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151.
152.
Qi Wang Cui Min Tingting Yan Hefang Pu Yinqiang Xin Shuangquan Zhang Lan Luo Zhimin Yin 《World journal of microbiology & biotechnology》2011,27(11):2603-2610
l-glutamine (Gln) is an important conditionally necessary amino acid in human body and potential demand in food or medicine
industry is expected. High efficiency of l-Gln production by coupling genetic engineered bacterial glutamine synthetase (GS) with yeast alcoholic fermentation system
has been developed. We report here first the application of small ubiquitin-related modifier (SUMO) fusion technology to the
expression and purification of recombinant Bacillus subtilis GS. In order to obtain GS with high Gln-forming activity, safety and low cost for food and pharmaceutics industry, 0.1% (w/v)
lactose was selected as inducer. The fusion protein was expressed in totally soluble form in E. coli, and expression was verified by SDS–PAGE and western blot analysis. The fusion protein was purified to 90% purity by nickel
nitrilo-triacetic acid (Ni–NTA) resin chromatography with a yield of 625 mg per liter fermentation culture. After the SUMO/GS
fusion protein was cleaved by the SUMO protease, the cleaved sample was reapplied to a Ni–NTA column. Finally, about 121 mg
recombinant GS was obtained from 1 l fermentation culture with no less than 96% purity. The recombinant purified GS showed
great transferase activity (23 U/mg), with 25 U recombinant GS in a 50 ml reaction system, a biosynthesis yield of 27.5 g/l l-Gln was detected by high pressure liquid chromatography (HPLC) or thin-layer chromatography. Thus, the application of SUMO
technology to the expression and purification of GS potentially could be employed for the industrial production of l-Gln. 相似文献
153.
Lan T Liu W Xie X Xu S Huang K Peng J Shen X Liu P Wang L Xia P Huang H 《Molecular endocrinology (Baltimore, Md.)》2011,25(12):2094-2105
Diabetic nephropathy is characterized by accumulation of glomerular extracellular matrix proteins, such as fibronectin (FN). Here, we investigated whether sphingosine kinase (SphK)1 pathway is responsible for the elevated FN expression in diabetic nephropathy. The SphK1 pathway and FN expression were examined in streptozotocin-induced diabetic rat kidney and glomerular mesangial cells (GMC) exposed to high glucose (HG). FN up-regulation was concomitant with activation of the SphK1 pathway as reflected in an increase in the expression and activity of SphK1 and sphingosine 1-phosphate (S1P) production in both diabetic kidney and HG-treated GMC. Overexpression of wild-type SphK1 (SphK(WT)) significantly induced FN expression, whereas treatment with a SphK inhibitor, N,N-dimethylsphingosine, or transfection of SphK1 small interference RNA or dominant-negative SphK1 (SphK(G82D)) abolished HG-induced FN expression. Furthermore, addition of exogenous S1P significantly induced FN expression in GMC with an induction of activator protein 1 (AP-1) activity. Inhibition of AP-1 activity by curcumin attenuated the S1P-induced FN expression. Finally, by inhibiting SphK1 activity, both N,N-dimethylsphingosine and SphK(G82D) markedly attenuated the HG-induced AP-1 activity. Taken together, these results demonstrated that the SphK1 pathway plays a critical role in matrix accumulation in GMC under diabetic condition, suggesting that the SphK1 pathway could be a potential therapeutic target for diabetic nephropathy. 相似文献
154.
Lin KW Huang AM Hour TC Yang SC Pu YS Lin CN 《Bioorganic & medicinal chemistry》2011,19(14):4274-4285
Twenty six 18β-glycyrrhetinic acid (GA) (1) derivatives 2-27 including twelve new GA derivatives 10, 11, 13-17, 21-25 were synthesized and evaluated for cytotoxicities against NTUB1 cells (human bladder cancer cell lines). seco-Compounds 9, 25, and 27 are the most potent compounds of this series, inhibiting cell growth of human NTUB1 cells with an IC(50) values of 2.34 ± 0.28, 4.76 ± 1.15, and 3.31 ± 0.61 μM, respectively. Exposure of NTUB1 to 25 for 24h significantly increased the production of reactive oxygen species (ROS). Flow cytometric analysis exhibited that treatment of NTUB1 with 25 did not induce cell cycle arrest but accompanied by an increase of apoptotic cell death in a dose-dependant manner after 24h. Mitochondrial membrane potential (MMP) decreased significantly in a dose-dependant manner when the NTUB1 cells were exposed to 25 for 24h. Marked collapse of the MMP suggested that dysfunction of the mitochondria may be involved in the oxidative burst and apoptosis induced by 25. Western blot analysis shows that NTUB1 cells treated with 25 increased the level of p-p53 in a dose-dependant manner. Further, NAC treatment prevented p53 phosphorylation stimulated by 25. These results suggested that 25 induced a mitochondrial-mediated apoptosis in NTUB1 cells through activation of p53, which are mainly mediated ROS generated by 25. 相似文献
155.
