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121.
Isolation and characterization of 101-beta-lysozyme that possesses the beta-aspartyl sequence at aspartic acid-101 总被引:1,自引:0,他引:1
H Yamada T Ueda R Kuroki T Fukumura T Yasukochi T Hirabayashi K Fujita T Imoto 《Biochemistry》1985,24(27):7953-7959
In the reaction of the intramolecular cross-linking between Lys-13 (epsilon-NH3+) and Leu-129 (alpha-COO-) in lysozyme using imidazole and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride [Yamada, H., Kuroki, R., Hirata, M., & Imoto, T. (1983) Biochemistry 22, 4551-4556], it was found that two-thirds of the protein (both the recovered and cross-linked lysozymes) showed a lower affinity than the rest against chitin-coated Celite, an affinity adsorbent for lysozyme. The protein with the reduced affinity was separated on chitin-coated Celite affinity chromatography and found to be slightly different from native lysozyme in the elution position of the tryptic peptide of Ile-98-Arg-112 on reversed-phase high-performance liquid chromatography. In contrast with native lysozyme, the limited hydrolysis of this abnormal tryptic peptide of Ile-98-Arg-112 in 6 N HCl at 110 degrees C gave a considerable amount of beta-aspartylglycine. Therefore, it was concluded that two-thirds of the protein obtained from this reaction possessed the beta-aspartylglycyl sequence at Asp-101-Gly-102. As a result, we obtained four lysozymes from this reaction, the derivative with the beta-aspartyl sequence at Asp-101 (101-beta-lysozyme), the cross-linked derivative between Lys-13 and Leu-129 (CL-lysozyme), the CL-lysozyme derivative with the beta-aspartyl sequence at Asp-101 (101-beta-CL-lysozyme), and native lysozyme. In the ethyl esterification of Asp-52 in lysozyme with triethyloxonium fluoroborate [Parsons, S. M., Jao, L., Dahlquist, F. W., Borders, C. L., Jr., Groff, T., Racs, J., & Raftery, M. A. (1969) Biochemistry 8, 700-712; Parsons, S. M., & Raftery, M. A. (1969) Biochemistry 8, 4199-4205], the same bond rearrangement was detected in the same ratio.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
122.
We have isolated two clones containing 19 kilobases (kb) of the human gene coding for a pro-alpha 1 (II) collagen chain from human lambda genomic DNA libraries. A 3' clone, HC2A, was selected by cross-hybridization with a cDNA clone containing sequences coding for the carboxy propeptide of chick type II procollagen. A second clone, HC2B, was obtained by screening the library with the 5' part of HC2A. The sequence analysis of exon 3 corresponding to the C propeptide reveals the presence of stretches of conserved nucleotides between the human and the chick type II procollagen genes. On Northern blots, the human collagen clone hybridizes strongly to a 5.5-kb RNA for the rat type II procollagen chain. Finally, studies of genomic DNAs from normal individuals reveal the presence of a HindIII and a BamHI polymorphic site at the 3' end of the gene. 相似文献
123.
Coordinate regulation of the levels of type III and type I collagen mRNA in most but not all mouse fibroblasts 总被引:20,自引:0,他引:20
A mouse genomic clone was isolated by cross-hybridization with a DNA fragment which codes for the NH2-propeptide of chick alpha1(III) collagen. The region of cross-hybridization within the mouse clone was localized, its sequence determined, and an exon coding for the NH2-propeptide of mouse alpha1(III) collagen was identified. This DNA fragment hybridizes to an RNA species of approximately 5300 nucleotides, slightly larger than the major alpha2(I) collagen RNA species. The mouse type III collagen probe was used to examine the effect of transformation on alpha1(III) collagen RNA levels in mouse fibroblasts. The levels of type III and type I collagen mRNA levels were compared in control and sarcoma virus-transformed murine cell lines, as well as in NIH 3T3 cells transformed by members of the human ras oncogenes. The levels of type III RNA decreased about 10-15-fold in Moloney sarcoma virus-transformed cells and in a cell line transformed with a v-mos-containing plasmid, but showed only a 50% decrease in a Kirsten murine sarcoma virus-transformed BALB 3T3 cell line, and increased 4-fold in a Rous sarcoma virus (RSV)-transformed BALB 3T3 cell line. In contrast, the levels of alpha2(I) collagen mRNA are 8- to 10-fold lower in all these cell lines when compared to untransformed cells. NIH 3T3 cells transformed with two human ras oncogenes showed decreased levels of alpha2(I) and alpha1(III) mRNAs. In contrast to the RSV-transformed mouse cell line, RSV-transformed chick embryo fibroblasts contained much smaller amounts of type III RNA than control chick embryo fibroblasts. We conclude that the levels of alpha1(III) and alpha2(I) collagen mRNA are often but not necessarily coordinately regulated by transformation in mouse cells. 相似文献
124.
