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51.
R. Guglielmone 《Cell and tissue research》1995,281(1):163-168
Although the cerebrospinal fluid-contacting neurons of the avian paraventricular organ exhibit considerable amounts of catecholamines, they show no tyrosine hydroxylase immunoreactivity. In the quail embryo, the development of these neurons has been studied using the paraformaldeyde-glutaraldeyde method for the fluorescence-histochemical localization of catecholamines. The timing of the appearance of catecholamine fluorescence in cerebrospinal fluid-contacting neurons and that in catecholamine-containing neurons of the brainstem have been compared. The first neurons displaying catecholamine fluorescence are found within the locus coeruleus and the nucleus subcoeruleus ventralis on the 5.5th day of incubation. Catecholaminergic neuronal groups of the medulla and mesencephalon can be identified by embryonic day 7, and fluorescent cerebrospinal fluid-contacting neurons of the hypothalamic paraventricular organ can be first recognized at the 8th day of incubation. If the catecholamine content of cerebrospinal fluid-contacting neurons that lack tyrosine hydroxylase depends upon an uptake mechanism, it may be significant that, in fluorescence-histochemical preparations, these neurons can be identified 1–3 days later than those in which catecholamines are synthesized and from which catecholamines are released at an earlier developmental stage. Moreover, cerebrospinal fluid-contacting neurons that have previously been shown to be tyrosine-hydroxylase immunoreactive, and that lie at the spinal-medullary junction display a different developmental pattern. By fluorescence histochemistry, they can be detected only by embryonic day 10.5. The chemical, developmental and topographical differences suggest that the catecholamine-containing cerebrospinal fluid-contacting elements of the paraventricular organ and those of the spinal cord represent two different subsets of cerebrospinal fluid-contacting neurons whose respective functional roles remain to be investigated. 相似文献
52.
Background
The deposition of aggregated β-amyloid peptide senile plaques and the accumulation of arginine within the astrocytes in the brain of an Alzheimer's patient are classic observations in the neuropathology of the disease. It would be logical, in the aetiology and pathogenesis, to investigate arginine-metabolising enzymes and their intimate association with amyloid peptides.Methods
Neuronal nitric oxide synthase (nNOS) was isolated, purified and shown, through fluorescence quenching spectroscopy and fluorescence resonance energy transfer (FRET), to interact with structural fragments of Aβ1–40 and be catalytic towards amyloid fibril formation.Results
Only one binding site on the enzyme was available for binding. Two amyloid peptide fragments of Aβ1–40 (Aβ17–28 and Aβ25–35) had Stern–Volmer values (KSV) of 0.111 μM−1 and 0.135 μM−1 indicating tight binding affinity to nNOS and easier accessibility to fluor molecules during binding. The polarity of this active site precludes binding of the predominantly hydrophobic amyloid peptide fragments contained within Aβ17–28 and within two glycine zipper motifs [G-X-X-X-G-X-X-X-G] [Aβ29–37] and bind to the enzyme at a site remote to the active region.Conclusions
The interaction and binding of Aβ17–28 and Aβ25–35 to nNOS causes the movement of two critical tryptophan residues of 0.77 nm and 0.57 nm respectively towards the surface of the enzyme.General significance
The binding of Aβ-peptide fragments with nNOS has been studied by spectrofluorimetry. The information and data presented should contribute towards understanding the mechanism for deposition of aggregated Aβ-peptides and fibrillogenesis in senile plaques in an AD brain. 相似文献53.
Bidwell P Blackwell DJ Hou Z Zima AV Robia SL 《The Journal of biological chemistry》2011,286(40):35044-35050
To investigate the mechanism of regulation of sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) by phospholamban (PLB), we expressed Cerulean-SERCA and yellow fluorescent protein (YFP)-PLB in adult rabbit ventricular myocytes using adenovirus vectors. SERCA and PLB were localized in the sarcoplasmic reticulum and were mobile over multiple sarcomeres on a timescale of tens of seconds. We also observed robust fluorescence resonance energy transfer (FRET) from Cerulean-SERCA to YFP-PLB. Electrical pacing of cardiac myocytes elicited cytoplasmic Ca(2+) elevations, but these increases in Ca(2+) produced only modest changes in SERCA-PLB FRET. The data suggest that the regulatory complex is not disrupted by elevations of cytosolic calcium during cardiac contraction (systole). This conclusion was also supported by parallel experiments in heterologous cells, which showed that FRET was reduced but not abolished by calcium. Thapsigargin also elicited a small decrease in PLB-SERCA binding affinity. We propose that PLB is not displaced from SERCA by high calcium during systole, and relief of functional inhibition does not require dissociation of the regulatory complex. The observed modest reduction in the affinity of the PLB-SERCA complex with Ca(2+) or thapsigargin suggests that the binding interface is altered by SERCA conformational changes. The results are consistent with multiple modes of PLB binding or alternative binding sites. 相似文献
54.
