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831.
Jie Li Rui Wen Parkeer Andersen Yuping Liang Qing Li Wei Xiao Zongbin Cui 《Molecular and cellular biochemistry》2010,343(1-2):173-182
Ubiquitination is an important post-translational protein modification that functions in diverse cellular processes of all eukaryotic organisms. Conventional Lys48-linked poly-ubiquitination leads to the degradation of specific proteins through 26S proteasomes, while Lys63-linked polyubiquitination appears to regulate protein activities in a non-proteolytic manner. To date, Ubc13 is the only known ubiquitin-conjugating enzyme capable of poly-ubiquitinating target proteins via Lys63-linked chains, and this activity absolutely requires a Ubc variant (Uev or Mms2) as a co-factor. However, Lys63-linked poly-ubiquitination and error-free DNA damage tolerance in zebrafish are yet to be defined. Here, we report molecular cloning and functional characterization of two zebrafish ubc13 genes, ubc13a and ubc13b. Analysis of their genomic structure, nucleotide and protein sequence indicates that the two genes are highly conserved during evolution and derived from whole genome duplication. Zebrafish Ubc13 proteins are able to physically interact with yeast or human Mms2 and both zebrafish ubc13 genes are able to functionally complement the yeast ubc13 null mutant for spontaneous mutagenesis and sensitivity to DNA damaging agents. In addition, upon DNA damage, the expression of zebrafish ubc13a and ubc13b is induced during embryogenesis and zebrafish Ubc13 is associated with nuclear chromatin. These results suggest the involvement of Lys63-linked poly-ubiquitylation in DNA damage response in zebrafish. 相似文献
832.
Hongbin Zhang Jie Wu Yu Zhang Na Fu Jie Wang Shujin Zhao 《Molecular biology reports》2010,37(7):3089-3095
A prokaryotic expression system has been used to produce recombinant human bone morphogenetic protein-2 (rhBMP-2). However,
low rhBMP-2 yields and protein loss during purification and renaturation are the hurdles in the clinical application. Previous
studies have indicated that variables such as temperature, host cell, salt concentration, and culture time affect the final
rhBMP-2 yield. The optimization of these conditions in an Escherichia coli culture yielded 28.258 mg of rhBMP-2 per liter of culture. To reduce rhBMP-2 loss during purification and renaturation, we
performed purification before renaturation in the prokaryotic expression system instead of using the traditional renaturation-before-purification
approach. rhBMP-2 was separated on a Sephacryl S-300 HR column and eluted from a DEAE-Sepharose Fast Flow column. The collected
protein was refolded by dialysis with urea buffer, which was followed by dialysis with ultrapure water. The purified rhBMP-2
dimer significantly increased alkaline phosphatase (ALP) activity and osteogenic activity in the femoral muscle and showed
the same level of bone-forming activity as natural BMP-2. This optimized procedure for expression and renaturation of rhBMP-2
has potential clinical applications. 相似文献
833.
Jia-peng Bao Wei-ping Chen Jie Feng Peng-fei Hu Zhong-li Shi Li-dong Wu 《Molecular biology reports》2010,37(7):3265-3272
Leptin has been shown to play a crucial role in the regulation of body weight. There is also evidence that this adipokine
plays a key role in the process of osteoarthritis. However, the precise role of leptin on articular cartilage metabolism is
not clear. We investigate the role of leptin on articular cartilage in vivo in this study. Recombinant rat leptin (100 μg)
was injected into the knee joints of rats, 48 h later, messenger RNA (mRNA) expression and protein levels of basic fibroblast
growth factor (bFGF), vascular endothelial growth factor (VEGF), matrix metalloproteinases 2 and 9 (MMP-2, MMP-9), cathepsin
D, and collagen II from articular cartilage were analyzed by real-time quantitative polymerase chain reaction (PCR) and western
blot. Two important aggrecanases ADAMTS-4 and -5 (a disintegrin and metalloproteinase with thrombospondin motifs 4 and 5)
were also analyzed by real-time quantitative PCR. Besides, articular cartilage was also assessed for proteoglycan/GAG content
by Safranin O staining. Leptin significantly increased both gene and protein levels of MMP-2, MMP-9, cathepsin D, and collagen
II, while decreased bFGF markedly in cartilage. Moreover, the gene expression of ADAMTS-4 and -5 were markedly increased,
and histologically assessed depletion of proteoglycan in articular cartilage was observed after treatment with leptin. These
results strongly suggest that leptin plays a catabolic role on cartilage metabolism and may be a disadvantage factor involve
in the pathological process of OA. 相似文献
834.
