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Superparamagnetic annexin-V conjugated microbeads are able to eliminate spermatozoa with externalized phosphatidylserine,
a membrane feature of apoptotic cells as well as spermatozoa with deteriorated plasma membrane. Our objective was to evaluate
the effects of annexin-V Magnetic-Activated Cell Sorting (MACS) in cryopreservation–thawing protocols and on integrity of
sperm mitochondrial transmembrane potential and mitochondrial integrity survival rate (MSR). Mature spermatozoa of 10 healthy
donors were prepared by density gradient centrifugation and divided into 2 aliquots afterwards. The first one was subjected
to annexin-V MACS followed by cryopreservation and thawing, while the second was cryopreserved–thawed without MACS to serve
as control. Annexin-negative sperm separated by MACS showed significantly higher levels of intact mitochondria following cryopreservation–thawing
(45.4±8.6%) compared to sperm that were not separated (15.8±4.6%, p<0.01). Separating a distinctive population of non-apoptotic spermatozoa with intact membranes may optimize cryopreservation–thawing
outcome. MACS using annexin-V microbeads enhances the percentage of spermatozoa with intact transmembrane mitochondrial potential
and mitochondrial integrity survival rates following cryopreservation. 相似文献
94.
Mullins C Hartnell LM Wassarman DA Bonifacino JS 《Molecular & general genetics : MGG》1999,262(3):401-412
The adaptor protein (AP) complexes AP-1, AP-2, and AP-3 mediate coated vesicle formation and sorting of integral membrane
proteins in the endocytic and late exocytic pathways in mammalian cells. A search of the Drosophila melanogaster expressed sequence tag (EST) database identified orthologs of family members mammalian medium (μ) chain families μ1, μ2,
and μ3, of the corresponding AP complexes, and δ-COP, the analogous component of the coatomer (COPI) complex. The Drosophila
orthologs exhibit a high degree of sequence identity to mammalian medium chain and δ-COP proteins. Northern analysis demonstrated
that medium chain and δ-COP mRNAs are expressed uniformly throughout fly development. Medium chain and δ-COP genes were cytologically
mapped and the μ3 gene was found to localize to a region containing the pigmentation locus carmine (cm). Analysis of genomic DNA of the cm
1
mutant allele indicated the presence of a large insertion in the coding region of the μ3 gene and Northern analysis revealed
no detectable μ3 mRNA. Light microscopy of the cm
1
mutant showed a reduction in primary, secondary, and tertiary pigment granules in the adult eye. These findings provide evidence
of a role for μ3 in the sorting processes required for pigment granule biogenesis in Drosophila.
Received: 7 June 1999 / Accepted: 4 July 1999 相似文献
95.
The localization of a protein in a cell is closely correlated with its biological function. With the explosion of protein sequences entering into DataBanks, it is highly desired to develop an automated method that can fast identify their subcellular location. This will expedite the annotation process, providing timely useful information for both basic research and industrial application. In view of this, a powerful predictor has been developed by hybridizing the gene ontology approach [Nat. Genet. 25 (2000) 25], functional domain composition approach [J. Biol. Chem. 277 (2002) 45765], and the pseudo-amino acid composition approach [Proteins Struct. Funct. Genet. 43 (2001) 246; Erratum: ibid. 44 (2001) 60]. As a showcase, the recently constructed dataset [Bioinformatics 19 (2003) 1656] was used for demonstration. The dataset contains 7589 proteins classified into 12 subcellular locations: chloroplast, cytoplasmic, cytoskeleton, endoplasmic reticulum, extracellular, Golgi apparatus, lysosomal, mitochondrial, nuclear, peroxisomal, plasma membrane, and vacuolar. The overall success rate of prediction obtained by the jackknife cross-validation was 92%. This is so far the highest success rate performed on this dataset by following an objective and rigorous cross-validation procedure. 相似文献
96.
