘色反应法

一般认为,用血清学方法鉴定苏芸金杆菌变种的方法较好,但也要参考生理生化、酯酶型等方法。我们用菌落碘色反应法经过几年的反复试验表明,对苏芸金杆菌变种来说,该法可做为用其它方法鉴定前的初始鉴别方法。     

Neurospora的一种新的、快速而有效的转化方法

叙述了一种新、快速、有效而可靠的转化Neurospora crassa的方法。本方法用醋酸锂处理萌发的分生孢子,再同DNA保温,然后用聚乙二醇(PEG)处理,然后短时间 热冲击,最后铺选择平板测定。报道了得到高转化率的最适条件。用环状和线状质粒DNA以及基因组DNA都可以得到转化。用相对不纯的DNA制备物如用快速微量制备方法得到的DNA也能进行转化,尽管转化率大大降低。这种转化方法简单而可靠,可大大节省时间和材料。     

小鼠近端肾小管上皮细胞原代培养及鉴定

本文旨在建立更有效的小鼠肾小管上皮细胞体外培养及鉴定方法。将小鼠肾脏皮髓质分离,取肾皮质将其充分剪碎,用II型胶原酶消化结合筛网过滤的方法获得小鼠近端肾小管上皮细胞,细胞培养在DMEM中。用倒置显微镜观察细胞形态及生长情况,用流式细胞仪检测细胞增殖能力,用CCK-8检测方法测定活力,用免疫荧光方法鉴定肾小管上皮细胞的纯度。结果显示,免疫荧光鉴定显示95%以上的细胞表达上皮细胞标志蛋白CK18,90%以上的细胞表达近端肾小管上皮细胞标志蛋白Villin、AQP1和SGLT2。细胞可传至第五代,随着传代次数增加,细胞增殖能力逐渐降低。以上结果提示,本研究成功建立了培养高纯度小鼠肾小管上皮细胞的方法,用这一改良方法获得的肾小管上皮细胞可用于后续的离体实验研究。     

关于统计学符号的书写方法

1 书写方法 :①样本的算术平均数用英文小写 ｘ表示 ,不用大写 Ｘ表示 ,也不同Ｍｅａｎ或Ｍ (中位数仍用Ｍ) ;②标准差用英文小写ｓ,不用ＳＤ ;③标准误用英文小写ｓ ｘ ,不用ＳＥ ,也不用ＳＥＭ ;④ｔ检验用英文小写ｔ;⑤Ｆ检验用英文大写Ｆ ;⑥卡方检验用希文小写 χ2 ;⑦相关系数用英文小写ｒ;⑧自由度用希文小写υ (钮 ) ;⑨样本数用英文小写ｎ ;⑩概率用英文大写Ｐ ; 以上符号均用斜体。2 统计学处理必须标明检验方法 ,对结论性指标最好给出具体的检验值 ,如ｔ值、χ2 值、Ｑ值等 ,然后列出Ｐ值。3 关于“差异”的描述方法 :使用…     

“根系分布具有向水性”的证明实验

初中《植物学》课本中用在沙箱中培养幼苗的方法,证明根系向水生长。我在教学中改用了用试管培养幼苗的方法,结果证明这个方法简单易行,现象明显,每个学生都可以动手。     

用聚合酶链式反应（PCR）检测马铃薯纺锤块茎类病毒

用DNA合成仪合成两个马铃薯纺锤块茎类病毒(Potato spindle tuber viroid, PSTVd)特异性引物,从感病的马铃薯块茎组织的核酸抽提液中,用反转录酶合成PSTVd eDNA,然后用PCR法进行扩增,扩增产物用电泳检测,建立了用PCR法检测PSTVd的新方法。结果表明,该方法特异性强,灵敏度可达0.15pg,比现有其它检测方法高,而且样品用量少。     

从松针中提取混合氨基酸方法研究

本文主要是讨论从马尾松（Pinus mossoniana Lamb)针叶中提取混合基酸的方法。研究结果表明,用方法I和方法Ⅱ从马尾松针叶中提取18种混合氨基酸,平均收率为10％－14％;用方法Ⅱ从马尾松针叶中提取18种氨基酸的总量是用方法1从同量松针叶中提取18种氨基酸的总量的1．5倍以上。     