升振山姜的选育和生物学特性研究 总被引:2,自引:1,他引:1
采用杂交育种技术,以姜科(Zingiberaceae)植物草豆蔻(Alpinia hainanensis K.Schumann)为亲本,母本于1975年从广东引种,具乳白色小苞片;父本于1983年从广西引种,具淡粉红色小苞片.经多年多代分离、筛选和鉴定,从其杂交后代中选育出花卉新品种升振山姜(A.hainanensis... 相似文献
156.
检测旋毛虫感染大鼠血清中的总IgE、特异性IgE和观察IgE介导的肥大细胞脱颗粒,并进一步探讨抗体依赖的(肥大)细胞介导的细胞毒性(Antibody dependent cell mediated cytotoxicity,ADCC)在旋毛虫病免疫机理中的作用。采用雄性Wistar大鼠为旋毛虫感染的动物模型,将90只大鼠随机分为10组。试验时,以ELISA双抗体夹心法和间接法分别动态检测总IgE和特异性IgE;肥大细胞脱颗粒试验采用直接法;然后采用细胞培养法观察免疫血清对肥大细胞杀伤旋毛虫肌幼虫作用的影响。在免疫血清存在时,无论感染鼠还是正常鼠的肥大细胞对旋毛虫幼虫均有杀伤作用,但以感染鼠的作用更强。肥大细胞在ADCC效应机制中对杀伤旋毛虫肌幼虫发挥了重要的作用。 相似文献
157.
158.
159.
Chen J Li Q Zhang Y Yang P Zong Y Qu S Liu Z 《Journal of physiology and biochemistry》2011,67(2):153-163
The reported data indicate that oleic acid (OA) decreases cholesterol absorption. To explore the underlying mechanisms, the
effects of OA on the expression of cholesterol transport-related proteins (NPC1L1, ABCG5/8, ACAT2, MTP) and the unfolded protein
response (UPR) pathway were studied in CaCo-2 enterocytes by incubating CaCo-2 cells with taurocholate micelles or taurocholate
micelles containing different concentrations of OA (0.25–1.0 mM). We show that OA effectively induces XBP1 mRNA splicing,
a key component of the UPR signaling, and the expression of BiP and mature ATF6 proteins in a concentration-dependent manner,
leading to the induction of endoplasmic reticulum (ER) stress and activation of the UPR. Interestingly, OA decreases NPC1L1
expression in a dose-dependent manner while it has no effects on ABCG5 and MTP mRNA level or SREBP-2, ABCG8, and ACAT2 protein
level. In CaCo-2 cells treated with 1.0 mM OA, both the NPC1L1 mRNA level and the NPC1L1 protein expression in brush-border
membrane fractions were decreased by 39% and 37%, respectively (P < 0.01). A dose of 1 mM dithiothreitol (DTT), a positive control for ER stress induction, also decreases NPC1L1 mRNA and
protein expression by 27% and 23%, respectively (P < 0.05). Furthermore, 4-phenyl-butyric acid, an UPR inhibitor, blocks OA- and DTT-induced reduction on NPC1L1 mRNA and protein
levels. The results suggest that OA down-regulates NPC1L1 mRNA and protein expression via the induction of the UPR, which
may play an important role in reducing intestinal cholesterol absorption. 相似文献
160.
Cui S Zhang D Jiang S Pu H Hu Y Guo H Chen M Su T Zhu C 《Fish & shellfish immunology》2011,31(2):173-181
Macrophage migration inhibitory factor (MIF) is an important cytokine and plays a crucial role as a pivotal regulator of innate immunity. In this study, a MIF cDNA was identified and characterized from the pearl oyster Pinctada fucata (designated as PoMIF). The full-length of PoMIF was 1544 bp and consisted of a 5'-untranslated region (UTR) of 45 bp, a 3'-UTR of 1139 bp with a polyadenylation signal (AATAAA) at 12 nucleotides upstream of the poly (A) tail. The open reading frame (ORF) of PoMIF was 360 bp which encoded a polypeptide of 120 amino acids with an estimated molecular mass of 13.3 kDa and a predicted pI of 6.1. SMART analysis showed that PoMIF contained the catalytic-sites P2 and K33 for tautomerase activity, a motif C??GSV?? for oxidoreductase activity and a MIF family signature D??PCGSVEVYSIGALG??. Homology analysis revealed that the PoMIF shared 40.3-65.5% similarity and 26.9-45.0% identity to other known MIF sequences. PoMIF mRNA was constitutively expressed in seven selected tissues of healthy pearl oysters, with the highest expression level in digestive gland. Eight hours after P. fucata was injected with Vibrio alginolyticus, the expression of PoMIF mRNA was significantly up-regulated in digestive gland, gills, hemocytes and intestine. The cDNA fragment encoding mature protein of PoMIF was subcloned to expression vector pRSET and transformed into Escherichia coli BL21 (DE3). The recombinant PoMIF (rPoMIF) was expressed and purified under optimized conditions. Function analysis showed that rPoMIF had oxidoreductase activity and could utilize dithiothreitol (DTT) as reductant to reduce insulin. 相似文献