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71.
Cecilia Nigro Alessia Leone Michele Longo Immacolata Prevenzano Thomas H. Fleming Antonella Nicolò Luca Parrillo Rosa Spinelli Pietro Formisano Peter P. Nawroth Francesco Beguinot Claudia Miele 《生物化学与生物物理学报:疾病的分子基础》2019,1865(1):73-85
Impaired angiogenesis leads to long-term complications and is a major contributor of the high morbidity in patients with Diabetes Mellitus (DM). Methylglyoxal (MGO) is a glycolysis byproduct that accumulates in DM and is detoxified by the Glyoxalase 1 (Glo1). Several studies suggest that MGO contributes to vascular complications through mechanisms that remain to be elucidated. In this study we have clarified for the first time the molecular mechanism involved in the impairment of angiogenesis induced by MGO accumulation.Angiogenesis was evaluated in mouse aortic endothelial cells isolated from Glo1-knockdown mice (Glo1KD MAECs) and their wild-type littermates (WT MAECs). Reduction in Glo1 expression led to an accumulation of MGO and MGO-modified proteins and impaired angiogenesis of Glo1KD MAECs. Both mRNA and protein levels of the anti-angiogenic HoxA5 gene were increased in Glo1KD MAECs and its silencing improved both their migration and invasion. Nuclear NF-?B-p65 was increased 2.5-fold in the Glo1KD as compared to WT MAECs. Interestingly, NF-?B-p65 binding to HoxA5 promoter was also 2-fold higher in Glo1KD MAECs and positively regulated HoxA5 expression in MAECs. Consistent with these data, both the exposure to a chemical inhibitor of Glo1 “SpBrBzGSHCp2” (GI) and to exogenous MGO led to the impairment of migration and the increase of HoxA5 mRNA and NF-?B-p65 protein levels in microvascular mouse coronary endothelial cells (MCECs).This study demonstrates, for the first time, that MGO accumulation increases the antiangiogenic factor HoxA5 via NF-?B-p65, thereby impairing the angiogenic ability of endothelial cells. 相似文献
72.
Rosa Spinelli Pasqualina Florese Luca Parrillo Federica Zatterale Michele Longo Vittoria DEsposito Antonella Desiderio Annika Nerstedt Birgit Gustafson Pietro Formisano Claudia Miele Gregory Alexander Raciti Raffaele Napoli Ulf Smith Francesco Beguinot 《Aging cell》2022,21(3)
Senescence of adipose precursor cells (APC) impairs adipogenesis, contributes to the age‐related subcutaneous adipose tissue (SAT) dysfunction, and increases risk of type 2 diabetes (T2D). First‐degree relatives of T2D individuals (FDR) feature restricted adipogenesis, reflecting the detrimental effects of APC senescence earlier in life and rendering FDR more vulnerable to T2D. Epigenetics may contribute to these abnormalities but the underlying mechanisms remain unclear. In previous methylome comparison in APC from FDR and individuals with no diabetes familiarity (CTRL), ZMAT3 emerged as one of the top‐ranked senescence‐related genes featuring hypomethylation in FDR and associated with T2D risk. Here, we investigated whether and how DNA methylation changes at ZMAT3 promote early APC senescence. APC from FDR individuals revealed increases in multiple senescence markers compared to CTRL. Senescence in these cells was accompanied by ZMAT3 hypomethylation, which caused ZMAT3 upregulation. Demethylation at this gene in CTRL APC led to increased ZMAT3 expression and premature senescence, which were reverted by ZMAT3 siRNA. Furthermore, ZMAT3 overexpression in APC determined senescence and activation of the p53/p21 pathway, as observed in FDR APC. Adipogenesis was also inhibited in ZMAT3‐overexpressing APC. In FDR APC, rescue of ZMAT3 methylation through senolytic exposure simultaneously downregulated ZMAT3 expression and improved adipogenesis. Interestingly, in human SAT, aging and T2D were associated with significantly increased expression of both ZMAT3 and the P53 senescence marker. Thus, DNA hypomethylation causes ZMAT3 upregulation in FDR APC accompanied by acquisition of the senescence phenotype and impaired adipogenesis, which may contribute to FDR predisposition for T2D. 相似文献
73.
