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41.
42.
Comparison of substrate specificities of transglutaminases using synthetic peptides as acyl donors 总被引:2,自引:0,他引:2
Transglutaminase (TGase) is an enzyme that catalyzes acyl transfer reactions between primary amines and Gln residues in proteins or peptides. Substrate specificities of TGase, Ca2+-independent microbial transglutaminase (MTGase), and Ca2+-dependent tissue type transglutaminase from guinea pig liver (GTGase) and fish, Red sea bream (Pagrus major), liver (FTGase), for acyl donors were investigated using synthetic peptides containing Gln residues and Gln analogues with different lengths of side chain. MTGase dose not recognize the Gln analogues as a substrate and has strict substrate specificities toward L-Gln. Substrate peptides with a variety of sequences around the Gln residue, GXXQXXG (X=G, A, S, L, V, F, Y, R, N, E, L) were synthesized and used as acyl donors. As an acyl acceptor, the fluorescent reagent monodancyl cadaverine was used and the reactions analyzed with RP-HPLC. Substitution of the C-terminal of a Gln residue with a hydrophobic amino acid accelerated the reaction by GTGase and FTGase. N-terminal substitution of Gln residues had similar effects on the reaction by MTGase. 相似文献
43.
The extracellular Lipases A and C produced by Geotrichum sp. FO401B have a preference for the sn-1,3 and sn-2 positions of triglyceride, respectively. Total production of these lipases was increased by plant oils and tributyrin. Butyl Toyopearl column chromatography demonstrated that only Lipase C was produced in the presence of tributyrin. Lipase C hydrolysed natural fats except sardine oil preferentially at the sn-2 position, but it showed little stereoselectivity for triolein. 相似文献
44.
Coexistence of gland mucous cell-type mucin and lysozyme in gastric gland mucous cells 总被引:2,自引:1,他引:1
Hidaka E Ota H Katsuyama T Nakayama J Momose M Hidaka H Ishii K Murata F Tsuyama S Kurihara M Ishihara K Hotta K 《Histochemistry and cell biology》2000,113(2):91-98
Class III mucin, identified by paradoxical concanavalin A staining, is confined to gastric gland mucous cells and is an essential
component of the gastric surface mucous gel layer. The pretreatment required has hampered the application of this method to
electron microscopic studies. Antibody HIK1083 reacts selectively with class III mucins. The present study was undertaken
to explore, electron microscopically, the immunoreactivity of the human stomach to HIK1083. We examined normal mucosa from
resected human stomachs (five cases; formalin-fixed, paraffin-embedded) and gastric biopsy specimens from patients with early
gastric cancer [nine cases; glutaraldehyde- and osmium-fixed, epoxy-embedded (seven cases) and half-strength Karnovsky’s solution-fixed,
Lowicryl K4M-embedded (two cases)]. Immunostaining with HIK1083 and anti-lysozyme antibody was examined under light and electron
microscopes. Gland mucous cells were labeled with HIK1083, and lysozyme was detected in some gland mucous cells and surface
mucous cells. Electron microscopically, the secretory granules of gland mucous cells contained a single electron-dense core.
HIK1083-positive mucins and lysozyme coexisted in the secretory granules of gastric gland mucous cells. HIK1083-reactive mucins
and lysozyme were distributed in the matrix and in the dense core of these secretory granules, respectively. HIK1083 can be
used for electron immunohistochemistry.
Accepted: 1 December 1999 相似文献
45.
46.
Genomic organization, chromosomal localization and regulation of expression of the neuronal nuclear matrix protein NRP/B in human brain tumors 总被引:5,自引:0,他引:5
The nuclear matrix and its role in cell physiology are largely unknown, and the discovery of any matrix constituent whose expression is tissue- and/or cell-specific offers a new avenue of exploration. Studies of the novel neuronal nuclear matrix protein, NRP/B, reveal that it is an early and highly specific marker of neuronal induction and development in vertebrates, since its expression is restricted mainly to the developing and mature nervous system. These studies also show that NRP/B is involved in neuronal differentiation. To further examine the structure-function of NRP/B, we have cloned and characterized the murine Nrp/b gene. The murine gene consists of four exons interrupted by three introns that span 7.6kb of DNA. The complete open reading frame is localized in exon 3, suggesting that NRP/B is highly conserved during evolution. Chromosomal analysis shows that NRP/B is localized to chromosome 13 in mouse and chromosome 5q12-13 in human.Since our previous studies demonstrated that NRP/B is expressed in primary hippocampal neurons but not in primary astrocytes, we have characterized NRP/B mRNA and protein expression in various brain cell lines and in human brain tumors. Abundant expression of NRP/B mRNA and protein was observed in human neuroblastoma cell lines (IMR32, SKN-MC, SKN-SH), in glioblastoma cell lines (A172, T98G, U87-MG, U118-MG, U138-MG, and U373-MG), in neuroglioma (H4) and astrocytoma cell lines (CCF-STTG1 and SW1088). Confocal analysis of NRP/B in U87-MG glioblastoma cells indicated nuclear localization of NRP/B. NRP/B expression was also observed in human primary brain tumors including glioblastoma multiformae and astrocytomas (total of five cases). These results suggest that NRP/B expression is upregulated in human brain tumors including glioblastomas and astrocytomas, while under normal conditions NRP/B expression is restricted to neurons. This study implicates a role for NRP/B in brain tumor development. 相似文献
47.
