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61.
The association of Nef with a cellular serine/threonine kinase and its enhancement of infectivity are viral isolate dependent. 总被引:3,自引:3,他引:0 下载免费PDF全文
The nef genes of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) encode a 27- to 34-kDa myristoylated protein which induces downregulation of CD4 surface levels and enhances virus infectivity. In adult macaques, Nef has been implicated in pathogenesis and disease progression. Both HIV-1 SF2 Nef and SIVmac239 Nef have been shown to associate with a cellular serine/threonine kinase. We tested five functional Nef isolates to examine whether this kinase association is a property conserved among different isolates. HIV-1 SF2 and 248 and SIVmac239 Nef proteins were found associated with the kinase. HIV-1 NL4-3 and 233 Nef proteins were found weakly associated or not associated with the kinase. All five Nef isolates efficiently downregulated CD4 cell surface expression, suggesting that the association with this cellular kinase is not required for Nef to downregulate CD4. Comparison of the SF2 and NL4-3 isolates shows a differential ability of Nef to enhance infectivity that suggests a possible correlation between kinase association and enhancement of infectivity. 相似文献
62.
Characterization of inhibition of M2 ion channel activity by BL-1743, an inhibitor of influenza A virus. 下载免费PDF全文
Q Tu L H Pinto G Luo M A Shaughnessy D Mullaney S Kurtz M Krystal R A Lamb 《Journal of virology》1996,70(7):4246-4252
The influenza A virus M2 integral membrane protein has ion channel activity that can be inhibited by the antiviral drug amantadine. Recently, a spirene-containing compound, BL-1743 (2-[3-azaspiro (5,5)undecanol]-2-imidazoline), that inhibits influenza virus growth was identified (S. Kurtz, G. Lao, K. M. Hahnenberger, C. Brooks, O. Gecha, K. Ingalls, K.-I. Numata, and M. Krystal, Antimicrob. Agents Chemother. 39:2204-2209, 1995). We have examined the ability of BL-1743 to inhibit the M2 ion channel when expressed in oocytes of Xenopus laevis. BL-1743 inhibition is complete as far as can be measured by electrophysiological methods and is reversible, with a reverse reaction rate constant of 4.0 x 10(-3) s(-1). In contrast, amantadine inhibition is irreversible within the time frame of the experiment. However, BL-1743 inhibition and amantadine inhibition have similar properties. The majority of isolated influenza viruses resistant to BL-1743 are also amantadine resistant. In addition, all known amino acid changes which result in amantadine resistance also confer BL-1743 resistance. However, one BL-1743-resistant virus isolated, designated M2-I35T, contained the change Ile-35-->Thr. This virus is >70-fold more resistant to BL-1743 and only 10-fold more resistant to amantadine than the wild-type virus. When the ion channel activity of M2-I35T was examined in oocytes, it was found that M2-I35T is BL-1743 resistant but is reversibly inhibited by amantadine. These findings suggest that these two drugs interact differently with the M2 protein transmembrane pore region. 相似文献
63.
64.
用菠菜甜菜碱醛脱氢酶 ( BADH)免疫巴比西 ( BALB/c)小鼠 ,将其脾细胞与骨髓瘤细胞 SP2 /O-Ag1 4融合 ,在 1 92孔中 ,有约 1 4 %孔生长的杂交瘤细胞 ,用间接酶联免疫方法 ( ELISA)检测表现为阳性。选择其中 2 G3和 2 D10 细胞系 ,用有限稀释法进行克隆化培养 ,约 2 0 %克隆化细胞为强阳性。选择其中 2 G3- H3细胞株注射到 BALB/c小鼠腹腔中诱导腹水 ,腹水的单抗效价为 1∶ 1 0 3。应用 BADH单抗检查了大麦、水稻、高粱、小麦幼苗的叶片和根的粗提物 ,均呈阳性反应 ,表明 BADH除在光合组织中存在外 ,在非光合组织中也可能存在。讨论了非光合组织 BADH的意义 相似文献
65.
棕色固氮菌缺失nifZ基因的突变种固氮酶MoFe蛋白的纯化和性质 总被引:1,自引:0,他引:1
采用 52℃下加热 6 min,后经 DEAE- 52、Sephacryls S- 2 0 0和 Q- Sepharose等柱层析方法 ,分离纯化了棕色固氮菌 (Azotobacter vinelandii)缺失 nif Z基因突变种固氮酶 Mo Fe(Δnif Z Mo Fe)蛋白 ,其纯度达到电泳纯。Δnif Z Mo Fe蛋白的固氮活性为 2 83nmol C2 H2 还原 / (min·mg蛋白 ) ,远低于野生种 Mo Fe蛋白。Δnif Z Mo Fe蛋白对氧更敏感 ;热稳定性略低于野生种。Δnif Z Mo Fe蛋白的可见光吸收光谱与野生种 Mo Fe蛋白极为相似。其圆二色谱和磁圆二色谱在 450~ 550 nm与野生种 Mo Fe蛋白显著不同 ,表明其 P- cluster及其周围环境与野生种 Mo Fe蛋白有所差异。这亦可能是造成缺失 nif Z突变种 Mo Fe蛋白固氮活性低的原因。 相似文献
66.
67.
