Replacements of Pro86 in phage T4 lysozyme extend an alpha-helix but do not alter protein stability

To investigate the relation between protein stability and the predicted stabilities of individual secondary structural elements, residue Pro86 in an alpha-helix in phage T4 lysozyme was replaced by ten different amino acids. The x-ray crystal structures of seven of the mutant lysozymes were determined at high resolution. In each case, replacement of the proline resulted in the formation of an extended alpha-helix. This involves a large conformational change in residues 81 to 83 and smaller shifts that extend 20 angstroms across the protein surface. Unexpectedly, all ten amino acid substitutions marginally reduce protein thermostability. This insensitivity of stability to the amino acid at position 86 is not simply explained by statistical and thermodynamic criteria for helical propensity. The observed conformational changes illustrate a general mechanism by which proteins can tolerate mutations.     

Frame shift mutations near the beginning of the lysozyme gene of bacteriophage T4

A pair of frame shift mutations in the lysozyme gene of bacteriophage T4 results in the substitution of a glutamyl-tyrosyl sequence for the asparagine residue that is the penultimate amino-terminal amino acid in the lysozyme of the wild-type strain. One of the mutations has been identified as the insertion of two bases, the other as the insertion of a single base.     

转溶菌酶基因水稻稻米毒理及致畸作用试验

通过小鼠急性毒性试验、大鼠长期毒性试验、小鼠微核试验和精子致畸试验对转溶菌酶基因水稻稻米的毒性及致畸作用进行了初步的评价.转基因大米淀粉灌胃小鼠,剂量相当于成人每日食用2 250 g,连续7 d观察,动物全部存活,表现正常,未见动物出现中毒反应,小鼠灌胃转基因大米的最大耐受量MTD≥37.5 g/kg.转基因大米粉分别以15 g/kg/d和7.5 g/kg/2个剂量,相当于成人日食用量900 g和450 g,连续94 d灌胃大鼠,灌胃期间动物的行为活动、外观体征、饮食、粪便、体重均无异常.5项血液学检查和13项生化指标检测及处死1/2的动物尸体解剖观察、脏器系数及脏器病理学检查,结果均属正常,与空白对照无显著差异;剩余1/2的动物留存观察2周,各项观察指标、血液、生化及病理检测也无异常.转基因大米粉以15 g/kg和7.5 g/kg 2个剂量分别灌胃小鼠,未发现对小鼠骨髓细胞微核和精子畸形发生率有不良影响.本试验结果提示携带溶菌酶基因、NPT-Ⅱ标记基因等外源DNA的转基因大米无明显的毒性和致畸作用.     

Aromatic-aromatic interaction: a mechanism of protein structure stabilization

Analysis of neighboring aromatic groups in four biphenyl peptides or peptide analogs and 34 proteins reveals a specific aromatic-aromatic interaction. Aromatic pairs (less than 7 A between phenyl ring centroids) were analyzed for the frequency of pair type, their interaction geometry (separation and dihedral angle), their nonbonded interaction energy, the secondary structural locations of interacting residues, their environment, and their conservation in related molecules. The results indicate that on average about 60 percent of aromatic side chains in proteins are involved in aromatic pairs, 80 percent of which form networks of three or more interacting aromatic side chains. Phenyl ring centroids are separated by a preferential distance of between 4.5 and 7 A, and dihedral angles approaching 90 degrees are most common. Nonbonded potential energy calculations indicate that a typical aromatic-aromatic interaction has energy of between -1 and -2 kilocalories per mole. The free energy contribution of the interaction depends on the environment of the aromatic pair. Buried or partially buried pairs constitute 80 percent of the surveyed sample and contribute a free energy of between -0.6 and -1.3 kilocalories per mole to the stability of the protein's structure at physiologic temperature. Of the proteins surveyed, 80 percent of these energetically favorable interactions stabilize tertiary structure, and 20 percent stabilize quaternary structure. Conservation of the interaction in related molecules is particularly striking.     

Binding of HTLV-III/LAV to T4+ T cells by a complex of the 110K viral protein and the T4 molecule

Human T-lymphotropic virus type III (HTLV-III) or lymphadenopathy-associated virus (LAV) is tropic for human T cells with the helper-inducer phenotype, as defined by reactivity with monoclonal antibodies specific for the T4 molecule. Treatment of T4+ T cells with monoclonal antibodies to T4 antigen blocks HTLV-III/LAV binding, syncytia formation, and infectivity. Thus, it has been inferred that the T4 molecule itself is a virus receptor. In the present studies, the surfaces of T4+ T cells were labeled radioactively, and then the cells were exposed to virus. After the cells were lysed, HTLV-III/LAV antibodies were found to precipitate a surface protein with a molecular weight of 58,000 (58K). By blocking and absorption experiments, this 58K protein was identified as the T4 molecule. No cell-surface structures other than the T4 molecule were involved in the antibody-antigen complex formation. Two monoclonal antibodies, each reactive with a separate epitope of the T4 molecule, were tested for their binding capacities in the presence of HTLV-III/LAV. When HTLV-III/LAV was bound to T4+ T cells, the virus blocked the binding of one of the monoclonal antibodies, T4A (OKT4A), but not of the other, T4 (OKT4). When HTLV-III/LAV was internally radiolabeled and bound to T4+ T cells which were then lysed, a viral glycoprotein of 110K (gp110) coprecipitated with the T4 molecule. The binding of gp110 to the T4 molecule may thus be a major factor in HTLV-III/LAV tropism and may prove useful in developing therapeutic or preventive measures for the acquired immune deficiency syndrome.     