Monoclonal antibodies against pertussis toxin subunits 总被引:2,自引:0,他引:2
Massimo Bigio Roberta Rossi Daniele Nucci Maria Giuseppina Borri Guido Antoni Antonella Bartoloni Rino Rappuoli 《FEMS microbiology letters》1988,51(1):7-11
Abstract Twenty monoclonal antibodies (mAbs) reacting with cholera toxin (CT) of Vibrio cholerae strain 569B were characterized in cross-section and GM1 ganglioside inhibition assays. MAbs were characterized by reaction with CT and Escherichia coli heat-labile porcine strain (LTp ) and human strain (LTh) enterotoxins, and by GM1 ganglioside inhibition of mAb binding. Eight of 10 CT-A specific and 3 of 10 CT-B-specific mAbs cross-reacted with LTh and LTp. GM1 ganglioside inhibited reactions of the CT-B cross-reacting antibodies. Results showed that these epitodes common to the B subunit of CT and LT are located in or near the GM1 ganglioside binding region, and that the GM1 ganglioside-binding region of LT differs from that of CT. 相似文献
74.
Imamura T Huang J Dalle S Ugi S Usui I Luttrell LM Miller WE Lefkowitz RJ Olefsky JM 《The Journal of biological chemistry》2001,276(47):43663-43667
The insulin and the endothelin type A (ETA) receptor both can couple into the heterotrimeric G protein alpha(q/11) (Galpha(q/11)), leading to Galpha(q/11) tyrosine phosphorylation, phosphatidylinositol 3-kinase activation, and subsequent stimulation of glucose transport. In this study, we assessed the potential role of Src kinase in ET-1 signaling to glucose transport in 3T3-L1 adipocytes. Src kinase inhibitor PP2 blocked ET-1-induced Src kinase activity, Galpha(q/11) tyrosine phosphorylation, and glucose transport stimulation. To determine which Src family kinase member was involved, we microinjected anti-c-Src, -c-Fyn, or -c-Yes antibody into these cells and found that only anti-c-Yes antibody blocked GLUT4 translocation (70% decreased). Overexpression or microinjection of a dominant negative mutant (K298M) of Src kinase also inhibited ET-1-induced Galpha(q/11) tyrosine phosphorylation and GLUT4 translocation. In co-immunoprecipitation experiments, we found that beta-arrestin 1 associated with the ETA receptor in an agonist-dependent manner and that beta-arrestin 1 recruited Src kinase to a molecular complex that included the ETA receptor. Microinjection of beta-arrestin 1 antibody inhibited ET-1- but not insulin-stimulated GLUT4 translocation. In conclusion, 1) the Src kinase Yes can induce tyrosine phosphorylation of Galpha(q/11) in response to ET-1 stimulation, and 2) beta-arrestin 1 and Src kinase form a molecular complex with the ETA receptor to mediate ET-1 signaling to Galpha(q/11) with subsequent glucose transport stimulation. 相似文献
75.
Céline Di Battista Antonella Amicucci Chiara Guidi Luana Bertini Davide Sisti Vilberto Stocchi 《Biotechnology Techniques》1999,13(5):331-335
A rapid procedure has been developed to isolate DNA from the ectomycorrhizae of Tuber spp. for use in PCR experiments. The method described is fast and sensitive and can overcome the amplification problems that can arise in the presence of inhibitors. For this reason it can be used to type ectomycorrhizae even starting from a single root tip and make mycorrhizae identification much more rapid. 相似文献
76.
Dalle S Smith P Blache P Le-Nguyen D Le Brigand L Bergeron F Ashcroft FM Bataille D 《The Journal of biological chemistry》1999,274(16):10869-10876
Using the MIN6 B-cell line, we investigated the hypothesis that miniglucagon, the C-terminal () fragment processed from glucagon and present in pancreatic A cells, modulates insulin release, and we analyzed its cellular mode of action. We show that, at concentrations ranging from 0.01 to 1000 pM, miniglucagon dose-dependently (ID50 = 1 pM) inhibited by 80-100% the insulin release triggered by glucose, glucagon, glucagon-like peptide-1-(7-36) amide (tGLP-1), or glibenclamide, but not that induced by carbachol. Miniglucagon had no significant effects on cellular cAMP levels. The increase in 45Ca2+ uptake induced by depolarizing agents (glucose or extracellular K+), by glucagon, or by the Ca2+channel agonist Bay K-8644 was blocked by miniglucagon at the doses active on insulin release. Electrophysiological experiments indicated that miniglucagon induces membrane hyperpolarization, probably by opening potassium channels, which terminated glucose-induced electrical activity. Pretreatment with pertussis toxin abolished the effects of miniglucagon on insulin release. It is concluded that miniglucagon is a highly potent and efficient inhibitor of insulin release by closing, via hyperpolarization, voltage-dependent Ca2+ channels linked to a pathway involving a pertussis toxin-sensitive G protein. 相似文献
77.
