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181.
Adrien W. Schmid Diego Chiappe V��r��ne Pignat Valerie Grimminger Ivan Hang Marc Moniatte Hilal A. Lashuel 《The Journal of biological chemistry》2009,284(19):13128-13142
Tissue transglutaminase (tTG) has been implicated in the pathogenesis of
Parkinson disease (PD). However, exactly how tTG modulates the structural and
functional properties of α-synuclein (α-syn) and contributes to
the pathogenesis of PD remains unknown. Using site-directed mutagenesis
combined with detailed biophysical and mass spectrometry analyses, we sought
to identify the exact residues involved in tTG-catalyzed cross-linking of
wild-type α-syn and α-syn mutants associated with PD. To better
understand the structural consequences of each cross-linking reaction, we
determined the effect of tTG-catalyzed cross-linking on the oligomerization,
fibrillization, and membrane binding of α-syn in vitro. Our
findings show that tTG-catalyzed cross-linking of monomeric α-syn
involves multiple cross-links (specifically 2-3). We subjected tTG-catalyzed
cross-linked monomeric α-syn composed of either wild-type or Gln →
Asn mutants to sequential proteolysis by multiple enzymes and peptide mapping
by mass spectrometry. Using this approach, we identified the glutamine and
lysine residues involved in tTG-catalyzed intramolecular cross-linking of
α-syn. These studies demonstrate for the first time that
Gln79 and Gln109 serve as the primary tTG reactive
sites. Mutating both residues to asparagine abolishes tTG-catalyzed
cross-linking of α-syn and tTG-induced inhibition of α-syn
fibrillization in vitro. To further elucidate the sequence and
structural basis underlying these effects, we identified the lysine residues
that form isopeptide bonds with Gln79 and Gln109. This
study provides mechanistic insight into the sequence and structural basis of
the inhibitory effects of tTG on α-syn fibrillogenesis in vivo,
and it sheds light on the potential role of tTG cross-linking on modulating
the physiological and pathogenic properties of α-syn.Parkinson disease
(PD)2 is a progressive
movement disorder that is caused by the loss of dopaminergic neurons in the
substantia nigra, the part of the brain responsible for controlling movement.
Clinically, PD is manifested in symptoms that include tremors, rigidity, and
difficulty in initiating movement (bradykinesia). Pathologically, PD is
characterized by the presence of intraneuronal, cytoplasmic inclusions known
as Lewy bodies (LB), which are composed primarily of the protein
“α-synuclein” (α-syn)
(1) and are seen in the
post-mortem brains of PD patients with the sporadic or familial forms of the
disease (2). α-Syn is a
presynaptic protein of 140 residues with a “natively” unfolded
structure (3). Three missense
point mutations in α-syn (A30P, E46K, and A53T) are associated with the
early-onset, dominant, inherited form of PD
(4,
5). Moreover, duplication or
triplication of the α-syn gene has been linked to the familial
form of PD, suggesting that an increase in α-syn expression is
sufficient to cause PD. Together, these findings suggest that α-syn
plays a central role in the pathogenesis of PD.The molecular and cellular determinants that govern α-syn
oligomerization and fibrillogenesis in vivo remain poorly understood.
In vitro aggregation studies have shown that the mutations associated
with PD (A30P, E46K, and A53T) accelerate α-syn oligomerization, but
only E46K and A53T α-syn show higher propensity to fibrillize than
wild-type (WT) α-syn
(6-8).
This suggests that oligomerization, rather than fibrillization, is linked to
early-onset familial PD (9).
Our understanding of the molecular composition and biochemical state of
α-syn in LBs has provided important clues about protein-protein
interactions and post-translational modifications that may play a role in
modulating oligomerization, fibrillogenesis, and LB formation of the protein.