Implantation of genetically engineered fibroblasts into mice: implications for gene therapy

In a variety of human genetic diseases, replacement of the absent or defective protein provides significant therapeutic benefits. As a model for a somatic cell gene therapy system, cultured murine fibroblasts were transfected with a human growth hormone (hGH) fusion gene and cells from one of the resulting clonal lines were subsequently implanted into various locations in mice. Such implants synthesized and secreted hGH, which was detectable in the serum. The function of the implants depended on their location and size, and on the histocompatibility of the donor cells with their recipients. The expression of hGH could be modified by addition of regulatory effectors, and, with appropriate immunosuppression, the implants survived for more than 3 months. This approach to gene therapy, here termed "transkaryotic implantation," is potentially applicable to many genetic diseases in that the transfected cell line can be extensively characterized prior to implantation, several anatomical sites are suitable for implantation, and regulated expression of the gene of therapeutic interest can be obtained.     

An engineered pathway for the formation of protein disulfide bonds

We have engineered a pathway for the formation of disulfide bonds. By imposing evolutionary pressure, we isolated mutations that changed thioredoxin, which is a monomeric disulfide reductase, into a [2Fe-2S] bridged dimer capable of catalyzing O2-dependent sulfhydryl oxidation in vitro. Expression of the mutant protein in Escherichia coli with oxidizing cytoplasm and secretion via the Tat pathway restored disulfide bond formation in strains that lacked the complete periplasmic oxidative machinery (DsbA and DsbB). The evolution of [2Fe-2S] thioredoxin illustrates how mutations within an existing scaffold can add a cofactor and markedly change protein function.     

Turnover of rat liver tyrosine transaminase: stabilization after inhibition of protein synthesis

Turnover of the rat liver tyrosine transaminase in vivo was measured by a label and chase procedure under conditions where the amount of enzyme undergoes no change. Half-life of the (14)C-labeled enzyme in this basal condition was found to be 1.5 +/- 0.3 hours. Inhibitors of protein synthesis (cycloheximide or puromycin) do not appreciably influence the basal enzyme level over a 5-hour period, although these drugs will block hormonal induction of this enzyme. In pulse-labeling experiments, cycloheximide blocked transaminase synthesis almost completely. The conclusion that enzyme degradation, as well as synthesis, must be blocked when protein synthesis is stopped was confirmed in experiments showing that labeled enzyme is stable in the liver of rats treated with cycloheximide The participation of a continuously synthesized polypeptide in the degradative phase of transaminase turnover is suggested.     

Efficient incorporation of human CD4 protein into avian leukosis virus particles

Virus envelope (Env) proteins are thought to contain specific signals for selective uptake by virus particles. In the course of attempting to define these signals by testing virus incorporation of CD4-Env chimeric proteins, normal human CD4 was found to be efficiently and selectively assembled into avian leukosis virus particles in quail cells. Viruses bearing CD4 at their surface may be useful reagents in the design of retrovirus-mediated gene therapy for the acquired immune deficiency syndrome.     

将野生二粒小麦的大粒和籽粒高蛋白质含量性状向普通小麦转移

将引自以色列的4份野生二粒小麦与普通小麦杂交、回交。从F5、BC1F4、BC2F3代中选出的128个外部形态性状已基本稳定的株系中，选出千粒重高于对应普遍小麦亲本（扬麦5号和扬麦158）5g以上的株系17个，籽粒蛋白质含量比普通小麦亲本高4％以上的株系12个，其中有5个株系的千粒重和籽粒蛋白质含量均显著高于普通小麦。表明野生二粒小麦可用于提高普通小麦栽培品种千粒重和籽粒蛋白质含量，且可以实现两者的同步改良。     

Fusidic acid: inhibition of factor T2 in reticulocyte protein synthesis

The steroid antibiotic fusidic acid inhibits reticulocyte protein synthesis. This inhibition appears to be due to interference with the activity of the T(2) supernatant fraction, and strengthens the proposition that T(2) is functionally analogous to the G-factor of bacterial protein synthesis, which is also specifically inhibited by this antibiotic.     

