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Human mast cells (MCs) contain TG-rich cytoplasmic lipid droplets (LDs) with high arachidonic acid (AA) content. Here, we investigated the functional role of adipose TG lipase (ATGL) in TG hydrolysis and the ensuing release of AA as substrate for eicosanoid generation by activated human primary MCs in culture. Silencing of ATGL in MCs by siRNAs induced the accumulation of neutral lipids in LDs. IgE-dependent activation of MCs triggered the secretion of the two major eicosanoids, prostaglandin D2 (PGD2) and leukotriene C4 (LTC4). The immediate release of PGD2 from the activated MCs was solely dependent on cyclooxygenase (COX) 1, while during the delayed phase of lipid mediator production, the inducible COX-2 also contributed to its release. Importantly, when ATGL-silenced MCs were activated, the secretion of both PGD2 and LTC4 was significantly reduced. Interestingly, the inhibitory effect on the release of LTC4 was even more pronounced in ATGL-silenced MCs than in cytosolic phospholipase A2-silenced MCs. These data show that ATGL hydrolyzes AA-containing TGs present in human MC LDs and define ATGL as a novel regulator of the substrate availability of AA for eicosanoid generation upon MC activation.  相似文献   
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Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (cas) genes constitute the adaptive immune system in bacteria and archaea. Although the CRISPR-Cas systems have been hypothesized to encode potential toxins, no experimental data supporting the hypothesis are available in the literature. In this work, we provide the first experimental evidence for the presence of a toxin gene in the type I-A CRISPR system of hyperthermophilic archaeon Sulfolobus. csa5, under the control of its native promoter in a shuttle vector, could not be transformed into CRISPR-deficient mutant Sulfolobus solfataricus Sens1, demonstrating a strong toxicity in the cells. A single-amino-acid mutation destroying the intersubunit bridge of Csa5 attenuated the toxicity, indicative of the importance of Csa5 oligomerization for its toxicity. In line with the absence of Csa5 toxicity in S. solfataricus InF1 containing functional CRISPR systems, the expression of csa5 is repressed in InF1 cells. Induced from the arabinose promoter in Sens1 cells, Csa5 oligomers resistant to 1% SDS co-occur with chromosome degradation and cell death, reinforcing the connection between Csa5 oligomerization and its toxicity. Importantly, a rudivirus was shown to induce Csa5 expression and the formation of SDS-resistant Csa5 oligomers in Sulfolobus cells. This demonstrates that the derepression of csa5 and the subsequent Csa5 oligomerization take place in native virus-host systems. Thus, csa5 is likely to act as a suicide gene under certain circumstances to inhibit virus spreading.  相似文献   
5.
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is associated with autosomal dominant hypercholesterolemia, a state of elevated levels of LDL (low-density lipoprotein) cholesterol. Autosomal dominant hypercholesterolemia can result in severe implications such as stroke and coronary heart disease. The inhibition of PCSK9 function by therapeutic antibodies that block interaction of PCSK9 with the epidermal growth factor-like repeat A domain of LDL receptor (LDLR) was shown to successfully lower LDL cholesterol levels in clinical studies. Here we present data on the identification, structural and biophysical characterization and in vitro and in vivo pharmacology of a PCSK9 antibody (mAb1). The X-ray structure shows that mAb1 binds the module 1 of the C-terminal domain (CTD) of PCSK9. It blocks access to an area bearing several naturally occurring gain-of-function and loss-of-function mutations. Although the antibody does not inhibit binding of PCSK9 to epidermal growth factor-like repeat A, it partially reverses PCSK9-induced reduction of the LDLR and LDL cholesterol uptake in a cellular assay. mAb1 is also effective in lowering serum levels of LDL cholesterol in cynomolgus monkeys in vivo. Complete loss of PCSK9 is associated with insufficient liver regeneration and increased risk of hepatitis C infections. Blocking of the CTD is sufficient to partially inhibit PCSK9 function. Antibodies binding the CTD of PCSK9 may thus be advantageous in patients that do not tolerate complete inhibition of PCSK9.  相似文献   
6.
Eukaryotic protein kinases are typically strictly controlled by second messenger binding, protein/protein interactions, dephosphorylations or similar processes. None of these regulatory mechanisms is known to work for protein kinase CK2 (former name “casein kinase 2”), an acidophilic and constitutively active eukaryotic protein kinase. CK2 predominantly exists as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2α) complexed to a dimer of non-catalytic subunits (CK2β). One model of CK2 regulation was proposed several times independently by theoretical docking of the first CK2 holoenzyme structure. According to this model, the CK2 holoenzyme forms autoinhibitory aggregates correlated with trans-autophosphorylation and driven by the down-regulatory affinity between an acidic loop of CK2β and the positively charged substrate binding region of CK2α from a neighboring CK2 heterotetramer. Circular trimeric aggregates in which one-half of the CK2α chains show the predicted inhibitory proximity between those regions were detected within the crystal packing of the human CK2 holoenzyme. Here, we present further in vitro support of the “regulation-by-aggregation” model by an alternative crystal form in which CK2 tetramers are arranged as approximately linear aggregates coinciding essentially with the early predictions. In this assembly, the substrate binding region of every CK2α chain is blocked by a CK2β acidic loop from a neighboring tetramer. We found these crystals with CK2Andante that contains a CK2β variant mutated in a CK2α-contact helix and described to be responsible for a prolonged circadian rhythm in Drosophila. The increased propensity of CK2Andante to form aggregates with completely blocked active sites may contribute to this phenotype.  相似文献   
7.
Parkinson's disease (PD) and diabetes belong to the most common neurodegenerative and metabolic syndromes, respectively. Epidemiological links between these two frequent disorders are controversial. The neuropathological hallmarks of PD are protein aggregates composed of amyloid‐like fibrillar and serine‐129 phosphorylated (pS129) α‐synuclein (AS). To study if diet‐induced obesity could be an environmental risk factor for PD‐related α‐synucleinopathy, transgenic (TG) mice, expressing the human mutant A30P AS in brain neurons, were subjected after weaning to a lifelong high fat diet (HFD). The TG mice became obese and glucose‐intolerant, as did the wild‐type controls. Upon aging, HFD significantly accelerated the onset of the lethal locomotor phenotype. Coinciding with the premature movement phenotype and death, HFD accelerated the age of onset of brainstem α‐synucleinopathy as detected by immunostaining with antibodies against pathology‐associated pS129. Amyloid‐like neuropathology was confirmed by thioflavin S staining. Accelerated onset of neurodegeneration was indicated by Gallyas silver‐positive neuronal dystrophy as well as astrogliosis. Phosphorylation of the activation sites of the pro‐survival signaling intermediate Akt was reduced in younger TG mice after HFD. Thus, diet‐induced obesity may be an environmental risk factor for the development of α‐synucleinopathies. The molecular and cellular mechanisms remain to be further elucidated.