Although glucose-6-phosphate isomerase (GPI) plays an important role in glycolysis of both the prokaryotes and eukaryotes,
studies on the GPI have not been involved in the halotolerant, unicellular green alga Dunaliella salina (D. salina). In this study, a 2,338 bp of full-length cDNA cloned using rapid amplification of cDNA end (RACE) technique contained an
open reading frame (ORF) of 1,980 bp encoding 660 amino acids, which has a predicted molecular weight of 73.3 kD and pI of
6.22 and shares high homology with other organisms. The cloned full-length cDNA was heterologously expressed in Escherichia coli and the recombinant GPI proteins purified using Ni-NTA His Bind column were consistent with the anticipated size of ~75 kD.
Predicted 2D and 3D structures of GPI proteins possessed potential active motifs including “GEPGTNGQHSFYQLIHQG” and “VQGFIWGINSFDQWGVELGK”,
and critical active site residues, such as Ser 241, Ser 296, Thr 298, Thr 301, Arg 358, Glu 444, His 475 and Lys 600. Real
time quantitative RT-PCR demonstrated that the expression level of the GPI gene from D. salina (DsGPI) was induced by 3.5 M NaCl with 14-fold higher than that by 1.5 M NaCl (P < 0.01), but inhibited by the light with 4-fold lower than that in the dark (P < 0.05). It is concluded that the cloned GPI gene is indeed from D. salina and may respond to salt and light. 相似文献
835.
Qingyu Lang Haoxing Zhang Jie Li Fang Xie Yifeng Zhang Bo Wan Long Yu 《Molecular biology reports》2010,37(3):1577-1583
The Aurora kinases play a critical role in mitosis and have been suggested as promising targets for cancer therapy due to
their frequent overexpression in a variety of tumors. Compared with established inhibitors of cell division such as the anti-tubulins,
novel agents target mitotic enzymes and show similar efficacy but with fewer side effects. Several small-molecule inhibitors
of Aurora kinases have been developed as anticancer agents, some of which have progressed to early clinical evaluation. Here
we identified 3-hydroxyflavone as a novel Aurora B inhibitor through high throughput screening. 3-Hydroxyflavone showed potent
inhibition to Aurora B with the IC50 on a nanomolar basis in the enzyme-based kinase activity assay. In the cell-based western blotting analysis, 3-hydroxyflavone
dramatically decreased the phosphorylation level of Histone H3 on the site of serine 10, demonstrating the potent endogenous
Aurora B activity inhibition in cell level. The followed cell image analysis provided the consist result. To make it clear
whether 3-hydroxyflavone inhibited Aurora B by direct binding or not, SPR analysis was carried out to measure the affinity
of interaction between Aurora B protein and 3-hydroxyflavone and the result proved the binding with high affinity. Usually
Aurora activity suppression induced cancer cell proliferation inhibition. Colony formation and cell viability with/without
treatment of 3-hydroxyflavone were measured using CCK-8. The growth suppression under 3-hydroxyflavone present and the growth
recovery after being released gave strong evidence that presence of 3-hydroxyflavone efficiently inhibited the fast growth
of cancer cells. 相似文献
836.
837.
Wei-Ping Chen Jia-Peng Bao Peng-Fei Hu Jie Feng Li-Dong Wu 《Molecular biology reports》2010,37(8):3967-3972
This study investigated the effects of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) on cartilage degradation in an experimental model of osteoarthritis (OA). Thirty-two male New Zealand rabbits underwent unilateral anterior cruciate ligament transection (ACLT) on left knee joints to induce OA and were randomly divided into two groups (n = 16), the TSA group was injected intra-articularly with 0.3 ml TSA [250 ng/ml in the dimethylsulphoxide (DMSO)], the OA group received DSMO since 4 weeks after operation once a week for 5 weeks. Rabbits were killed seven days after the last injection. Left knee cartilage was harvested for morphological, histological and genetic analysis. Another ten rabbits were used for normal control and received no injection. The TSA group showed less cartilage degradation as compared to the OA group assessed by morphological and histological evaluation. Gene expression of matrix metalloproteinase-1 (MMP-1), MMP-3, MMP-13, and interleukin-1 (IL-1) was increased significantly in the OA group compared to the normal group. The elevated expression was reduced by TSA. Our results suggest that TSA could be considered as a potential agent for treatment for OA. 相似文献
838.
