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71.
Luo H Wang Y Wang H Yang J Yang Y Huang H Yang P Bai Y Shi P Fan Y Yao B 《Applied microbiology and biotechnology》2009,82(3):453-461
Using degenerate polymerase chain reaction (PCR) and thermal asymmetric interlaced PCR, a 1,347-bp full-length complementary
DNA fragment encompassing the gene man5A, which encodes a 429-amino acid β-mannanase with a calculated mass of 46.8 kDa, was cloned from acidophilic Bispora sp. MEY-1. The deduced amino acid sequence (catalytic domain) displayed highest identity (54.1%) with the Emericella nidulans endo-β-1,4-d-mannanase, a member of the glycoside hydrolase family 5. Recombinant MAN5A was overexpressed in Pichia pastoris, and its activity in the culture medium reached 500 U ml−1. The enzyme was acidophilic, with highest activity at pH 1.0–1.5, lower than any known mannanases, and optimal temperature
for activity was 65°C. MAN5A had good pH adaptability, excellent thermal and pH stability, and high resistance to both pepsin
and trypsin. The specific activity, K
m, and V
max for locust bean gum substrate was 3,373 U mg−1, 1.56 mg ml−1, and 6,587.6 μmol min−1 mg−1, respectively. The enzymatic activity was not significantly affected by ions such as Ca2+, Cr3+, Co2+, Zn2+, Na+, K+, and Mg2+ and enhanced by Ni2+, Fe3+, Mn2+ and Ag+. These favorable properties make MAN5A a potential candidate for use in various industrial applications. 相似文献
72.
Ping Li ;Chenfang Dong ;Yanjun Lei ;Bing Shan ;Xinli Xiao ;Huiying Jiang ;Xin Wang ;Chen Gao ;Qi Shi ;Kun Xu ;Chan Tian ;Jun Han ;Xiaoping Dong 《Acta biochimica et biophysica Sinica》2009,(1):42-53
Doppel (Dpl) is a prion (PrP)-like protein due to the structural and biochemical similarities; however, the natural functions of Dpl and PrP remain unclear. In this study, a 531-bp human PRND gene sequence encoding Dpl protein was amplified from human peripheral blood leucocytes. Furl-length and various truncated human Dpl and PrP proteins were expressed and purified from Escherichia coil Supplement of the full-length Dpl onto human neuroblastoma cell SH-SY5Y induced remarkable cytotoxicity, and the region responsible for its cytotoxicity was mapped at the middle segment of Dpl [amino acids (aa) 81-122]. Interestingly, DpMnduced cytotoxicity was antagonized by the presence of full- length wild-type PrP. Analysis on fragments of PrP mutants showed that the N-terminal fragment (aa 23- 90) of PrP was responsible for the protective activity. A truncated PrP (PrPA32-121) with similar secondary structure as Dpl induced DpMike cytotoxicity on SH- SY5Y cells. Furthermore, binding of copper ion could enhance the antagonizing effect of PrP on Dpi-induced cytotoxicity. Apoptosis assays revealed that cytotoxicity induced by Dpl occurred through an apoptotic mechanism. These results suggested that the function of Dpl is antagonistic to PrP rather than synergistic. 相似文献
73.
A new xylanase from thermoacidophilic Alicyclobacillus sp. A4 with broad-range pH activity and pH stability 总被引:1,自引:0,他引:1
Yingguo Bai Jianshe Wang Zhifang Zhang Peilong Yang Pengjun Shi Huiying Luo Kun Meng Huoqing Huang Bin Yao 《Journal of industrial microbiology & biotechnology》2010,37(2):187-194
We have identified a highly pH-adaptable and stable xylanase (XynA4) from the thermoacidophilic Alicyclobacillus sp. A4, a strain that was isolated from a hot spring in Yunnan Province, China. The gene (xynA4) that encodes this xylanase was cloned, sequenced, and expressed in Escherichia coli. It encodes a 338-residue polypeptide with a calculated molecular mass of 42.5 kDa. The deduced amino acid sequence is most
similar to (53% identity) an endo-1,4-β-xylanase from Geobacillus stearothermophilus that belongs to family 10 of the glycoside hydrolases. Purified recombinant XynA4 exhibited maximum activity at 55°C and
pH 7.0, had broad pH adaptability (>40% activity at pH 3.8–9.4) and stability (retaining >80% activity after incubation at
pH 2.6–12.0 for 1 h at 37°C), and was highly thermostable (retaining >90% activity after incubation at 60°C for 1 h at pH
7.0). These properties make XynA4 promising for application in the paper industry. This is the first report that describes
cloning and expression of a xylanase gene from the genus Alicyclobacillus. 相似文献
74.
