高比强〔r-32P〕ATP的合成

高比强〔r—~(32)p〕ATP是核酸结构功能研究中常用标记化合物,它在DNA重组、分子杂交和核酸的结构分析中起着重要作用。下面我们从《Methods in Enzymology》Vol 65摘译合成高比强〔r—~(32)p)ATP的方法供大家参考。 10倍浓度反应液(贮于-20℃,每100μl为一份):     

DNA5′端~(32)P标记方法

在DNA的结构与功能研究中,其5′端~(32)P标记是常用的有效技术。下面我们从《Methods in Enzymolgy》Vol 65中摘译了DNA 5′端脱磷及~(32)P标记方法,供大家参考。1.用磷酸单酯酶除去DNA链5′末端磷     

The radiolysis of tryptophan and leucine with32Pβ-radiation

Summary We have extended earlier experiments on the radiolysis of DL-tryptophan using³²P-radiation to longer reaction times, observing complete destruction of the tryptophan by secondary, non-radiolytic processes. We have also undertaken the irradiation of DL-leucine with³²P's at -196°, achieving radiolyses to the extents of ca. 20–30%, but observing no concomittant asymmetric bias. The implications of these observations are discussed with regard to the Vester-Ulbricht mechanism for the origin of optical activity.     

~(32)P诱变龟裂链霉菌初报

有关~(32)P诱变抗生素产生菌的报道很少。我们曾于73年底探索过该诱变因子对龟裂链霉菌(Streptomyces rimosus)的诱变作用,实验结果如下。将龟裂链霉菌8-1768菌株的孢子悬液在含有NaH_2~(32)PO_4(比放射性为0.244毫居里/毫     

植物中~(32)P的Cerenkov效应测量

放射性同位素应用技术,已成为植物、植物生理学研究中不可缺少的手段。用这种技术在研究植物的各种代谢过程中,发挥了巨大的作用。目前,Cerenkov计数的方法已经广泛     

Phosphorus distribution inside Chinese fir seedlings under different P supplies based on 32 P tracer北大核心CSCD

Aim The objective of this study was to determine the amount and distribution of exogenous phosphorus (P) in different organs, as well as their changes in Chinese fir (Cunninghamia lanceolata) under different P supply levels. The results could be used as scientific base for selecting P-efficient genotypes. Methods Seedlings of two Chinese fir genotypes (M1 and M4), both with high P use efficiency, were treated with different P supply levels and quantified by using 32 P isotope tracer for P distributions in different organs. The seedlings used in this study were selected by our team through previous research as the experimental materials. Important findings We found that the distribution of exogenous P in M1 and M4 was the highest in the roots and the lowest in the stems, and at an intermedia level in the needles. The 32 P content of each organ under the same treatment was ranked as root > stem > needle on the horizontal projection plane. The exogenous P content by the roots, stems and needles of M1 and M4 under low-P treatment appeared lower than that under the high-P treatment. The blackening degree of low-P image of roots, stems and needles under the same treatment was also lower than that under high-P treatment. The content of exogenous P in these organs under the low-P treatment increased slowly, indicating that the low-P stress significantly affected the absorption and accumulation of P in the seedlings. P allocation rates in the roots of M1 and M4 showed an initial decreasing and increasing later under low-P stress, while under the high-P treatment, the root P level increased first and stabilizing later. These findings indicate that M1 and M4 could adapt to external low-P stress through redistribution of P within the plants by transferring P from roots to above-ground parts at the early stage under low P stress. With the extension of stressing time, P from above-ground parts was shifted to roots. However, the distribution of exogenous P in M1 and M4 was significantly different under the low P treatment. The distribution of exogenous P from the beginning to the end of M1 was greater in the roots than that in above-ground parts, while M4 showed a similar pattern in early stages but a higher rate toward the above-ground parts later. This indicates that M1 has a higher degree of strengthening P circulation in vivo than M4 with low P stress, i.e. the tendency of P transfer from above-ground parts to roots is stronger in M1 than in M4. © Chinese Journal of Plant Ecology.     

