大肠杆菌精氨酰－tRNA合成酶的Lys306为酶活力所必需

将大肠杆菌精氨酰ｔＲＮＡ合成酶（ＡｒｇＲＳ）上Ｌｙｓ３０６用基因点突变的方法分别变为Ａｌａ和Ａｒｇ的密码子;得到变种基因ａｒｇｓ３０６ＫＡ和ａｒｇｓ３０６ＫＲ。变种基因重组在ｐＵＣ１８上,转化到大肠杆菌ＴＧ１中,转化子中ＡｒｇＲＳ及其变种ＡｒｇＲＳ３０６ＫＡ和ＡｒｇＲＳ３０６ＫＲ所表达的蛋白量至少为ＴＧ１表达ＡｒｇＲＳ蛋白量的１００倍。细胞粗抽提液中ＡｒｇＲＳ的比活ＴＧ１、转化子ｐＵＣ１８－ａｒｇｓ、ｐＵＣ１８－ａｒｇｓ３０６ＫＡ和ｐＵＣ１８－ａｒｇｓ３０６ＫＲ分别为１．６５、２１０、１．８和３８单位／毫克。结果表明ＡｒｓＲＳ的Ｌｙｓ３０６为Ａｌａ取代使活力完全丧失;若被Ａｒｇ取代,则活力丧失８０％以上。Ｌｙｓ３０６为ＡｒｇＲＳ活力所必需。     

萝卜抗真菌蛋白1（Rs—AFP1）在大肠杆菌中的融合表达及其对大丽轮 …

利用聚合酶链式反应（ＰＣＲ）获得了萝卜（Ｒａｑｈａｎｕｓ ｓａｔｉｖｕｓ Ｌ．）抗真菌蛋白１（Ｒｓ－ＡＦＰ１）基因编码区核苷酸序列。将整个阅读框架片段和云除了Ｎ－端信号肽序列的片段分别装入原核表达载体ｐＥＴ－３２ｇ（＋）中,在大肠杆菌中表达,发现带有信号 的Ｒｓ－ＡＦＰ１不能在大肠杆菌中表达,而当这一序列去除后,表达出约２７ｋＤ的Ｒｓ－ＡＦＰ１的融合蛋白。用凝血酶处理融合蛋白以云除Ｎ－端Ｈｉｓ．ｔ     

蛇毒蛋白Echistatin的C端突变基因的构建,表达及其活性研究

通过ＰＣＲ定点突变的技术,将蛇毒蛋白Ｅｃｈｉｓｔａｔｉｎ基因的Ｃ端进行了突变（Ａｌａ４８→Ａｒｇ４８→,Ｔｈｒ４９→Ｖａｌ４９）,模拟纤维蛋白Ｎ端的四肽（Ｇｌｙ－Ｐｒｏ－Ａｒｇ－Ｖａｌ）,以期增加Ｅｃｓ（Ｅｃｈｉｓｔａｔｉｎ）的活性,突变的基因重组到表达质粒ｐＪＣ２６４上,经ＩＰＴＧ诱导,以ＣｈｅＹ－Ｅｃｓ融合蛋白方式进行了表达,表达量占菌体总蛋白的１５￣２０％,Ｓｅｐｈａｄｅｘ Ｇ－７５初步纯化     

