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991.
Shibukaw A Yoshikawa Y Kimura T Kuroda Y Nakagawa T Wainer IW 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2002,768(1):189-197
Plasma protein binding of N-desethyloxybytynin (DEOXY), a major active metabolite of oxybutynin (OXY), was investigated quantitatively and enantioselectively using high-performance frontal analysis (HPFA). An on-line HPLC system which consists of HPFA column, extraction column and analytical column was developed to determine the unbound concentrations of DEOXY enantiomers in human plasma, in human serum albumin (HSA) solutions, and in human alpha1-acid glycoprotein (AGP) solutions. DEOXY is bound in human plasma strongly and enantioselectively. The unbound drug fraction in human plasma samples containing 5 microM (R)- or (S)-DEOXY was 1.19 +/- 0.001 and 2.33 +/- 0.044%, respectively. AGP plays the dominant role in this strong and enantioselective plasma protein binding of DEOXY. The total binding affinity (nK) of (R)-DEOXY and (S)-DEOXY to AGP was 2.97 x 10(7) and 1.31 x 10(7) M(-1), respectively, while the nK values of (R)-DEOXY and (S)-DEOXY to HSA were 7.77 x 10(3) and 8.44 x 10(3) M(-1), respectively. While the nK value of (S)-DEOXY is weaker than that of (S)-OXY (1.53 x 10(7) M(-1)), the nK value of (R)-DEOXY is 4.33 times stronger than that of (R)-OXY (6.86 x I0(6) M(-1)). This suggests that the elimination of an ethyl group weakens the binding affinity of the (S)-isomer because of the decrease in hydrophobicity, while the binding affinity of the (R)-isomer is enhanced by the decrease in steric hindrance. The total binding affinity of DEOXY to HSA is much lower than that of DEOXY-AGP binding as well as OXY-HSA binding (2.64 x 10(4) and 2.19 x 10(4) M(-1) for (R)-OXY and (S)-OXY, respectively). The study on competitive binding between OXY and DEOXY indicated that DEOXY enantiomers and OXY enantiomers are all bound competitively at the same binding site of AGP molecule. 相似文献
992.
Requirement for Activation of the Serine-Threonine Kinase Akt (Protein Kinase B) in Insulin Stimulation of Protein Synthesis but Not of Glucose Transport 总被引:17,自引:6,他引:11 下载免费PDF全文
Tadahiro Kitamura Wataru Ogawa Hiroshi Sakaue Yasuhisa Hino Shoji Kuroda Masafumi Takata Michihiro Matsumoto Tetsuo Maeda Hiroaki Konishi Ushio Kikkawa Masato Kasuga 《Molecular and cellular biology》1998,18(7):3708-3717
A wide variety of biological activities including the major metabolic actions of insulin is regulated by phosphatidylinositol (PI) 3-kinase. However, the downstream effectors of the various signaling pathways that emanate from PI 3-kinase remain unclear. Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, is thought to be one such downstream effector. A mutant Akt (Akt-AA) in which the phosphorylation sites (Thr308 and Ser473) targeted by growth factors are replaced by alanine has now been shown to lack protein kinase activity and, when overexpressed in CHO cells or 3T3-L1 adipocytes with the use of an adenovirus vector, to inhibit insulin-induced activation of endogenous Akt. Akt-AA thus acts in a dominant negative manner in intact cells. Insulin-stimulated protein synthesis, which is sensitive to wortmannin, a pharmacological inhibitor of PI 3-kinase, was abolished by overexpression of Akt-AA without an effect on amino acid transport into the cells, suggesting that Akt is required for insulin-stimulated protein synthesis. Insulin activation of p70 S6 kinase was inhibited by ~75% in CHO cells and ~30% in 3T3-L1 adipocytes, whereas insulin-induced activation of endogenous Akt was inhibited by 80 to 95%, by expression of Akt-AA. Thus, Akt activity appears to be required, at least in part, for insulin stimulation of p70 S6 kinase. However, insulin-stimulated glucose uptake in both CHO cells and 3T3-L1 adipocytes was not affected by overexpression of Akt-AA, suggesting that Akt is not required for this effect of insulin. These data indicate that Akt acts as a downstream effector in some, but not all, of the signaling pathways downstream of PI 3-kinase. 相似文献
993.
