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71.
Huynh TT Huynh VT Harmon MA Phillips MA 《The Journal of biological chemistry》2003,278(41):39794-39800
The parasitic protozoa Trypanosoma brucei utilizes a novel cofactor (trypanothione, T(SH)2), which is a conjugate of GSH and spermidine, to maintain cellular redox balance. gamma-Glutamylcysteine synthetase (gamma-GCS) catalyzes the first step in the biosynthesis of GSH. To evaluate the importance of thiol metabolism to the parasite, RNAi methods were used to knock down gene expression of gamma-GCS in procyclic T. brucei cells. Induction of gamma-GCS RNAi with tetracycline led to cell death within 4-6 days post-induction. Cell death was preceded by the depletion of the gamma-GCS protein and RNA and by the loss of the cellular pools of GSH and T(SH)2. The addition of GSH (80 microM) to cell cultures rescued the RNAi cell death phenotype and restored the intracellular thiol pools to wild-type levels. Treatment of cells with buthionine sulfoximine (BSO), an enzyme-activated inhibitor of gamma-GCS, also resulted in cell death. However, the toxicity of the inhibitor was not reversed by GSH, suggesting that BSO has more than one cellular target. BSO depletes intracellular thiols to a similar extent as gamma-GCS RNAi; however, addition of GSH did not restore the pools of GSH and T(SH)2. These data suggest that BSO also acts to inhibit the transport of GSH or its peptide metabolites into the cell. The ability of BSO to inhibit both synthesis and transport of GSH likely makes it a more effective cytotoxic agent than an inhibitor with a single mode of action. Finally the potential for the T(SH)2 biosynthetic enzymes to be regulated in response to reduced thiol levels was studied. The expression levels of ornithine decarboxylase and of S-adenosylmethionine decarboxylase, two essential enzymes in spermidine biosynthesis, remained constant in induced gamma-GCS RNAi cell lines. 相似文献
72.
Ten Hagen KG Tran DT Gerken TA Stein DS Zhang Z 《The Journal of biological chemistry》2003,278(37):35039-35048
Here we report the cloning and functional characterization of eight members of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase gene family from Drosophila melanogaster (polypeptide GalNAc transferase = pgant1-8). Full-length cDNAs were isolated from a Drosophila embryonic library based on homology to known ppGaNTases. Alignments with characterized mammalian isoforms revealed strong sequence similarities between certain fly and mammalian isoforms, highlighting putative orthologues between the species. In vitro activity assays demonstrated biochemical transferase activity for each gene, with three isoforms requiring glycosylated substrates. Comparison of the activities of Drosophila and mammalian orthologues revealed conservation of substrate preferences against a panel of peptide and glycopeptide substrates. Furthermore, Edman degradation analysis demonstrated that preferred sites of GalNac addition were also conserved between certain fly and mammalian orthologues. Semi-quantitative PCR amplification of Drosophila cDNA revealed expression of most isoforms at each developmental stage, with some isoforms being less abundant at certain stages relative to others. In situ hybridization to Drosophila embryos revealed specific staining of pgant5 and pgant6 in the salivary glands and pgant5 in the developing hindgut. Additionally, pgant5 and pgant6 expression within the egg chamber was restricted to the follicle cells, cells known to be involved in egg formation and subsequent embryonic patterning. The characterization reported here provides additional insight into the use of this model system to dissect the biological role of this enzyme family in vivo during both fly and mammalian development. 相似文献
73.
Cleavage of the matricellular protein SPARC by matrix metalloproteinase 3 produces polypeptides that influence angiogenesis 总被引:8,自引:0,他引:8
Sage EH Reed M Funk SE Truong T Steadele M Puolakkainen P Maurice DH Bassuk JA 《The Journal of biological chemistry》2003,278(39):37849-37857
SPARC, a matricellular protein that affects cellular adhesion and proliferation, is produced in remodeling tissue and in pathologies involving fibrosis and angiogenesis. In this study we have asked whether peptides generated from cleavage of SPARC in the extracellular milieu can regulate angiogenesis. Matrix metalloproteinase (MMP)-3, but not MMP-1 or 9, showed significant activity toward SPARC. Limited digestion of recombinant human (rhu)SPARC with purified catalytic domain of rhuMMP-3 produced three major fragments, which were sequenced after purification by HPLC. Three synthetic peptides (Z-1, Z-2, and Z-3) representing motifs from each fragment were tested in distinct assays of angiogenesis. Peptide Z-1 (3.9 kDa, containing a Cu2+-binding sequence KHGK) exhibited a biphasic effect on [3H]thymidine incorporation by cultured endothelial cells and stimulated vascular growth in the chick chorioallantoic membrane (CAM). In contrast, peptides Z-2 (6.1 kDa, containing Ca2+-binding EF hand-1) and Z-3 (2.2 kDa, containing neither Cu2+-binding motifs nor EF hands), inhibited cell proliferation in a concentration-dependent manner and exhibited no effects on vessel growth in the CAM. Reciprocal results were obtained in a migration assay in native collagen gels: peptide Z-1 was ineffective over a range of concentrations, whereas Z-2 or Z-3 stimulated cell migration. Therefore, proteolysis of SPARC by MMP-3 produced peptides that regulate endothelial cell proliferation and/or migration in vitro in a mutually exclusive manner. One of these peptides containing KHGK also demonstrated a concentration-dependent effect on angiogenesis. 相似文献
74.
