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61.
62.
Chloroplast to chromoplast
development involves new synthesis and plastid localization of
nuclear-encoded proteins, as well as changes in the organization of
internal plastid membrane compartments. We have demonstrated that
isolated red bell pepper (Capsicum annuum) chromoplasts
contain the 75-kD component of the chloroplast outer envelope
translocon (Toc75) and are capable of importing chloroplast precursors
in an ATP-dependent fashion, indicating a functional general import
apparatus. The isolated chromoplasts were able to further localize the
33- and 17-kD subunits of the photosystem II O2-evolution
complex (OE33 and OE17, respectively), lumen-targeted precursors that
utilize the thylakoidal Sec and ΔpH pathways, respectively, to the
lumen of an internal membrane compartment. Chromoplasts contained the
thylakoid Sec component protein, cpSecA, at levels comparable to
chloroplasts. Routing of OE17 to the lumen was abolished by ionophores,
suggesting that routing is dependent on a transmembrane ΔpH. The
chloroplast signal recognition particle pathway precursor major
photosystem II light-harvesting chlorophyll a/b protein
failed to associate with chromoplast membranes and instead accumulated
in the stroma following import. The Pftf (plastid
fusion/translocation factor), a
chromoplast protein, integrated into the internal membranes of
chromoplasts during in vitro assays, and immunoblot analysis indicated
that endogenous plastid fusion/translocation factor was also an
integral membrane protein of chromoplasts. These data demonstrate that
the internal membranes of chromoplasts are functional with respect to
protein translocation on the thylakoid Sec and ΔpH
pathways.Plastids are developmentally related organelles capable of
interconversion among a variety of structurally and biochemically
distinct forms in response to both environmental and tissue-specific
cues (Whatley, 1978; Thomson and Whatley, 1980). Formation of
chromoplasts in many fruits is one striking example of this plasticity.
Heavily pigmented, photosynthetically inactive chromoplasts frequently
develop from chloroplasts during ripening. This conversion involves
dramatic changes in the organization and composition of the internal
plastid compartment, which include the loss of proteins involved in
carbon fixation in the stroma and replacement with
chromoplast-specific proteins, the breakdown of the photosynthetic
thylakoid membranes and loss of proteins involved in light capture and
electron transfer, and, in some cases, the formation of new membranes
(Spurr and Harris, 1968; Camara and Brangeon, 1981; Piechulla et al.,
1987; Kuntz et al., 1989).Chromoplast formation is an active rather than simply a degradative
process. New proteins, specific to or enhanced in chromoplasts, are
synthesized and compartmentalized in the plastid (Camara et al., 1995;
Price et al., 1995). Most chromoplast proteins are predicted to be
nuclear encoded, translated on cytoplasmic ribosomes, and
posttranslationally imported into the plastid, as are nuclear-encoded
chloroplast proteins. Import of chloroplast proteins occurs via a
general import machinery that appears to mediate translocation of most
or all proteins that are delivered to the chloroplast stroma, either as
a final destination or as an intermediate location (Cline and Henry,
1996; Robinson and Mant, 1997; Schnell, 1998). Proteins are targeted to
the general import pathway by an N-terminal extension that is
cleaved upon import, resulting in the appearance of a processed
protein of reduced Mr. Presumably, the
import of proteins into chromoplasts is accomplished by the same
machinery that is responsible for import of proteins into chloroplasts,
although this has never been directly examined.In some chromoplasts an extensive set of internal membranes
accumulates, replacing the thylakoids. For example, in the
fibrillar-type chromoplast of bell pepper (Capsicum annuum),
the photosynthetic membranes are replaced by membranous sheets and
vesicles in addition to the carotenoid-rich plastoglobules and fibrils
(Spurr and Harris, 1968; Laborde and Spurr, 1973; Camara and Brangeon,
1981; Deruere et al., 1994). The often extensive internal membranes are
the site of synthesis of keto-xanthophylls, which constitute the major
carotenoids of red fruit (Bouvier et al., 1994).Our interests are in the biogenesis of the internal membranes of
plastids, in particular the proteins that are integral to the bilayer,
as well as those located in the luminal compartment formed by the
bilayer. In chloroplasts, proteins destined for the thylakoid membrane
or lumen are routed from the stroma into the thylakoid membrane and
lumen by one of at least four distinct mechanisms: the ΔpH,
chloroplast SRP, thylakoid Sec pathways, and an apparently spontaneous
insertion mechanism (for review, see Cline and Henry, 1996; Robinson
and Mant, 1997; Schnell, 1998). In view of the extensive internal
membrane system of bell pepper chromoplasts, one would expect the
presence of proteins and accompanying translocation machinery in these
membranes. However, no chromoplast-specific proteins have been
conclusively demonstrated to be either integral or luminal to these
membranes.One protein, Pftf (plastid
fusion/translocation factor),
predicted to be membrane anchored by sequence analysis, has been
purified from the stromal compartment of pepper chromoplasts (Hugueney
et al., 1995). This raised the possibility that mature chromoplasts
lack the ability to localize proteins into/across internal membranes.