黄地老虎颗粒体病毒DNA片段的克隆与颗粒体蛋白基因的定位

用ｐＵＣ１９质粒作载体,克隆了黄地老虎颗粒体病毒（Ａｇｒｏｌｉｓｓｅｇｅｔｕｍｇｒａｎｕｌｏｓｉｓｖｉｒｕｓ,简称ＡｓＧＶ）ＤＮＡＰｓｔＩ－Ｄ．Ｅ．Ｆ．Ｇ．Ｈ．Ｊ．Ｋ．等７个片段。以［￣（３２）Ｐ］－ｄＣＴＰ标记的油桐尺蠖核型多角体病毒（Ｂｕｚｕｒａｓｕｐｐｒｅｓｓａｒｉａｎｕｃｌｃａｒｐｏｌｙｈｅｄｒｏｓｉｓｖｉｒｕｓ简称ＢｓＮＰＶ）多角体蛋白基因为探针,在３７℃条件下对ＡｓＧＶ）颗粒体蛋白基因进行了定位,将其分别定位于ＢｓｌⅡ－Ｓ或ＴＰｓＴＩ－Ａ或Ｂ和ＥｃｉＲＩ－Ａ片段上。     

Epstein—Barr病毒膜抗原基因免疫的研究

将Ｅｐｓｔｅｉｎ－Ｂａｒｒ病毒（ＥＢＶ）膜抗原（ＭＡ）ＢＬＬＦ１基因,插入含有ＣＭＶ启动子的真核表达载体ｐｃＤＮＡ３下游ＢａｍＨＩ位点,构建成真核表达质粒ｐｃＤＮＡ３－ＭＡ。将纯化的ＤＮＡ注射Ｂａｌｂ／ｃ小鼠股四头肌。经免疫的动物产生抗ＥＢ病毒ＭＡ特异性的抗体和中和抗体,依赖抗体细胞介导的细胞毒作用（ＡＤＣＣ）,特异性Ｔ淋巴细胞增生性反应及细胞毒性Ｔ淋巴细胞（ＣＴＬ）杀伤作用。基因免疫与基因－蛋白     

酶母K.cicerisporus基因组中有启动子功能的DNA片段的克隆

利用以３′－氨基糖苷磷酸转移酶基因（ＡＰＨＩ）为报道基因的一套启动子探针质粒ｐＳＫ－ｋａｎ４０１、ｐＳＫ－ｋａｎ１１０５、ｐＳＫ－ｋａｎ１２３８,从酵母Ｋｌｙｖｅｒｏｍｙｃｅｓ ｃｉｃｅｒｉｓｐｏｒｕｓ基因组中克隆到８个有较强启动功能的ＤＮＡ片段,分析出３′端ＤＮＡ序列,并在酵母Ｋｌｕｙｖｅｒｏ８ｍｙｃｅｓｅ ｌａｃｔｉｓ中通过检测报告基因与３－磷酸甘油醛脱氢酶基因（ＡＰＤＨ）的ｍＲＮＡ表达量的比     

人类T细胞白血病病毒I型pX基因反式调节和致白血病机理的研究进展

人类Ｔ细胞白血病病毒Ｉ型（ＨＴＬＶ－Ｉ）是成人Ｔ细胞白血病（ＡＴＬ）的病因。ＨＴＬＶ－Ｉ基因组中的ｐＸ基因区域编码ｐ４０＾ｔａｘ和ｐ２７＾ｒｅｘ蛋白,前者可反式激活病毒的ＬＴＲ和ＩＬ－２Ｒ及ｃ－ｆｏｓ、ＴＧＦβ１等细胞基因,加强转录过程,在白血病发生中发挥重要作用。ｐ２７＾ｒｅｘ蛋白则在转录后水平进行反馈性调节。白血病的发生机理是多因子参加的多步骤过程。     

hsp70与c—myc,c—fos和p50的结构及功能联系

热休克蛋白７０基因启动子部位有ｍｙｃ原癌基因产物的两个结构位点,分别位于ｈｓｐ７０基因５＇上游－１６０和－２３０碱基处,ｍｙｃ蛋白可以与其结合调节ｈｓｐ７０基因的转录活性,ｃ－ｆｏｓ基因启动与ｈｓｐ７０启动子区域存在着一种共同的反式作用因子结合区,可能受一种共同的特异性反式作用的调节,野生型Ｐ５３对ｈｓｐ７０启动子的抑制可能与ＣＣＡＡｂｏｘ结合因子有关;ｈｓｐ７０resume     