We screened clones for thioredoxin reductase genes with a degenerate PCR-based strategy and have isolated two novel cDNA clones from a mouse thymocyte cDNA library. These encode two distinct thioredoxin reductases (TrxR1 and TrxR2) with 499 and 527 amino acid (aa) residues and calculated molecular masses of 54.5 kDa and 56.8 kDa respectively. These proteins share 90% and 50% aa sequence identity with those of previously cloned human TrxR, containing the redox-active cysteines, FAD binding domain, and the selenocysteine (SeCys) insertion sequence, which is composed of a putative stem-loop sequence located in the 3'-untranslated region (UTR). TrxR2 showing less homology to human TrxR has a mitochondrial translocation signal and a mitochondrial prepeptide protease cleavage site in the N-terminal domain. Transient expression experiments of each gene as fusion proteins with Xpress-tagged protein in NIH 3T3 cells indicated that TrxR1 was localized in the nucleus and cytoplasm and TrxR2 in the mitochondria. Furthermore, we mapped the TrxR1 gene to chromosome 10 (placed 1.71 cR from D10Mit42, lod>3.0) and the TrxR2 gene to chromosome 16 (placed 22.56 cR from D16Mit34, lod>3.0). Thus, the mouse has at least two distinct nuclear genes for TrxR that have different translocation sites in the cell. 相似文献
48.
49.
Artificial sequences of the 153 amino acids have been designed to fit the main-chain framework of the sperm whale myoglobin (Mb) structure based on a knowledge-based 3D-1D compatibility method. The previously designed artificial globin (DG1) folded into a monomeric, compact, highly helical and globular form with overall dimensions similar to those of the target structure, but it lacked structural uniqueness at the side-chain level [Isogai, Y., Ota, M., Fujisawa, T. , Izuno, H., Mukai, M., Nakamura, H., Iizuka, T., and Nishikawa, K. (1999) Biochemistry 38, 7431-7443]. In this study, we redesigned hydrophobic sites of DG1 to improve the structural specificity. Several Leu and Met residues in DG1 were replaced with beta-branched amino acids, Ile and Val, referring to the 3D profile of DG1 to produce three redesigned globins, DG2-4. These residue replacements resulted in no significant changes of their compactness and alpha-helical contents in the absence of denaturant, whereas they significantly affected the dependence of the secondary structure on the concentration of guanidine hydrochloride. The analyses of the denaturation curves revealed higher global stabilities of the designed globins than that of natural apoMb. Among DG1-4, DG3, in which 11 Leu residues of DG1 are replaced with seven Ile and four Val residues, and one Met residue is replaced with Val, displayed the lowest stability but the most cooperative folding-unfolding transition and the most dispersed NMR spectrum with the smallest line width. The present results indicate that the replacements of Leu (Met) with the beta-branched amino acids at appropriate sites reduce the freedom of side-chain conformation and improve the structural specificity at the expense of stability. 相似文献
50.
(1) Superoxide dismutase activity in polymorphonuclear cells from human blood is considerably lower than that in lymphocytes. Macrophages from ascites show the middle level between the other two cells. (2) In myelocytic, monocytic, and lymphocytic leukemia cells, the enzyme activities are increased compared to those in the corresponding normal cells. (3) Gel electrophoresis patterns of all normal cells reveal bands corresponding to the cytosol and mitochondrial bands reported in previous studies. However, the mitochondrial Mn-containing superoxide dismutase activities are diminished or absent in leukemia cells. CN-insensitive superoxide dismutase activity in leukemia cells is not detected under the conditions. 相似文献