In addition to ubiquitination
(10), phosphorylation
(11,
12), nitration
(13,
14), and C-terminal truncation
(15,
16), analysis of post-mortem
brain tissues from PD and Lewy bodies in dementia patients has confirmed the
colocalization of tissue transglutaminase (tTG)-catalyzed cross-linked
α-syn monomers and higher molecular aggregates in LBs within
dopaminergic neurons (17,
18). Tissue transglutaminase
catalyzes a calcium-dependent transamidating reaction involving glutamine and
lysine residues, which results in the formation of a covalent cross-link via
ε-(γ-glutamyl) lysine bonds
(Fig. 2F). To date,
seven different isoforms of tTGs have been reported, of which only tTG2 seems
to be expressed in the human brain
(19), whereas tTG1 and tTG3
are more abundantly found in stratified squamous epithelia
(20). Subsequent
immuno-histochemical, colocalization, and immunoprecipitation studies have
shown that the levels of tTG and cross-linked α-syn species are
increased in the substantia nigra of PD brains
(17). These findings, combined
with the known role of tTG in cross-linking and stabilizing bimolecular
assemblies, led to the hypothesis that tTG plays an important role in the
initiation and propagation of α-syn fibril formation and that it
contributes to fibril stability in LBs. This hypothesis was initially
supported by in vitro studies demonstrating that tTG catalyzes the
polymerization of the α-syn-derived non-amyloid component (NAC) peptide
via intermolecular covalent cross-linking of residues Gln79 and
Lys80 (21) and by
other studies suggesting that tTG promotes the fibrillization of amyloidogenic
proteins implicated in the pathogenesis of other neurodegenerative diseases
such as Alzheimer disease, supranuclear palsy, Huntington disease, and other
polyglutamine diseases
(22-24).
However, recent in vitro studies with full-length α-syn have
shown that tTG catalyzes intramolecular cross-linking of monomeric α-syn
and inhibits, rather than promotes, its fibrillization in vitro
(25,
26). The structural basis of
this inhibitory effect and the exact residues involved in tTG-mediated
cross-linking of α-syn, as well as structural and functional
consequences of these modifications, remain poorly understood.Open in a separate windowFIGURE 2.tTG-catalyzed cross-linking of α-syn involves one to three
intramolecular cross-links. A-C, MALDI-TOF/TOF analysis of native
(—) and cross-linked (- - -) α-syn, showing that most
tTG-catalyzed cross-linking products of WT or disease-associated mutant forms
of α-syn are intramolecularly linked (predominant peak with two
cross-links), and up to three intramolecular cross-links can occur (left
shoulder). The abbreviations M and m/cl are
used to designate native and cross-linked α-synuclein, respectively.
D and E, kinetic analysis of α-syn (A30P)
cross-linking monitored by MALDI-TOF and SDS-PAGE. F, schematic
depiction of the tTG-catalyzed chemical reaction (isodipeptide formation)
between glutamine and lysine residues.In this study, we have identified the primary glutamine and lysine residues
involved in tTG-catalyzed, intramolecularly cross-linked monomeric α-syn
and investigated how cross-linking these residues affects the oligomerization,
fibrillization, and membrane binding of α-syn in vitro. Using
single-site mutagenesis and mass spectrometry applied to exhaustive
proteolytic digests of native and cross-linked monomeric α-syn, we
identified Gln109 and Gln79 as the major tTG substrates.
We demonstrate that the altered electrophoretic mobility of the
intramolecularly cross-linked α-syn in SDS-PAGE occurs as a result of
tTG-catalyzed cross-linking of Gln109 to lysine residues in the N
terminus of α-syn, which leads to the formation of more compact
monomers. Consistent with previous studies, we show that intramolecularly
cross-linked α-syn forms off-pathway oligomers that are distinct from
those formed by the wild-type protein and that do not convert to fibrils
within the time scale of our experiments (3-5 days). We also show that
membrane-bound α-syn is a substrate of tTG and that intramolecular
cross-linking does not interfere with the ability of monomeric α-syn to
adopt an α-helical conformation upon binding to synthetic membranes.
These studies provide novel mechanistic insight into the sequence and
structural basis of events that allow tTG to inhibit α-syn
fibrillogenesis, and they shed light on the potential role of tTG-catalyzed
cross-linking in modulating the physiological and pathogenic properties of
α-syn. 相似文献
182.
