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991.
Yoshihiro Kuroda Yoshitaka Maeda Shinichi Sawa Kiyohiro Shibata Kazuhide Miyamoto Terumichi Nakagawa 《Journal of peptide science》2003,9(4):212-220
Pathogenic prion proteins (PrP(Sc)) are thought to be produced by alpha-helical to beta-sheet conformational changes in the normal cellular prion proteins (PrP(C)) located solely in the caveolar compartments. In order to inquire into the possible conformational changes due to the influences of hydrophobic environments within caveolae, the secondary structures of prion protein peptides were studied in various kinds of detergents by CD spectra. The peptides studied were PrP(129-154) and PrP(192-213); the former is supposed to assume beta-sheets and the latter alpha-helices, in PrP(Sc). The secondary structure analyses for the CD spectra revealed that in buffer solutions, both PrP(129-154) and PrP(192-213) mainly adopted random-coils (approximately 60%), followed by beta-sheets (30%-40%). PrP(129-154) showed no changes in the secondary structures even in various kinds of detergents such as octyl-beta-D-glucopyranoside (OG), octy-beta-D-maltopyranoside (OM). sodium dodecyl sulfate (SDS), Zwittergent 3-14 (ZW) and dodecylphosphocholine (DPC). In contrast, PrP(192-213) changed its secondary structure depending on the concentration of the detergents. SDS, ZW, OG and OM increased the alpha-helical content, and decreased the beta-sheet and random-coil contents. DPC also increased the alpha-helical content, but to a lesser extent than did SDS, ZW, OG or OM. These results indicate that PrP(129-154) has a propensity to adopt predominantly beta-sheets. On the other hand, PrP(192-213) has a rather fickle propensity and varies its secondary structure depending on the environmental conditions. It is considered that the hydrophobic environments provided by these detergents may mimic those provided by gangliosides in caveolae, the head groups of which consist of oligosaccharide chains containing sialic acids. It is concluded that PrP(C) could be converted into a nascent PrP(Sc) having a transient PrP(Sc) like structureunder the hydrophobic environments produced by gangliosides. 相似文献
992.
Abstract: Incorporation of radiolabelled amino acids into proteins of Euglena gracilis revealed that the amount of labelled protein depends on the conditions of illumination and temperature of cultivation. Protein synthesis was generally lower under dark conditions except at 37 °C. The largest amounts of labelled protein were measured at 21 °C and decreased at higher and lower temperatures. By separating the labelled proteins of the membraneous cell fraction from subcultures under a range of culture conditions, the synthesis of some specific proteins was found to be light- and/or temperature-dependent. On incubating cells taken at different times during a light/dark cycle and under constant conditions, a circadian rhythm of 35S-methionine- as well as 35S-cysteine-incorporation was detected. Thereby the cells incorporated ten-times less cysteine than methionine. Protein synthesis always peaked during the last quarter of the daily light phase, confirming the rhythmic rise in total protein. The length of the rhythm period, approximately 24 h, was nearly independent of the applied temperature in the range of 16 to 27 °C. 相似文献
993.
Jean A. Boutin 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1996,684(1-2)
Protein kinases form a large family of enzymes that play a major role in a number of live processes. The study of their action is important for the understanding of the transformation mechanisms and of the normal and pathological growth events. The quality of an enzyme assay is often the key point of an enzymatic study. It must be flexible and compatible with various experimental conditions, such as those for the purification process, the screening of inhibitors and the substrate specificity studies. As will be shown in the present review, two categories of substrates, peptidic and proteic, should be distinguished. The use of peptide substrates facilitates the determination of the recognition requirements of the enzyme and of the kinetic effects of even minute variations in their sequence. These linear peptide structures are assumed to mimic a complex interaction between the enzyme and a proteic substrate in which distant amino acids in the sequence are vicinal in the folded substrate. Less amenable to a systematic study, but probably more adequate to investigate the natural substrate of a given kinase, are the proteic substrates. Obviously the tools to measure protein kinase activities are not the same in these two cases. The main difficulty in assaying protein kinases is the use of labelles γ-ATP, mostly at large excess concentration, since the final product of the reaction has to be separated from the non-reacted labelled ATP. In the case of peptide substrates, the difficulty is to separate them from ATP basing on differences of molecular mass. Despite the efforts of many investigators to rely upon differences in solubility, in charges or in “affinity”, this separation, which is crucial for the assay, is still an unsolved experimental problem. Chromatographic, as well as electrophoretic assays appeared relatively late in this domain, and more work in assessing new methodologies might bring new breakthroughs in the next few years. Specific, simple and reliable kinase assays are still a major challenge. Their improvement will help to conduct specificity studies, to elucidate complex growth mechanisms in which they are involved and to discover more selective potent inihibitors. 相似文献
994.
以玉米根微粒体为材料进行的微量放射配体结合(MRLB)实验表明玉米根微粒体膜上存在着ABA结合位点,ABA与ABA结合蛋白(ABA-BP)结合的最适pH为6.5,结合反应对温度(0℃和25℃)不太敏感,ABA与ABA-BP的结合反应是一个动态平衡过程,5min即可达最大结合(Bmax)的50%,30min达到最大结合,1h内基本保持不变。胰蛋白酶处理表明此结合位点为蛋白质,冻融实验则表明此蛋白与ABA的结合不仅要求其自身具有特定构象而且需要有一定的膜脂环境,DTT处理实验结果显示ABA-BP中可能存在着二硫键。逆境处理可以提高玉米根微粒体膜对ABA的结合活性,盐胁迫、渗透胁迫、干旱胁迫和热冲激处理分别使结合活性上升34.9%、17.8%、23.1%和13.3%。 相似文献
995.
