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91.
Tagaya Y Osaki A Miura A Okada S Ohshima K Hashimoto K Yamada M Satoh T Shimizu H Mori M 《Protein and peptide letters》2012,19(9):997-1004
Nucleobindin-2 is a 420 amino acid EF-hand Ca2+ binding protein that can be further processed to generate an 82 amino terminal peptide termed Nesfatin-1. To examine the function of secreted Nucleobindin-2 in adipocyte differentiation, cultured 3T3-L1 cells were incubated with either 0 or 100 nM of GST, GST-Nucleobindin-2, prior to and during the initiation of adipocyte differentiation. Nucleobindin-2 treatment decreased neutral lipid accumulation (Oil-Red O staining) and expression of several marker genes for adipocyte differentiation (PPARγ, aP2, and adipsin). When Nucleobindin- 2 was constitutively secreted into cultured medium, cAMP content and insulin stimulated CREB phosphorylation were significantly reduced. On the other hand, intracellularly overexpressed Nucleobindin-2 failed to affect cAMP content and CREB phosphorylation. Taken together, these data indicate that secreted Nucleobindin-2 is a suppressor of adipocyte differentiation through inhibition of cAMP production and insulin signal. 相似文献
92.
Butler MO Imataki O Yamashita Y Tanaka M Ansén S Berezovskaya A Metzler G Milstein MI Mooney MM Murray AP Mano H Nadler LM Hirano N 《PloS one》2012,7(1):e30229
Background
Using in vivo mouse models, the mechanisms of CD4+ T cell help have been intensively investigated. However, a mechanistic analysis of human CD4+ T cell help is largely lacking. Our goal was to elucidate the mechanisms of human CD4+ T cell help of CD8+ T cell proliferation using a novel in vitro model.Methods/Principal Findings
We developed a genetically engineered novel human cell-based artificial APC, aAPC/mOKT3, which expresses a membranous form of the anti-CD3 monoclonal antibody OKT3 as well as other immune accessory molecules. Without requiring the addition of allogeneic feeder cells, aAPC/mOKT3 enabled the expansion of both peripheral and tumor-infiltrating T cells, regardless of HLA-restriction. Stimulation with aAPC/mOKT3 did not expand Foxp3+ regulatory T cells, and expanded tumor infiltrating lymphocytes predominantly secreted Th1-type cytokines, interferon-γ and IL-2. In this aAPC-based system, the presence of autologous CD4+ T cells was associated with significantly improved CD8+ T cell expansion in vitro. The CD4+ T cell derived cytokines IL-2 and IL-21 were necessary but not sufficient for this effect. However, CD4+ T cell help of CD8+ T cell proliferation was partially recapitulated by both adding IL-2/IL-21 and by upregulation of IL-21 receptor on CD8+ T cells.Conclusions
We have developed an in vitro model that advances our understanding of the immunobiology of human CD4+ T cell help of CD8+ T cells. Our data suggests that human CD4+ T cell help can be leveraged to expand CD8+ T cells in vitro. 相似文献93.
Tomoko Ichiyanagi Kenji Ichiyanagi Miho Miyake Hiroyuki Sasaki 《Nucleic acids research》2013,41(2):738-745
DNA methylation is a well-characterized epigenetic modification involved in gene regulation and transposon silencing in mammals. It mainly occurs on cytosines at CpG sites but methylation at non-CpG sites is frequently observed in embryonic stem cells, induced pluriotent stem cells, oocytes and the brain. The biological significance of non-CpG methylation is unknown. Here, we show that non-CpG methylation is also present in male germ cells, within and around B1 retrotransposon sequences interspersed in the mouse genome. It accumulates in mitotically arrested fetal prospermatogonia and reaches the highest level by birth in a Dnmt3l-dependent manner. The preferential site of non-CpG methylation is CpA, especially CpApG and CpApC. Although CpApG (and CpTpG) sites contain cytosines at symmetrical positions, hairpin-bisulfite sequencing reveals that they are hemimethylated, suggesting the absence of a template-dependent copying mechanism. Indeed, the level of non-CpG methylation decreases after the resumption of mitosis in the neonatal period, whereas that of CpG methylation does not. The cells eventually lose non-CpG methylation by the time they become spermatogonia. Our results show that non-CpG methylation accumulates in non-replicating, arrested cells but is not maintained in mitotically dividing cells during male germ-cell development. 相似文献
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Yuriko Uehara Katsutoshi Oda Yuji Ikeda Takahiro Koso Shingo Tsuji Shogo Yamamoto Kayo Asada Kenbun Sone Reiko Kurikawa Chinami Makii Otoe Hagiwara Michihiro Tanikawa Daichi Maeda Kosei Hasegawa Shunsuke Nakagawa Osamu Wada-Hiraike Kei Kawana Masashi Fukayama Keiichi Fujiwara Tetsu Yano Yutaka Osuga Tomoyuki Fujii Hiroyuki Aburatani 《PloS one》2015,10(6)
Ovarian clear cell carcinoma (CCC) is generally associated with chemoresistance and poor clinical outcome, even with early diagnosis; whereas high-grade serous carcinomas (SCs) and endometrioid carcinomas (ECs) are commonly chemosensitive at advanced stages. Although an integrated genomic analysis of SC has been performed, conclusive views on copy number and expression profiles for CCC are still limited. In this study, we performed single nucleotide polymorphism analysis with 57 epithelial ovarian cancers (31 CCCs, 14 SCs, and 12 ECs) and microarray expression analysis with 55 cancers (25 CCCs, 16 SCs, and 14 ECs). We then evaluated PIK3CA mutations and ARID1A expression in CCCs. SNP array analysis classified 13% of CCCs into a cluster with high frequency and focal range of copy number alterations (CNAs), significantly lower than for SCs (93%, P < 0.01) and ECs (50%, P = 0.017). The ratio of whole-arm to all CNAs was higher in CCCs (46.9%) than SCs (21.7%; P < 0.0001). SCs with loss of heterozygosity (LOH) of BRCA1 (85%) also had LOH of NF1 and TP53, and LOH of BRCA2 (62%) coexisted with LOH of RB1 and TP53. Microarray analysis classified CCCs into three clusters. One cluster (CCC-2, n = 10) showed more favorable prognosis than the CCC-1 and CCC-3 clusters (P = 0.041). Coexistent alterations of PIK3CA and ARID1A were more common in CCC-1 and CCC-3 (7/11, 64%) than in CCC-2 (0/10, 0%; P < 0.01). Being in cluster CCC-2 was an independent favorable prognostic factor in CCC. In conclusion, CCC was characterized by a high ratio of whole-arm CNAs; whereas CNAs in SC were mainly focal, but preferentially caused LOH of well-known tumor suppressor genes. As such, expression profiles might be useful for sub-classification of CCC, and might provide useful information on prognosis. 相似文献
98.