DNA5′端~(32)P标记方法

在DNA的结构与功能研究中,其5′端~(32)P标记是常用的有效技术。下面我们从《Methods in Enzymolgy》Vol 65中摘译了DNA 5′端脱磷及~(32)P标记方法,供大家参考。 1.用磷酸单酯酶除去DNA链5′末端磷     

λ-Carrageenan P32 Is a Potent Inhibitor of Rabies Virus Infection

Rabies, caused by rabies virus (RABV), is an acute, fatal encephalitic disease that affects many warm-blooded mammals. Currently, post-exposure prophylaxis regimens are effective for most rabies cases, but once the clinical signs of the disease appear, current treatment options become ineffective. Carrageenan has been reported as a potent inhibitor of many viruses. In this study, the λ-carrageenan (λ-CG) P32 was investigated for its potential role in inhibiting RABV infection. Our results show that P32 specifically inhibits the replication of several RABV strains but not vesicular stomatitis virus in multiple cell lines and shows low cytotoxicity. P32 mainly abrogated viral replication during the early stage of the post-adsorption period. Further studies demonstrated that P32 could affect not only viral internalization but also viral uncoating by blocking cell fusion mediated by RABV glycoprotein. Moreover, P32 can fully inhibit RABV infection in vitro during the post-adsorption period, whereas heparin and heparan sulfate, which possess similar structures to P32, showed significant but not complete inhibition of RABV infectivity. Collectively, our results indicate that λ-CG P32 is a promising agent that can inhibit RABV infection mainly by inhibiting viral internalization and glycoprotein-mediated cell fusion and can be used for the development of novel anti-RABV drugs.     

温州蜜柑果实发育期叶片和果皮吸收32P的研究

应用同位素示踪技术,研究了宫川温州蜜柑果实发育期叶片、果皮对³²P的吸收速率和转化形式。结果表明:在湖南气候条件下,叶片、果皮能以不同速率利用其表面³²P并进一步转化为蛋白质、核酸、醚溶性磷和磷脂。果皮吸收³²P的性能弱于叶片,没有出现第二次吸收高峰,而是随油胞数量增加、蜡质层加厚、细胞老化而逐渐减弱,且吸收的³²P绝大部分仍滞留于果皮中。     

Studies on the specific activity of [γ-32P]ATP in adipose and other tissue preparations incubated with medium containing [32P]phosphate

1. The specific activity of the γ-³²P position of ATP was measured in various tissue preparations by two methods. One employed HPLC and the enzymatic conversion of ATP to glucose 6-phosphate and ADP. The other was based on the phosphorylation of histone by catalytic subunit of cAMP-dependent protein kinase (Hawkins, P.T., Michell, R.H. and Kirk, C.J. (1983) Biochem. J. 210, 717–720). The HPLC method also allowed the incorporation of ³²P into the (α + β)-positions of ATP to be determined. 2. In rat epididymal fat-pad pieces and fat-cell preparations the specific activity of [γ-³²P]ATP attained a steady-state value after 1–2 h incubation in medium containing 0.2 mM [³²P]phosphate. Addition of insulin or the β-agonist isoprenaline increased this value by 5–10% within 15 min. 3. Under these conditions the steady-state specific activity of [γ-³²P]ATP was 30–40% of the initial specific activity of the medium [³²P]phosphate. However, if allowance was made for the change in medium phosphate specific activity during incubations the equilibration of the γ-phosphate position of ATP with medium phosphate was greater than 80% in both preparations. The change in medium phosphate specific activity was a combination of the expected equilibration of [³²P]phosphate with exchangeable intracellular phosphate pools plus the net release of substantial amounts of tissue phosphate. At external phosphate concentrations of less than 0.6 mM the loss of tissue phosphate to the medium was the major factor in the change in medium phosphate specific activity. 4. It is concluded that little advantage is gained in employing external phosphate concentrations of less than 0.6 mM in experiments concerned with the incorporation of phosphate into proteins and other intracellular constituents. Indeed, a low external phosphate concentration may cause depletion of important intracellular phosphorus-containing components.     