Miao Yin Liang Zhang Xiao-ming Sun Liu-feng Mao Jie Pan 《Molecular biology reports》2010,37(4):2049-2054
To investigate the effect of apolipoprotein E (apoE) on cytokine expression profile of the liver of young mice, quantitative
RT-PCR (qRT-PCR) assay and cytokine antibody array for multiplex analysis of 62 cytokines have been used to analyze characteristics
of expression of cytokines in the liver of 6-week-old apoE-null (apoE−/−) mice. The levels of plasma cytokines were also analyzed. The mRNA level of IL-1β, IL-2, IL-6, ICAM-1, VCAM-1, MCP-1, NF-κB
(p65), IFN-γ and IκB-α were increased significantly in apoE−/− mice comparative to wild-type (WT) mice. IL-4, IL-10 and GM-CSF, however, were slightly decreased. Compared with WT, levels
of 21 cytokines altered twofold or more in apoE−/− mice, including 10 cytokines increased and 11 decreased. Expression patterns of IL-1β, IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-γ
and VCAM-1 showed identical trend between cytokine antibody array and qRT-PCR analysis. Moreover, levels of IL-1β, IFN-γ and
IL-6 in the plasma were elevated, while IL-4 was lightly decreased in apoE−/− mice compared to those in WT mice. These results implied that promotion of type I immune response in the liver of young apoE−/− mice due to alteration of these cytokines, and the phenotypes may be caused by the regulation of NF-κB. The inflammation
and lipid metabolism dysfunction in the liver cooperated in dysfunction of the liver in young apoE−/− mice. 相似文献
839.
Xin-Hong Guo Ke-Qin Deng Jie Wang Da-Shi Yu Qiong Zhao Xuan-Ming Liu 《Molecular biology reports》2010,37(2):763-769
The homozygous T-DNA mutant of the PP2CA2 gene in Arabidopsis thaliana was identified at DNA and RNA levels. The semi-quantitative RT-PCR analysis showed expression of PP2CA2 was induced by NaCl and ABA. When grown in presence of increasing concentration of exogenous ABA the pp2ca2 mutant showed a significant loss of ABA sensitivity in terms of seed germination, efficiency of post-germination growth and
root growth. In presence of all ABA and NaCl concentrations tested the germination percentage of wild-type seeds was lower
than that of mutant ppca2 seeds. Furthermore, in the presence of exogenous ABA, the pp2ca2 seeds showed higher germination percentages than wild-type at different stages of development and the pp2ca2 seedlings showed a reduced inhibition of root growth compared with wild-type plants. The above results indicated that the
pp2ca2 was an ABA-hyposensitive mutant. 相似文献
840.
Elymus L. is the largest genus in Triticeae, containing about 150 species with four recognized genome donors (St, H, P, and W).
Traditionally, the genome compound of this genus is identified based on cytological data. Recently, molecular phylogenetic
analysis was used to investigate its genomic combination. Here we describe a restriction fragment length polymorphism (RFLP)
assay based on digesting alcohol dehydrogenase (Adh) amplicons with two restriction enzyme combinations, EcoRI–HindIII and EcoRI–PstI, which easily can be used to distinguish Elymus and its closely related genera genomes. The method includes only four steps: (1) amplifying nuclear Adh genes with universal primers; (2) purifying and cloning PCR products; (3) digesting plasmids with restriction enzymes that
identify a given genome; (4) running the digested products on an agarose gel and identify the sample based on the restriction
profiles. Results showed that: (1) PCR products ranged from 1,200 to 2,000 bp; (2) Adh2 gene was amplified from all the tested genomes; Adh1 gene was amplified from almost all of the tested genomes except the W genome; Adh3 gene was amplified only from the St genome; (3) the EcoRI–HindIII combination was effective to distinguish different Adh gene types (Adh1, Adh2, and Adh3); (4) the Adh2–EcoRI–PstI fragments could be used to distinguish Elymus and its closely related genera genomes. Therefore, This RFLP assay provides an inexpensive and simple means of identifying
Elymus genomes. 相似文献