幽门螺杆菌热休克蛋白A基因的克隆,表达及免疫原性 …

幽门螺杆菌的感染可以诱发人体产生胃炎和消化性溃疡,其组成成分热休克蛋白Ａ可刺激机体产生保护性的免疫反应,用ＰＣＲ方法从幽门螺杆菌的染色体ＤＮＡ上扩增出ＨｓｐＡ基因片段,将其插入原核表达载体ｐＥＴ－２２ｂ（＋）中,并在ＢＬ２１（ＤＥ３）大肠杆菌表达。经测序Ｈｓｐ基因片段有３５４ｂｐ组成,可编码１１８个氨基酸残基的多肽。     

酵母K.cicerisporus基因组中有启动子功能的DNA片段的克隆

利用以３′－氨基糖苷磷酸转移酶基因（ＡＰＨＩ）为报告基因的一套启动子探针质粒ｐＳＫ－ｋａｎ４０１、ｐＳＫ－ｋａｎ１１０５、ｐＳＫ－ｋａｎ１２３８,从酵母Ｋｌｕｙｖｅｒｏｍｙｃｅｓｃｉｃｅｒｉｓｐｏｒｕｓ基因组中克隆到８个有较强启动功能的ＤＮＡ片段,分析出３′端ＤＮＡ序列,并在酵母Ｋｌｕｙｖｅｒｏｍｙｃｅｓｌａｃｔｉｓ中通过检测报告基因与３－磷酸甘油醛脱氢酶基因（ＧＡＰＤＨ）的ｍＲＮＡ表达量的比值,确定它们的功能强度,其中ｐＳＫ－ｋａｎ４０１－４１和ｐＳＫ－ｋａｎ１１０５－５１两个克隆的插入片段具有较强的启动子功能,并证明这两个片段在酵母Ｋ．ｃｉｃｅｒｉｓｐｏｒｕｓ中也有启动子功能。     

Presenilins, the Endoplasmic Reticulum, and Neuronal Apoptosis in Alzheimer's Disease

Abstract: Many cases of autosomal dominant inherited forms of early-onset Alzheimer's disease are caused by mutations in the genes encoding presenilin-1 (PS-1; chromosome 14) and presenilin-2 (PS-2; chromosome 1). PSs are expressed in neurons throughout the brain wherein they appear to be localized primarily to the endoplasmic reticulum (ER) of cell bodies and dendrities. PS-1 and PS-2 show high homology and are predicted to have eight transmembrane domains with the C terminus, N terminus, and a loop domain all on the cytosolic side of the membrane; an enzymatic cleavage of PSs occurs at a site near the loop domain. The normal function of PSs is unknown, but data suggest roles in membrane trafficking, amyloid precursor protein processing, and regulation of ER calcium homeostasis. Homology of PSs to the C. elegans gene sel-12 , which is involved in Notch signaling, and phenotypic similarities of PS-1 and Notch knockout mice suggest a developmental role for PSs in the nervous system. When expressed in cultured cells and transgenic mice, mutant PSs promote increased production of a long form of amyloid β-peptide (Aβ1-42) that may possess enhanced amyloidogenic and neurotoxic properties. PS mutations sensitize cultured neural cells to apoptosis induced by trophic factor withdrawal, metabolic insults, and amyloid β-peptide. The mechanism responsible for the proapoptotic action of mutant PSs may involve perturbed calcium release from ER stores and increased levels of oxidative stress. Recent studies of apoptosis in many different cell types suggest that ER calcium signaling can modulate apoptosis. The evolving picture of PS roles in neuronal plasticity and Alzheimer's disease is bringing to the forefront the ER, an organelle increasingly recognized as a key regulator of neuronal plasticity and survival.     

Langevin dynamics of peptides: the frictional dependence of isomerization rates of N-acetylalanyl-N'-methylamide.

