全文获取类型
收费全文 | 4123篇 |
免费 | 206篇 |
国内免费 | 2篇 |
专业分类
4331篇 |
出版年
2023年 | 10篇 |
2022年 | 29篇 |
2021年 | 56篇 |
2020年 | 34篇 |
2019年 | 44篇 |
2018年 | 69篇 |
2017年 | 53篇 |
2016年 | 69篇 |
2015年 | 140篇 |
2014年 | 168篇 |
2013年 | 279篇 |
2012年 | 269篇 |
2011年 | 291篇 |
2010年 | 144篇 |
2009年 | 158篇 |
2008年 | 252篇 |
2007年 | 253篇 |
2006年 | 191篇 |
2005年 | 255篇 |
2004年 | 228篇 |
2003年 | 219篇 |
2002年 | 201篇 |
2001年 | 52篇 |
2000年 | 61篇 |
1999年 | 68篇 |
1998年 | 66篇 |
1997年 | 53篇 |
1996年 | 40篇 |
1995年 | 30篇 |
1994年 | 36篇 |
1993年 | 32篇 |
1992年 | 44篇 |
1991年 | 46篇 |
1990年 | 33篇 |
1989年 | 46篇 |
1988年 | 29篇 |
1987年 | 29篇 |
1986年 | 22篇 |
1985年 | 22篇 |
1984年 | 39篇 |
1983年 | 17篇 |
1982年 | 18篇 |
1981年 | 15篇 |
1980年 | 10篇 |
1979年 | 10篇 |
1977年 | 18篇 |
1976年 | 10篇 |
1974年 | 10篇 |
1973年 | 12篇 |
1970年 | 9篇 |
排序方式: 共有4331条查询结果,搜索用时 15 毫秒
171.
172.
Furochi H Tamura S Mameoka M Yamada C Ogawa T Hirasaka K Okumura Y Imagawa T Oguri S Ishidoh K Kishi K Higashiyama S Nikawa T 《FEBS letters》2007,581(30):5743-5750
Intact osteoactivin, a novel type I membrane glycoprotein, were shed at a dibasic motif in the juxtamembrane region in C2C12 myoblasts. Extracellular fragments were secreted into the culture media by a putative metalloprotease. Extracellular fragments of osteoactivin, but not control protein, induced matrix metalloprotease-3 (MMP-3) expression in NIH-3T3 fibroblasts. Epidermal growth factor (ERK) kinase inhibitors inhibited the osteoactivin-mediated MMP-3 expression, whereas the extracellular fragment of osteoactivin activated ERK1/2 and p38 in the mitogen-activated protein kinase pathway. Our results suggest that the extracellular fragments of osteoactivin produced by shedding act as a growth factor to induce MMP-3 expression via the ERK pathway in fibroblasts. 相似文献
173.
Tanaka S Saito K Chon H Matsumura H Koga Y Takano K Kanaya S 《The Journal of biological chemistry》2007,282(11):8246-8255
The crystal structure of an active site mutant of pro-Tk-subtilisin (pro-S324A) from the hyperthermophilic archaeon Thermococcus kodakaraensis was determined at 2.3 A resolution. The overall structure of this protein is similar to those of bacterial subtilisin-propeptide complexes, except that the peptide bond linking the propeptide and mature domain contacts with the active site, and the mature domain contains six Ca2+ binding sites. The Ca-1 site is conserved in bacterial subtilisins but is formed prior to autoprocessing, unlike the corresponding sites of bacterial subtilisins. All other Ca2+-binding sites are unique in the pro-S324A structure and are located at the surface loops. Four of them apparently contribute to the stability of the central alphabetaalpha substructure of the mature domain. The CD spectra, 1-anilino-8-naphthalenesulfonic acid fluorescence spectra, and sensitivities to chymotryptic digestion of this protein indicate that the conformation of pro-S324A is changed from an unstable molten globule-like structure to a stable native one upon Ca2+ binding. Another active site mutant, pro-S324C, was shown to be autoprocessed to form a propeptide-mature domain complex in the presence of Ca2+. The CD spectra of this protein indicate that the structure of pro-S324C is changed upon Ca2+ binding like pro-S324A but is not seriously changed upon subsequent autoprocessing. These results suggest that the maturation process of Tk-subtilisin is different from that of bacterial subtilisins in terms of the requirement of Ca2+ for folding of the mature domain and completion of the folding process prior to autoprocessing. 相似文献
174.