T Matsuura Y Takeuchi M Kojima S Ueda H Yamada Y Nojyo K Ushijima Y Sano 《Acta anatomica》1985,123(4):207-219
Distributional and morphological features, especially characteristics of the ramification of serotonin-containing supraependymal fibers (SEF), were studied in the ventricular systems of mammals (mouse, rat, guinea pig, rabbit, cat, dog, monkey) by means of a modified peroxidase antiperoxidase technique, using antiserotonin antiserum prepared in our laboratory. SEF were present in all ventricular systems, except on the third ventricle floor and in the choroid plexus. The density of SEF was higher in the smaller species. In the rat, light- and scanning electron microscopical SEF were almost completely abolished 1 week after intraventricular administration of 5,6-dihydroxytryptamine. Ramification of SEF was complicated; the SEF formed a true network with frequent anastomosing. In the ventricular system of rats rendered hydrocephalic by kaolin administration, the mode of axonal branching in the supraependymal plexus could best be analyzed by the scanning electron microscope because the meshes of the plexus were spread out. 相似文献
125.
Squalene analogs such as lycopersene, geranylfarnesyl, digeranyl, and 2-hydroxy-2,3-dihydrosqualene and terpene alcohol derivatives such as farnesyl benzyl ether, farnesyl pivalate, geranylgeranyl pivalate, geranyl pivalate, and geranyl benzyl ether were oxidized by Corynebacterium sp. strain SY-79, which was isolated from soil by using squalene as a carbon source. Lycopersene and geranylfarnesyl gave no major product. Digeranyl, geranyl benzyl ether, and geranyl pivalate gave terminal oxidation products, and 2-hydroxy-2,3-dihydrosqualene, farnesyl benzyl ether, farnesyl pivalate, and geranylgeranyl pivalate were degraded to give lower molecular carboxylic acids. Strain SY-79 showed promising oxidative activities toward acyclic terpenes, although the metabolites obtained were variable, depending upon the structure of the substrate. 相似文献
126.
Antileukemic effect of coral-prostanoids clavulones from the stolonifer Clavularia viridis on human myeloid leukemia (HL-60) cells 总被引:1,自引:0,他引:1
A Honda Y Yamamoto Y Mori Y Yamada H Kikuchi 《Biochemical and biophysical research communications》1985,130(2):515-523
To elucidate the biological activities of coral-prostanoids, clavulones, discovered from the Japanese stolonifer Clavularia viridis, we examined the effect of clavulone on the cell growth of human cancer (human promyelocytic leukemia (HL-60) cells and HeLa cells) and normal (Chang liver cells and lung fibroblasts) cells in vitro. Clavulone showed strong antiproliferative and cytotoxic activities in the human cells and it had some selectivity to leukemic (HL-60) cells over other HeLa cells or normal cells on the basis of the IC50 values and cytotoxic effect of the cells. The IC50 value of clavulone in the HL-60 cells was about 0.4 microM (0.2 micrograms/ml). Over 1.0 microM (0.5 micrograms/ml), clavulone showed a significant cytotoxic activity on the HL-60 cells. The data on DNA synthesis and flow cytometric analysis revealed that clavulone arrests the cells in the G1-phase and inhibits the cell growth of HL-60 cells by inhibiting S-phase DNA synthesis. These results suggest that clavulone has a potent antileukemic effect on HL-60 cells. 相似文献
127.
