硫和6-苄氨基腺嘌呤对水稻幼苗硝酸还原酶活性的影响

缺硫培养6天的水稻幼苗,其叶片和根中的硝酸还原酶(NR)活性明显下降。用1pPm 的6-苄氨基腺嘌呤(6-BA)处理培养了10天的水稻幼苗根系,24小时后缺硫培养的水稻幼苗叶片和根系的 NR 活性升高,加硫培养的水稻幼苗叶片和根中的 NR 活性下降。用~(35)S示踪发现,6-BA 可降低加硫幼苗对~(35)S 的吸收和转化,但促进缺硫幼苗对~(35)S 的转化。     

长叶车前花叶病毒(HRV_(sh))外壳蛋白赖氨酸残基的修饰

我们曾报道长叶车前花叶病毒上海分离株(简称HRVsh)的外壳蛋白有二个赖氨酸残基,在PH8.5无变性剂存在的条件下,完整病毒颗粒表面的赖氨酸残基可与三硝基苯磺酸(TNPS)起反应,反应后的TNP-HRVsh病毒颗粒的感染力丧失达90％以上。 本文又进行了甲基乙亚胺甲酯(MEI)对HRVsh赖氨酸残基的修饰反应,修饰后的MEI-HRVsh病毒颗粒的感染力也同样丧失90％以上。 从三硝基苯磺酸修饰的病毒颗粒(TNP-HRVsh)中分离得到的RNA能与天然的HRVsh的外壳蛋白重建病毒颗粒,并具有感染力,说明修饰过程中核酸并不受影响。 进一步用同位素~(35)S,~(32)P双标记病毒,再以TNPS修饰标记的病毒,得到(~(35)S,~(32)P)-HRVsh及TNP-(~(35)S,~(32)P)-HRVsh。将两者分别接种于系统寄主青菜(Brassica chinensis)的一片叶片,一天后在非接种叶片上都可测得~(35)S,~(32)P的放射计数。其中,(~(35)S,~(32)P)-HRVsh的~(35)S/~(32)P比值降低了,而TNP-(~(35)S,~(32)P)-HRVsh的~(35)S/~(32)P比值保持不变。说明HRVsh外壳蛋白赖氨酸残基的修饰并不影响病毒颗粒进入寄主细胞,以及在寄主细胞间的转移。同位素双标记的结果表明,其感染力丧失的原因可能是由于上述修饰作用阻止了病毒在感染中所必须的脱壳过程。     

~(33)P:一种新的分子生物学放射性示踪物

在选择放射性示踪物时,人们主要考虑敏感性、分辨率和安全性。作为分子生物学实验常用的放射性示踪物,~(32)P 和~(35)S 各有其不足之处。~(32)P 因为能量高、放射比活性大因而其操作极不安全,且有分辨率不够高之缺陷,用于序列测定极不理想,而且由于其半衰期太短,操作很不方便。~(35)S 虽补充了~(32)P 的不足,但由于其放射比活性低,检测敏感性不够高,对于单拷贝基因的检测往往是爱莫能助。最近,在国外出现了一种新的放射性示踪物——~(33)P。与其他两种常用的同位素~(32)P 和~(35)S 相比,~(33)P     

~(35)S标记DNA探针检测真核单拷贝基因

本室采用国产[α-~(35)S]dATP制备DNA探针,用于检测真核单拷贝基因并初步获得了满意结果。本文还叙述了~(35)S取代~(32)P中标记DNA探针和Southern吸印杂交时,适宜的反应条件,优点及其意义。     