Kudou D Misaki S Yamashita M Tamura T Takakura T Yoshioka T Yagi S Hoffman RM Takimoto A Esaki N Inagaki K 《Journal of biochemistry》2007,141(4):535-544
l-Methionine gamma-lyase (EC 4.4.1.11, MGL_Pp) from Pseudomonas putida is a multifunctional enzyme, which belongs to the gamma-family of pyridoxal-5'-phosphate (PLP) dependent enzymes. In this report, we demonstrate that the three-dimensional structure of MGL_Pp has been completely solved by the molecular replacement method to an R-factor of 20.4% at 1.8 A resolution. Detailed information of the overall structure of MGL_Pp supplies a clear picture of the substrate- and PLP-binding pockets. Tyr59 and Arg61 of neighbouring subunits, which are strongly conserved in other gamma-family enzymes, contact the phosphate group of PLP. These residues are important as the main anchor within the active site. Lys240, Asp241 and Arg61 of one partner monomer and Tyr114 and Cys116 of the other partner monomer form a hydrogen-bond network in the MGL active site which is specific for MGLs. It is also suggested that electrostatic interactions at the subunit interface are involved in the stabilization of the structural conformation. The detailed structure will facilitate the development of MGL_Pp as an anticancer drug. 相似文献
175.
Hiyoshi W Sasaki T Takayama-Watanabe E Takai H Watanabe A Onitake K 《Journal of experimental zoology. Part A, Ecological genetics and physiology》2007,307(6):301-311
The acrosome reaction of newt sperm is induced at the surface of egg jelly and the acrosome-reacted sperm acquire the ability to bind to the vitelline envelope. However, because the substance that induces the acrosome reaction has not been identified, the mechanism by which the acrosome-reacted sperm bind to the vitelline envelope remains unclear. We found here that a Dolichos biforus agglutinin (DBA) specifically mimicked the acrosome reaction immediately upon its addition in the presence of milimolar level Ca(2+). Fluorescein isothiocyanate-labeled DBA bound specifically to the acrosomal cap of the intact sperm in the presence of a Ca(2+)-chelating agent, EDTA, suggesting that binding of DBA to the native receptor for the egg jelly substance on the acrosomal region took the place of the egg jelly substance-induced acrosome reaction. In contrast, the sperm that had been acrosome reacted by DBA treatment did not bind to the vitelline envelope of the egg whose jelly layers were removed. Subsequent addition of jelly extract caused the sperm binding to vitelline envelope, indicating that the egg jelly of the newt contains substances that are involved in not only inducing the acrosome reaction but also binding to the vitelline envelope. This is the first demonstration of the involvement of egg jelly substance in the binding of acrosome-reacted sperm to the vitelline envelope. 相似文献
176.
Dingjiang Liu Melanie Cocco Masazumi Matsumura Da Ren Bridget Becker Richard L. Remmele Jr. David N. Brems 《Biomolecular NMR assignments》2007,1(1):93-94
Here we report the NMR resonance assignments for the reduced form of human IgG1 CH3 domain, a 26 kDa dimer in solution (residues 341–447). The assignments have been deposited in the BioMagResBank with a BMRB
accession number of 15204. 相似文献
177.
Ohtsubo T Ohya Y Nakamura Y Kansui Y Furuichi M Matsumura K Fujii K Iida M Nakabeppu Y 《DNA Repair》2007,6(6):760-769
Accumulation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in DNA is associated with mutagenesis and cell death. Little attention has been given to the biological significance of 8-oxo-dG accumulation in cardiovascular tissues during the different stage of hypertension and its prevention. We thus investigated the levels and localization of both 8-oxo-dG accumulation and expression of MTH1, which hydrolyzes 8-oxo-dGTP to prevent its incorporation into DNA, in the thoracic aorta prepared from stroke-prone spontaneously hypertensive rats (SHRSP) and age-matched Wister-Kyoto rats (WKY), aged 5-32 weeks. HPLC-MS/MS analysis revealed that the levels of nuclear 8-oxo-dG in the aorta increased significantly in SHRSP, but not WKY, with aging. Immunohistochemical study revealed that both TUNEL reactivity and 8-oxo-dG immunoreactivity were increased in smooth muscle cells (SMC) and endothelial cells (EC) of the aorta with aging, and they exhibited similar distributions in serial sections. The number of 8-oxo-dG and TUNEL positive cells in EC, but not in SMC, was significantly higher in SHRSP than WKY at 32 weeks of age. In contrast, the expression levels of Mth1mRNA and MTH1 protein in the aorta were similarly decreased both in SHRSP and WKY with aging. However, the number of MTH1 expressing EC was remarkably increased in the older SHRSP compared to the younger ones or age-matched WKY. Hypertension significantly increased not only 8-oxo-dG accumulation but also the expression of MTH1 in EC of the aorta during aging. While accumulation of 8-oxo-dG in SMC of the aorta was slightly increased, the expression of MTH1 protein in SMC was rather decreased by hypertension. We thus suggest that MTH1 may protect EC in the aorta from the oxidative damage increased by hypertension. 相似文献
178.