Yoshikazu Furusawa Shinji Yamada Shunsuke Itai Takuro Nakamura Miyuki Yanaka Masato Sano Hiroyuki Harada Masato Fukui Mika K. Kaneko Yukinari Kato 《Biochemistry and Biophysics Reports》2019
Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, and bovine podoplanin (PDPN), a lymphatic endothelial cell marker, have been established in our previous studies. However, mAbs against horse PDPN (horPDPN), which are useful for immunohistochemical analysis, remain to be developed. In the present study, mice were immunized with horPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/horPDPN), and hybridomas producing mAbs against horPDPN were screened using flow cytometry. One of the mAbs, PMab-219 (IgG2a, kappa), specifically detected CHO/horPDPN cells via flow cytometry and recognized horPDPN protein using Western blotting. Furthermore, PMab-219 strongly stained CHO/horPDPN via immunohistochemistry. These findings suggest that PMab-219 is useful for investigating the function of horPDPN. 相似文献
99.
Hiroyuki Kimura Saki Yamauchi Hidekazu Kawashima Kenji Arimitsu Yusuke Yagi Yuji Nakamoto Kaori Togashi Masahiro Ono Hideo Saji 《Bioorganic & medicinal chemistry letters》2018,28(17):2949-2952
The tripeptide formyl–Met–Leu–Phe (fMLF) is a prototype of N-formylated chemotactic peptides for neutrophils owing to its ability to bind and activate the G protein-coupled formyl peptide receptor (FPR). Here, we developed an 18F-labeled fMLF derivative targeting FPR as a positron emission tomography (PET) imaging probe for bacterial infections. The study demonstrates that the fMLF derivative fMLFXYk(FB)k (X?=?Nle) has a high affinity for FPR (Ki?=?0.62?±?0.13?nM). The radiochemical yield and purity of [18F]fMLFXYk(FB)k were 16% and >96%, respectively. The in vivo biodistribution study showed that [18F]fMLFXYk(FB)k uptake was higher in the bacterial infected region than in the non-infected region. We observed considerably higher infection-to-muscle ratio of 4.6 at 60?min after [18F]fMLFXYk(FB)k injection. Furthermore, small-animal PET imaging studies suggested that [18F]fMLFXYk(FB)k uptake in the bacterial infected region was clearly visualized 60?min after injection. 相似文献
100.
Ozaki T Nakazawa M Yamashita T Sorimachi H Hata S Tomita H Isago H Baba A Ishiguro S 《Biochimica et biophysica acta》2012,1822(11):1783-1795
Mitochondrial μ-calpain initiates apoptosis-inducing factor (AIF)-dependent apoptosis in retinal photoreceptor degeneration. Mitochondrial μ-calpain inhibitors may represent therapeutic targets for the disease. Therefore, we sought to identify inhibitors of mitochondrial calpains and determine their effects in Royal College of Surgeons' (RCS) rats, an animal model of retinitis pigmentosa (RP). We synthesized 20-mer peptides of the C2-like (C2L) domain of μ-calpain. Two μ-calpain peptides N2 and N9 inhibited mitochondrial μ-calpain activity (IC(50); 892 and 498nM, respectively), but not other proteases. Western blotting showed that 50μM of both μ-calpain peptides caused specific degradation of mitochondrial μ-calpain. Three-dimensional structure of calpains suggested that the peptides N2 and N9 corresponded to the regions forming salt bridges between the protease core domain 2 and the C2L domain. We determined the inhibitory regions of μ-calpain peptides N2 and N9 using 10-mers, and one peptide, N2-10-2, inhibited the activity of mitochondrial μ-calpain (IC(50); 112nM). We next conjugated the peptide N2-10-2 to the C-terminal of HIV-1 tat (HIV), a cell-penetrating peptide. Using isolated rat liver mitochondria, 50μM HIV-conjugated μ-calpain N2-10-2 peptide (HIV-Nμ, IC(50); 285nM) significantly inhibited AIF truncation. The intravitreal injection of 20mM HIV-Nμ also prevented retinal photoreceptor apoptosis determined by TUNEL staining, and preserved retinal function assessed by electroretinography in RCS rats. Topical application of 40mM HIV-Nμ also prevented apoptosis of retinal photoreceptors in RCS rats. Our results demonstrate that HIV-Nμ, a peptide inhibitor of mitochondrial μ-calpain, offers a new modality for treating RP. 相似文献