山羊痘病毒P32基因序列分析及其B细胞表位预测

目的：比较山羊痘病毒不同分离株间P32基因的同源性,并对其B细胞表位进行预测。方法：选取GenBank上10株不同山羊痘毒株P32基因序列,采用DNAsis MAX序列分析软件对该基因序列进行同源性分析,并以B细胞表位分析参数及蛋白质二级结构分析数据,综合预测P32蛋白B细胞表位。结果：10株不同山羊痘毒株P32基因核苷酸及氨基酸序列同源性分别为99.4%和98.4%;在P32蛋白的肽链中,19-60、64-80、98-118、138-182和214-275区段亲水性强,17-45、64-75、88-97、110-128、152-165和201-251区段柔韧性好,150-172、200-255区段抗原指数高,而31-59、101-119、142-145、165-172和215-252区段表面可及性高,42-56、159-181、200-208和227-259区段。结论：不同山羊痘病毒株间P32基因具有较高的同源性,P32蛋白227-251区段可能是B细胞表位优势区,为P32蛋白功能的深入研究与新型疫苗的设计提供一定的基础。     

兴安落叶松幼苗对~（32）P的吸收与累积研究

利用３２Ｐ示踪法研究兴安落叶松幼苗在侧根形成前对磷的吸收利用及其在体内的转运。结果表明：兴安落叶松幼苗在侧根形成前能吸收利用外源磷。且由根到茎转运较快,由茎到叶吸收转运缓慢,各部位３２Ｐ累积量依次是根＞茎＞叶,３２Ｐ在根部的累积主要是无机磷形式,在茎、叶部主要是有机磷形式。该时期幼苗酸性磷酸酶活性较低,但地上部酶活性略高,地下部较低．     

应用32P标记喜马拉雅旱獭研究其寄生蚤的散布情况

1973年7—8月和1974年4—9月在甘肃南部肃南县皇城区草原上,应用³²P标记喜马拉雅旱獭,用放射自显影的方法确定跳蚤的散布情况。共获得五种跳蚤,其中 Callopsylla dolabris和Oropsyllasilantiewi是旱獭的主要寄生蚤。旱獭和跳蚤种群间接触的频度取决于旱獭本身在栖息地内的分布、被旱獭所占用洞穴的数量以及媒介跳蚤的数量。在旱獭密度不高而蚤指数较高的情况下,以及存在大量隐蔽所的条件下,其交在比较频繁,标记蚤分布的范围较大,跳蚤的交换也较多。我们于标记后的第14天就发现距原洞口470米的标记蚤。艾鼬和草原鸟由于经常窜旱獭洞或洒居于旱獭洞内,所以它们的寄生蚤同旱獭蚤亦经常发生交换,并被标记,这在旱獭鼠疫病流行期间是值得引起注意的问题。     

RNA3’-末端~(32)P标记及其核苷酸简易测定

近几年来,遗传的物质基础,即脱氧核糖核酸(DNA)序列分析技术,有了重大突破。对核酸的结构与功能、基因表达、调控等研究起了巨大的推动作用。核糖核酸(RNA)序列分析工作,在经典方法的基础上,借鉴于DNA序列分析方法,也得到了迅速的发展。简易直读技术也广泛应用于RNA序列研究中。DNA或RNA序列分析,多采用体外放射性同位素     

羊痘病毒P32基因真核表达载体的构建、表达及其免疫原性

通过PCR方法扩增全长P32基因和截去跨膜区的P32基因(MP32),将其分别克隆到真核表达载体pcDNA3.1( )和已插入CpG序列的pcDNA3.1-CpG中,构建pcDNA3.1-P32、pcDNA3.1-CpG-P32和pcDNA3.1-CpG-MP32质粒;用脂质体法转染BHK-21细胞,通过间接免疫荧光(IFA)试验验证其表达效果;经肌肉免疫注射健康BALB/c小鼠,用间接ELISA法检测抗体;在免疫后的第3、5周取免疫小鼠的脾细胞,用流式细胞仪检测CD4 和CD8 T细胞亚群.结果所构建的真核表达载体在BHK-21细胞中都能表达P32蛋白;免疫小鼠血清在免疫第2周后均能检测到羊痘特异性IgG抗体;免疫组小鼠脾脏CD4 T细胞数目和CD4 /CD8 T细胞比值明显高于对照组.结果提示,所构建的真核载体可诱导小鼠产生特异性体液免疫应答,并能刺激小鼠产生较强的细胞免疫应答.     

A simple, rapid preparation ofα-[32P]- labelled adenosine diphosphate

Hexokinase (EC 2.7.1.1) will convert commercially available α-[³²P]-labelled ATP into α-[³²P]-labelled ADP. A simple, rapid isolation procedure for the α-[³²P]-labelled ADP is described and this synthetic method can be used for the preparation of other α-[³²P]-labelled nucleoside diphosphates.