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51.
Kristine von Bargen Mirella Scraba Ina Krmer Maren Ketterer Christian Nehls Sina Krokowski Urska Repnik Michaela Wittlich Anna Maaser Pia Zapka Madeleine Bunge Martin Schlesinger Gitta Huth Annette Klees Philipp Hansen Andreas Jeschke Gerd Bendas Olaf Utermhlen Gareth Griffiths Thomas Gutsmann Jens Wohlmann Albert Haas 《Cellular microbiology》2019,21(1)
Professional phagocytic cells such as macrophages are a central part of innate immune defence. They ingest microorganisms into membrane‐bound compartments (phagosomes), which acidify and eventually fuse with lysosomes, exposing their contents to a microbicidal environment. Gram‐positive Rhodococcus equi can cause pneumonia in young foals and in immunocompromised humans. The possession of a virulence plasmid allows them to subvert host defence mechanisms and to multiply in macrophages. Here, we show that the plasmid‐encoded and secreted virulence‐associated protein A (VapA) participates in exclusion of the proton‐pumping vacuolar‐ATPase complex from phagosomes and causes membrane permeabilisation, thus contributing to a pH‐neutral phagosome lumen. Using fluorescence and electron microscopy, we show that VapA is also transferred from phagosomes to lysosomes where it permeabilises the limiting membranes for small ions such as protons. This permeabilisation process is different from that of known membrane pore formers as revealed by experiments with artificial lipid bilayers. We demonstrate that, at 24 hr of infection, virulent R. equi is contained in a vacuole, which is enriched in lysosome material, yet possesses a pH of 7.2 whereas phagosomes containing a vapA deletion mutant have a pH of 5.8 and those with virulence plasmid‐less sister strains have a pH of 5.2. Experimentally neutralising the macrophage endocytic system allows avirulent R. equi to multiply. This observation is mirrored in the fact that virulent and avirulent R. equi multiply well in extracts of purified lysosomes at pH 7.2 but not at pH 5.1. Together these data indicate that the major function of VapA is to generate a pH‐neutral and hence growth‐promoting intracellular niche. VapA represents a new type of Gram‐positive virulence factor by trafficking from one subcellular compartment to another, affecting membrane permeability, excluding proton‐pumping ATPase, and consequently disarming host defences. 相似文献
52.
Cottrell SE Distler J Goodman NS Mooney SH Kluth A Olek A Schwope I Tetzner R Ziebarth H Berlin K 《Nucleic acids research》2004,32(1):e10
DNA methylation-based biomarkers have been discovered that could potentially be used for the diagnosis of cancer by detection of circulating, tumor-derived DNA in bodily fluids. Any methylation detection assay that would be applied to these samples must be capable of detecting small amounts of tumor DNA in the presence of background normal DNA. We have developed a real-time PCR assay, called HeavyMethyl, that is well suited for this application. HeavyMethyl uses methylation-specific oligonucleotide blockers and a methylation-specific probe to achieve methylation-specific amplification and detection. We tested the assays on unmethylated and artificially methylated DNA in order to determine the limit of detection. After careful optimization, our glutathione-S-transferase pi1 and Calcitonin assays can amplify as little as 30 and 60 pg of methylated DNA, respectively, and neither assay amplifies unmethylated DNA. The Calcitonin assay showed a highly significant methylation difference between normal colon and colon adenocarcinomas, and methylation was also detected in serum DNA from colon cancer patients. These assays show that HeavyMethyl technology can be successfully employed for the analysis of very low concentrations of methylated DNA, e.g. in serum of patients with tumors. 相似文献
53.
Transrectal color Doppler sonography was used to investigate uterine and umbilical blood flow during pregnancy (duration, 46-48 weeks) in four mares. The resistance index (RI) and blood flow volume (VOL) of the uterine arteries ipsilateral and contralateral to the conceptus, and the presence of an early diastolic notch in the Doppler wave, were evaluated every 4 week throughout pregnancy. Fetal blood flow was calculated semiquantitatively every 2 week (from 20 to 40 weeks), using the RI of the umbilical arteries. During the entire period of investigation, there were no significant individual variations in uterine RI and VOL nor differences between the two uterine arteries. Mean RI decreased by more than half during pregnancy from 0.89 +/- 0.01 to 0.39 +/- 0.03, and mean VOL increased almost 400-fold from 69 +/- 37 to 27,467 +/- 8851 ml/min. There were relationships (P<0.0001) between week of pregnancy (x) and RI as well as VOL. These were described by the equations RI=0.938-0.150 ln(x) and VOL (ml/min)=7.621x(2.157). Log transformed total estrogen (TE) were related to RI (r=-0.879; P<0.05) as well as to VOL (r=0.888; P<0.05). The notch in the Doppler wave of the uterine artery disappeared between 18 and 26 weeks. There was a correlation (P<0.0001) between week of gestation (x) and RI values of the umbilical arteries; this was described by the equation RI=1.763-0.071x+0.001x2. Further studies are needed to determine whether transrectal color Doppler sonography could be used to identify mares at risk of abortion. 相似文献
54.
