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61.
Tropical peat swamp forests are important and endangered ecosystems, although little is known of their microbial diversity
and ecology. We used molecular and enzymatic techniques to examine patterns in prokaryotic community structure and overall
microbial activity at 0-, 10-, 20-, and 50-cm depths in sediments in a peat swamp forest in Malaysia. Denaturing gradient
gel electrophoresis profiles of amplified 16S ribosomal ribonucleic acid (rRNA) gene fragments showed that different depths
harbored different bacterial assemblages and that Archaea appeared to be limited to the deeper samples. Cloning and sequencing
of longer 16S rRNA gene fragments suggested reduced microbial diversity in the deeper samples compared to the surface. Bacterial
clone libraries were largely dominated by ribotypes affiliated with the Acidobacteria, which accounted for at least 27–54%
of the sequences obtained. All of the sequenced representatives from the archaeal clone libraries were Crenarchaeota. Activities
of microbial extracellular enzymes involved in carbon, nitrogen, and phosphorus cycling declined appreciably with depth, the
only exception being peroxidase. These results show that tropical peat swamp forests are unusual systems with microbial assemblages
dominated by members of the Acidobacteria and Crenarchaeota. Microbial communities show clear changes with depth, and most
microbial activity is likely confined to populations in the upper few centimeters, the site of new leaf litter fall, rather
than the deeper, older, peat layers. 相似文献
62.
Matthew J. Betzenhauser Larry E. Wagner II Hyung Seo Park David I. Yule 《The Journal of biological chemistry》2009,284(24):16156-16163
ATP is known to increase the activity of the type-1 inositol 1,4,5-trisphosphate receptor (InsP3R1). This effect is attributed to the binding of ATP to glycine rich Walker A-type motifs present in the regulatory domain of the receptor. Only two such motifs are present in neuronal S2+ splice variant of InsP3R1 and are designated the ATPA and ATPB sites. The ATPA site is unique to InsP3R1, and the ATPB site is conserved among all three InsP3R isoforms. Despite the fact that both the ATPA and ATPB sites are known to bind ATP, the relative contribution of these two sites to the enhancing effects of ATP on InsP3R1 function is not known. We report here a mutational analysis of the ATPA and ATPB sites and conclude neither of these sites is required for ATP modulation of InsP3R1. ATP augmented InsP3-induced Ca2+ release from permeabilized cells expressing wild type and ATP-binding site-deficient InsP3R1. Similarly, ATP increased the single channel open probability of the mutated InsP3R1 to the same extent as wild type. ATP likely exerts its effects on InsP3R1 channel function via a novel and as yet unidentified mechanism.Inositol 1,4,5-trisphosphate receptors (InsP3R)3 are a family of large, tetrameric, InsP3-gated cation channels. The three members of this family (InsP3R1, InsP3R2, and InsP3R3) are nearly ubiquitously expressed and are localized primarily to the endoplasmic reticulum (ER) membrane (1–3). Numerous hormones, neurotransmitters, and growth factors bind to receptors that stimulate phospholipase C-induced InsP3 production (4). InsP3 subsequently binds to the InsP3R and induces channel opening. This pathway represents a major mechanism for Ca2+ liberation from ER stores (5). All three InsP3R isoforms are dynamically regulated by cytosolic factors in addition to InsP3 (1). Ca2+ is perhaps the most important determinant of InsP3R activity besides InsP3 itself and is known to regulate InsP3R both positively and negatively (6). ATP, in concert with InsP3 and Ca2+, also regulates InsP3R as do numerous kinases, phosphatases, and protein-binding partners (7–10). This intricate network of regulation allows InsP3R activity to be finely tuned by the local cytosolic environment (9). As a result, InsP3-induced Ca2+ signals can exhibit a wide variety of spatial and temporal patterns, which likely allows Ca2+ to control many diverse cellular processes.Modulation of InsP3-induced Ca2+ release (IICR) by ATP and other nucleotides provides a direct link between intracellular Ca2+ signaling and the metabolic state of the cell. Metabolic fluctuations could, therefore, impact Ca2+ signaling in many cell types given that InsP3R are expressed in all cells (11, 12). Consistent with this, ATP has been shown to augment IICR in many diverse cell types including primary neurons (13), smooth muscle cells (14), and exocrine acinar cells (15) as well as in immortalized cell lines (16–18). The effects of ATP on InsP3R function do not require hydrolysis because non-hydrolyzable ATP analogues are as effective as ATP (7, 14). ATP is thought to bind to distinct regions in the central, coupling domain of the receptors and to facilitate channel opening (2, 19). ATP is not required for channel gating, but instead, increases InsP3R activity in an allosteric fashion by increasing the open probability of the channel in the presence of activating concentrations of InsP3 and Ca2+ (7, 8, 20).Despite a wealth of knowledge regarding the functional effects of ATP