应用~(35)硫对 NaHSO_3-穿心莲内酯代谢的研究

本文是用放射性同位素~(35)硫标记NaHSO_3-穿心莲内酯做动物实验.以阐明NaHSO_3(~(35)S)-穿心莲内酯在体内的代谢过程。动物实验是用NaHSO_3(~(35)S)-穿心莲内酯注入大白鼠。实验用的药物是用NaHSO_3(~(35)S)溶液用加成反应标记于穿心莲内酯的第12位碳上。标记率达92%。标记化合物的性能和浓度与治疗用商品注射针剂NaHSO_3-穿心莲内酯相同。大小便标本是注射后连续按一定间隔时间取出的,组织标本是按一定间隔时杀死的动物身上取出的。标本的放射性是用液体闪烁计数器测量的。标记化合物在大白鼠的体内代谢表明,NaHSO_3(~(35)S)-穿心莲内酯进入体内后,在中枢神经系统中主要选择性地蓄积于脊髓中,并且呈下行性蓄积,可因此导致肌紧张度减低和外周血管扩张,引起体温下降。在脏器中此药较明显地蓄积于十二指肠、直肠,这是临床采用此药治疗肠炎和菌痢的有力依据。实验也指出,此药在体内迅速吸收,迅速排泄,72小时内在尿粪中总排除量达86.2%,建议临床酌情增加剂量或次数。NaHSO_3(~(35)S)-穿心莲内酯大多在尿中主要以原形排出,从而考虑对泌尿系统感染可能有一定疗效。     

应用~(35)硫对NaHSO_3-穿心莲内酯代谢的研究

本文是用放射性同位素~(35)硫标记NaHSO_3-穿心莲内酯做动物实验,以阐明NaHSO_3(~(35)S)-穿心莲内酯在体内的代谢过程。动物实验是用NaHSO_3(~(35)S)-穿心莲内酯注入大白鼠。实验用的药物是用NaHSO_3(~(35)S)溶液用加成反应标记于穿心莲内酯的第12位碳上。标记率达92%。标记化合物的性能和浓度与治疗用商品注射针剂NaHSO_3-穿心莲内酯相同。大小便标本是注射后连续按一定间隔时间取出的,组织标本是按一定间隔时杀死的动物身上取出的。标本的放射性是用液体闪烁计数器测量的。标记化合物在大白鼠的体内代谢表明,NaHSO_3(~(35)S)-穿心莲内酯进入体内后,在中枢神经系统中主要选择性地蓄积于脊髓中,并且呈下行性蓄积,可因此导致肌紧张度减低和外周血管扩张,引起体温下降。在脏器中此药较明显地蓄积于十二指肠、直肠,这是临床采用此药治疗肠炎和菌痢的有力依据。实验也指出,此药在体内迅速吸收,迅速排泄,72小时内在尿粪中总排除量达86.2%,建议临床酌情增加剂量或次数。NaHSO_3(~(35)S)-穿心莲内酯大多在尿中主要以原形排出,从而考虑对泌尿系统感染可能有一定疗效。     

螺旋藻乙醇酸氧化酶(GO)的研究

采用比色法对鄂尔多斯高原碱湖的钝顶螺旋藻(S1)与国外引进的钝顶螺旋藻(S2)和极大螺旋藻(S3)的乙醇酸氧化酶(GO)进行了比较研究。结果表明:在25℃、pH 8.0条件下,S1、S2和S3的GO活性分别为70.9 U/gFW、59.6 U/gFW和80.9 U/gFW;最适温度均为30℃;在0℃~35℃(30℃)范围内比较稳定;最适pH值分别为8.6、8.2和8.4;pH值稳定范围,S1为7.6~10.0、S2为8.0~9.0;S3为8.0~8.6。S1的GO对温度和pH适应范围最宽,且在低温、高温、强酸和强碱下的活性均比引进种的高。     

Virulence Phenotyping and Molecular Characterization of a Low-pathogenicity Isolate of Listeria monocytogenes from Cow's Milk