核磁共振氢谱法在芹亚科植物化学分类中的简便应用

用核磁共振红谱（1H－NMR）方法对芹亚科14种植物的香豆素类成分进行了检测,然后用常规植物化学方法加以验证。结果表明,1H-NMR方法可以简便应用于芹亚科植物的化学分类。     

亚洲玉米螟产卵量增长曲线的一种新的拟合方法

本文用多项式方法与微分理论,提出逻辑斯蒂曲线的拟合新方法。应用这个方法建立了亚洲玉米螟产卵量动态模型,并阐述了计算参数方法。结果与其它方法相比,其优点是计算方便,而且拟合程度精确。用新方程作为预报虫害,可以提高预报效果。     

用热变性温度法测定细菌DNA中GC含量

用热变性温度法测定细菌DNA中GC含量,是一种重复性高,仪器较易解决,操作较简便的方法。本文介绍此方法中所用仪器的改装,测定操作过程和应注意事项。     

Identification of the domains required for direct interaction of the helicase-like and polymerase-like RNA replication proteins of brome mosaic virus.

Brome mosaic virus is a positive-strand RNA virus whose RNA replication requires viral protein 1a, which has putative helicase and capping functions, and 2a, which has putative polymerase function. Since domains of related sequence are conserved in a wide range of plus-strand RNA viruses, analysis of 1a and 2a function should have applicability to many other viruses. We have recently demonstrated that 1a and 2a form a complex in vivo and in vitro. Using immune coprecipitation and mutant polypeptides made in reticulocyte lysates, we have now mapped both the 1a and 2a domains necessary for complex formation. The sequences needed to bind 2a map to the carboxy-terminal helicase-like domain of 1a. Truncated polypeptides containing this domain were able to bind to 2a, while several small insertions in the helicase-like domain disrupted binding. The sequence required for binding 1a lies within a 115-residue subset of the 2a N-terminal segment preceding the polymerase-like domain. Truncations or fusion polypeptides containing this segment can bind 1a. We also determined that highly purified 2a protein made in insect cells can form a complex with highly purified 1a helicase-like domain made in Escherichia coli, suggesting that no other factor is required to mediate 1a-2a interaction. Previous genetic analyses of 1a and 2a are consistent with this mapping and show that the newly defined 1a and 2a binding regions are required for RNA synthesis. The locations of these interacting regions are discussed with regard to models of viral replication and the evolution of positive-strand RNA virus genomes.     

The polymerase-like core of brome mosaic virus 2a protein, lacking a region interacting with viral 1a protein in vitro, maintains activity and 1a selectivity in RNA replication.

Brome mosaic virus (BMV), a member of the alphavirus-like super-family of positive-strand RNA viruses, encodes two proteins required for viral RNA replication: 1a and 2a. 1a contains m7G methyltransferase- and helicase-like domains, while 2a contains a polymerase (pol)-like core flanked by N- and C-terminal extensions. Genetic studies show that BMV RNA replication requires 1a-2a compatibility implying direct or indirect 1a-2a interaction in vivo. In vitro, la interacts with the N-terminal 125-amino-acid segment of 2a preceding the pol-like core, and prior deletion studies suggested that this 2a segment was essential for RNA replication. We have now used protein fusions and deletions to explore possible parallels between noncovalent 1a-2a interaction and covalent fusion of similar protein domains in tobacco mosaic virus and to see whether the N-terminal 2a-1a interaction was the primary basis for 1a-2a compatibility in vivo. We found that 2a can function as part of a tobacco mosaic virus-like 1a-2a fusion and that a 2a segment (amino acids 162 to 697) comprising the pol-like core was sufficient to provide 2a functions in such a fusion. Unexpectedly, the unfused 2a core segment also supported RNA replication when it and wild-type la were expressed as separate proteins. Moreover, in gene reassortant experiments with the related cowpea chlorotic mottle virus, the unfused 2a core segment showed the same 1a compatibility requirements as did wild-type BMV 2a. Thus, the pol-like core of 2a must interact with la in a way that is selective and essential for RNA synthesis, and 1a-2a interactions are more complex than the single, previously mapped interaction of the N-terminal 2a segment with 1a.     