75.
Wang G Meng K Luo H Wang Y Huang H Shi P Pan X Yang P Yao B 《Applied microbiology and biotechnology》2011,91(6):1561-1570
An esterase-encoding gene, estR5, was directly obtained from the genomic DNA of goat rumen contents. The 555-bp full-length gene encodes a 184-residue polypeptide
(EstR5) without putative signal peptide. Deduced EstR5 shared the highest identity (50%) to a putative arylesterase from Ruminococcaceae bacterium D16. Phylogenetic analysis indicated that EstR5 was closely related with microbial esterases of gastrointestinal source.
A comparison of the conserved motifs shared with GDSL proteins revealed that EstR5 could be grouped into the GDSL family and
was further classified into the subfamily of SGNH hydrolases. The gene estR5 was expressed in Escherichia coli BL21 (DE3) and purified to electrophoretic homogeneity. Recombinant EstR5 exhibited highest catalytic efficiency towards α-naphthyl
acetate followed by phenyl acetate and p-nitrophenyl acetate and had no activity towards PNP esters with acyl chains longer than C8. The enzyme exhibited optimal
activity at around 60°C and pH 8.0, was stable at pH ranging from 6.0 to 11.0 and was slightly activated by detergent Tween,
Nonidet P-40, and Triton X-100. These properties suggest that EstR5 has great potential for basic research and industrial
applications. To our knowledge, this is the first arylesterase obtained from rumen microenvironment. 相似文献
76.
Shaohui Cui Bing Wang Yinan Zhao Huiying Chen Huiqin Ding Defu Zhi Shubiao Zhang 《Biotechnology letters》2014,36(1):1-7
For studying the mechanism of cationic liposome-mediated transmembrane routes for gene delivery, various inhibitors of endocytosis were used to treat human throat epidermis cancer cells, Hep-2, before transfection with Lipofectamine 2000/pGFP-N2 or Lipofectamine 2000/pGL3. To eliminate the effect of inhibitor toxicity on transfection, the RLU/survival rate was used to represent the transfection efficiency. Chlorpromazine and wortmannin, clathrin inhibitors, decreased transfection efficiency by 44 % (100 μM) and 31 % (100 nM), respectively. At the same time, genistein, a caveolin inhibitor, decreased it by 30 % (200 μM). Thus combined transmembrane routes through the clathrin and caveolae-mediated pathways were major mechanisms of cell uptake for the cationic liposome-mediated gene delivery. After entering the cells, microtubules played an important role on gene delivery as vinblastine, a microtubulin inhibitor, could reduce transfection efficiency by 41 % (200 nM). 相似文献
77.