Baldwin J Krebs C Saleh L Stelling M Huynh BH Bollinger JM Riggs-Gelasco P 《Biochemistry》2003,42(45):13269-13279
The diiron(II) cluster in the R2 subunit of Escherichia coli ribonucleotide reductase (RNR) activates oxygen to generate a mu-oxodiiron(III) cluster and the stable tyrosyl radical that is critical for the conversion of ribonucleotides to deoxyribonucleotides. Like those in other diiron carboxylate proteins, such as methane monooxygenase (MMO), the R2 diiron cluster is proposed to activate oxygen by formation of a peroxodiiron(III) intermediate followed by an oxidizing high-valent cluster. Substitution of key active site residues results in perturbations of the normal oxygen activation pathway. Variants in which the active site ligand, aspartate (D) 84, is changed to glutamate (E) are capable of accumulating a mu-peroxodiiron(III) complex in the reaction pathway. Using rapid freeze-quench techniques, this intermediate in a double variant, R2-W48A/D84E, was trapped for characterization by M?ssbauer and X-ray absorption spectroscopy. These samples contained 70% peroxodiiron(III) intermediate and 30% diferrous R2. An Fe-Fe distance of 2.5 A was found to be associated with the peroxo intermediate. As has been proposed for the structures of the higher valent intermediates in both R2 and MMO, carboxylate shifts to a mu-(eta(1),eta(2)) or a mu-1,1 conformation would most likely be required to accommodate the short 2.5 A Fe-Fe distance. In addition, the diferrous form of the enzyme present in the reacted sample has a longer Fe-Fe distance (3.5 A) than does a sample of anaerobically prepared diferrous R2 (3.4 A). Possible explanations for this difference in detected Fe-Fe distance include an O(2)-induced conformational change prior to covalent chemistry or differing O(2) reactivity among multiple diiron(II) forms of the cluster. 相似文献
75.
76.
Huynh QK Kishore N Mathialagan S Donnelly AM Tripp CS 《The Journal of biological chemistry》2002,277(15):12550-12558
Nuclear factor-kappaB activation depends on phosphorylation and degradation of its inhibitor protein, IkappaB. The phosphorylation of IkappaBalpha on Ser(32) and Ser(36) is initiated by an IkappaB kinase (IKK) complex that includes a catalytic heterodimer composed of IkappaB kinase 1 (IKK-1) and IkappaB kinase 2 (IKK-2) as well as a regulatory adaptor subunit, NF-kappaB essential modulator. Recently, two related IkappaB kinases, TBK-1 and IKK-i, have been described. TBK-1 and IKK-i show sequence and structural homology to IKK-1 and IKK-2. TBK-1 and IKK-i phosphorylate Ser(36) of IkappaBalpha. We describe the kinetic mechanisms in terms of substrate and product inhibition of the recombinant human (rh) proteins, rhTBK-1, rhIKK-I, and rhIKK-1/rhIKK-2 heterodimers. The results indicate that although each of these enzymes exhibits a random sequential kinetic mechanism, the effect of the binding of one substrate on the affinity of the other substrate is significantly different. ATP has no effect on the binding of an IkappaBalpha peptide for the rhIKK-1/rhIKK-2 heterodimer (alpha = 0.99), whereas the binding of ATP decreased the affinity of the IkappaBalpha peptide for both rhTBK-1 (alpha = 10.16) and rhIKK-i (alpha = 62.28). Furthermore, the dissociation constants of ATP for rhTBK-1 and rhIKK-i are between the expected values for kinases, whereas the dissociation constants of the IkappaBalpha peptide for each IKK isoforms is unique with rhTBK-1 being the highest (K(IkappaBalpha) = 69.87 microm), followed by rhIKK-i (K(IkappaBalpha) = 5.47 microm) and rhIKK-1/rhIKK-2 heterodimers (K(IkappaBalpha) = 0.12 microm). Thus this family of IkappaB kinases has very unique kinetic properties. 相似文献
77.
beta -Amyloid peptide activates alpha 7 nicotinic acetylcholine receptors expressed in Xenopus oocytes 总被引:3,自引:0,他引:3
The alpha7 nicotinic acetylcholine receptor is highly expressed in hippocampus and in cholinergic projection neurons from the basal forebrain, structures that are particularly vulnerable to the ravages of Alzheimer's disease. Previous work suggests that beta-amyloid peptide can interact with alpha7 nicotinic acetylcholine receptors, although the nature of this interaction has not been well characterized. To test whether beta-amyloid peptide can activate alpha7 nicotinic acetylcholine receptors, we expressed these receptors in Xenopus oocytes and performed two-electrode voltage clamp recordings, characterizing the response to beta-amyloid peptide 1-42 applied at concentrations ranging from 1 pm to 100 nm. In alpha7-expressing oocytes, beta-amyloid peptide 1-42 elicits inward currents at low concentrations (1-100 pm), whereas at higher concentrations (nm), less effective receptor activation is observed, indicative of receptor desensitization. Preincubation with the alpha7-selective agents, the antagonist methyllycaconatine, and the agonist 4-OH-GTS-21 blocked beta-amyloid peptide-induced receptor activation. beta-amyloid peptide 1-42 at low concentrations was able to activate the L250T mutant alpha7 receptor. The endogenous Ca(2+)-activated chloride current in Xenopus oocytes is recruited upon receptor activation since replacing Ca(2+) with Ba(2+) in the recording solution reduced current amplitude. Thus, when beta-amyloid peptide activation of alpha7 receptors occurs, these currents are comprised, at least in part, of Ca(2+). 相似文献
78.