Takahashi Yoshihiro Khairun Hisam Bin Nasir Akiko Ito Hiroyuki Kanzaki Hideo Matsumura Hiromasa Saitoh Shizuko Fujisawa Sophien Kamoun Ryohei Terauchi 《Plant signaling & behavior》2007,2(5):396-398
A high-throughput in planta overexpression screen of a Nicotiana benthamiana cDNA library identified a mitogen activated protein kinase kinase (MAPKK), NbMKK1, as a potent inducer of hypersensitive response (HR)-like cell death. NbMKK1-mediated cell death was attenuated in plants whereby expression of NbSIPK, an ortholog of tobacco SIPK and Arabidopsis AtMPK6, was knocked down by virus-induced gene silencing (VIGS), suggesting that NbMKK1 functions upstream of NbSIPK. In accordance with this result, NbMKK1 phosphorylated NbSIPK in vitro, and furthermore NbMKK1 and NbSIPK physically interacted in yeast two-hybrid assay. VIGS of NbMKK1 in N. benthamiana resulted in a delay of Phytophthora infestans INF1 elicitin-mediated HR as well as in the reduction of resistance against a non-host pathogen Pseudomonas cichorii. Our data of NbMKK1, together with that of LeMKK4,1 demonstrate the presence of a novel defense signaling pathway involving NbMKK1/LeMKK4 and SIPK.Key Words: MAPK, defense, cell death, in planta screenMitogen activated protein kinase (MAPK) cascades are highly conserved signaling pathways in eukaryotes, comprising three tiered classes of protein kinase, MAPKKK (MAPKK kinase), MAPKK and MAPK, that sequentially relay phosphorylation signals.2 The Arabidopsis genome carries genes for 20 MAPKs, 10 MAPKKs3 and more than 25 MAPKKKs.4 In plants, MAPK signaling is known to function in various biotic4,5 and abiotic6 stress responses and cytokinesis.7 In defense signaling, extensive research has been carried out for two tobacco MAPKs, SIPK8 (salicylic-acid-induced protein kinase; hereafter designated as NtSIPK) and WIPK9 (wound-induced protein kinase = NtWIPK), and their orthologs in Arabidopsis10 (AtMPK6 and ATMPK3, respectively), partly because kinase activities of these two MAPKs are easy to detect by an in gel kinase assay using myeline basic protein (MBP) as substrate.11 Both NtSIPK and NtWIPK are activated by the interaction between host resistance (R)- gene and cognate avirulence gene of pathogen11,12 and elicitor perception by host cells.13,14 Shuqun Zhang and his group showed that an upstream kinase of both NtSIPK and NtWIPK is NtMEK2.15 Transient overexpression of constitutively active NtMEK2 caused phosphorylation of NtSIPK and NtWIPK, resulting in rapid HR-like cell death in tobacco leaves.15 Later, the same lab showed that overexpression of NtSIPK alone also caused HR-like cell death.16 The downstream target proteins of NtSIPK and AtMPK6 are being identified and include 1-aminocyclopropane-1-carboxylic acid sythase-6 (ACS-6).17,18 Although recent studies identified another MAPK cascade (NtMEK1 → Ntf6) involved in defense responses19,20 we can still say that the current research focus of MAPK defense signaling centers around the cascade comprising [NtMEK2→ NtSIPK/NtWIPK→ target proteins] of tobacco and its orthologous pathways in other plant species.In an effort to search for plant genes involved in HR-like cell death, we have been employing a high-throughput in planta expression screen of N. benthamiana cDNA libraries. In this experimental system, a cDNA library was made in a binary potato virus X (PVX)-based expression vector pSfinx.21 The cDNA library was transferred to Agrobacterium tumefaciens, and 40,000 of the bacterial colonies were individually inoculated by toothpicks onto leaf blades of N. benthamiana leaves. The phenotype around the inoculated site was observed 1–2 weeks following the inoculation. This rapid screen identified 30 cDNAs that caused cell death after overexpression, including genes coding for ubiquitin proteins, RNA recognition motif (RRM) containing proteins, a class II ethylene-responsive element binding factor (EREBP)-like protein22 and a MAPKK protein (this work). Such an in planta screening technique has been used before for the isolation of fungal21 and oomycete23,24 elicitors and necrosis inducing genes, but not for isolation of plant genes. Overexpression screening of cDNA libraries is a common practice in prokaryotes, yeast and amimal cells,25,26 so it is a surprise that this approach has not been systematically applied in plants. Given its throughput, we propose that this virus-based transient overexpression system is a highly efficient way to isolate novel plant genes by functional screen.27 Since overexpression frequently causes non-specific perturbation of signaling, genes identified by overexpression should be further validated by loss-of-function assays, for instance, VIGS.28Overexpression of the identified MAPKK gene, NbMKK1, triggered a rapid generation of H2O2, followed by HR-like cell death in