Effects of Wnt proteins on cell proliferation and apoptosis in HEK293 cells

Wnt proteins and Wnt signalings have been implicated in a variety of development and cell processes, while aberrant activation of Wnt signaling is linked to a range of cancers in many tissues. In this study, we used the HEK293 cell line to investigate the effects of Wnt3a and Wnt5a on proliferation and apoptosis in a serum starvation culture. After Wnt3a and Wnt5a proteins were expressed, they both promoted the proliferation of HEK293 cells under serum starvation. After 48h of serum starvation, both Wnt3a and Wnt5a inhibited serum starvation-induced apoptosis of HEK293 cells and continued up to 96h. We demonstrated that Wnt3a and Wnt5a can promote proliferation of HEK293 cells and inhibit serum starvation-induced apoptosis, which implies that Wnt3a and Wnt5a can maintain the survival of HEK293 cells under stress, and also provide a novel insight into the role of Wnt3a and Wnt5a and their related signalings in carcinogenesis.     

A quantitative assay for the non-covalent association between apolipoprotein[a] and apolipoprotein B: an alternative measure of Lp[a] assembly

Increasing evidence suggests that the assembly of lipoprotein[a] (Lp[a]) proceeds in two steps. In the first step, non-covalent interactions between apolipoprotein[a] (apo[a]) and apolipoprotein B (apoB) of low density lipoprotein (LDL) form a dissociable apo[a]:LDL complex. In the second step, a covalent disulfide linkage forms the stable Lp[a] particle. Several methods are currently used to study the assembly of Lp[a], however, these methods are laborious, time-consuming, and not suitable for a high throughput screening. We report here the development of a rapid and simple assay based on the binding of labeled LDL to a Lp[a]/apo[a] substrate which is immobilized on the surface of a microtiter plate. Quantification of bound LDL provides a measure of the extent of complex formation. Labeled LDL bound to both Lp[a] and apo[a] substrates with similar affinity. Plasma lipoproteins containing apoB as well as free apo[a] were capable of competing with LDL binding. The binding of LDL to Lp[a]/apo[a] was inhibited by L-proline and lysine analogs, which are known to inhibit the non-covalent association between apo[a] and apoB. Using this method we have found that nicotinic acid and captopril are able to inhibit the association of apo[a] with apoB. This method is compatible with automation and can be applied to a high throughput screening of inhibitors of Lp[a] formation.     

Molecular cloning and expression of the col2a1a and col2a1b genes in the medaka, Oryzias latipes

The Col2a1 gene is expressed in notochord, otic vesicle, cartilaginous tissue and the anlage of endochondral bone during development in higher vertebrates. Type II collagen, a homotrimeric product of the Col2a1 gene, functions as a key regulatory protein for cartilage development and endochondral ossification. In medaka and zebrafish, a single homolog of the col2a1 gene has been identified. However, it is necessary to note that many genes are duplicated in teleost fishes. To clarify function of col2a1 genes in teleost fishes and to further understand the process of cartilage development and endochondral ossification, we cloned and mapped the gene loci of two col2a1 orthologs in medaka. The proteins encoded by both medaka col2a1a and col2a1b genes were highly conserved (85.3% and 82.6%) relative to human COL2A1, but synteny was not observed. We also examined the expression patterns of col2a1a and col2a1b during embryonic development. Whole-mount insitu hybridization data suggests that expression patterns of both medaka co2a1a and col2a1b genes are similar to that of zebrafish co2a1 in the early embryonic stages. In medaka, the two col2a1 genes show a closely correlated pattern of spatial and temporal expression. In late embryonic stages, however, there were differences in both expression patterns in the pectoral fin. This study is the first report of two homologs of col2a1 in teleosts and also the first examination of col2a1a and col2a1b expression patterns in this group.     