Roy S Chakravarty D Cortez V De Mukhopadhyay K Bandyopadhyay A Ahn JM Raj GV Tekmal RR Sun L Vadlamudi RK 《Molecular cancer research : MCR》2012,10(1):25-33
Breast cancer metastasis is a major clinical problem. The molecular basis of breast cancer progression to metastasis remains poorly understood. PELP1 is an estrogen receptor (ER) coregulator that has been implicated as a proto-oncogene whose expression is deregulated in metastatic breast tumors and whose expression is retained in ER-negative tumors. We examined the mechanism and significance of PELP1-mediated signaling in ER-negative breast cancer progression using two ER-negative model cells (MDA-MB-231 and 4T1 cells) that stably express PELP1-shRNA. These model cells had reduced PELP1 expression (75% of endogenous levels) and exhibited less propensity to proliferate in growth assays in vitro. PELP1 downregulation substantially affected migration of ER-negative cells in Boyden chamber and invasion assays. Using mechanistic studies, we found that PELP1 modulated expression of several genes involved in the epithelial mesenchymal transition (EMT), including MMPs, SNAIL, TWIST, and ZEB. In addition, PELP1 knockdown reduced the in vivo metastatic potential of ER-negative breast cancer cells and significantly reduced lung metastatic nodules in a xenograft assay. These results implicate PELP1 as having a role in ER-negative breast cancer metastasis, reveal novel mechanism of coregulator regulation of metastasis via promoting cell motility/EMT by modulating expression of genes, and suggest PELP1 may be a potential therapeutic target for metastatic ER-negative breast cancer. 相似文献
183.
184.
Valerie Cortez Katherine Odem-Davis R. Scott McClelland Walter Jaoko Julie Overbaugh 《PLoS pathogens》2012,8(3)
Identifying naturally-occurring neutralizing antibodies (NAb) that are cross-reactive against all global subtypes of HIV-1 is an important step toward the development of a vaccine. Establishing the host and viral determinants for eliciting such broadly NAbs is also critical for immunogen design. NAb breadth has previously been shown to be positively associated with viral diversity. Therefore, we hypothesized that superinfected individuals develop a broad NAb response as a result of increased antigenic stimulation by two distinct viruses. To test this hypothesis, plasma samples from 12 superinfected women each assigned to three singly infected women were tested against a panel of eight viruses representing four different HIV-1 subtypes at matched time points post-superinfection (∼5 years post-initial infection). Here we show superinfected individuals develop significantly broader NAb responses post-superinfection when compared to singly infected individuals (RR = 1.68, CI: 1.23–2.30, p = 0.001). This was true even after controlling for NAb breadth developed prior to superinfection, contemporaneous CD4+ T cell count and viral load. Similarly, both unadjusted and adjusted analyses showed significantly greater potency in superinfected cases compared to controls. Notably, two superinfected individuals were able to neutralize variants from four different subtypes at plasma dilutions >1∶300, suggesting that their NAbs exhibit elite activity. Cross-subtype breadth was detected within a year of superinfection in both of these individuals, which was within 1.5 years of their initial infection. These data suggest that sequential infections lead to augmentation of the NAb response, a process that may provide insight into potential mechanisms that contribute to the development of antibody breadth. Therefore, a successful vaccination strategy that mimics superinfection may lead to the development of broad NAbs in immunized individuals. 相似文献
185.
Annie J. Valuska Katherine A. Leighty Gina M. Ferrie Valerie D. Nichols Cheryl L. Tybor Chelle Plassé Tamara L. Bettinger 《Zoo biology》2013,32(2):216-221
Mixed‐species exhibits offer a variety of benefits but can be challenging to maintain due to difficulty in managing interspecific interactions. This is particularly true when little has been documented on the behavior of the species being mixed. This was the case when we attempted to house three species of turaco (family: Musophagidae) together with other species in a walk‐through aviary. To learn more about the behavior of great blue turacos, violaceous turacos, and white‐bellied gray go‐away birds, we supplemented opportunistic keeper observations with systematic data collection on their behavior, location, distance from other birds, and visibility to visitors. Keepers reported high levels of aggression among turacos, usually initiated by a go‐away bird or a violaceous turaco. Most aggression occurred during feedings or when pairs were defending nest sites. Attempts to reduce aggression by temporarily removing birds to holding areas and reintroducing them days later were ineffective. Systematic data collection revealed increased social behavior, including aggression, during breeding season in the violaceous turacos, as well as greater location fidelity. These behavioral cues may be useful in predicting breeding behavior in the future. Ultimately, we were only able to house three species of turaco together for a short time, and prohibitively high levels of conflict occurred when pairs were breeding. We conclude that mixing these three turaco species is challenging and may not be the most appropriate housing situation for them, particularly during breeding season. However, changes in turaco species composition, sex composition, or exhibit design may result in more compatible mixed‐turaco species groups. Zoo Biol. 32:216–221, 2013. © 2012 Wiley Periodicals, Inc. 相似文献
186.