荧光法研究血清白蛋白与药物的结合作用 总被引:3,自引:0,他引:3
本文应用荧光光谱法,观测了药物分子头孢菌素Ⅳ、异烟肼、维生素B_6和氟哌酸对白蛋白荧光的猝灭。由Lineweaver-Burk双倒数作图法,确定了药物与白蛋白作用的离解常数。并通过Forster偶极-偶极无辐射能量转移机理确定了药物分子氟哌酸在人血清白蛋白中与色氨酸残基之间的距离R为2.55nm,由这一距离确定了药物分子可能进入的区域和位置。 相似文献
55.
利用分离的叶绿体作实验材料,发现华山松(Pinus armandi Franch.)南方种源的4阶导数吸收光谱在680nm处峰值较大,在670nm处峰值较小,而北方种源中出现了在680nm处峰值较在670nm峰值小的类群,推断北方种群反应中心活力有下降趋势。南、北种源之间的低温(77K)荧光发射光谱有明显差异,PSⅠ、PSⅡ发射峰位置出现地理变动。低温荧光激发光谱分析表明,地理变异主要发生在叶绿素a的分子状态上。研究还表明,完整的针叶因为有角质层、松脂等物质干扰,检测不出光谱的差异,不是理想的实验材料。 相似文献
56.
Peter Müller Karl-Rudolf Erlemann Karin Müller Juan J. Calvete Edda Töpfer-Petersen Kathleen Marienfeld A. Herrmann 《European biophysics journal : EBJ》1998,27(1):33-41
PDC-109 is the major protein of bovine seminal plasma. It binds to the bovine sperm surface at ejaculation and modulates
sperm capacitation. PDC-109 displays phosphorylcholine- and heparin-binding activities which are thought to account for its
sperm surface coating and glycosaminoglycan-induced sperm capacitating activities, respectively. We have characterized the
interaction of isolated PDC-109 with membranes of phospholipid vesicles using a biophysical approach. Our results show that
PDC-109 interacts not only with the solvent-exposed phosphorylcholine head group but also with the hydrophobic core of liposomes.
Binding of PDC-109 to membranes is a very rapid, biphasic process with half times of less than one second. Maximal binding
of PDC-109 to small unilamellar vesicles was achieved with a stoichiometric ratio of 10–11 phosphatidylcholine molecules/PDC-109
molecule. Incorporation of phosphatidylethanolamine or phosphatidylserine into phosphatidylcholine vesicles reduced the binding
of PDC-109, suggesting that both the density of phosphorylcholine groups and the surface charge determine the interaction
of the seminal plasma protein with the surface of the membrane. Electron spin resonance measurements showed that binding of
PDC-109 to phosphatidylcholine vesicles caused a rigidification of the membrane. The relevance of the data for describing
the role of PDC-109 in the modulation of sperm capacitation is discussed.
Received: 16 June 1997 / Accepted: 10 September 1997 相似文献
57.
Early events of apoptosis following HSV-1 infection were investigated at the single-cell level using intensified fluorescence digital-imaging microscopy. The results provide evidence that infection of differentiated ND7 neuronlike cells by HSV-1 triggers detectable alterations indicative of physiological changes associated with the early stages of apoptosis. Less than 1 h after infection with HSV-1 (KOS strain) or K26GFP (GFP being fused to HSV-1 capsid protein VP26) we observed (i) moderate decrease in mitochondrial membrane potential (about 20%), (ii) exposure of phosphatidyl serine, (iii) morphological change in the mitochondria that became spherical instead of filamentous, and (iv) activation of caspase-8. Within 3 h changes reverted to normal, which indicated that apoptosis was counteracted very early following HSV-1 infection. Similar results were obtained with KOS-TK27GFP, lacking TK and UL24 proteins, suggesting that TK and UL24 play no role in apoptosis. In Vero cells mitochondrial changes characteristic of the apoptotic process were not observed following HSV-1 infection. The UV-inactivated K26GFP had the capacity to induce apoptosis in neuronlike cells. This real-time multiparametric analysis, in combination with relevant viral mutants, could be a useful approach for dissecting the roles of various viral genes in modulating apoptotic pathways during infection. 相似文献
58.