Diagnostic polymorphisms in the mitochondrial cytochrome b gene allow discrimination between cattle,sheep, goat,roe buck and deer by PCR-RFLP 总被引:3,自引:0,他引:3
Background
As an alternative to direct DNA sequencing of PCR products, random PCR-RFLP is an efficient technique to discriminate between species. The PCR-RFLP-method is an inexpensive tool in forensic science, even if the template is degraded or contains only traces of DNA from various species. 相似文献55.
Galantino-Homer HL Florman HM Storey BT Dobrinski I Kopf GS 《Molecular reproduction and development》2004,67(4):487-500
Mammalian sperm capacitation is the obligatory maturational process leading to the development of the fertilization-competent state. Heparin is known to be a unique species-specific inducer of bovine sperm capacitation in vitro and glucose a unique inhibitor of this induction. Heparin-induced capacitation of bovine sperm has been shown to correlate with protein kinase A (PKA)-dependent protein tyrosine phosphorylation driven by an increase in intracellular cAMP. This study examines the possible roles of cyclic nucleotide phosphodiesterase (PDE) activity and intracellular alkalinization on bovine sperm capacitation and the protein tyrosine phosphorylation associated with it. Measurement of whole cell PDE kinetics during capacitation reveals neither a substantial change with heparin nor one with glucose: PDE activity is effectively constitutive in maintaining intracellular cAMP levels during capacitation. In contrast to a transient increase in intracellular pH, a sustained increase in medium pH by switching from 5% CO(2)/95% air incubation to 1% CO(2)/99% air incubation over 4 hr in the absence of heparin resulted in an increase in protein tyrosine phosphorylation and in the extent of induced acrosome reaction comparable to that observed following heparin-induced capacitation in 5% CO(2). These results suggest that increased bicarbonate-dependent adenylyl cyclase activity, driven by alkalinization, increases intracellular cAMP and so increases PKA activity mediating protein tyrosine phosphorylation. Quantitative analysis of the lactic acid production rate by bovine sperm glycolysis accounts fully for intracellular acidification sufficient to offset heparin-induced alkalinization, thus inhibiting capacitation. The mechanism by which heparin uniquely induces intracellular alkalinization in bovine sperm leading to capacitation remains obscure, inviting future investigation. 相似文献
56.
Schulz R Aker S Belosjorow S Konietzka I Rauen U Heusch G 《American journal of physiology. Heart and circulatory physiology》2003,285(5):H2084-H2090
In hearts with chronic left ventricular (LV) systolic dysfunction secondary to hypertension or myocardial infarction, MAPK phosphorylation and/or activity are increased. Whether other settings of LV dysfunction not associated with ischemia-reperfusion are also characterized by increased MAPK phosphorylation or activity is unknown. After 3 wk of rapid LV pacing (400 beats/min), eight rabbits displayed clinical signs of heart failure (HF), and echocardiography revealed an increase in LV end-diastolic diameter from 15.6 +/- 0.7 (means +/- SE) to 18.8 +/- 0.7 mm and a reduced shortening fraction from 31 +/- 1to10 +/- 2% (both P < 0.05). Morphological alterations in HF included increased numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cardiomyocytes, extent of fibrosis, and cross-sectional cardiomyocyte area. Total p38 MAPK did not differ between failing and normal hearts (n = 8). However, p38 MAPK phosphorylation [164,488 +/- 29,323 vs. 43,565 +/- 14,817 arbitrary units (AU), P < 0.05, densitometry] and the activities of p38 MAPK-alpha and -beta were increased in failing compared with normal hearts (149,441 +/- 38,381 and 170,430 +/- 32,952 vs. 68,815 +/- 28,984 and 81,788 +/- 22,774 AU, respectively, both P < 0.05). In failing compared with normal hearts, total and phosphorylated JNK46 and JNK54 MAPK were increased, whereas total and phosphorylated ERK MAPK remained unchanged. In pacing-induced HF, p38 and JNK MAPK phosphorylation as well as p38 MAPK activity was increased. Further studies will have to define whether or not chronic specific blockade of MAPK activity can interfere with apoptosis/fibrosis and thereby attenuate the progression of HF. 相似文献
57.