Structure and protein composition of the bacteriophage T2L connector]

Methods of isolating structural bacteriophage T2 fragments containing: 1. a fragment consisting of a connector, tail tube and contracted sheath; 2. a fragment consisting of a free head, a connector and contracted sheath; 3. a fraction of some free tail tube and some free connectors; 4. a fraction of some free tail, free connectors and free fibers. The following parameters of connector consisting from a neck and a sleeve, which in its turn consists of a cap and a leg, are determined by means of electrone microscopy: 1) the length and the diameter of a cap and a sleeve being 45 and 145 A respectively; 2) the length and the diameter of a sleeve leg being 45 and 85 A respectively; 3) the length and the diameter of a connector neck being 85 and 70 A respectively. Polyacrylamide gel electrophoresis revealed in connectors proteins having molecular weight of 14 000, 15 000, 26 000 and 35 000 daltons.     

Identification of the 7-hydroxymethyl chlorophyll a reductase of the chlorophyll cycle in Arabidopsis

The interconversion of chlorophyll a and chlorophyll b, referred to as the chlorophyll cycle, plays a crucial role in the processes of greening, acclimation to light intensity, and senescence. The chlorophyll cycle consists of three reactions: the conversions of chlorophyll a to chlorophyll b by chlorophyllide a oxygenase, chlorophyll b to 7-hydroxymethyl chlorophyll a by chlorophyll b reductase, and 7-hydroxymethyl chlorophyll a to chlorophyll a by 7-hydroxymethyl chlorophyll a reductase. We identified 7-hydroxymethyl chlorophyll a reductase, which is the last remaining unidentified enzyme of the chlorophyll cycle, from Arabidopsis thaliana by genetic and biochemical methods. Recombinant 7-hydroxymethyl chlorophyll a reductase converted 7-hydroxymethyl chlorophyll a to chlorophyll a using ferredoxin. Both sequence and biochemical analyses showed that 7-hydroxymethyl chlorophyll a reductase contains flavin adenine dinucleotide and an iron-sulfur center. In addition, a phylogenetic analysis elucidated the evolution of 7-hydroxymethyl chlorophyll a reductase from divinyl chlorophyllide vinyl reductase. A mutant lacking 7-hydroxymethyl chlorophyll a reductase was found to accumulate 7-hydroxymethyl chlorophyll a and pheophorbide a. Furthermore, this accumulation of pheophorbide a in the mutant was rescued by the inactivation of the chlorophyll b reductase gene. The downregulation of pheophorbide a oxygenase activity is discussed in relation to 7-hydroxymethyl chlorophyll a accumulation.     

Analysis of the binding of fluorescent C5a and C3a to human peripheral blood leukocytes

Fluorescein-labeled human C5a and C3a were prepared and utilized to analyze the binding of C5a and C3a to human neutrophils and mononuclear cells. The fluorescein derivatives of C5a (Fl-C5a) and C3a (Fl-C3a) contained approximately one fluorescein molecule per molecule of protein. Fl-C5a retained biologic activity as determined by neutrophil O2- production, enzyme release, receptor binding, and reaction with rabbit anti-C5a antibody. Fl-C3a was biologically active as measured by contraction of guinea pig ileal strips, and maintained 87% of its antigenic character when reacted with rabbit anti-human C3a. The binding of Fl-C5a and Fl-C3a to human neutrophils and mononuclear cells was assessed with the use of flow cytometry. Fl-C5a bound to greater than 90% of neutrophils, with an average ED50 ranging from 2.8 to 6.8 nM, depending on the method of analysis. Fl-C5a binding to neutrophils was specific and was not inhibited by the presence of formyl-methionyl-leucyl-phenylalanine (f-MLP), C3a, or casein. Fl-C5a binding was totally blocked by an excess of C5a. C5a des arg partially inhibited the binding of Fl-C5a to neutrophils, but was 1000-fold less effective than C5a. Similar experiments with mononuclear cells showed that Fl-C5a was bound by monocytes but not by lymphocytes. Fl-C5a binding to monocytes was blocked totally by C5a but not by C3a or f-MLP. Comparative binding studies with neutrophils, monocytes, and lymphocytes showed that Fl-C5a was bound by an average of 93% +/- 4 of neutrophils, 68% +/- 9 of monocytes, and 6% +/- 3 of lymphocytes. Fl-C3a did not show significant binding to neutrophils, monocytes, or lymphocytes. These studies demonstrate that fluorescein derivatives of C5a and C3a can be prepared with retention of biologic activity, and provide a means to evaluate the binding of C5a to individual cells.