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61.
Xiaolin Pei Hongyu Zhang Lijun Meng Gang Xu Lirong Yang Jianping Wu 《Process Biochemistry》2013,48(12):1921-1927
A nitrile hydratase (NHase) gene from Aurantimonas manganoxydans was cloned and expressed in Escherichia coli BL21 (DE3). A downstream gene adjacent to the β-subunit was necessary for the functional expression of the recombinant NHase. The structural gene order of the Co-type NHase was α-subunit beyond β-subunit, different from the order typically reported for Co-type NHase genes. The NHase exhibited adequate thermal stability, with a half-life of 1.5 h at 50 °C. The NHase efficiently hydrated 3-cyanopyridine to produce nicotinamide. In a 1-L reaction mixture, 3.6 mol of 3-cyanopyridine was completely converted to nicotinamide in four feedings, exhibiting a productivity of 187 g nicotinamide/g dry cell weight/h. An industrial auto-induction medium was applied to produce the recombinant NHase in 10-L fermenter. A glycerol-limited feeding method was performed, and a final activity of 2170 U/mL culture was achieved. These results suggested that the recombinant NHase was efficiently cloned and produced in E. coli. 相似文献
62.
Aims
To purify and primarily characterize an anti‐Alicyclobacillus bacteriocin produced by Bifidobacterium animalis subsp. animalis CICC 6165, suggested to be named bificin C6165.Methods and Results
During purification of the bificin C6165, optimal recovery was achieved with ammonium sulfate precipitation followed by two chromatographic steps. Mass spectrometry analyses revealed a distinctive peak corresponding to a molecular mass of 3395·1 Da. This bacteriocin was heat stable, effective after refrigerated storage and freeze–thaw cycles. The primary mode of action of bificin C6165 is most probably due to pore formation, as indicated by the efflux of K+ from metabolically active cells of Alicyclobacillus acidoterrestris. In the presence of 10 mmol l?1 gadolinium, bificin C6165 did not affect cells of Alicyclobacillus acidoterrestris. This suggests that the mode of action of bificin C6165 relies on a net negatively charged cell surface.Conclusions
Bificin C6165 is indeed a novel bacteriocin and it exhibited remarkable potency for Alicyclobacillus control.Significance and Impact of the Study
Application of bacteriocins in preservation of fruit juices has seldom been studied. Bificin C6165 may be an alternative method to control juice spoilage by this Alicyclobacillus acidoterrestris and meet increasing consumer demand for nature and artificial chemical additive‐free food products. 相似文献63.
Shufeng Li Xiaofeng Huang Dapeng Zhang Qilai Huang Guoshun Pei Lixiang Wang Wenhui Jiang Qingang Hu Renxiang Tan Zi-Chun Hua 《PloS one》2013,8(11)
Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase critically involved in cancer metastasis. We found an elevation of FAK expression in highly metastatic melanoma B16F10 cells compared with its less metastatic partner B16F1 cells. Down-regulation of the FAK expression by either small interfering RNA or dominant negative FAK (FAK Related Non-Kinase, FRNK) inhibited the B16F10 cell migration in vitro and invasiveness in vivo. The mechanism by which FAK activity is up-regulated in highly metastatic cells remains unclear. In this study, we reported for the first time that one of the Est family proteins, PEA3, is able to transactivate FAK expression through binding to the promoter region of FAK. We identified a PEA3-binding site between nucleotides −170 and +43 in the FAK promoter that was critical for the responsiveness to PEA3. A stronger affinity of PEA3 to this region contributed to the elevation of FAK expression in B16F10 cells. Both in vitro and in vivo knockdown of PEA3 gene successfully mimicked the cell migration and invasiveness as that induced by FAK down-regulation. The activation of the well-known upstream of PEA3, such as epidermal growth factor, JNK, and ERK can also induce FAK expression. Furthermore, in the metastatic human clinic tumor specimens from the patients with human primary oral squamous cell carcinoma, we observed a strong positive correlation among PEA3, FAK, and carcinoma metastasis. Taking together, we hypothesized that PEA3 might play an essential role in the activation of the FAK gene during tumor metastasis. 相似文献
64.