Atsuo Taniguchi Masayuki Hakoda Hisashi Yamanaka Chihiro Terai Keiji Hikiji Ryuji Kawaguchi Noriko Konishi Sadao Kashiwazaki N. Kamatani 《Human genetics》1998,102(2):197-202
Adenine phosphoribosyltransferase (APRT) is a purine metabolic enzyme and a homozygous deficiency in this enzyme causes 2,8-dihydroxyadenine
urolithiasis. Various germline abnormalities have been described, but we report here a unique type of germline mutation in
a homozygous individual (SY) who had excreted 2,8-dihydroxyadenine crystals. In SY, TCA was substituted for the physiological
stop codon TGA. This base substitution generates a new HinfI restriction site, and, using the polymerase chain reaction and subsequent digestion by this enzyme, it was confirmed that
SY is homozygous for the base substitution. This base change is unique in that it generates an open reading frame that extends
to the poly(A) addition site. The amount of mRNA in transformed B cells from SY was approximately a quarter of that in control
subjects and no APRT proteins were detected. In eukaryotes, unlike in prokaryotes, no rescue systems for defective polypeptide
termination caused by a missing stop codon have been found. Therefore, the outcome of the defect of SY is unclear from present
knowledge about termination of polypeptide synthesis. Investigations into the mechanisms of the absence of protein in the
cells of SY may lead to a better understanding of the physiological and nonphysiological termination of polypeptide synthesis
in eukaryotic cells.
Received: 26 August 1997 / Accepted: 5 November 1997 相似文献
994.
The chl a specific absorption coefficients [a* (λ), m2·mg chl a ? 1] were examined in chemostat culture of the Prymnesiophyceae Isochrysis galbana (Parke) under a 12:12‐h light:dark cycle at low light (75 μmol photons·m ? 2·s ? 1) and high light (500 μmol photons· m ? 2·s ? 1) conditions. Other associated measurements such as pigment composition, cell density, and diameter as the measure of cell size were also made at the two light regimes every 2 h for 2 days to confirm the periodicity. A distinct diel variability was observed for the a* (λ) with maxima near dawn and minima near dusk. The magnitude of diel variation in a* (440) was 15% at low light and 22% at high light. Pronounced diel patterns were observed for cell size with minima near dawn and maxima near dusk. The magnitude of diel variation in cell size was 9.3% at low light and 21% at high light. The absorption efficiency factors [Q a (440)] were determined by reconstruction using intracellular concentrations of pigments and cell size. The Q a (440) also showed a distinct diel variability, with minima near dawn and maxima near dusk. The diel variation in a* (λ) and Q a (λ) was primarily caused by changes in cell size due to growth, although there was some influence from diel variations in the intracellular pigment concentrations. The results presented here indicated that diel variation in a* (λ) was an important component of the optical characterization of phytoplankton. 相似文献
995.
996.
This paper describes a monoclonal antibody that recognizes a molecule whose expression is mostly restricted to some of the forebrain areas that control singing behavior in adult estrildine species studied, including the zebra, Bengalese, and spice finches. When the song system displays extreme sexual dimorphism, as in these species, antibody staining occurs only in the male's song nuclei. However, protein expression is identical in both sexes of estrildine finches, in which females also have a well-developed song system. Canaries appear to lack the protein, but it can be induced in female zebra finches by early estrogen treatment. Antibody staining patterns in the zebra finch show that the protein's expression is developmentally regulated to coincide with the abrupt increase in the volume and cell size of the male's or the estrogen-treated female's song system. 相似文献
997.