We have examined the effects of soluble collagen on the function of fibronectin in baby hamster kidney (BHK) cells. Collagen and its purified alpha1(l) chain noncompetitively inhibited cell spreading on substrates precoated with fibronectin or a 75,000-D cell-binding fragment of fibronectin. Neither preincubation of cells with collagen followed by washing nor the addition of collagen to previously spread cells had any inhibitory effect on cell spreading, which indicates a requirement for the concurrent presence of collagen during the process of spreading. Treatment of collagen or alpha1(l) chain with collagenase abolished the inhibitory effect on fibronectin-mediated cell spreading. However, direct attachment of BHK cells to fibronectin-coated or 75,000-D fragment-coated substrates was not inhibited by collagen or by the alpha1(l) chain. Moreover, the binding of [3H]fibronectin or the 3'-75,000-D fragment to cell surfaces was not inhibited by the presence of soluble collagen, whereas soluble fibronectin inhibited binding. Although the binding of [3H]fibronectin-coated beads to BHK cell surfaces was also not inhibited by collagen, the phagocytosis of such beads was inhibited by the presence of collagen. On the other hand, soluble fibronectin partially inhibited the binding of fibronectin-coated beads but did not inhibit phagocytosis of the beads that did bind. The mechanism of the inhibition of fibronectin function by collagen and the possible interactions of two different kinds of receptors on the cell surface are discussed. 相似文献
128.
W T Chen E Hasegawa T Hasegawa C Weinstock K M Yamada 《The Journal of cell biology》1985,100(4):1103-1114
129.
Purification and physicochemical characterization of murine T cell replacing factor (TRF) 总被引:9,自引:0,他引:9
K Takatsu N Harada Y Hara Y Takahama G Yamada K Dobashi T Hamaoka 《Journal of immunology (Baltimore, Md. : 1950)》1985,134(1):382-389
Murine T cell replacing factor (TRF) was purified from a cellfree supernatant of a T cell hybridoma (B151K12) that constitutively produces TRF. Two assay systems for TRF activity were employed: 1) induction of anti-DNP IgG PFC responses in cultures of splenic B cells from DNP-KLH-primed BALB/c mice, and 2) induction of IgM PFC in chronic B cell leukemic cells (BCL1). The purification scheme consisted of ammonium sulfate precipitation, DEAE-cellulose chromatography, Blue-Sepharose chromatography, hydroxylapatite chromatography, gel permeation with fast protein liquid chromatography (FPLC), and disc polyacrylamide gel electrophoresis. Overall, TRF was purified approximately 34,000-fold with a maximum 3.8% recovery of activity, and the specific activity of the purified TRF was approximately 9.6 X 10(4) U/mg. The TRF that is active in these systems is distinct from the other lymphokines such as IL 1, IL 2, BCGFI (now known as BSFp1), and gamma-interferon. The TRF is extremely hydrophobic, with an apparent m.w. of 50,000 to 60,000 on gel permeation chromatography and 18,000 on SDS-PAGE under reducing conditions. Highly purified B151-TRF abrogated the activity by treatment with trypsin but not with RNase. Moreover, it bound to lima bean agglutinin-Sepharose specific for N-acetylgalactosamine residues, indicating that B151-TRF is a glycosylated glycoprotein containing N-acetylgalactosamine residues. The role of N-acetylgalactosamine residues on TRF activity was additionally substantiated by the fact that the addition of appropriate amounts of N-acetylgalactosamine in the assay systems for TRF preferentially induced a profound suppression for TRF-mediated PFC responses. 相似文献
130.
Haruki Yamada Shūichi Yanahira Hiroaki Kiyohara Jong-Chol Cyong Yasuo Otsuka 《Phytochemistry》1985,25(1):129-132
The water-soluble major polysaccharides from the seed of Coix lacryma-jobi var. ma-yuen eluted as a broad peak by gel filtration on Sepharose CL-2B. The mixture (CS-Glucan) was resolved into 7 glucans by HPLC on the column of Asahi-Pak GS-510 + GS-320. Similarities were observed between M, shown in the gel filtration profile and the elution volume in HPLC. Methylation analysis indicated that the ethanol-fractionated CS-glucan contained 4-O- and 4,6-di-O-substituted glucosyl residues. 1H and 13C NMR data accorded with the results of methylation analysis, and the glycosidic linkages were shown to have an α-configuration. Thus, CS-glucan contained (1 → 4) linked α-d-glucans to which are attached glucosyl side chains at O-6 of the main chain in a similar way to amylopectin. Each purified glucan was shown to have different absorption maxima ( > 550 nm or 530 nm) in the iodine reaction. The results of the methylation analysis and of the pullulanase digestion suggest that the 550 nm-glucan has a lower branching frequency and shorter side chains than the 530 nm-glucan. Although CS-glucan was found to have weak anti-complementary activity, HPLC-purified > 550 nm-glucan was found to be more potent than the 530 nm-glucan. Thus CS-glucan is highly heterogeneous, and the glucans which form a tight complex when tested with iodine, generally tend to have considerable anti-complementary activity. 相似文献