Katariina M. Hutterer Robert W. Hong Jonathon Lull Xiaoyang Zhao Tian Wang Rex Pei M. Eleanor Le Oleg Borisov Rob Piper Yaoqing Diana Liu Krista Petty Izydor Apostol Gregory C. Flynn 《MABS-AUSTIN》2013,5(4):608-613
Manufacturing-induced disulfide reduction has recently been reported for monoclonal human immunoglobulin gamma (IgG) antibodies, a widely used modality in the biopharmaceutical industry. This effect has been tied to components of the intracellular thioredoxin reduction system that are released upon cell breakage. Here, we describe the effect of process parameters and intrinsic molecule properties on the extent of reduction. Material taken from cell cultures at the end of production displayed large variations in the extent of antibody reduction between different products, including no reduction, when subjected to the same reduction-promoting harvest conditions. Additionally, in a reconstituted model in which process variables could be isolated from product properties, we found that antibody reduction was dependent on the cell line (clone) and cell culture process. A bench-scale model using a thioredoxin/thioredoxin reductase regeneration system revealed that reduction susceptibility depended on not only antibody class but also light chain type; the model further demonstrates that the trend in reducibility was identical to DTT reduction sensitivity following the order IgG1λ > IgG1κ > IgG2λ > IgG2κ. Thus, both product attributes and process parameters contribute to the extent of antibody reduction during production. 相似文献
65.
Matthew J. Cooper Nathan J. Cox Eric I. Zimmerman Brian J. Dewar James S. Duncan Martin C. Whittle Thien A. Nguyen Lauren S. Jones Sreerupa Ghose Roy David M. Smalley Pei Fen Kuan Kristy L. Richards Richard I. Christopherson Jian Jin Stephen V. Frye Gary L. Johnson Albert S. Baldwin Lee M. Graves 《PloS one》2013,8(6)
Protein kinases play key roles in oncogenic signaling and are a major focus in the development of targeted cancer therapies. Imatinib, a BCR-Abl tyrosine kinase inhibitor, is a successful front-line treatment for chronic myelogenous leukemia (CML). However, resistance to imatinib may be acquired by BCR-Abl mutations or hyperactivation of Src family kinases such as Lyn. We have used multiplexed kinase inhibitor beads (MIBs) and quantitative mass spectrometry (MS) to compare kinase expression and activity in an imatinib-resistant (MYL-R) and -sensitive (MYL) cell model of CML. Using MIB/MS, expression and activity changes of over 150 kinases were quantitatively measured from various protein kinase families. Statistical analysis of experimental replicates assigned significance to 35 of these kinases, referred to as the MYL-R kinome profile. MIB/MS and immunoblotting confirmed the over-expression and activation of Lyn in MYL-R cells and identified additional kinases with increased (MEK, ERK, IKKα, PKCβ, NEK9) or decreased (Abl, Kit, JNK, ATM, Yes) abundance or activity. Inhibiting Lyn with dasatinib or by shRNA-mediated knockdown reduced the phosphorylation of MEK and IKKα. Because MYL-R cells showed elevated NF-κB signaling relative to MYL cells, as demonstrated by increased IκBα and IL-6 mRNA expression, we tested the effects of an IKK inhibitor (BAY 65-1942). MIB/MS and immunoblotting revealed that BAY 65-1942 increased MEK/ERK signaling and that this increase was prevented by co-treatment with a MEK inhibitor (AZD6244). Furthermore, the combined inhibition of MEK and IKKα resulted in reduced IL-6 mRNA expression, synergistic loss of cell viability and increased apoptosis. Thus, MIB/MS analysis identified MEK and IKKα as important downstream targets of Lyn, suggesting that co-targeting these kinases may provide a unique strategy to inhibit Lyn-dependent imatinib-resistant CML. These results demonstrate the utility of MIB/MS as a tool to identify dysregulated kinases and to interrogate kinome dynamics as cells respond to targeted kinase inhibition. 相似文献
66.
Qing Tian Li Pei Xun Zhang Xiao Feng Yin Na Han Yu Hui Kou Jiu Xu Deng Bao Guo Jiang 《PloS one》2013,8(11)
Functional recovery is usually poor following peripheral nerve injury when reinnervation is delayed. Early innervation by sensory nerve has been indicated to prevent atrophy of the denervated muscle. It is hypothesized that early protection with sensory axons is adequate to improve functional recovery of skeletal muscle following prolonged denervation of mixed nerve injury. In this study, four groups of rats received surgical denervation of the tibial nerve. The proximal and distal stumps of the tibial nerve were ligated in all animals except for those in the immediate repair group. The experimental groups underwent denervation with nerve protection of peroneal nerve (mixed protection) or sural nerve (sensory protection). The experimental and unprotected groups had a stage II surgery in which the trimmed proximal and distal tibial nerve stumps were sutured together. After 3 months of recovery, electrophysiological, histological and morphometric parameters were assessed. It was detected that the significant muscle atrophy and a good preserved structure of the muscle were observed in the unprotected and protective experimental groups, respectively. Significantly fewer numbers of regenerated myelinated axons were observed in the sensory-protected group. Enhanced recovery in the mixed protection group was indicated by the results of the muscle contraction force tests, regenerated myelinated fiber, and the results of the histological analysis. Our results suggest that early axons protection by mixed nerve may complement sensory axons which are required for promoting functional recovery of the denervated muscle natively innervated by mixed nerve. 相似文献
67.