on InsP3R function, there is relatively little known about the molecular determinants of these actions. ATP is thought to exert effects on channel function by direct binding to glycine-rich regions containing the consensus sequence GXGXXG that are present in the receptors (2). These sequences were first proposed to be ATP-binding domains due to their similarity with Walker A motifs (21). The neuronal S2+ splice variant of InsP3R1 contains two such domains termed ATPA and ATPB. A third site, ATPC, is formed upon removal of the S2 splice site (2, 22). The ATPB site is conserved in InsP3R2 and InsP3R3, while the ATPA and ATPC sites are unique to InsP3R1. Our prior work examining the functional consequences of mutating these ATP-binding sites has yielded unexpected results. For example, mutating the ATPB site in InsP3R2 completely eliminated the enhancing effects of ATP on this isoform while mutating the analogous site in InsP3R3 failed to alter the effects of ATP (23). This indicated the presence of an additional locus for ATP modulation of InsP3R3. In addition, mutation of the ATPC in the S2− splice variant of InsP3R1 did not alter the ability of ATP to modulate Ca2+ release, but instead impaired the ability of protein kinase A to phosphorylate Ser-1755 of this isoform (22).The ATPA and ATPB sites in InsP3R1 were first identified as putative nucleotide-binding domains after the cloning of the full-length receptor (24). Early binding experiments with 8-azido-[α-32P]ATP established that ATP cross-linked with receptor purified from rat cerebellum at one site per receptor monomer (19). Later, more detailed, binding experiments on trypsinized recombinant rat InsP3R1 showed cross-linking of ATP to two distinct regions of the receptor that corresponded with the ATPA and ATPB sites (17). We and others (16, 22, 23) have also reported the binding of ATP analogues to purified GST fusions of small regions of InsP3R1 surrounding the ATPA and ATPB sites. It is widely accepted, in the context of the sequence similarity to Walker A motifs and biochemical data, that the ATPA and ATPB sites are the loci where ATP exerts its positive functional effects on InsP3R1 function (1–3, 16). Furthermore, the higher affinity of the ATPA site to ATP is thought to confer the higher sensitivity of InsP3R1 to ATP versus InsP3R3, which contains the ATPB site exclusively (25, 26). The purpose of this study, therefore, was to examine the contributions of the ATPA and ATPB sites to ATP modulation of the S2+ splice variant of InsP3R1. We compared the effects of ATP on InsP3R1 and on ATP-binding site mutated InsP3R1 using detailed functional analyses in permeabilized cells and in single channel recordings. Here we report that InsP3R1 is similar to InsP3R3 in that ATP modulates IICR even at maximal InsP3 concentrations and that neither the ATPA nor the ATPB site is required for this effect. 相似文献
63.
Bruno G. Madeira Mário M. Espírito-Santo Santos D’?ngelo Neto Yule R. F. Nunes G. Arturo Sánchez Azofeifa G. Wilson Fernandes Mauricio Quesada 《Plant Ecology》2009,201(1):291-304
We investigated changes in species composition and structure of tree and liana communities along a successional gradient in
a seasonally dry tropical forest. There was a progressive increase in tree richness and all tree structural traits from early
to late stages, as well as marked changes in tree species composition and dominance. This pattern is probably related to pasture
management practices such as ploughing, which remove tree roots and preclude regeneration by resprouting. On the other hand,
liana density decreased from intermediate to late stages, showing a negative correlation with tree density. The higher liana
abundance in intermediate stage is probably due to a balanced availability of support and light availability, since these
variables may show opposite trends during forest growth. Predicted succession models may represent extremes in a continuum
of possible successional pathways strongly influenced by land use history, climate, soil type, and by the outcomes of tree–liana
interactions. 相似文献
64.
65.
David I. Yule 《The international journal of biochemistry & cell biology》2010,42(11):1757-1761
Pancreatic acinar cells are classical exocrine gland cells. The apical regions of clusters of coupled acinar cells collectively form a lumen which constitutes the blind end of a tube created by ductal cells – a structure reminiscent of a “bunch of grapes”. When activated by neural or hormonal secretagogues, pancreatic acinar cells are stimulated to secrete a variety of proteins. These proteins are predominately inactive digestive enzyme precursors called “zymogens”. Acinar cell secretion is absolutely dependent on secretagogue-induced increases in intracellular free Ca2+. The increase in [Ca2+]i has precise temporal and spatial characteristics as a result of the exquisite regulation of the proteins responsible for Ca2+ release, Ca2+ influx and Ca2+ clearance in the acinar cell. This brief review discusses recent studies in which transgenic animal models have been utilized to define in molecular detail the components of the Ca2+ signaling machinery which contribute to these characteristics. 相似文献
66.