A low-pathogenicity isolate of Listeria monocytogenes from cow's milk,as screened in mouseand chicken embryonated egg models,was examined for virulence-related phenotypic traits.Correspondingvirulence genes (iap,prfA,plcA,hly,mpl,actA,plcB,InlA and InlB) were compared with L.monocytogenesreference strains 10403S and EGD to elucidate the possible molecular mechanisms of low virulence.Al-though L.monocytogenes H4 exhibited similar patterns to strain 10403S in terms of hemolytic activity,invitro growth and invasiveness and even had higher adhesiveness,faster intracellular growth and higherphospholipase activity in vitro,it was substantially less virulent than the strain 10403S in mouse and chickenembryo models (50% lethal dose:10~(8.14) vs.10~(5.49) and 10~(6.73) vs.10~(1.9),respectively).The genes prfA,plcA andmpl were homologous among L.monocytogenes strains H4,10403S and EGD (>98%).Genes iap,hly,plcB,InlA and InIB of L.monocytogenes 10403S had higher homology to those of strain EGD (>98%) than isolateH4.The homology of the gene hly between strain 10403S and isolate H4 was 96.9% at the nucleotide level,but 98.7% at the amino acid level.The actA gene of isolate H4 had deletions of 105 nucleotides correspondingto 35 amino acid deletions falling Within the proline-rich region.Taken together,this study presents someclues as to reduced virulence to mice and chicken embryos of the isolate H4 probably as a result of deletionmutations of actA.     

疑难问题解答一例

问:在噬菌体侵染细菌的实验中,怎样知道进入细菌细胞的是噬菌体的DNA而不是蛋白质呢?答:噬菌体是有蛋白质的外壳包裹着DNA的简单结构.它侵染细菌时,进入细胞的是DNA还是蛋白质呢?美国科学家赫尔希(Hersher)和蔡斯(Chase)在1952年做了如下的实验回答了这个问题.他首先将噬菌体培养在含有用~(32)P同位素标记的磷酸盐培养基上,结果DNA被~(32)P标记.而蛋白质一般只是由C、H、O、N、S元素组成,而不含P,故蛋白质不会被标记.他又将噬菌体放在含有~(35)S同位素标记的硫酸盐培养基里培养,结果噬菌体的蛋白质被~(35)S标记上.而DNA没有被标记.     

一株果胶酶产生菌HDYM-02的分离鉴定及系统发育分析

对1株从沤麻液中分离的果胶酶产生菌HDYM-02进行了形态、生理生化特征及16S rDNA序列分析,结果表明该菌株为革兰染色阳性,菌体大小为(1~1.2)×(2.7~2.9)μm,最适生长温度为33~35℃之间,最适生长pH为6.5~7.0。以该菌株16S rDNA序列同源性为基础,比对分析发现其与蜡状芽胞杆菌(Bacillus cereus)的同源性高达99.5%。     

硫酸乙酰肝素蛋白聚糖对培养人脐静脉内皮细胞合成蛋白聚糖的影响

以[35S」-Na2SO4为示踪物,观察人正常主动脉中的硫酸乙酸肝素蛋白聚糖（HSPG）对培养的第一代人脐静脉内皮细胞（hUVEC）合成蛋白聚糖（PG）的影响．用解聚提取法及离子交换柱层析分离人主动脉HSPG．35S-PGs的混合物用离子交换及凝胶过滤柱层析法分离35S-HSPG,35S-硫酸软骨素-硫酸皮肤素PG（35S-CSDSPG）及35S-硫酸皮肤素PG（35S-DSPG）．结果发现实验组（加HSPG）与对照组（未加HSPG）相比,hU-VEC的35S-PGs总量（培养液+细胞层）无差别,但实验组培养液中35S-PGs总量升高、35S-DSPG、35S-CSDSPG及其相对百分含量均升高,而35S-HSPG及其百分含量降低．细胞层的35S-PGs,35S-HSPG及其相对百分含量降低,35S-DSPG及其相对百分含量升高,而CSDSPG未见差别．     

In Vivo Biosynthesis of 35S-Substance P from [35S]Methionine in the Rat Striatum and Its Transport to the Substantia Nigra