Tai‐Long Pan Tung‐Ho Wu Pei‐Wen Wang Yann‐Lii Leu Nardnisa Sintupisut Chun‐Hsun Huang Fang‐Rong Chang Yang‐Chang Wu 《Proteomics》2013,13(15):2297-2311
Hepatic ischemia‐reperfusion (IR) injury is a common clinical problem and ROS may be a contributing factor on IR injury. The current study evaluates the potential protective effect of saffron ethanol extract (SEE) in a rat model upon hepatic IR injury. Caspases 3 and terminal deoxynucleotidyl transferase‐mediated dUTP biotin nick end labeling (TUNEL) results showed increased cell death in the IR samples; reversely, minor apoptosis was detected in the SEE/IR group. Pretreatment with SEE significantly restored the content of antioxidant enzymes (SOD1 and catalase) and remarkably inhibited the intracellular ROS concentration in terms of reducing p47phox translocation. Proteome tools revealed that 20 proteins were significantly modulated in protein intensity between IR and SEE/IR groups. Particularly, SEE administration could attenuate the carbonylation level of several chaperone proteins. Network analysis suggested that saffron extract could alleviate IR‐induced ER stress and protein ubiquitination, which finally lead to cell apoptosis. Taken together, SEE could reduce hepatic IR injury through modulating protein oxidation and our results might help to develop novel therapeutic strategies against ROS‐caused diseases. 相似文献
68.
Tai‐Long Pan Pei‐Wen Wang Yu‐Chiang Hung Chun‐Hsun Huang Kun‐Ming Rau 《Proteomics》2013,13(23-24):3411-3423
Cervix cancer is the second most common cancer among women worldwide, whereas paclitaxel, the first line chemotherapeutic drug used to treat cervical cancer, shows low chemosensitivity on the advanced cervical cancer cell line. Tanshinone IIA (Tan IIA) exhibited strong growth inhibitory effect on CaSki cells (IC50 = 5.51 μM) through promoting caspase cascades with concomitant upregulating the phosphorylation of p38 and JNK signaling. Comprehensive proteomics revealed the global protein changes and the network analysis implied that Tan IIA treatment would activate ER stress pathways that finally lead to apoptotic cell death. Moreover, ER stress inhibitor could alleviate Tan IIA caused cell growth inhibition and ameliorate C/EBP‐homologous protein as well as apoptosis signal‐regulating kinase 1 mediated cell death. The therapeutic interventions targeting the mitochondrial‐related apoptosis and ER stress responses might be promising strategies to conquer paclitaxel resistance. 相似文献
69.
Fang Liu Yonghua Lu Wenhai Yu Qiang Fu Pei Wang Hai Ming 《Plasmonics (Norwell, Mass.)》2013,8(2):1279-1284
Herein, a flexible surface-enhanced Raman spectroscopy (SERS) substrate composed of nanodot-arrayed Ag film and Ag nanocubes was fabricated through a simple method. The large-area nanodot-arrayed Ag film was produced at low cost and high reproducibility. The experimental results show that the coupled structure produces a much stronger SERS signal than the Ag nanocube alone and the isolated nanodot-arrayed Ag film. Furthermore, the coupling effect is sensitive to geometrical parameter of the period of the dot-array. Numerical simulations are performed to verify the electric field enhancement of the composite SERS substrate and support the experimental results. 相似文献
70.
Hepatocellular carcinoma is one of the most common malignant neoplasms in the world and is the main cause of death in patients with liver cirrhosis. Surgical intervention is not suitable for majority of hepatocellular carcinoma. Investigation of the effective targeting to the tumor cells is essential for both primary tumors and metastases. Tumor specific cytotoxic T lymphocytes (CTL) have been considered to be the attractive vehicles for delivering therapeutic agents toward various tumor diseases. This study was to explore the distribution pattern of CTL carrying the lentiviral vectors with the characteristic of adenoviral E1 gene under the control of the cell activation-dependent CD40 ligand promoter (Lenti/hCD40L/E1AB). Following transduction with adenoviral vectors containing chimeric type 5 and type 35 fiber proteins (Ad5/35-TRAIL), these CTLs produced infectious virus when exposed to HepG2 cells. We assessed the therapeutic ability of CTLs using MTT, Western blot and colony formation assay. The novel CTL harboring Lenti/hCD40L/E1AB and Ad5/35-TRAIL caused proliferation inhibition and significant apoptosis in hepatocellular carcinoma cell lines. Thus, the novel CTL may be useful for the development of gene therapy approaches to hepatocellular carcinoma. 相似文献