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71.
The proton translocating membrane-bound inorganic pyrophosphatase of Rhodospirillum rubrum S1, has been solubilized with good yield from chromatophores using Triton X-100 (9–10 oxyethylene groups) in the presence of high concentrations of MgCl2 and ethyleneglycol. The enzyme has been purified 80-fold by hydroxylapatite column chromatography, to a state of near homogeneity, according to polyacrylamide-gelelectrophoresis. The enzyme appears to be a very hydrophobic integrally bound membrane protein. Phospholipids or Triton X-100 reconstitutes the enzyme activity after solubilization and purification. The purified enzyme preparation has a specific activity of 24 units. Both the purified and the chromatophore-bound enzyme are inhibited by N-ethylmaleimide, 4-chloro-7-nitrobenzo-2-oxo-1,3-diazol (NBF-Cl), sodium fluoride, imidodiphosphate, methylenediphosphonate and the antibiotic Dio-9 (energy-transfer inhibitor). In the solubilized state the purified enzyme is not stimulated by uncouplers or inhibited by dicyclohexylcarbodiimide in contrast to the chromatophore-bound pyrophosphatase. When reconstituted into liposomes the purified enzyme regains the stimulation by uncouplers. 相似文献
72.
Runar Gjerp Solstad Chun Li Johan Isaksson Jostein Johansen Johan Svenson Klara Stensv?g Tor Haug 《PloS one》2016,11(3)
The global problem of microbial resistance to antibiotics has resulted in an urgent need to develop new antimicrobial agents. Natural antimicrobial peptides are considered promising candidates for drug development. Echinoderms, which rely on innate immunity factors in the defence against harmful microorganisms, are sources of novel antimicrobial peptides. This study aimed to isolate and characterise antimicrobial peptides from the Edible sea urchin Echinus esculentus. Using bioassay-guided purification and cDNA cloning, three antimicrobial peptides were characterised from the haemocytes of the sea urchin; two heterodimeric peptides and a cysteine-rich peptide. The peptides were named EeCentrocin 1 and 2 and EeStrongylocin 2, respectively, due to their apparent homology to the published centrocins and strongylocins isolated from the green sea urchin Strongylocentrotus droebachiensis. The two centrocin-like peptides EeCentrocin 1 and 2 are intramolecularly connected via a disulphide bond to form a heterodimeric structure, containing a cationic heavy chain of 30 and 32 amino acids and a light chain of 13 amino acids. Additionally, the light chain of EeCentrocin 2 seems to be N-terminally blocked by a pyroglutamic acid residue. The heavy chains of EeCentrocins 1 and 2 were synthesised and shown to be responsible for the antimicrobial activity of the natural peptides. EeStrongylocin 2 contains 6 cysteines engaged in 3 disulphide bonds. A fourth peptide (Ee4635) was also discovered but not fully characterised. Using mass spectrometric and NMR analyses, EeCentrocins 1 and 2, EeStrongylocin 2 and Ee4635 were all shown to contain post-translationally brominated Trp residues in the 6 position of the indole ring. 相似文献
73.
Veronika Damkova Veronika Paskova Hana Bandouchova Klara Hilscherova Jana Sedlackova Ladislav Novotny Lucie Peckova Frantisek Vitula Miroslav Pohanka Jiri Pikula 《Harmful algae》2011,10(6):612-618
Previous studies demonstrated the toxic effects of cyanobacteria in Japanese quails (Coturnix coturnix japonica) in various experimental set-ups including acute, sub-chronic and reproduction toxicity, co-exposures with toxic metals and the Newcastle vaccination. This study aimed to assess the testicular toxicity of a complex cyanobacterial biomass administered to Japanese quails in the feed for eight weeks (daily dose of 61.62 μg microcystins pro toto including 26.54 μg MC-RR, 7.62 μg MC-YR and 27.39 μg MC-LR). There was no mortality in the controls or the biomass-exposed birds. However, males exposed to cyanobacteria in the feed showed moderate to marked atrophy of the seminiferous tubular epithelium with only sparse numbers of the developmental stages of spermatozoa and Sertoli cells. Biomass-exposed birds had elevated catalase activities but decreased glutathione peroxidase activities and surprisingly lower levels of lipid peroxides in the testes. The other biochemical parameters studied (i.e., glutathione level and glutathione reductase, glutathione-S-transferase and superoxide dismutase activities) showed no differences. The cell defensive system protecting testicular tissue from the damage associated with toxic effects of the complex cyanobacterial biomass seemed to be insufficient and partly depleted after the chronic exposure of male birds to this biomass. 相似文献
74.