To address this question we developed a method for isolating protein
import-competent chromoplasts from bell peppers. Immunoblotting
confirmed that these chromoplasts contain known translocation machinery
components. Chromoplasts were assayed in vitro for their ability to
import and localize passenger proteins from the three known
protein-machinery-dependent thylakoid-targeting pathways. We found
mature chromoplasts to be capable of membrane targeting of proteins
that utilize the thylakoidal Sec and ΔpH pathways but not capable of
inserting a membrane protein, LHCP, which utilizes the chloroplast SRP
pathway. Pftf was inserted into the membranes of these chromoplasts in
a manner similar to that observed in chloroplasts, and resident Pftf
was also found to be integrally associated with chromoplast membranes.
The precise role of these pathways in the formation of bell pepper
chromoplasts remains to be fully elucidated. 相似文献
63.
Griffith JL Coleman LE Raymond AS Goodson SG Pittard WS Tsui C Devine SE 《Genetics》2003,164(3):867-879
Retroviruses and their relatives, the long terminal repeat (LTR) retrotransposons, carry out complex life cycles within the cells of their hosts. We have exploited a collection of gene deletion mutants developed by the Saccharomyces Genome Deletion Project to perform a functional genomics screen for host factors that influence the retrovirus-like Ty1 element in yeast. A total of 101 genes that presumably influence many different aspects of the Ty1 retrotransposition cycle were identified from our analysis of 4483 homozygous diploid deletion strains. Of the 101 identified mutants, 46 had significantly altered levels of Ty1 cDNA, whereas the remaining 55 mutants had normal levels of Ty1 cDNA. Thus, approximately half of the mutants apparently affected the early stages of retrotransposition leading up to the assembly of virus-like particles and cDNA replication, whereas the remaining half affected steps that occur after cDNA replication. Although most of the mutants retained the ability to target Ty1 integration to tRNA genes, 2 mutants had reduced levels of tRNA gene targeting. Over 25% of the gene products identified in this study were conserved in other organisms, suggesting that this collection of host factors can serve as a starting point for identifying host factors that influence LTR retroelements and retroviruses in other organisms. Overall, our data indicate that Ty1 requires a large number of cellular host factors to complete its retrotransposition cycle efficiently. 相似文献
64.
Single nucleotide polymorphisms (SNPs) that map to gaps in the human SNP map 总被引:4,自引:0,他引:4 下载免费PDF全文
Tsui C Coleman LE Griffith JL Bennett EA Goodson SG Scott JD Pittard WS Devine SE 《Nucleic acids research》2003,31(16):4910-4916
An international effort is underway to generate a comprehensive haplotype map (HapMap) of the human genome represented by an estimated 300000 to 1 million ‘tag’ single nucleotide polymorphisms (SNPs). Our analysis indicates that the current human SNP map is not sufficiently dense to support the HapMap project. For example, 24.6% of the genome currently lacks SNPs at the minimal density and spacing that would be required to construct even a conservative tag SNP map containing 300 000 SNPs. In an effort to improve the human SNP map, we identified 140 696 additional SNP candidates using a new bioinformatics pipeline. Over 51 000 of these SNPs mapped to the largest gaps in the human SNP map, leading to significant improvements in these regions. Our SNPs will be immediately useful for the HapMap project, and will allow for the inclusion of many additional genomic intervals in the final HapMap. Nevertheless, our results also indicate that additional SNP discovery projects will be required both to define the haplotype architecture of the human genome and to construct comprehensive tag SNP maps that will be useful for genetic linkage studies in humans. 相似文献
65.
66.