Haiqiang Lu Huiying Luo Pengjun Shi Huoqing Huang Kun Meng Peilong Yang Bin Yao 《Applied microbiology and biotechnology》2014,98(5):2155-2163
The gene man5XZ3 from Aspergillus nidulans XZ3 encodes a multimodular β-mannanase of glycoside hydrolase family 5 that consists of a family 1 carbohydrate-binding module (CBM1), a Thr/Ser-rich linker region, and a catalytic domain. Recombinant Man5XZ3 and its two truncated derivatives, Man5ΔCBM (removing the CBM1) and Man5ΔCL (removing both the CBM1 and linker region), were produced in Pichia pastoris and showed significant variance in the secondary structure. The three enzymes had similar biochemical properties, such as optimal pH and temperature (pH 5.0 and 80 °C) and excellent pH stability at pH 4.0–10.0. Removal of the CBM1 alone could improve the thermostability of Man5XZ3, but further removal of the linker region resulted in worse thermostability. Man5XZ3 retained greater enzyme activity in the presence of an organic solvent (acetone), two detergents (SDS and Triton X-100), and a chaotropic agent (urea) compared with Man5ΔCBM and Man5ΔCL. This study provides an excellent β-mannanase candidate favorable for various industries and primarily demonstrates the relationship between enzyme structure and function. 相似文献
78.
Previously, we showed that the encapsulation and release properties of a liposomal ciprofloxacin formulation could be modified post manufacture, by addition of surfactant in concert with osmotic swelling of the liposomes. This strategy may provide more flexibility and convenience than the alternative of manufacturing multiple batches of liposomes differing in composition to cover a wide range of release profiles. The goal of this study was to develop a surfactant-associated liposomal ciprofloxacin (CFI) formulation possessing good long-term stability which could be delivered as an inhaled aerosol. Preparations of 12.5 mg/ml CFI containing 0.4% polysorbate 20 were formulated between pH 4.7 and 5.5. These formulations, before and after mesh nebulization, and after refrigerated storage for up to 2 years, were characterized in terms of liposome structure by cryogenic transmission electron microscopy (cryo-TEM) imaging, vesicle size by dynamic light scattering, pH, drug encapsulation by centrifugation-filtration, and in vitro release (IVR) performance. Within the narrower pH range of 4.9 to 5.2, these formulations retained their physicochemical stability after 2-year refrigerated storage, were robust to mesh nebulization, and formed respirable aerosols with a volume mean diameter (VMD) of 3.7 μm and a geometric standard deviation (GSD) of 1.7. This study demonstrates that it may be possible to provide a range of release profiles by simple addition of surfactant to a liposomal formulation post manufacture, and that these formulations may retain their physicochemical properties after long-term refrigerated storage and following aerosolization by mesh nebulizer.KEY WORDS: ciprofloxacin, drug delivery, liposome, nebulized aerosol, surfactant 相似文献
79.
Xu Zhu Ju Zhang Huiying Sun Cuicui Jiang Yusheng Dong Qiang Shan Siyuan Su Yingying Xie Ningzhi Xu Xiaomin Lou Siqi Liu 《The Journal of biological chemistry》2014,289(44):30567-30577
Deciphering the inositol-requiring enzyme 1 (IRE1) signaling pathway is fundamentally important for understanding the unfolded protein response (UPR). The ubiquitination of proteins residing on the endoplasmic reticulum (ER) membrane has been reported to be involved in the UPR, although the mechanism has yet to be fully elucidated. Using immunoprecipitation and mass spectrometry, IRE1 was identified as a substrate of the E3 ligase CHIP (carboxyl terminus of HSC70-interacting protein) in HEK293 cells under geldanamycin-induced ER stress. Two residues of IRE1, Lys545 and Lys828, were targeted for Lys63-linked ubiquitination. Moreover, in CHIP knockdown cells, IRE1 phosphorylation and the IRE1-TRAF2 interaction were nearly abolished under ER stress, which may be due to lacking ubiquitination of IRE1 on Lys545 and Lys828, respectively. The cellular responses were evaluated, and the data indicated that CHIP-regulated IRE1/TRAF2/JNK signaling antagonized the senescence process. Therefore, our findings suggest that CHIP-mediated ubiquitination of IRE1 contributes to the dynamic regulation of the UPR. 相似文献
80.
Huifang Hua Huiying Luo Yingguo Bai Kun Wang Canfang Niu Huoqing Huang Pengjun Shi Caihong Wang Peilong Yang Bin Yao 《PloS one》2014,9(11)