Hsu S Borke JL Lewis JB Singh B Aiken AC Huynh CT Schuster GS Caughman GB Dickinson DP Smith AK Osaki T Wang XF 《Cell proliferation》2002,35(3):183-192
A human oral tumour progression model was established that consists of normal epithelial cells and three cell lines representing stages from dysplastic to metastatic cells. To investigate the impact of exogenous transforming growth factor-beta 1 on this model system, we analysed the responsiveness of those cells to transforming growth factor-beta 1 and explored the potential mechanism underlying the transforming growth factor-beta 1 activity. We found that the growth of all cell types, regardless of their stage of tumour progression, is inhibited by transforming growth factor-beta 1, although to different degrees. Transforming growth factor-beta 1 induced the expression of cyclin-dependent kinase inhibitors p15(INK4B), p21WAF1/(CIP1) and p27(KIP1). In contrast, transforming growth factor-beta 1 was found to stimulate the invasive potential of one cell type that represents the most advanced stage of tumour phenotype, suggesting that the impact of transforming growth factor-beta 1 on functional features of tumour cells other than cellular proliferation may play a significant role in the process of oral tumour progression. 相似文献
79.
Protein unfolding transitions in an intrinsically unstable annexin domain: molecular dynamics simulation and comparison with nuclear magnetic resonance data
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Unfolding transitions of an intrinsically unstable annexin domain and the unfolded state structure have been examined using multiple approximately 10-ns molecular dynamics simulations. Three main basins are observed in the configurational space: native-like state, compact partially unfolded or intermediate compact state, and the unfolded state. In the native-like state fluctuations are observed that are nonproductive for unfolding. During these fluctuations, after an initial loss of approximately 20% of the core residue native contacts, the core of the protein transiently completely refolds to the native state. The transition from the native-like basin to the partially unfolded compact state involves approximately 75% loss of native contacts but little change in the radius of gyration or core hydration properties. The intermediate state adopts for part of the time in one of the trajectories a novel highly compact salt-bridge stabilized structure that can be identified as a conformational trap. The intermediate-to-unfolded state transition is characterized by a large increase in the radius of gyration. After an initial relaxation the unfolded state recovers a native-like topology of the domain. The simulated unfolded state ensemble reproduces in detail experimental nuclear magnetic resonance data and leads to a convincing complete picture of the unfolded domain. 相似文献
80.
Use of expression constructs to dissect the functional domains of the CHS/beige protein: identification of multiple phenotypes 总被引:2,自引:0,他引:2
Ward DM Shiflett SL Huynh D Vaughn MB Prestwich G Kaplan J 《Traffic (Copenhagen, Denmark)》2003,4(6):403-415
The Chediak-Higashi Syndrome (CHS) and the orthologous murine disorder beige are characterized at the cellular level by the presence of giant lysosomes. The CHS1/Beige protein is a 3787 amino acid protein of unknown function. To determine functional domains of the CHS1/Beige protein, we generated truncated constructs of the gene/protein. These truncated proteins were transiently expressed in Cos-7 or HeLa cells and their effect on membrane trafficking was examined. Beige is apparently a cytosolic protein, as are most transiently expressed truncated Beige constructs. Expression of the Beige construct FM (amino acids 1-2037) in wild-type cells led to enlarged lysosomes. Similarly, expression of a 5.5-kb region (amino acids 2035-3787) of the carboxyl terminal of Beige (22B) also resulted in enlarged lysosomes. Expression of FM solely affected lysosome size, whereas expression of 22B led to alterations in lysosome size, changes in the Golgi and eventually cell death. The two constructs could be used to further dissect phenotypes resulting from loss of the Beige protein. CHS or beigej fibroblasts show an absence of nuclear staining using a monoclonal antibody directed against phosphatidylinositol 4,5 bisphosphate [PtdIns(4,5) P2]. Transformation of beige j fibroblasts with a YAC containing the full-length Beige gene resulted in the normalization of lysosome size and nuclear PtdIns(4,5)P2 staining. Expression of the carboxyl dominant negative construct 22B led to loss of nuclear PtdIns(4,5)P2 staining. Expression of the FM dominant negative clone did not alter nuclear PtdIns(4,5) P2 localization. These results suggest that the Beige protein interacts with at least two different partners and that the Beige protein affects cellular events, such as nuclear PtdIns(4,5)P2 localization, in addition to lysosome size. 相似文献