Mogi M Yang J Lambert JF Colvin GA Shiojima I Skurk C Summer R Fine A Quesenberry PJ Walsh K 《The Journal of biological chemistry》2003,278(40):39068-39075
Akt is an important regulator of cell survival, growth, and glucose metabolism in many cell types, but the role of this signaling molecule in hematopoietic stem cells is poorly defined. Side population (SP) cells are enriched for hematopoietic stem cell activity and are defined by their ability to efficiently efflux Hoechst 33342. Bone marrow from Akt1-null mice exhibited a reduced SP fraction. However, bone marrow cellularity, growth factor-responsive progenitor cultures, and engraftable stem cells were normal in these mice. Treatment of bone marrow with LY294002, an inhibitor of the Akt effector protein phosphatidylinositol 3-kinase, led to a reversible loss of the SP fraction. Bcrp1, which encodes the Hoechst dye transporter, was translocated from the membrane to the intracellular compartment under conditions that promote the SP-depleted state. Lentivirus-mediated overexpression of Akt1 in bone marrow markedly increased the SP fraction, whereas there was no effect on bone marrow from Bcrp(-/-) mice. These data suggest that Akt signaling modulates the SP cell phenotype by regulating the expression of Bcrp1. 相似文献
67.
68.
Fluorescein-labeled dextran concentration is increased in BAL fluid after ANTU-induced edema in rats
Guery Benoit P.; Nelson Steve; Viget Nathalie; Fialdes Patrice; Summer Warren R.; Dobard Elizabeth; Beaucaire Gilles; Mason Carol M. 《Journal of applied physiology》1998,85(3):842-848
Several methodologies have been developed to assessalveolocapillary membrane permeability in acute lung injury. Thepurpose of this study was to determine the reliability of FITC-dextran compared with radioactive tracers to assess lung permeability alterations. After intraperitoneal administration of-naphthylthiourea (ANTU, 50 mg/kg) or DMSO-ANTU vehicle, the animalswere euthanized and their lungs were studied in an isolated-lungpreparation. FITC-dextran or radiolabeled tracers were added to theperfusate. At 2 h the bronchoalveolar lavage (BAL) fluid from the ANTUgroup showed a significantly greater amount of fluorescence in thesupernatant after centrifugation of BAL fluid compared with the DMSOgroup. Consistent results were observed with the radioactive tracers: there was an increase in extravascular albumin space and extravascular lung water compared with the control group. No cleavage of the FITCfrom the dextran molecule was evident by chromatography comparing samples recovered from the BAL fluid to the pure FITC-dextran molecule.In conclusion, measurement of FITC-dextran in the supernatant of BALfluid after intravascular administration is a reliable method ofassessing lung permeability changes in vivo and ex vivo. 相似文献
69.
A total of 239 green frogs Rana clamitans, collected between June 3 and August 27, 1998 from 6 locations in southwestern Michigan, was examined for helminths. Of the 26 helminth taxa found, the larval cestode Mesocestoides sp. had the highest mean intensity, followed by the larval trematode Fibricola sp. Of the helminths that mature in frogs, Haematoloechus varioplexus had the highest prevalence and Gorgodera amplicava had the highest mean intensity. Frogs from 118th Pond had the highest species richness (20), mean helminth species richness (5.2), and mean helminth abundance (153.7). Frogs from Constantine East had the highest mean helminth species diversity (0.8778) and evenness (0.6033), followed by frogs from 118th Pond. In all comparisons of mean helminth community species richness, abundance, diversity, and evenness, adult frogs had significantly higher or higher values than did juveniles at each location. Jaccard's coefficients of similarity for the helminth communities for location pairs ranged from 0.545 to 0.823. Nine and 2 core helminth taxa occurred at the local and regional levels, respectively. The differences in several helminth community measures in green frogs among locations stress the importance of local ecological conditions on helminth community structure. 相似文献
70.
Summer E. Young Matthew T. Duvernay Michael L. Schulte Craig W. Lindsley Heidi E. Hamm 《PloS one》2013,8(6)
Protease activated receptor-4 (PAR4) is one of the thrombin receptors on human platelets and is a potential target for the management of thrombotic disorders. We sought to develop potent, selective, and novel PAR4 antagonists to test the role of PAR4 in thrombosis and hemostasis. Development of an expedient three-step synthetic route to access a novel series of indole-based PAR4 antagonists also necessitated the development of a platelet based high-throughput screening assay. Screening and subsequent structure activity relationship analysis yielded several selective PAR4 antagonists as well as possible new scaffolds for future antagonist development. 相似文献