The aim of the study was to evaluate the exclusivity and/or preference of macroinvertebrate taxa for river habitats. Indicator
species analysis and random forests methods were applied to the data set of macroinvertebrate samples taken from 58 sampling
points. Samples were classified according to habitat types defined by the position in a river channel and local hydraulic
characteristics. 86 macroinvertebrate taxa were included in the analyses. High indicative values for habitats (importance
value ≥50 and/or indicator value ≥40) were identified for 26 taxa. The results of both methods can be considered similar.
Merged habitats of channel margin (margin of main channel and side arms) were mainly defined by “negative” indicator taxa
(correct classification of given samples was caused by non-occurrence and low abundances of certain taxa in this habitat).
In general, there was only a small group of taxa preferring these habitats. Taxa were not fully habitat specific because they
mostly occurred in two or three habitat types. This could be the result of autecological plasticity of individual taxa and
the connectivity among habitats. According to the experience from this case study, it can be concluded that both random forests
and IndVal methods are suitable for the detection of indicative species, and random forests method has some additional advantages. 相似文献
75.
Luijsterburg MS Lindh M Acs K Vrouwe MG Pines A van Attikum H Mullenders LH Dantuma NP 《The Journal of cell biology》2012,197(2):267-281
Nucleotide excision repair (NER) is the principal pathway that removes helix-distorting deoxyribonucleic acid (DNA) damage from the mammalian genome. Recognition of DNA lesions by xeroderma pigmentosum group C (XPC) protein in chromatin is stimulated by the damaged DNA-binding protein 2 (DDB2), which is part of a CUL4A-RING ubiquitin ligase (CRL4) complex. In this paper, we report a new function of DDB2 in modulating chromatin structure at DNA lesions. We show that DDB2 elicits unfolding of large-scale chromatin structure independently of the CRL4 ubiquitin ligase complex. Our data reveal a marked adenosine triphosphate (ATP)-dependent reduction in the density of core histones in chromatin containing UV-induced DNA lesions, which strictly required functional DDB2 and involved the activity of poly(adenosine diphosphate [ADP]-ribose) polymerase 1. Finally, we show that lesion recognition by XPC, but not DDB2, was strongly reduced in ATP-depleted cells and was regulated by the steady-state levels of poly(ADP-ribose) chains. 相似文献
76.
Pott C Steinritz D Bölck B Mehlhorn U Brixius K Schwinger RH Bloch W 《American journal of physiology. Cell physiology》2006,290(5):C1437-C1445
In endothelial cells, two ways of endothelial nitric oxide (NO) synthase (eNOS) activation are known: 1) translocation and 2) Akt-dependent phosphorylation of the enzyme at Ser1177 (Ser1177 eNOS). We have recently shown that agonist-induced Ser1177 eNOS phosphorylation also occurs in human myocardium (10). In this study, we investigated the Ca2+ dependency of these two mechanisms in human atrium. Therefore, atrial tissue was obtained from patients who underwent coronary artery bypass operations. In immunohistochemical experiments, the translocated form of eNOS and phosphorylated Ser1177 eNOS were labeled using specific antibodies. eNOS translocation was measured in the absence and presence of the Ca2+ chelator BAPTA before and after application of BRL 37344 (BRL), a 3-adrenoceptor agonist that increases eNOS activity (34). In the absence of BAPTA, BRL time dependently increased the staining intensity of translocated eNOS, whereas in the presence of BAPTA, this effect was blunted. In contrast, BRL clearly increased the staining of phosphorylated Ser1177 eNOS even in the presence of BAPTA. This observation was confirmed using Western blot analysis. Using the NO-sensitive dye diaminofluorescein, we have demonstrated that BRL induced a strong NO release. This effect was completely abolished in the presence of BAPTA but was unaffected by LY-292004, an inhibitor of phosphatidylinositol 3-kinase activity and eNOS phosphorylation. Although Ca2+ dependent, neither the translocation of eNOS nor NO release was changed by the adenylate cyclase activator forskolin. In conclusion, 1) in human atrial myocardium, BRL-induced eNOS translocation but not Ser1177 eNOS phosphorylation is dependent on intracellular Ca2+. 2) In atrial myocardium, eNOS-translocation and not Ser1177 eNOS phosphorylation is responsible for generating the main amount of NO. 3) Although Ca2+ dependent, eNOS translocation and NO release could not be mimicked by adenylate cyclase activation as a mediator of -adrenergic stimulation. 3-adrenoceptor; BRL 37344; cardiomyocyte; heart; Ca2+ regulation 相似文献
77.