The rate constant for the transition between the equatorial and axial conformations of N-acetylalanyl-N'-methylamide has been determined from Langevin dynamics (LD) simulations with no explicit solvent. The isomerization rate is maximum at collision frequency gamma = 2 ps-1, shows diffusive character for gamma greater than or equal to 10 ps-1, but does not approach zero even at gamma = 0.01 ps-1. This behavior differs from that found for a one-dimensional bistable potential and indicates that both collisional energy transfer with solvent and vibrational energy transfer between internal modes are important in the dynamics of barrier crossing for this system. It is suggested that conformational searches of peptides be carried out using LD with a collision frequency that maximizes the isomerization rate (i.e., gamma approximately 2 ps-1). This method is expected to be more efficient than either molecular dynamics in vacuo (which corresponds to LD with gamma = 0) or molecular dynamics in solvent (where dynamics is largely diffusive).     

An alternative spliced mouse presenilin-2 mRNA encodes a novel γ-secretase inhibitor

The γ-secretase, composed of presenilin-1 (PS1) or presenilin-2 (PS2), nicastrin (NCT), anterior pharynx-defective phenotype 1 (APH-1), and PEN-2, is critical for the development of Alzheimer’s disease (AD). PSs are autoproteolytically cleaved, producing an N-terminal fragment (NTF) and a hydrophilic loop domain-containing C-terminal fragment. However, the role of the loop domain in the γ-secretase complex assembly remains unknown. Here, we report a novel PS2 isoform generated by alternative splicing, named PS2β, which is composed of an NTF with a hydrophilic loop domain. PS2β disturbed the interaction between NCT and APH-1, resulting in the inhibition of amyloid-β production. We concluded that PS2β may inhibit γ-secretase activity by affecting the γ-secretase complex assembly.

Structured summary

MINT-7025654: APH1 (uniprotkb:Q96BI3) physically interacts (MI:0218) with PEN2 (uniprotkb:Q9NZ42), PS2 beta (uniprotkb:Q61144-2) and PS1 (uniprotkb:P49769) by anti tag coimmunoprecipitation (MI:0007)MINT-7025631: APH1 (uniprotkb:Q96BI3) physically interacts (MI:0218) with NCT (uniprotkb:Q92542), PEN2 (uniprotkb:Q9NZ42) and PS1 (uniprotkb:P49769) by anti tag coimmunoprecipitation (MI:0007)     

Cellular Expression and Proteolytic Processing of Presenilin Proteins Is Developmentally Regulated During Neuronal Differentiation

Abstract: We have determined the expression of the Alzheimer's disease-associated proteins presenilin-1 and presenilin-2 in primary cultures of rat hippocampal neurons. Neurons highly express presenilin-1 and presenilin-2, whereas both proteins were not detected in astrocytes. Further, we have analyzed the subcellular localization and expression in rat hippocampal neurons during development. Although presenilin proteins were localized predominantly to the endoplasmic reticulum in nonneuronal cells transfected with presenilin cDNAs, in neurons, presenilin proteins were also found in compartments not staining with antibodies to grp78(BiP). Presenilin-1 and presenilin-2 were predominantly detected in vesicular structures within the somatodendritic compartment with much less expression in axons. Polarized distribution of presenilin-1 and presenilin-2 differs slightly, with more presenilin-2 expressed in axons compared with presenilin-1. Presenilin expression was found to be developmentally regulated. Presenilin expression strongly increased during neuronal differentiation until full morphological polarization and then declined. No full-length presenilin-1 or presenilin-2 could be detected within cell lysates. At early developmental stages the expected ～34-kDa N-terminal proteolytic fragment of presenilin-1 and the ～38-kDa fragment of presenilin-2 were detected. Later during differentiation we predominantly detected a ～38-kDa fragment for presenilin-1 and a ～42-kDa fragment for presenilin-2. By epitope mapping, we show that these slower migrating peptides represent N-terminal proteolytic fragments, cleaved C-terminal to the conventional site of processing. It is noteworthy that both presenilin-1 and presenilin-2 undergo alternative proteolytic cleavage at the same stage of neuronal differentiation. Regulation of presenilin expression and proteolytic processing might have implications for the pathological as well as the biological function of presenilins during aging in the human brain.     