Wataru Masuda Matthew J. Betzenhauser David I. Yule 《The Journal of biological chemistry》2010,285(48):37927-37938
Ca2+ release through inositol 1,4,5-trisphosphate receptors (InsP3R) can be modulated by numerous factors, including input from other signal transduction cascades. These events shape the spatio-temporal characteristics of the Ca2+ signal and provide fidelity essential for the appropriate activation of effectors. In this study, we investigate the regulation of Ca2+ release via InsP3R following activation of cyclic nucleotide-dependent kinases in the presence and absence of expression of a binding partner InsP3R-associated cGMP kinase substrate (IRAG). cGMP-dependent kinase (PKG) phosphorylation of only the S2+ InsP3R-1 subtype resulted in enhanced Ca2+ release in the absence of IRAG expression. In contrast, IRAG bound to each InsP3R subtype, and phosphorylation of IRAG by PKG attenuated Ca2+ release through all InsP3R subtypes. Surprisingly, simply the expression of IRAG attenuated phosphorylation and inhibited the enhanced Ca2+ release through InsP3R-1 following cAMP-dependent protein kinase (PKA) activation. In contrast, IRAG expression did not influence the PKA-enhanced activity of the InsP3R-2. Phosphorylation of IRAG resulted in reduced Ca2+ release through all InsP3R subtypes during concurrent activation of PKA and PKG, indicating that IRAG modulation is dominant under these conditions. These studies yield mechanistic insight into how cells with various complements of proteins integrate and prioritize signals from ubiquitous signaling pathways. 相似文献
67.
Bruce JI Giovannucci DR Blinder G Shuttleworth TJ Yule DI 《The Journal of biological chemistry》2004,279(13):12909-12917
Parotid acinar cells exhibit rapid cytosolic calcium signals ([Ca2+]i) that initiate in the apical region but rapidly become global in nature. These characteristic [Ca2+]i signals are important for effective fluid secretion, which critically depends on a synchronized activation of spatially separated ion fluxes. Apically restricted [Ca2+]i signals were never observed in parotid acinar cells. This is in marked contrast to the related pancreatic acinar cells, where the distribution of mitochondria has been suggested to contribute to restricting [Ca2+]i signals to the apical region. Therefore, the aim of this study was to determine the mitochondrial distribution and the role of mitochondrial Ca2+ uptake in shaping the spatial and temporal properties of [Ca2+]i signaling in parotid acinar cells. Confocal imaging of cells stained with MitoTracker dyes (MitoTracker Green FM or MitoTracker CMXRos) and SYTO dyes (SYTO-16 and SYTO-61) revealed that a majority of mitochondria is localized around the nucleus. Carbachol (CCh) and caged inositol 1,4,5-trisphosphate-evoked [Ca2+]i signals were delayed as they propagated through the nucleus. This delay in the CCh-evoked nuclear [Ca2+]i signal was abolished by inhibition of mitochondrial Ca2+ uptake with ruthenium red and Ru360. Likewise, simultaneous measurement of [Ca2+]i with mitochondrial [Ca2+] ([Ca2+]m), using fura-2 and rhod-FF, respectively, revealed that mitochondrial Ca2+ uptake was also inhibited by ruthenium red and Ru360. Finally, at concentrations of agonist that evoke[Ca2+]i oscillations, mitochondrial Ca2+ uptake, and a nuclear [Ca2+] delay, CCh also evoked a substantial increase in NADH autofluorescence. This autofluorescence exhibited a predominant perinuclear localization that was also sensitive to mitochondrial inhibitors. These data provide evidence that perinuclear mitochondria and mitochondrial Ca2+ uptake may differentially shape nuclear [Ca2+] signals but more importantly drive mitochondrial metabolism to generate ATP close to the nucleus. These effects may profoundly affect a variety of nuclear processes in parotid acinar cells while facilitating efficient fluid secretion. 相似文献
68.