Klara Stefflova Matthew C. Dulik Athma A. Pai Amy H. Walker Charnita M. Zeigler-Johnson Serigne M. Gueye Theodore G. Schurr Timothy R. Rebbeck 《PloS one》2009,4(11)
Background
Population history can be reflected in group genetic ancestry, where genomic variation captured by the mitochondrial DNA (mtDNA) and non-recombining portion of the Y chromosome (NRY) can separate female- and male-specific admixture processes. Genetic ancestry may influence genetic association studies due to differences in individual admixture within recently admixed populations like African Americans.Principal Findings
We evaluated the genetic ancestry of Senegalese as well as European Americans and African Americans from Philadelphia. Senegalese mtDNA consisted of ∼12% U haplotypes (U6 and U5b1b haplotypes, common in North Africa) while the NRY haplotypes belonged solely to haplogroup E. In Philadelphia, we observed varying degrees of admixture. While African Americans have 9–10% mtDNAs and ∼31% NRYs of European origin, these results are not mirrored in the mtDNA/NRY pools of European Americans: they have less than 7% mtDNAs and less than 2% NRYs from non-European sources. Additionally, there is <2% Native American contribution to Philadelphian African American ancestry and the admixture from combined mtDNA/NRY estimates is consistent with the admixture derived from autosomal genetic data. To further dissect these estimates, we have analyzed our samples in the context of different demographic groups in the Americas.Conclusions
We found that sex-biased admixture in African-derived populations is present throughout the Americas, with continual influence of European males, while Native American females contribute mainly to populations of the Caribbean and South America. The high non-European female contribution to the pool of European-derived populations is consistently characteristic of Iberian colonization. These data suggest that genomic data correlate well with historical records of colonization in the Americas. 相似文献78.
Discovery of antimicrobial peptides (AMP) is to a large extent based on screening of fractions of natural samples in bacterial growth inhibition assays. However, the use of bacteria is not limited to screening for antimicrobial substances. In later steps, bioengineered "bugs" can be applied to both production and characterization of AMPs. Here we describe the idea to use genetically modified Escherichia coli strains for both these purposes. This approach allowed us to investigate SpStrongylocins 1 and 2 from the purple sea urchin Strongylocentrotus purpuratus only based on sequence information from a cDNA library and without previous direct isolation or chemical synthesis of these peptides. The recombinant peptides are proved active against all bacterial strains tested. An assay based on a recombinant E. coli sensor strain expressing insect luciferase, revealed that SpStrongylocins are not interfering with membrane integrity and are therefore likely to have intracellular targets. 相似文献
79.
Imaging apoptotic cells or tissues after cancer therapy in situ would be a very useful tool for assessing proper treatment conditions and therapeutic outcome. By combining therapeutic and imaging functions, we have designed a multifunctional, membrane-permeable, and cancer-specific agent that triggers and images apoptosis in targeted cells. We chose photodynamic therapy (PDT) as an appropriate cancer treatment modality and caspase 3 as an apoptosis-specific imaging target. This targeted photodynamic therapy agent with a built-in apoptosis sensor (TaBIAS) induces photodamage only to target cells and simultaneously identifies those that are apoptotic by its near-infrared fluorescence. It contains a fluorescent photosensitizer used as an anticancer drug and a cancer-associated folate receptor homing molecule connected to a caspase 3 cleavable peptide linker that has a fluorescence quencher on the opposing site. We demonstrated that PDT-triggered cleavage of the peptide linker by caspase 3, one of the key executioner caspases, results in a detectable increase in fluorescence in folate receptor-overexpressing cancer cells and tumors. The presence of apoptosis was confirmed in vitro by flow cytometry and ex vivo by Apoptag assay, supporting the ability of TaBIAS to specifically induce and image apoptosis in situ. 相似文献
80.
Mollinari C Reynaud C Martineau-Thuillier S Monier S Kieffer S Garin J Andreassen PR Boulet A Goud B Kleman JP Margolis RL 《Developmental cell》2003,5(2):295-307
Passenger proteins migrate from inner centromeres to the spindle midzone during late mitosis, and those described to date are essential both for proper chromosome segregation and for completion of cell cleavage. We have purified and cloned the human passenger protein TD-60, and we here report that it is a member of the RCC1 family and that it binds preferentially the nucleotide-free form of the small G protein Rac1. Using siRNA, we further demonstrate that the absence of TD-60 substantially suppresses overall spindle assembly, blocks cells in prometaphase, and activates the spindle assembly checkpoint. These defects suggest TD-60 may have a role in global spindle assembly or may be specifically required to integrate kinetochores into the mitotic spindle. The latter is consistent with a TD-60 requirement for recruitment of the passenger proteins survivin and Aurora B, and suggests that like other passenger proteins, TD-60 is involved in regulation of cell cleavage. 相似文献