Dynamic properties of Na+ ions in models of ion channels: a molecular dynamics study.

We present simulation results for the effective diffusion coefficients of a sodium ion in a series of model ion channels of different diameters and hydrophobicities, including models of alamethicin, a leucine-serine peptide, and the M2 helix bundle of the nicotinic acetylcholine receptor. The diffusion coefficient, which in the simulations has a value of 0.15(2) A2ps-1 in bulk water, is found to be reduced to as little as 0.02(1) A2ps-1 in the narrower channels, and to about 0.10(5) A2ps-1 in wider channels such as the nicotinic acetylcholine receptor. It is anticipated that this work will be useful in connection with calculations of channel conductivity using such techniques as the Poisson-Nernst-Planck equation, Eyring rate theory, or Brownian dynamics.     

Subcellular localization of presenilins during mouse preimplantation development.

The genes defective in familial Alzheimer's disease encode the proteins presenilin 1 and 2 (PS1 and 2). Expression of presenilins (PSs) and their proteolytic processing are regulated during neuronal development. Even though these proteins are detected and regulated mainly in Golgi and endoplasmic reticulum, their subcellular distribution during the development is not known. The present study aimed to investigate the localization of PSs and their role during early developmental stage using mouse embryo model. At preimplantation stage, PSs were detected not only in cytoplasm, but also in the nucleus from oocyte to 2.5 dpc (day postcoitum), then disappeared in the nucleus at blastocyst stage (3.5 dpc). Antisense against PS1 and PS2 decreased the transition to blastocyst stage, whereas each antisense alone had no effect. Treatment with lactacystin (26S proteosome inhibitor), which arrest cell cycle at M phase, redistributed PSs into centrosome-kinetochore microtubule. PS2 overexpression in HEK 293 cell arrested cell cycle at S phase. These data suggest that PSs play key roles in cell division and differentiation during early development.     

Characterization of detergent-insoluble complexes containing the familial Alzheimer's disease-associated presenilins

Many cases of early-onset familial Alzheimer's disease have been linked to mutations within two genes encoding the proteins presenilin-1 and presenilin-2. The presenilins are 48-56-kDa proteins that can be proteolytically cleaved to generate an N-terminal fragment (approximately 25-35 kDa) and a C-terminal fragment (approximately 17-20 kDa). The N- and C-terminal fragments of presenilin-1, but not full-length presenilin-1, were readily detected in both human and mouse cerebral cortex and in neuronal and glioma cell lines. In contrast, presenilin-2 was detected almost exclusively in cerebral cortex as the full-length molecule with a molecular mass of 56 kDa. The association of the presenilins with detergent-insoluble, low-density membrane microdomains, following the isolation of these structures from cerebral cortex by solubilization in Triton X-100 and subsequent sucrose density gradient centrifugation, was also examined. A minor fraction (10%) of both the N- and C-terminal fragments of presenilin-1 was associated with the detergent-insoluble, low-density membrane microdomains, whereas a considerably larger proportion of full-length presenilin-2 was present in the same membrane microdomains. In addition, a significant proportion of full-length presenilin-2 was present in a high-density, detergent-insoluble cytoskeletal pellet enriched in beta-actin. The presence of the presenilins in detergent-insoluble, low-density membrane microdomains indicates a possible role for these specialized regions of the membrane in the lateral separation of Alzheimer's disease-associated proteins within the lipid bilayer and/or in the distinct functions of these proteins.     