Signaling by chimeric erythropoietin-TGF-beta receptors: homodimerization of the cytoplasmic domain of the type I TGF-beta receptor and heterodimerization with the type II receptor are both required for intracellular signal transduction. 总被引:4,自引:1,他引:3 下载免费PDF全文
Transforming growth factor-beta (TGF-beta) affects multiple cellular functions through the type I and type II receptor Ser/Thr kinases (TbetaRI and TbetaRII). Analysis of TGF-beta signaling pathways has been hampered by the lack of cell lines in which both TbetaRI and TbetaRII are deleted, and by the inability to study signal transduction by TbetaRI independently of TbetaRII since TbetaRI does not bind TGF-beta directly. To overcome these problems, we constructed and expressed chimeric receptors with the extracellular domain of the erythropoietin receptor (EpoR) and the cytoplasmic domains of TbetaRI or TbetaRII. When expressed in Ba/F3 cells, which do not express EpoR, Epo induces the formation of a heteromeric complex between cell surface EpoR-TbetaRI and EpoR-TbetaRII chimeras. Neither the EpoR-TbetaRI nor the EpoR-TbetaRII chimera interacts with endogenous TGF-beta receptors. Ba/F3 cells expressing both EpoR-TbetaRI and EpoR-TbetaRII chimeras, but not EpoR-TbetaRI or EpoR-TbetaRII alone, undergo Epo-induced growth arrest. When expressed in Ba/F3 cells in the absence of the EpoR-TbetaRII chimera, EpoR-TbetaRI(T204D), a chimeric receptor with a point mutation in the GS domain of TbetaRI that is autophosphorylated constitutively, triggers growth inhibition in response to Epo. Thus, both homo- and heterodimerization of the cytoplasmic domain of the type I TGF-beta receptor are required for intracellular signal transduction leading to inhibition of cell proliferation. These chimeric receptors provide a unique system to study the function and signal transduction of individual TGF-beta receptor subunits independently of endogenous TGF-beta receptors. 相似文献
68.
Y Luo S O Marx H Kiyokawa A Koff J Massagu A R Marks 《Molecular and cellular biology》1996,16(12):6744-6751
The potent antiproliferative activity of the macrolide antibiotic rapamycin is known to involve binding of the drug to its cytosolic receptor, FKBP12, and subsequent interaction with targets of rapamycin, resulting in inhibition of p70 S6 kinase (p70S6K). However, the downstream events that lead to inhibition of cell cycle progression remain to be elucidated. The antiproliferative effects of rapamycin are associated with prevention of mitogen-induced downregulation of the cyclin-dependent kinase inhibitor p27Kip1, suggesting that the latter may play an important role in the growth pathway targeted by rapamycin. Murine BC3H1 cells, selected for resistance to growth inhibition by rapamycin, exhibited an intact p70S6K pathway but had abnormally low p27 levels that were no longer responsive to mitogens or rapamycin. Fibroblasts and T lymphocytes from mice with a targeted disruption of the p27Kip1 gene had impaired growth-inhibitory responses to rapamycin. These results suggest that the ability to regulate p27Kip1 levels is important for rapamycin to exert its antiproliferative effects. 相似文献
69.
DNA ligases play a pivotal role in DNA replication, repair and recombination. Reactions catalyzed by DNA ligases consist of three steps: adenylation of the ligase in the presence of ATP or NAD+, transferring the adenylate moiety to the 5'-phosphate of the nicked DNA substrate (deadenylation) and sealing the nick through the formation of a phosphodiester bond. Thermus thermophilus HB8 DNA ligase (Tth DNA ligase) differs from mesophilic ATP-dependent DNA ligases in three ways: (i) it is NAD+ dependent; (ii) its optimal temperature is 65 instead of 37 degrees C; (iii) it has higher fidelity than T4 DNA ligase. In order to understand the structural basis underlying the reaction mechanism of Tth DNA ligase, we performed site-directed mutagenesis studies on nine selected amino acid residues that are highly conserved in bacterial DNA ligases. Examination of these site-specific mutants revealed that: residue K118 plays an essential role in the adenylation step; residue D120 may facilitate the deadenylation step; residues G339 and C433 may be involved in formation of the phosphodiester bond. This evidence indicates that a previously identified KXDG motif for adenylation of eukaryotic DNA ligases [Tomkinson, A.E., Totty, N.F., Ginsburg, M. and Lindahl, T. (1991) Proc. Natl. Acad. Sci. USA, 88, 400-404] is also the adenylation site for NAD+-dependent bacterial DNA ligases. In a companion paper, we demonstrate that mutations at a different Lys residue, K294, may modulate the fidelity of Tth DNA ligase. 相似文献
70.
Hong Luo Benoît Van Coppenolle Marc Seguin Marc Boutry 《Molecular breeding : new strategies in plant improvement》1995,1(1):51-63
Using fourteen random mitochondrial DNA probes, we have examined restriction fragment length polymorphism (RFLP) in wild and cultivatedHevea brasiliensis. A total of 395 accessions, including 345 from various prospectings collected in Brazil, Colombia and Peru and 50 cultivated clones, were analyzed. Two other species (H. benthamiana andH. pauciflora) were also included in the study for comparison. The high level of mitochondrial polymorphism allowed us to divide all the accessions analyzed into 212 distinct genotypes. The genetic variability of cultivated clones was limited to four genotypes forming two clusters. In contrast, considerable genetic variation was found in the wild collections. In almost all cases, accessions displaying the same RFLP profile were restricted to the same geographical area (same or neighbor administrative districts). In addition, accessions whose genetic closeness was predicted by RFLP profiles were also clustered according to geographical origin. In a few cases, however, similar RFLP profiles were found for accessions originating from geographically distant districts. This discrepancy can be explained either by seed dispersion (by river) or possibly by similar genetic events occurring independently in different geographical locations. Chloroplast DNA RFLP was also analyzed in 217 accessions, representative of 126 distinct mitochondrial genotypes. Very few differences were found, indicating that the chloroplast genome is more highly conserved than the mitochondrial genome. 相似文献