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8.
Intraplantar injection of 0.4% formalin into the rat hind paw leads to a biphasic nociceptive response; an ‘acute’ phase (0–15 min) and ‘tonic’ phase (16–120 min), which is accompanied by significant phosphorylation of extracellular signal‐regulated kinase (ERK)1/2 in the contralateral striatum at 120 min post‐formalin injection. To uncover a possible relationship between the slow‐onset substance P (SP) release and increased ERK1/2 phosphorylation in the striatum, continuous infusion of SP into the striatum by reverse microdialysis (0.4 μg/mL in microdialysis fiber, 1 μL/min) was performed to mimic volume neurotransmission of SP. Continuous infusion for 3 h of SP reduced the duration of ‘tonic’ phase nociception, and this SP effect was mediated by neurokinin 1 (NK1) receptors since pre‐treatment with NK1 receptor antagonist CP96345 (10 μM) blocked the effect of SP infusion. However, formalin‐induced ‘tonic’ phase nociception was significantly prolonged following acute injection of the MAP/ERK kinase 1/2 inhibitor PD0325901 (100 pmol) by microinjection. The coinfusion of SP and PD0325901 significantly increased the ‘tonic’ phase of nociception. These data demonstrate that volume transmission of striatal SP triggered by peripheral nociceptive stimulation does not lead to pain facilitation but a significant decrease of tonic nociception by the activation of the SP‐NK1 receptor–ERK1/2 system.

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9.
Toxoplasma 3 main clonal lineages are designated as type I, II, and III; however, atypical and mixed genotypes were also reported. This study was conducted for detection of Toxoplasma gondii genotypes in rats (Rattus rattus) in Riyadh region, Saudi Arabia. PCR test on T. gondii B1 gene was conducted on ELISA IgM positive samples for confirmation of the infection. However, genetic analysis of the SAG2 locus was performed to determine T. gondii genotypes using PCR-RFLP technique. PCR test on T. gondii B1gene showed that 22 (81.5%) out of the 27 ELISA IgM positive samples have T. gondii DNA. Genotypic analysis shows that, of the total 22 PCR positive samples, only 13 (59.1%) were of type II, 7 (31.8%) were of type III, and 2 (9.1%) were of an unknown genotype. It is obvious that the prevalence of both type II and III is high in rats. No reports have been available on T. gondii genotypes among rats in Riyadh region, and only little is known about its seroprevalence in rats. Future studies on T. gondii genotypes in rats using multi-locus markers is needed in Riyadh region, Saudi Arabia for better understanding of T. gondii pathogenesis and treatment in humans and animals.  相似文献   
10.

Aims

Septic shock, the severe form of sepsis, is associated with development of progressive damage in multiple organs. Kidney can be injured and its functions altered by activation of coagulation, vasoactive-peptide and inflammatory processes in sepsis. Endothelin (ET)-1, a potent vasoconstrictor, is implicated in the pathogenesis of sepsis and its complications. Protease-activated receptors (PARs) are shown to play an important role in the interplay between inflammation and coagulation. We examined the time-dependent alterations of ET-1 and inflammatory cytokine, such as tumor necrosis factor (TNF)-α in kidney tissue in lipopolysaccharide (LPS)-induced septic rat model and the effects of PAR2 blocking peptide on the LPS-induced elevations of renal ET-1 and TNF-α levels.

Main methods

Male Wistar rats at 8 weeks of age were administered with either saline solution or LPS at different time points (1, 3, 6 and 10 h). Additionally, we treated LPS-administered rats with PAR2 blocking peptide for 3 h to assess whether blockade of PAR2 has a regulatory role on the ET-1 level in septic kidney.

Key findings

An increase in ET-1 peptide level was observed in kidney tissue after LPS administration time-dependently. Levels of renal TNF-α peaked (around 12-fold) at 1 h of sepsis. Interestingly, PAR2 blocking peptide normalized the LPS-induced elevations of renal ET-1 and TNF-α levels.

Significance

The present study reveals a distinct chronological expression of ET-1 and TNF-α in LPS-administered renal tissues and that blockade of PAR2 may play a crucial role in treating renal injury, via normalization of inflammation, coagulation and vaso-active peptide.  相似文献   
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