Desmosomes are cell junctions and cytoskeleton-anchoring structures of epithelia, the myocardium, and dendritic reticulum cells of lymphatic follicles whose major components are known. Using cultured HT-1080 SL-1 fibrosarcoma-derived cells and transfection of cDNAs encoding specific desmosomal components, we have determined a minimum ensemble of proteins sufficient to introduce de novo structures, which, by morphology and functional competence, are indistinguishable from authentic desmosomes. In a more refined analysis, the influence of the desmosomal proteins desmoplakin (Dp), plakoglobin (Pg), and plakophilin 2 (Pp2) on the lateral clustering of the desmosomal transmembrane-glycoprotein desmoglein 2 (Dsg) was examined. We found that for efficient clustering of desmoglein 2 and desmosome structure formation, all three major plaque proteins-desmoplakin, plakoglobin, and plakophilin 2- were necessary. Furthermore, in this cell model, plakophilin 2 was capable of directing desmoplakin to adhaerens junctions (AJ), whereas plakoglobin was crucial for the segregation of desmosomal and AJ components. These results are discussed with respect to the variability in cell junction composition observed in various nonepithelial tissues. 相似文献
97.
Allometric scaling relationships or quarter-power rules, as a universal biological law, can be viewed as having some genetic component, and the particular genes (or quantitative trait loci, QTL) underlying these allometric relationships can be mapped using molecular markers. We develop a mathematical and statistical model for mapping allometric QTL on the basis of nonlinear power functions using Taylors approximation theory. Simulation studies indicate that the QTL position and effect can be estimated using our model, but the estimation precision can be improved from the higher- over lower-order approximation when the sample size used and gene effects are small. The application of our approach in a real example from forest trees leads to successful detection of a QTL governing the allometric relationship between 3rd-year stem height and 3rd-year stem biomass. It is expected that our model will have broad implications for genetic, evolutionary, biomedical and breeding research. 相似文献
98.
Predicting subcellular localization of proteins by hybridizing functional domain composition and pseudo-amino acid composition 总被引:3,自引:0,他引:3
Recent advances in large-scale genome sequencing have led to the rapid accumulation of amino acid sequences of proteins whose functions are unknown. Since the functions of these proteins are closely correlated with their subcellular localizations, many efforts have been made to develop a variety of methods for predicting protein subcellular location. In this study, based on the strategy by hybridizing the functional domain composition and the pseudo-amino acid composition (Cai and Chou [2003]: Biochem. Biophys. Res. Commun. 305:407-411), the Intimate Sorting Algorithm (ISort predictor) was developed for predicting the protein subcellular location. As a showcase, the same plant and non-plant protein datasets as investigated by the previous investigators were used for demonstration. The overall success rate by the jackknife test for the plant protein dataset was 85.4%, and that for the non-plant protein dataset 91.9%. These are so far the highest success rates achieved for the two datasets by following a rigorous cross validation test procedure, further confirming that such a hybrid approach may become a very useful high-throughput tool in the area of bioinformatics, proteomics, as well as molecular cell biology. 相似文献
99.
100.
A role for CBS domain 2 in trafficking of chloride channel CLC-5 总被引:5,自引:0,他引:5
CLC-5 is a member of the CLC family of voltage-gated chloride channels. Mutations disrupting CLC-5 lead to Dent's disease, an X-linked renal tubular disorder, characterised by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis, and renal stones. Sequence analysis of CLC-5 reveals a 746 amino acid protein with an intracellular amino-terminus, transmembrane spanning domains, and two CBS domains within its intracellular carboxy-terminus. CBS domains have been implicated in intracellular targetting and trafficking as well as protein-protein interactions. We investigate subcellular localisation of three naturally occurring CLC-5 mutants which all lead to a truncated protein, disrupting the second CBS domain. These mutants are unable to traffic normally to acidic endosomes but are retained in perinuclear compartments, colocalising with the Golgi complex. This is the first identification of the cellular pathogenesis of CBS domain mutations of CLC-5. 相似文献