Replication-mediated instability of the GAA triplet repeat mutation in Friedreich ataxia 总被引:7,自引:5,他引:2
Pollard LM Sharma R Gómez M Shah S Delatycki MB Pianese L Monticelli A Keats BJ Bidichandani SI 《Nucleic acids research》2004,32(19):5962-5971
Friedreich ataxia is caused by the expansion of a polymorphic and unstable GAA triplet repeat in the FRDA gene, but the mechanisms for its instability are poorly understood. Replication of (GAA•TTC)n sequences (9–105 triplets) in plasmids propagated in Escherichia coli displayed length- and orientation-dependent instability. There were small length variations upon replication in both orientations, but large contractions were frequently observed when GAA was the lagging strand template. DNA replication was also significantly slower in this orientation. To evaluate the physiological relevance of our findings, we analyzed peripheral leukocytes from human subjects carrying repeats of similar length (8–107 triplets). Analysis of 9400 somatic FRDA molecules using small-pool PCR revealed a similar mutational spectrum, including large contractions. The threshold length for the initiation of somatic instability in vivo was between 40 and 44 triplets, corresponding to the length of a eukaryotic Okazaki fragment. Consistent with the stabilization of premutation alleles during germline transmission, we also found that instability of somatic cells in vivo and repeats propagated in E.coli were abrogated by (GAGGAA)n hexanucleotide interruptions. Our data demonstrate that the GAA triplet repeat mutation in Friedreich ataxia is destabilized, frequently undergoing large contractions, during DNA replication. 相似文献
78.
Angiopoietin decoy secreted at tumor site impairs tumor growth and metastases by inducing local inflammation and altering neoangiogenesis 总被引:3,自引:0,他引:3
Melani C Stoppacciaro A Foroni C Felicetti F Caré A Colombo MP 《Cancer immunology, immunotherapy : CII》2004,53(7):600-608
The extracellular domain of the receptor tyrosine kinase Tie2/TEK (exTEK) has been used as an angiopoietin decoy to study the role of angiopoietins in the tumor–host interactions, using a syngeneic model of experimental metastases and subcutaneous tumor. Soluble exTEK secreted by transfected tumor cells inhibited HUVECs from forming tubes in Matrigel. ExTEK-transfected C26 colon carcinoma and TS/A mammary tumor cells displayed reduced growth rate when injected subcutaneously, and reduced ability to form experimental metastases when injected intravenously. Immunohistochemical analysis of tumors and metastases showed increased leukocytes infiltration and signs of inflammation in exTEK-secreting compared to parental tumor, as well as impairment in neo-vessel growth and organization. However, while neoangiogenesis eventually rescued in the subcutis, it failed to organize in the experimental metastases of exTEK-secreting tumor, contributing to the hampering of metastatic growth and to increased mice survival. The reactive infiltrate of C26TEK contained a different percentage of leukocytes and was responsible for the tumor inhibition. In fact, leukopenia induced by -irradiation of recipient mice or injection into interferon gamma (IFN-) gene knockout (GKO) mice resulted in reduced mouse survival and an increased number of lung metastases. On the other hand, interleukin (IL)-12 treatment prolonged the survival of mice bearing subcutaneous C26TEK but not of those bearing lung metastases, suggesting that IL-12 could exert further antiangiogenic effects at the site where the tumor can restore neoangiogenesis. These results show in vivo that reduced angiopoietin availability at the tumor site induces a local inflammatory response and impairment of neoangiogenesis which act synergistically to limit tumor growth and metastasis.Abbreviations AEC
amino-ethylcarbazole
- ELISA
enzyme-linked immunosorbent assay
- HRP
horseradish peroxidase
- HUVEC
human umbilical vascular endothelial cell
- i.v.
intravenous
- s.c.
subcutaneous
- TBS
Tris-HCl buffered solution 相似文献
79.
Marangoni A Aldini R Sambri V Giacani L Di Leo K Cevenini R 《FEMS immunology and medical microbiology》2004,40(3):187-191
Stimulation of isolated rat Kupffer cells by viable Leptospira interrogans, Treponema pallidum and Borrelia garinii elicited cellular responses resulting in the release of different tumor necrosis factor alpha (TNF-alpha) levels, depending on the spirochetes. L. interrogans induced TNF-alpha levels higher than those achieved with B. garinii and T. pallidum (in this order), but lower than the levels achieved with lipopolysaccharide (LPS). In contrast to L. interrogans, pretreatment of borreliae and treponemes with polymyxin B did not substantially diminish the ability of B. garinii and T. pallidum to stimulate Kupffer cells. Purified T. pallidum lipoproteins TpN47, TmpA, TpN15-TpN17, and B. garinii OspA induced TNF-alpha responses comparable to that achieved by LPS. This response was almost insensitive to the action of polymyxin B. 相似文献