Summary An electron microscopic investigation of the extrapulmonary respiratory tract of embryos and chick of the domestic fowl (Gallus domesticus) has demonstrated for the first time in birds the presence here of a small number of epithelial cells characterised by an aminecontaining type of granule. These granular cells were scattered singly throughout the trachea, syrinx and primary bronchi and seemed more numerous in the caudal part of the airway. In favourable planes of section a small part of the cell was in contact with the luminal surface of the epithelium. The characteristic granular vesicles (approximate diameter 140 nm) appeared to be randomly distributed in the cytoplasm and there was no concentration of vesicles close to the plasma membrane. One of the cells was closely associated with an intraepithelial axon. By fluorescence microscopy, a small number of cells with a similar shape and distribution to the granular cells was observed after administration of 3,4-dihydroxyphenylalanine which may indicate the presence of an amine handlign mechanism in these cells. It is suggested that the granular cells belong to the APUD system of endocrine cells and that they may be modulated by the concentration of gas in the airways. 相似文献
59.
C. N. N′ soukpoé-Kossi C. St-Louis M. Beauregard M. Subirade R. Carpentier S. Hotchandani 《Journal of biomolecular structure & dynamics》2013,31(3):277-283
Abstract Resveratrol (Res), a polyphenolic compound found largely in the skin of red grape and wine, exhibits a wide range of pharmaceutical properties and plays a role in prevention of human cardiovascular diseases [Pendurthi et al., Arterioscler. Thromb. Vasc. Biol. 19, 419–426 (1999)]. It shows a strong affinity towards protein binding and used as inhibitor for cyclo- oxygenase and ribonuclease reductase. The aim of this study was to examine the interaction of resveratrol with human serum albumin (HSA) in aqueous solution at physiological conditions, using a constant protein concentration (0.3 mM) and various pigment contents μM to mM). FTIR, UV-Visible, CD, and fluorescence spectroscopic methods were used to determine the resveratrol binding mode, the binding constant and the effects of pigment complexation on protein secondary structure. Structural analysis showed that resveratrol bind non-specifically (H-bonding) via polypeptide polar groups with overall binding constant of KRes = 2.56× 105 M?1. The protein secondary structure, analysed by CD spectroscopy, showed no major alterations at low resveratrol concentrations (0.125 mM), whereas at high pigment content (1 mM), major increase of α-helix from 57% (free HSA) to 62% and a decrease of β-sheet from 10% (free HSA) to 7% occurred in the resveratrol-HSA complexes. The results indicate a partial stabilization of protein secondary structure at high resveratrol content. 相似文献
60.
Chia-Cheng Hung Shee-Uan Chen Shin-Yu Lin Mei-Ya Fang Yi-Yi Tsai Yu-Shih Yang Yi-Ning Su 《Analytical biochemistry》2010,400(1):69-77
Preimplantation genetic diagnosis (PGD) is employed increasingly to allow transfer of embryos to the uterus in assisted reproduction procedures. There are three stages of biopsy: polar bodies, one or two blastomeres from the cleavage-stage embryos, and trophectoderm cells (∼5 cells) from the blastocyst-stage embryos. Validation of polymerase chain reaction (PCR)-based assays are challenging because only limited genetic material can be obtained for PGD. In the current study, we modified a valid single-cell PCR protocol for PGD using real-time PCR assay with fluorescence resonance energy transfer (FRET) hybridization probes followed by melting curve analysis. We optimized and clinically applied the protocol, permitting molecular genetic analysis to amplify a specific region on the beta-globin (HBB) gene for a couple, carriers of two mutations: c.-78A>G and c.52A>T. Among a total of eight embryos obtained after ovarian stimulation, a single blastomere per embryo at the six- to eight-cell stage was biopsied. This PGD method showed that four embryos were unaffected, two embryos were selected for transfer, and one pregnancy was achieved. Finally, a healthy male baby was delivered at 38 weeks’ gestation. The results obtained using the new method, FRET hybridization probes, were compared with findings using an existing method, primer extension minisequencing. 相似文献