High-resolution fine mapping of ps-2, a mutated gene conferring functional male sterility in tomato due to non-dehiscent anthers

Functional male sterility is an important trait for the production of hybrid seeds. Among the genes coding for functional male sterility in tomato is the positional sterility gene ps-2. ps-2 is monogenic recessive, confers non-dehiscent anthers and is the most suitable for practical uses. In order to have tools for molecular-assisted selection (MAS) we fine mapped the ps-2 locus. This was done in an F₂ segregating population derived from the interspecific cross between a functionally male sterile line (ps-2/ps-2; Solanum lycopersicum) and a functionally male fertile line (S. pimpinellifolium). Here we report the procedure that has led to the high-resolution fine mapping of the ps-2 locus in a 1.65 cM interval delimited by markers T0958 and T0635 on the short arm of Chromosome 4. The presence of many COS markers in the local high-resolution map allowed us to study the synteny between tomato and Arabidopsis at the ps-2 locus region. No obvious candidate gene for ps-2 was identified among the known functional male sterility genes in Arabidopsis.     

Interactions between selected photosensitizers and model membranes: an NMR classification

Membrane interactions of porphyrinic photosensitizers (PSs) are known to play a crucial role for PS efficiency in photodynamic therapy (PDT). In the current paper, the interactions between 15 different porphyrinic PSs with various hydrophilic/lipophilic properties and phospholipid bilayers were probed by NMR spectroscopy. Unilamellar vesicles consisting of dioleoyl-phosphatidyl-choline (DOPC) were used as membrane models. PS-membrane interactions were deduced from analysis of the main DOPC 1H-NMR resonances (choline and lipid chain signals). Initial membrane adsorption of the PSs was indicated by induced changes to the DOPC choline signal, i.e. a split into inner and outer choline peaks. Based on this parameter, the PSs could be classified into two groups, Type-A PSs causing a split and the Type-B PSs causing no split. A further classification into two subgroups each, A1, A2 and B1, B2 was based on the observed time-dependent changes of the main DOPC NMR signals following initial PS adsorption. Four different time-correlated patterns were found indicating different levels and rates of PS penetration into the hydrophobic membrane interior. The type of interaction was mainly affected by the amphiphilicity and the overall lipophilicity of the applied PS structures. In conclusion, the NMR data provided valuable structural and dynamic insights into the PS-membrane interactions which allow deriving the structural constraints for high membrane affinity and high membrane penetration of a given PS.     

Specificity of presenilin‐1‐ and presenilin‐2‐dependent γ‐secretases towards substrate processing

The two presenilin‐1 (PS1) and presenilin‐2 (PS2) homologs are the catalytic core of the γ‐secretase complex, which has a major role in cell fate decision and Alzheimer's disease (AD) progression. Understanding the precise contribution of PS1‐ and PS2‐dependent γ‐secretases to the production of β‐amyloid peptide (Aβ) from amyloid precursor protein (APP) remains an important challenge to design molecules efficiently modulating Aβ release without affecting the processing of other γ‐secretase substrates. To that end, we studied PS1‐ and PS2‐dependent substrate processing in murine cells lacking presenilins (PSs) (PS1KO, PS2KO or PS1‐PS2 double‐KO noted PSdKO) or stably re‐expressing human PS1 or PS2 in an endogenous PS‐null (PSdKO) background. We characterized the processing of APP and Notch on both endogenous and exogenous substrates, and we investigated the effect of pharmacological inhibitors targeting the PSs activity (DAPT and L‐685,458). We found that murine PS1 γ‐secretase plays a predominant role in APP and Notch processing when compared to murine PS2 γ‐secretase. The inhibitors blocked more efficiently murine PS2‐ than murine PS1‐dependent processing. Human PSs, especially human PS1, expression in a PS‐null background efficiently restored APP and Notch processing. Strikingly, and contrary to the results obtained on murine PSs, pharmacological inhibitors appear to preferentially target human PS1‐ than human PS2‐dependent γ‐secretase activity.