Rats with a push-pull cannula implanted in the right striatum were used to study the biosynthesis of 35S-substance P (SP) from [35S]methionine and its transport to the ipsilateral substantia nigra. [35S]Methionine was delivered for 2, 3 or 5 h to the push-pull cannula. 35S-SP in striatal and nigral tissues was estimated after immunoadsorption and HPLC. Higher levels of 35S-SP in striatal homogenates were found after a 5-h labelling period. 35S-P biosynthesis was inhibited when cycloheximide was superfused together with [35S]methionine. The identity of 35S-SP was further checked by its conversion into 35S-SP sulphoxide. After a 5-h labelling period, 35S-SP was also recovered in the substantia nigra. This was not the case after hemisection of striato-nigral fibers. When rats were killed 15 or 24 h after the 5-h labelling period, 35S-SP levels in the substantia nigra were higher than those found just after 5-h labelling period, while the reverse was observed in the striatum.     

Role of the varicella-zoster virus gene product encoded by open reading frame 35 in viral replication in vitro and in differentiated human skin and T cells in vivo

Although genes related to varicella-zoster virus (VZV) open reading frame 35 (ORF35) are conserved in the herpesviruses, information about their contributions to viral replication and pathogenesis is limited. Using a VZV cosmid system, we deleted ORF35 to produce two null mutants, designated rOkaDelta35(#1) and rOkaDelta35(#2), and replaced ORF35 at a nonnative site, generating two rOkaDelta35/35@Avr mutants. ORF35 Flag-tagged recombinants were made by inserting ORF35-Flag at the nonnative Avr site as the only copy of ORF35, yielding rOkaDelta35/35Flag@Avr, or as a second copy, yielding rOka35Flag@Avr. Replication of rOkaDelta35 viruses was diminished in melanoma and Vero cells in a 6-day analysis of growth kinetics. Plaque sizes of rOkaDelta35 mutants were significantly smaller than those of rOka in melanoma cells. Infection of melanoma cells with rOkaDelta35 mutants was associated with disrupted cell fusion and polykaryocyte formation. The small plaque phenotype was not corrected by growth of rOkaDelta35 mutants in melanoma cells expressing the major VZV glycoprotein E, gE. The rOkaDelta35/35@Avr viruses displayed growth kinetics and plaque morphologies that were indistinguishable from those of rOka. Analysis with ORF35-Flag recombinants showed that the ORF35 gene product localized predominantly to the nuclei of infected cells. Evaluations in the SCIDhu mouse model demonstrated that ORF35 was required for efficient VZV infection of skin and T-cell xenografts, although the decrease in infectivity was most significant in skin. These mutagenesis experiments indicated that ORF35 was dispensable for VZV replication, but deleting ORF35 diminished growth in cultured cells and was associated with attenuated VZV infection of differentiated human skin and T cells in vivo.     

Toxoplasma gondii: expression pattern and detection of infection using full-length recombinant P35 antigen

A complete P35 surface antigen of Toxoplasma gondii was sequenced (GenBank ). Immunoblot found that it reacted specially with T. gondii acute infected sera, and the recombinant P35 signal was specific for P35 antigen. The P35-GST protein was used as antigen to detect 125 sera samples by double-sandwich ELISA. P35-IgM positive rate in a chronic infected group, a persistent IgM positive chronic group, a recently seroconvered group and an acute infected group were 4% (1 out of 25), 16% (4 out of 25), 88% (22 out of 25), and 100% (25 out of 25), respectively. The sensitivity and specificity of the recombinant full-length P35 antigen were 100 and 96%, respectively. The detailed expression patterns of P35 antigen were studied in 36 IgM and IgG positive sequential samples from 10 recently seroconvered patients. Results showed that the P35-IgM positive rate decreased as the time after the first seroconversion increased. P35-IgM positive samples in the first, second, third, fourth, and fifth month after the first seroconversion test were 90, 78, 57, 50, and 33%, respectively. P35-IgG positive samples in the first, second, third, fourth, fifth, sixth, and seventh month after the first seroconversion test were 70, 100, 100, 100, 67, 100, and 100%, respectively. All samples were P35-IgM negative after the fifth month, and P35-IgG negative after the seventh month from seroconversion. P35-IgM existed the shortest time and was a more specific marker for T. gondii acute infection than P35-IgG, IgM, and IgG to whole tachyzoites antigens.     