Sneyd J Tsaneva-Atanasova K Bruce JI Straub SV Giovannucci DR Yule DI 《Biophysical journal》2003,85(3):1392-1405
We construct a mathematical model of Ca(2+) wave propagation in pancreatic and parotid acinar cells. Ca(2+) release is via inositol trisphosphate receptors and ryanodine receptors that are distributed heterogeneously through the cell. The apical and basal regions are separated by a region containing the mitochondria. In response to a whole-cell, homogeneous application of inositol trisphosphate (IP(3)), the model predicts that 1), at lower concentrations of IP(3), the intracellular waves in pancreatic cells begin in the apical region and are actively propagated across the basal region by Ca(2+) release through ryanodine receptors; 2), at higher [IP(3)], the waves in pancreatic and parotid cells are not true waves but rather apparent waves, formed as the result of sequential activation of inositol trisphosphate receptors in the apical and basal regions; 3), the differences in wave propagation in pancreatic and parotid cells can be explained in part by differences in inositol trisphosphate receptor density; 4), in pancreatic cells, increased Ca(2+) uptake by the mitochondria is capable of restricting Ca(2+) responses to the apical region, but that this happens only for a relatively narrow range of [IP(3)]; and 5), at higher [IP(3)], the apical and basal regions of the cell act as coupled Ca(2+) oscillators, with the basal region partially entrained to the apical region. 相似文献
69.
Catimel B; Scott AM; Lee FT; Hanai N; Ritter G; Welt S; Old LJ; Burgess AW; Nice EC 《Glycobiology》1998,8(9):927-938
We describe a novel immobilization technique to investigate interactions
between immobilized gangliosides (GD3, GM1, and GM2) and their respective
antibodies, antibody fragments, or binding partners using an optical
biosensor. Immobilization was performed by direct injection onto a
carboxymethyldextran sensor chip and did not require derivatization of the
sensor surface or the ganglioside. The ganglioside appeared to bind to the
sensor surface by hydrophobic interaction, leaving the carbohydrate epitope
available for antibody or, in the case of GM1, cholera toxin binding. The
carboxyl group of the dextran chains on the sensor surface did not appear
to be involved in the immobilization as evidenced by equivalent levels of
immobilization following conversion of the carboxyl groups into acyl amino
esters, but rather the dextran layer provided a hydrophilic coverage of the
sensor chip which was essential to prevent nonspecific binding. This
technique gave better reactivity and specificity for anti- ganglioside
monoclonal antibodies (anti-GD3: KM871, KM641, R24; and anti-GM2: KM966)
than immobilization by hydrophobic interaction onto a gold sensor surface
or photoactivated cross-linking onto carboxymethydextran. This rapid
immobilization procedure has facilitated detailed kinetic analysis of
ganglioside/antibody interactions, with the surface remaining viable for a
large number of cycles (>125). Kinetic constants were determined from
the biosensor data using linear regression, nonlinear least squares and
equilibrium analysis. The values of kd, ka, and KAobtained by nonlinear
analysis (KAKM871 = 1.05, KM641 = 1.66, R24 = 0.14, and KM966 = 0.65 x
10(7) M- 1) were essentially independent of concentration and showed good
agreement with data obtained by other analytical methods.
相似文献
70.
Huangai Li Danfeng Zhang Ke Xie Yan Wang Qiansheng Liao Yiguo Hong Yule Liu 《Plant physiology》2021,187(4):2865
Virus-induced gene silencing (VIGS) is a versatile and attractive approach for functional gene characterization in plants. Although several VIGS vectors for maize (Zea mays) have been previously developed, their utilities are limited due to low viral infection efficiency, insert instability, short maintenance of silencing, inadequate inoculation method, or abnormal requirement of growth temperature. Here, we established a Cucumber mosaic virus (CMV)-based VIGS system for efficient maize gene silencing that overcomes many limitations of VIGS currently available for maize. Using two distinct strains, CMV-ZMBJ and CMV-Fny, we generated a pseudorecombinant-chimeric (Pr) CMV. Pr CMV showed high infection efficacy but mild viral symptoms in maize. We then constructed Pr CMV-based vectors for VIGS, dubbed Pr CMV VIGS. Pr CMV VIGS is simply performed by mechanical inoculation of young maize leaves with saps of Pr CMV-infected Nicotiana benthamiana under normal growth conditions. Indeed, suppression of isopentenyl/dimethylallyl diphosphate synthase (ZmIspH) expression by Pr CMV VIGS resulted in non-inoculated leaf bleaching as early as 5 d post-inoculation (dpi) and exhibited constant and efficient systemic silencing over the whole maize growth period up to 105 dpi. Furthermore, utilizing a ligation-independent cloning (LIC) strategy, we developed a modified Pr CMV-LIC VIGS vector, allowing easy gene cloning for high-throughput silencing in maize. Thus, our Pr CMV VIGS system provides a much-improved toolbox to facilitate efficient and long-duration gene silencing for large-scale functional genomics in maize, and our pseudorecombination-chimera combination strategy provides an approach to construct efficient VIGS systems in plants.A pseudorecombinant-chimeric Cucumber mosaic virus-based virus-induced gene silencing system rapidly and efficiently triggers persistent gene silencing in maize. 相似文献