N. benthamiana leaves (this work). NbMKK1-GFP fusion protein overexpression also caused cell death, and curiously NbMKK1-GFP was shown to localize consistently in the nucleus. Sequence comparison classified NbMKK1 to the Group D of MAPKKs about which little information is available. So far, a MAPKK, LeMKK4, from tomato belonging to the Group D MAPKKs, was shown to cause cell death after overexpression.1 Based on amino acid sequence similarity and phylogenetic analyses, LeMKK4 and NbMKK1 seem to be orthologs. To see whether NbMKK1 transduces signals through SIPK and WIPK, we performed NbMKK1 overexpression in N. benthamiana plants whereby the expression of either NbSIPK or NbWIPK (WIPK ortholog in N. benthamiana) was silenced by VIGS. NbMKK1 did not induce cell death in NbSIPK-silenced plants, suggesting that the NbMKK1 cell death signal is transmitted through NbSIPK. Indeed, NbMKK1 phosphorylated NbSIPK in vitro, and NbMKK1 and NbSIPK physically interacted in yeast two-hybrid assay. These results suggest that NbMKK1 interacts with NbSIPK, most probably with its N-terminal docking domain, and phosphorylates NbSIPK in vivo to transduce the cell death signal downstream.NbMKK1 exhibits constitutive expression in leaves. To determine the function of NbMKK1 in defense, we silenced NbMKK1 by VIGS, and such plants were challenged with Phytophthora infestans INF1 elicitin29 and Pseudomonas cichorii, a non-host pathogen. INF1-mediated HR cell death was remarkably delayed in NbMKK1-silenced plants. Likewise, plant defense against P. cichorii was compromised in NbMKK1-silenced plants. These results indicate that NbMKK1 is an important component of signaling of INF1-mediated HR and non-host resistance to P. cichorii.Together, our analyses of NbMKK1 and independent work from Greg Martin''s lab on LeMKK41 suggest that a Group D MAPKK, NbMKK1/LeMKK4, functions upstream of SIPK and transduces defense signals in these solanaceous plants (Fig. 1). In plants as well as in other eukaryotes, it is common that kinases have multiple partners. The work on these kinases fits this concept. A single MAPK (e.g., SIPK) is phosphorylated by multiple MAPKKs (e.g., NtMEK2 and NbMKK1), and a single MAPKK (e.g., NtMEK2) can phosphorylate multiple MAPKs (e.g., NtSIPK and NtWIPK).Open in a separate windowFigure 1Defense signaling through NbMKK1/LeMKK4. Two defense signal pathways involving NtMEK2 (indicated as MEK2) → WIPK/SIPK and NtMEK1(indicated as MEK1) → Ntf6 are well documented. By our and Pedley and Martin''s1 works, another novel MAPKK, NbMKK1/LeMKK4 was demonstrated to participate in defense signaling by phosphorylation of SIPK. 相似文献
179.
Hirotoshi Motoda Takayuki Sasaki Yoshio Kano Peter R Ryan Emmanuel Delhaize Hideaki Matsumoto Yoko Yamamoto 《Plant signaling & behavior》2007,2(6):467-472
The wheat ALMT1 gene encodes an aluminum (Al)-activated malate transport protein which confers Al-resistance. We investigated the membrane topology of this plasma-membrane localized protein with immunocytochemical techniques. Several green fluorescent protein (GFP)-fused and histidine (His)-tagged chimeras of ALMT1 were prepared based on a computer-predicted secondary structure and transiently expressed in cultured mammalian cells. Antibodies raised to polypeptide epitopes of ALMT1 were used in conjunction with the antibody to the His-tags to determine the topology of ALMT1. This study shows that the ALMT1 protein contains six transmembrane domains with the amino and carboxyl termini located on the extracellular side of the plasma membrane.Key Words: ALMT1, aluminum resistance, immunofluorescent staining, malate transporter, topology, Triticum aestivum 相似文献
180.
Cypripedium macranthos var. rebunense is an endangered plant endemic to Rebun Island, Japan. A proper understanding of genetic diversity is needed when conducting
conservation programs for rare and endangered species. We therefore examined the genetic diversity of C. macranthos var. rebunense using allozyme markers with a view to future conservation. Our study revealed that C. macranthos var. rebunense has relatively high genetic diversity (P was 0.62, n
a and n
e were 1.85 and 1.28 respectively, and H
o and H
e were 0.163 and 0.187, respectively) when compared with other plant taxa. The natural habitats of C. macranthos var. rebunense are geographically separated into northern and the southern populations. Disappearance of alleles and increase in homozygosity
expected as a result of the bottleneck effect were observed, particularly in the southern populations composed of a small
number of plants. As additional negative effects (inbreeding depression and further genetic drift) due to fragmentation are
predicted in these populations, the southern populations may show deterioration of genetic diversity in the near future. 相似文献