Pipecolic acid naturally occurs in microorganisms, plants, and animals, where it plays many roles, including the interactions between these organisms, and is a key constituent of many natural and synthetic bioactive molecules. This article provides a review of current knowledge on the natural occurrence of pipecolic acid and the known and potential significance of its L‐ and D‐enantiomers in different scientific disciplines. Knowledge gaps with perspectives for future research identified within this article include the roles of the L‐ versus the D‐enantiomer of pipecolic acid in plant resistance, nutrient acquisition, and decontamination of polluted soils, as well as rhizosphere ecology and medical issues. Chirality 25:823–831, 2013. © 2013 Wiley Periodicals, Inc. 相似文献
187.
188.
Macrophage colony stimulating factor prevents NMDA-induced neuronal death in hippocampal organotypic cultures 总被引:2,自引:0,他引:2
Macrophage colony stimulating factor (M-CSF) and its receptor are up-regulated in the brain in Alzheimer's disease (AD), in transgenic mouse models for AD, and experimental models for traumatic and ischemic brain injury. M-CSF induces activation and proliferation of microglial cells and expression of proinflammatory cytokines. We examined the role of M-CSF in excitotoxic neuronal cell death in organotypic hippocampal cultures. NMDA treatment induced neuronal apoptosis and caspase-3 activation in organotypic hippocampal cultures, whereas treatment with M-CSF protected hippocampal neurons from NMDA-induced apoptosis. Caspase-3 activation was inhibited by M-CSF treatment to the same degree as with the caspase inhibitor Z-VAD-FMK. These results suggest that M-CSF has neuroprotective properties through inhibition of caspase-3 that could promote neuronal survival after excitotoxic insult. The role of M-CSF in neurological disease should be reevaluated as a microglial activator with potentially neuroprotective effects. 相似文献
189.
Saranya Ravi Balu Chacko Hirotaka Sawada Philip A. Kramer Michelle S. Johnson Gloria A. Benavides Valerie O’Donnell Marisa B. Marques Victor M. Darley-Usmar 《PloS one》2015,10(4)
Platelet thrombus formation includes several integrated processes involving aggregation, secretion of granules, release of arachidonic acid and clot retraction, but it is not clear which metabolic fuels are required to support these events. We hypothesized that there is flexibility in the fuels that can be utilized to serve the energetic and metabolic needs for resting and thrombin-dependent platelet aggregation. Using platelets from healthy human donors, we found that there was a rapid thrombin-dependent increase in oxidative phosphorylation which required both glutamine and fatty acids but not glucose. Inhibition of fatty acid oxidation or glutamine utilization could be compensated for by increased glycolytic flux. No evidence for significant mitochondrial dysfunction was found, and ATP/ADP ratios were maintained following the addition of thrombin, indicating the presence of functional and active mitochondrial oxidative phosphorylation during the early stages of aggregation. Interestingly, inhibition of fatty acid oxidation and glutaminolysis alone or in combination is not sufficient to prevent platelet aggregation, due to compensation from glycolysis, whereas inhibitors of glycolysis inhibited aggregation approximately 50%. The combined effects of inhibitors of glycolysis and oxidative phosphorylation were synergistic in the inhibition of platelet aggregation. In summary, both glycolysis and oxidative phosphorylation contribute to platelet metabolism in the resting and activated state, with fatty acid oxidation and to a smaller extent glutaminolysis contributing to the increased energy demand. 相似文献
190.