58.
Germ cell transplantation in goats 总被引:19,自引:0,他引:19
Honaramooz A Behboodi E Blash S Megee SO Dobrinski I 《Molecular reproduction and development》2003,64(4):422-428
Transplantation of spermatogonial stem cells provides a unique approach for the study of spermatogenesis and manipulation of the male germ line. This technique may also offer an alternative to the currently inefficient methods of producing transgenic domestic animals. We have recently established the technique of spermatogonial transplantation, originally developed in laboratory rodents, in pigs, and this study was aimed to extend the technique to the goat. Isolated donor testis cells were infused into the seminiferous tubules of anesthetized recipient goats through an ultrasonographically-guided catheter inserted into the rete testis. Donor cells were obtained by enzymatic digestion of freshly collected testes from immature goats (either from the recipients' contralateral testis or from unrelated donors). Prior to transplantation, testis cells were labeled with a fluorescent marker to allow identification after transplantation. Recipient testes were examined for the presence and localization of labeled donor cells at 3-week intervals up to 12 weeks after transplantation. Labeled donor cells were found in the seminiferous tubules of all testes, comprising 10-35% of the examined tubules. Histological examination of the recipient testes did not reveal evident tissue damage, except for limited fibrotic changes at the site of needle insertion. Likewise there were no detectable local or systemic signs of immunologic reactions to the transplantations. These results indicate that germ cell transplantation is technically feasible in immature male goats and that donor-derived cells are retained in the recipient testis for at least three months and through puberty. This study represents the first report of germ cell transplantation in goats. 相似文献
59.
Fertility and germline transmission of donor haplotype following germ cell transplantation in immunocompetent goats 总被引:10,自引:0,他引:10
Honaramooz A Behboodi E Megee SO Overton SA Galantino-Homer H Echelard Y Dobrinski I 《Biology of reproduction》2003,69(4):1260-1264
Transplantation of spermatogonial stem cells into syngeneic or immunosuppressed recipient mice or rats can result in donor-derived spermatogenesis and fertility. Recently, this approach has been employed to introduce a transgene into the male germline. Germ-cell transplantation in species other than laboratory rodents, if successful, holds great promise as an alternative to the inefficient methods currently available to generate transgenic farm animals that can produce therapeutic proteins in their milk or provide organs for transplantation to humans. To explore whether germ-cell transplantation could result in donor-derived spermatogenesis and fertility in immunocompetent recipient goats, testis cells were transplanted from transgenic donor goats carrying a human alpha-1 antitrypsin expression construct to the testes of sexually immature wild-type recipient goats. After puberty, sperm carrying the donor-derived transgene were detected in the ejaculates of two out of five recipients. Mating of one recipient resulted in 15 offspring, one of which was transgenic for the donor-derived transgene. This is the first report of donor cell-derived sperm production and transmission of the donor haplotype to the next generation after germ-cell transplantation in a nonrodent species. Furthermore, these results indicate that successful germ-cell transplantation is feasible between immunocompetent, unrelated animals. In the future, transplantation of genetically modified germ cells may provide a more efficient alternative for production of transgenic domestic animals. 相似文献
60.
Molecular basis of scrapie strain glycoform variation 总被引:10,自引:0,他引:10
Transmissible spongiform encephalopathies (TSE) are characterized by the conversion of a protease-sensitive host glycoprotein, prion protein or PrP-sen, to a protease-resistant form (PrP-res). PrP-res molecules that accumulate in the brain and lymphoreticular system of the host consist of three differentially glycosylated forms. Analysis of the relative amounts of the PrP-res glycoforms has been used to discriminate TSE strains and has become increasingly important in the differential diagnosis of human TSEs. However, the molecular basis of PrP-res glycoform variation between different TSE agents is unknown. Here we report that PrP-res itself can dictate strain-specific PrP-res glycoforms. The final PrP-res glycoform pattern, however, can be influenced by the cell and significantly altered by subtle changes in the glycosylation state of PrP-sen. Thus, strain-specific PrP-res glycosylation profiles are likely the consequence of a complex interaction between PrP-res, PrP-sen, and the cell and may indicate the cellular compartment in which the strain-specific formation of PrP-res occurs. 相似文献