本文用PCR方法获得大肠杆菌热休克蛋白转录因子σ32的编码基因rpoH,并克隆在含有tac启动子的表达载体pUHE中,经IPTG诱导,在大肠杆菌中表达了C端融合有6个寡聚组氨酸的σ32。表达产物经金属螯合层析一步纯化,达到SDS-PAGE银染一条带纯度,氨基酸组成分析及N端序列分析结果与文献报道一致。35S细胞内参入实验表明:即使在较低的温度下,表达产物σ32(His)6也能导致热休克蛋白如GroEl、DnaK、Htp的大量合成. 相似文献
996.
猪精子中与卵透明带糖蛋白ZP3结合的蛋白质 总被引:3,自引:0,他引:3
依次经PSL-Sepharose亲和层析柱和纤维素CM-52离子交换层析柱,从猪精子的CHAPS抽提液分离得4个蛋白质组分。用固相透明带精蛋白结合试验(IZPGBA)检测;表明精子蛋白SP1和SP2具有结合透明带糖蛋白ZP3的活性,SP2并显示凝集血球的活性。精子蛋白SP1与卵预温育明显抑制精卵结合,抑制活性与加入的精子蛋白的浓度呈正相关。用生物素标记的ZP3和蛋白质印迹技术,证明SP1中的68kD精子蛋白与ZP3结合,提示68kD精子蛋白参与精卵结合。 相似文献
997.
Signal transduction pathways in guinea pig sperm 总被引:2,自引:0,他引:2
Trifluoperazine (TFP), the antagonist of calmodulin (CaM). significantly stimulated the capacitation and acrosome reaction of guinea pig spermatozoa at the concentration of 10-100μmol/L, independent of the external Ca2+. Forskolin, dbcAMP and caffeine evidently promoted the occurrence of acrosome reaction of spermatozoa at early capacitation stage (5 h) in nonsynchronous system but not in synchronous system. If the spermatozoa were capacitated for 15 h in synchronous system, the above three drugs significantly stimulated acrosome reaction in a Ca2+-independent manner. Protein kinase C activators, i.e. phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDB) did not influence the occurrence of acrosome reaction of spermatozoa at early capacitation stage, but significantly increased the acrosome reaction rate in capacitated spermatozoa in a Ca2+-independent manner. In contrast. PKC inhibitor staurosporine significantly inhibited the occurrence of acrosome reaction. 相似文献
998.
红原鸡与家鸡的亲缘关系研究 总被引:15,自引:2,他引:13
对中国红原鸡滇地亚种和海南亚种与我国茶花鸡,泰和鸡和寿光鸡等地方鸡种以及芦花鸡,洛岛红等外国鸡种进行了血型(3个位点,13个等位基因),蛋白质(酶)多态(5个位点,11个等位基因)和DNA指纹分析,结果表明,红原鸡与茶花鸡(原始型品种)的亲缘关系较近;与泰和鸡,寿光鸡,芦花鸡,洛岛红(进化型品种)的亲缘关系较远,呈红原鸡-茶花鸡-泰和鸡,寿光鸡或芦花鸡,洛岛红这样一个进化阶梯,以上结果与国外资料( 相似文献
999.
Insect-resistant transgenic cabbage plants and their progenies 总被引:3,自引:0,他引:3
An insecticidal crystal protein gene of Bacillus thuringiensis was transferred into cabbage genome with the method of Agrobacterium infection. Cotyledons with petioles as explants were cocultivated with Agrobacterial suspension. Calli generated at the basis of petiole were subjected to selection on the MS medium containing 15-30 mg/L kanamycin (Km). About 5% explants produced calli growing continuously on the selective medium. Green shoots appeared on these calli when they were transplanted onto medium with Km and 6-BA for plant differentiation. The shoots were separated and cultivated on medium with kanamycin. About 80% shoots were rooted. Non-transformed control calli could not give normal shoots and roots and brownized and died gradually. Larvae of Pieris rapae showed poisonous symptoms: growth inhibition and mortality when fed with the leaf of the transgenic plants. About 80% of regenerated plants showed positive hybridization bands when their DNA were probed with crystal protein sequence of Bacillu 相似文献
1000.
D. Buffard R. Esnault Á. Kondorosi 《World journal of microbiology & biotechnology》1996,12(2):175-188
During effective symbiosis, rhizobia colonize their hosts, and avoid plant defence mechanisms. To determine whether the host defence responses can be elicited by the symbiotic bacteria, specific markers involved in incompatible pathogenic interactions are required. The available markers of alfalfa defence mechanisms are described and their use in the study of the symbiotic interaction discussed. As defence-related gene expression in roots is not always related to defence mechanisms, other model systems have been established allowing confirmation of an important role of bacterial surface components in alfalfa-Rhizobium meliloti interactions. Nod factors at high concentrations have been shown to elicit defence-like responses in Medicago cell suspensions and roots. Elicitation of defence mechanisms by high levels of Nod factors in Rhizobium-infected roots may be a part of the mechanism by which nodulation is feed-back regulated.The authors are with the Institut des Sciences Végétales, CNRS, F-91198 Gif-sur-Yvette cédex, France. 相似文献