野生二粒小麦抗白粉病基因向普通小麦转移的研究

野生二粒小麦(资源代号:AS846)叶鞘被有茸毛,穗轴脆易断;对小麦白粉病、条锈病免疫.普通小麦与其杂交和回交,F₁回交结实率是杂交结实率的3～4倍;野生二粒小麦性状在F₁呈显性,BC₁开始分离;F₃形态指示性状出现中间类型,一直到高代还保留这种类型.形态指示性状与抗白粉性连锁不紧密,不能作为抗白粉性的选择标志.在普通小麦和野生二粒小麦的单交组合里,通过定向选择,选育出了农艺性状好,抗白粉性基本稳定的株系N9134-2-11-1.     

Evolution of the molecular machines for protein import into mitochondria

In creating mitochondria some 2 billion years ago, the first eukaryotes needed to establish protein import machinery in the membranes of what was a bacterial endosymbiont. Some of the preexisting protein translocation apparatus of the endosymbiont appears to have been commandeered, including molecular chaperones, the signal peptidase, and some components of the protein-targeting machinery. However, the protein translocases that drive protein import into mitochondria have no obvious counterparts in bacteria, making it likely that these machines were created de novo. The presence of similar translocase subunits in all eukaryotic genomes sequenced to date suggests that all eukaryotes can be considered descendants of a single ancestor species that carried an ancestral "protomitochondria."     

出雏前后蛋鸡和肉鸡血清中甲状腺激素水平的比较

用放射免疫分析法测定了出雏前后肉鸡（ＡＡ）和蛋鸡（伊莎）血清中总甲状腺激素（Ｔ３,Ｔ４）和游离甲状腺激素（ＦＴ３,ＦＴ４）的水平,结果表明：肉鸡和蛋鸡血清总Ｔ３水平在１４胚龄和１８胚龄时较低,１日龄时显著上升,肉鸡表现尤为突出。今后 Ｔ３水平的变化在品种间有所不同,肉鸡从１日龄到１０日龄斩下降,１日与５日龄差异显著;恧旧鸡１日龄至５日龄Ｔ３水平显著升高,除１日龄肉鸡Ｔ３水平显著高于蛋鸡外,其它日龄     

Atomic structure of PDE4: insights into phosphodiesterase mechanism and specificity

Cyclic nucleotides are second messengers that are essential in vision, muscle contraction, neurotransmission, exocytosis, cell growth, and differentiation. These molecules are degraded by a family of enzymes known as phosphodiesterases, which serve a critical function by regulating the intracellular concentration of cyclic nucleotides. We have determined the three-dimensional structure of the catalytic domain of phosphodiesterase 4B2B to 1.77 angstrom resolution. The active site has been identified and contains a cluster of two metal atoms. The structure suggests the mechanism of action and basis for specificity and will provide a framework for structure-assisted drug design for members of the phosphodiesterase family.     

高致病性猪繁殖与呼吸综合征病毒M蛋白CD4+T细胞表位优势区的鉴定

采用RT-PCR方法扩增猪繁殖与呼吸障碍综合征病毒(PRRSV)临床分离株的M基因(525 bp),通过设计4对引物,把M蛋白分成4段K1(78-112)、K2(98-131)、K3(118-153)、K4(139-174),分别扩增相应基因M1、K1、K2、K3和K4,连接pET-32a(+)表达载体,经鉴定后转化Escherichia coliBL21,经IPTG诱导表达,SDS-PAGE和Western-blot检测结果显示,分别在相对分子质量30.8×103和24.4×103位置出现目的条带,重组蛋白均可与阳性血清发生特异反应。将蛋白M1免疫BALB/c小鼠,于第三次免2周后从小鼠脾脏分离淋巴细胞,分别用K1、K2、K3、K4蛋白进行体外刺激。流式细胞仪检测分泌IFN-γ细胞因子的T细胞的频率,K1蛋白刺激组显著高于其他组和对照组(P≤0.01)。结论:M1蛋白K1(78-112)段为T细胞表位优势区。     

Transport of dsRNA into cells by the transmembrane protein SID-1

RNA interference (RNAi) spreads systemically in plants and nematodes to silence gene expression distant from the site of initiation. We previously identified a gene, sid-1, essential for systemic but not cell-autonomous RNAi in Caenorhabditis elegans. Here, we demonstrate that SID-1 is a multispan transmembrane protein that sensitizes Drosophila cells to soaking RNAi with a potency that is dependent on double-stranded RNA (dsRNA) length. Further analyses revealed that SID-1 enables passive cellular uptake of dsRNA. These data indicate that systemic RNAi in C. elegans involves SID-1-mediated intercellular transport of dsRNA.     

信息技术:走向农业生态系统的可持续管理

21世纪信息科学及其工程技术将加速生态学、分子生物学等现代生物科学的两个前沿学科及其工程技术的发展，在解决人类生存的环境问题和探寻生命本原问题中起推波助澜作用。农业的终极目标是实现对农业生态系统的可持续管理，现代信息工程技术是实现这个目标的核心技术，其中地理信息系统（GIS）、全球定位系统（GPS）和遥感（RS）等3S技术为实现农业生态系统的可持续管理提供了综合的、多维的、动态的，因而也是通晓的和精确的信息资源和技术控制方案，将促进农业真正通向可持续发展之路。