Yuki Nakayama Takashi Yamamoto Yoshiko Matsuda Shin-Ichi Abé 《Development, growth & differentiation》1998,40(6):599-608
To investigate the function of Wilms' tumor 1 (WT1) during spermatogenesis, cDNA for newt WT1 homolog was cloned and the expression of WT1 in newt testes was examined. The cDNA is 2089 bp in length and encodes 426 amino acid (aa) residues. The deduced aa sequence shares 76 and 79% homology with human and Xenopus WT1, respectively. Northern blot analysis shows that WT1 mRNA, 3.2 and 4.5kb in length, are expressed in the testis and kidney. Both WT1 mRNA species are detected in various stages of spermatogenesis, but the 3.2kb mRNA is highly expressed in spermatogonia and mature sperm stages, while the amount of 4.5kb mRNA is almost constant throughout spermatogenesis. In situ hybridization reveals that WT1 mRNA is localized in Sertoli cells. Moreover, immunohistochemical analysis shows that WT1 protein is highly expressed in the nuclei of Sertoli cells in early spermatogonia and mature sperm stages, but not in pericystic cells or germ cells. These results suggest that WT1 is involved in the regulation of gene expression in Sertoli cells, depending on the spermatogenic stage. 相似文献
998.
Yun Zhong Katsuhiko Enomoto Hiroshi Isomura Norimasa Sawada Takashi Minase Masahito Oyamada Yasuhiro Konishi Michio Mori 《Experimental cell research》1994,214(2)
An important function of the tight junction is to act as a selective barrier to ions and small molecules, although no molecule responsible for the barrier function has been identified. Here we report evidence that the localization of the 7H6 tight junction-associated antigen identified in our laboratory at tight junctions correlates with the barrier function of MDCK cells. MDCK cells in a confluent monolayer possessed a polarized morphology, having an apical plasma membrane and a basolateral membrane, which is separated from the former by tight junctions. MDCK cells expressed both ZO-1 and 7H6 antigen at tight junctions, which maintain a tight barrier as determined by resistance to lanthanum permeation and high transepithelial electrical resistance (TER, 1500 ohm-cm2). The 7H6 antigen disappeared as tight junctions became permeable to lanthanum with a decrease in TER (below 100 ohm-cm2) due to treatment with metabolic inhibitors (10 μm antimycin A and 10 mM 2-deoxyglucose) for 30 min, while leaving ZO-1 at the cell border. The 7H6 antigen appeared at tight junctions again as TER recovered to a high level (1500 ohm-cm2) within 3 h after withdrawal of metabolic inhibitors. In addition, we found that 7H6 antigen is a phosphorylated protein and that phosphorylation is closely related to the localization of 7H6 antigen in the area of tight junctions. 相似文献
999.
1000.
Toshimi Mitsuishi Yuki Akuzawa Shinobu Sato Jin Rui Kazue Kodama Kinji Inoue Satonori Kurashige 《Microbiology and immunology》1993,37(12):943-951
TtT/M-87 cell is a macrophage cell line established from thyrotropic pituitary tumor tissues in mouse. In this paper, we report the immunological properties of M-87 cells as a model of tumor-associated macrophage. Contrasting with resident peritoneal macrophages, M-87 cells constitutively secreted small but significant amounts of TNF-α and IL-1α, which were detectable in both biological assays (cytotoxic activity for L929 and co-mitogenic activity for Con A-induced T cell proliferation, respectively) and ELISA, and produced larger amounts of these cytokines upon stimulation with LPS. They expressed MHC class II molecules on their cell surface without stimulation by IFN-γ. The accessory or antigen-presenting cell activity in antibody-producing response of spleen lymphocytes to sheep red blood cells was shown to be much higher in M-87 cells than normal peritoneal macrophages. In addition, when normal spleen lymphocytes were cultured with allogeneic tumor cells, such as EL-4 and S-180, in the presence of M-87 cells, lymphocytes reactive to stimulator cells were activated to manifest inhibitory effect on the tumor cell growth and also to manifest specific cytotoxic effect on the allogeneic tumor cells. These results show that M-87 cells derived from tumor-associated tissue are activated macrophages and that they are inhibitory to tumor cell growth and augmentative in the induction of T-cell-mediated immune responses. 相似文献