Autoradiographic study of [35S]-cysteine and [1-14C]-cystine in pregnant and neonatal mice

The distribution and fate of 35S from [35S]-cysteine and 14C from [1-14C]-cystine, both precursors to taurine in 17-day pregnant mice and (1-day-old) neonates were investigated by whole-body autoradiography following IV injection for the pregnant mice and IP injection for the neonates. Survival intervals were 30 min, 3 hr. In the dam, 35S and 14C were both highly incorporated into the pancreas. As very low uptake of [35S]-taurine in the pancreas was found in a previous study, it is reasonable to suggest that most radioactivity found in the pancreas represents cysteine. In maternal brain, brown fat, and myocardium, optical density and relative ratio (ODs of 35S and 14C in each tissue and organ/ODs of 35S and 14C in the blood) of 35S were significantly higher than those of 14C, assuming that most of 35S might be present as taurine in these regions. In the developing brain, 35S was found mainly in the differentiating neurons of the cortical plate, including the primordial hippocampal cell layer, and of the cerebellar cortical plate. Relative ratios of 35S in these cerebral regions were significantly higher than those of 14C, suggesting that most of 35S represents taurine.     

The apoptotic suppressor P35 is required early during baculovirus replication and is targeted to the cytosol of infected cells.

The p35 gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) is required to block virus-induced apoptosis. The trans-dominant activity of p35 suppresses premature cell death and facilitates AcMNPV replication in a cell line- and host-specific manner. To characterize the p35 gene product (P35), a specific polyclonal antiserum was raised. As revealed by immunoblot analyses of wild-type AcMNPV-infected cells, P35 appeared early (8 to 12 h) and accumulated through the late stages of infection (24 to 36 h). Biochemical fractionation of cells both early and late in infection and indirect immunochemical staining demonstrated that P35 localized predominantly to the cytosol (150,000 x g supernatant); comparatively minor quantities of P35 were associated with intracellular membranes. The cytoplasmic localization of P35 was independent of virus infection. The functional significance of the early and late synthesis of P35 was examined by constructing recombinant viruses in which the timing and level of p35 expression were altered. Delaying P35 synthesis by placing p35 under exclusive control of a strong, very late promoter failed to suppress intracellular DNA fragmentation and apoptotic blebbing in most cells. Thus, earlier expression of p35 was required to block virus-induced apoptosis. Site-specific mutagenesis of the p35 promoter demonstrated that low levels of P35 were sufficient to block apoptosis, whereas higher levels were required to maintain wild-type virus gene expression. Consistent with an early role in infection, P35 was also detected in the budded form of AcMNPV. Because of the lack of sequence similarity and its cytosolic targeting, P35 may function in a manner that is mechanistically distinct from other apoptotic regulators, including Bcl-2 and the adenovirus E1B 19-kDa protein.     

Molecular characterization and phylogeny of U2AF35 homologs in plants

U2AF (U2 small nuclear ribonucleoprotein auxiliary factor) is an essential splicing factor with critical roles in recognition of the 3'-splice site. In animals, the U2AF small subunit (U2AF35) can bind to the 3'-AG intron border and promote U2 small nuclear RNP binding to the branch-point sequences of introns through interaction with the U2AF large subunit. Two copies of U2AF35-encoding genes were identified in Arabidopsis (Arabidopsis thaliana; atU2AF35a and atU2AF35b). Both are expressed in all tissues inspected, with atU2AF35a expressed at a higher level than atU2AF35b in most tissues. Differences in the expression patterns of atU2AF35a and atU2AF35b in roots were revealed by a promoter::beta-glucuronidase assay, with atU2AF35b expressed strongly in whole young roots and root tips and atU2AF35a limited to root vascular regions. Altered expression levels of atU2AF35a or atU2AF35b cause pleiotropic phenotypes (including flowering time, leaf morphology, and flower and silique shape). Novel slicing isoforms were generated from FCA pre-mRNA by splicing of noncanonical introns in plants with altered expression levels of atU2AF35. U2AF35 homologs were also identified from maize (Zea mays) and other plants with large-scale expressed sequence tag projects. A C-terminal motif (named SERE) is highly conserved in all seed plant protein homologs, suggesting it may have an important function specific to higher plants.     

Characterization of Cry34/Cry35 binary insecticidal proteins from diverse Bacillus thuringiensis strain collections

Bacillus thuringiensis crystal proteins of the Cry34 and Cry35 classes function as binary toxins showing activity on the western corn rootworm, Diabrotica virgifera virgifera LeConte. We surveyed 6,499 B. thuringiensis isolates by hybridization for sequences related to cry35A genes, identifying 78 strains. Proteins of the appropriate molecular mass (ca. 44 kDa) for Cry35 were observed in 42 of the strains. Full-length, or nearly full-length, sequences of 34 cry34 genes and 16 cry35 genes were also obtained from cloning, PCR analysis, and DNA sequencing. These included representatives of all known Cry34A, Cry34B, Cry35A, and Cry35B classes, as well as a novel Cry34A/Cry35A-like pair. Bioassay analysis indicated that cry35-hybridizing strains not producing a ca. 14-kDa protein, indicative of Cry34, were not active on corn rootworms, and that the previously identified Cry34A/Cry35A pairs were more active than the Cry34B/Cry35B pairs. The cry35-hybridizing B. thuringiensis strains were found in locales and materials typical for other B. thuringiensis strains. Comparison of the sequences with the geographic origins of the strains showed that identical, or nearly identical, sequences were found in strains from both Australasia and the Americas. Sequence similarity searches revealed that Cry34 proteins are similar to predicted proteins in Photorhabdus luminescens and Dictyostelium discoidium, and that Cry35Ab1 contains a segment similar to beta-trefoil domains that may be a binding motif. The binary Cry34/Cry35 B. thuringiensis crystal proteins thus appear closely related to each other, are environmentally ubiquitous, and share sequence similarities consistent with activity through membrane disruption in target organisms.     

Specific reaction of Met 35 in amyloid beta peptide with hypochlorous acid

Abstract

The reaction of the amyloid beta peptide (Aβ) with hypochlorous acid and hydroxyl radicals was analysed by spectrophotometry and mass spectrometry. N-acetylmethionine, Aβ25-35 and Aβ1-42 reacted rapidly with hypochlorous acid. The relative reaction rates of N-acetylmethionine and Aβ with hypochlorous acid was in the order N-acetylmethionine > Aβ25-35 > Aβ1-42. While the reaction of Aβ25-35 in the presence of a slight excess of hypochlorous acid resulted in complete conversion of Met35 to Met35 sulphoxide, Aβ1-42 required more than a 4-fold excess of hypochlorous acid for complete conversion of Met35. Identical products were obtained when Aβ25-35 and Aβ1-42 were treated with a hypochlorous acid generating system. Conversion of Met35 to Met35 sulphoxide in Aβ abolished the aggregation of Aβ25-35. Reaction of Aβ with hydroxyl radicals resulted in limited conversion of Met35 to Met35 sulphoxide. The specific reaction of Met35 in Aβ with hypochlorous acid to form Met35 sulphoxide has been analysed.     

超量表达IPT和KN1基因对转基因烟草生长发育的影响

为了比较异戊烯转移酶基因IPT和玉米homeobox基因KNI超量表达对植物生长发育的影响,将35S：：IPT和35S：：KNI分别导入烟草。观察发现,过量表达IPT和KNI基因对植株再生频率、形态、生长周期和顶端优势等方面的影响均相似,但在生根和开花结籽方面,35S：：IPT转基因植物所受影响更显著。细胞分裂素含量检测表明,35S：：IPT转基因植物叶片中细胞分裂素的含量高于35S：：KNI转基因植物。