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101.
A simple method for the simultaneous isolation of stellate cells and hepatocytes from rat liver tissue 总被引:6,自引:0,他引:6
Riccalton-Banks L Bhandari R Fry J Shakesheff KM 《Molecular and cellular biochemistry》2003,248(1-2):97-102
Hepatic stellate cells (HSCs), also referred to as Ito cells, perisinusiodal cells and fat-storing cells, have numerous vital functions. They are the main extracellular matrix-producing cells within the liver and are involved in the storage of retinol. HSCs are also known to secrete a number of liver mitogens. Current isolation techniques are cumbersome and most require a pronase digestion step, which destroys any hepatocytes present. We present a simple method for isolation and culture of hepatic stellate cells from the normally discarded washings from a two-step collagenase hepatocyte isolation, which has shown a yield of more than 1.5 × 106 viable HSCs after 5 days in culture. The cells were positively identified as HSCs by staining for two intermediate filaments (desmin and GFAP) and observing their distinct morphology from other liver cell types. This efficient method allows rapid and consistent isolation of stellate cells to give a culture that may be passaged several times. 相似文献
102.
May RJ Beenhouwer DO Scharff MD 《Journal of immunology (Baltimore, Md. : 1950)》2003,171(9):4905-4912
Cryptococcus neoformans causes a life-threatening meningoencephalitis in AIDS patients. Mice immunized with a glycoconjugate vaccine composed of the glucuronoxylomannan (GXM) component of the cryptococcal capsular polysaccharide conjugated to tetanus toxoid produce Abs that can be either protective or nonprotective. Because nonprotective Abs block the efficacy of protective Abs, an effective vaccine must focus the Ab response on a protective epitope. Mice immunized with peptide mimetics of GXM conjugated to keyhole limpet hemocyanin (KLH) with glutaraldehyde developed Abs to GXM. However, control peptides P315 and P24 conjugated to KLH also elicited Abs to GXM. GXM-binding Abs from mice immunized with P315-KLH were inhibited by KLH treated with glutaraldehyde (KLH-g), but not by P315. Furthermore, KLH-g inhibited binding of GXM by serum of mice immunized with GXM-TT, indicating that glutaraldehyde treatment of KLH reveals an epitope(s) that cross-reacts with GXM. Vaccination with KLH-g or unmodified KLH elicited Abs to GXM, but did not confer protection against C. neoformans, suggesting the cross-reactive epitope on KLH was not protective. This was supported by the finding that 4H3, a nonprotective mAb, cross-reacted strongly with KLH-g. Sera from mice immunized with either native KLH or KLH-g cross-reacted with several other carbohydrate Ags, many of which have been conjugated to KLH for vaccine development. This study illustrates how mAbs can be used to determine the efficacy of potential vaccines, in addition to describing the complexity of using KLH and glutaraldehyde in the development of vaccines to carbohydrate Ags. 相似文献
103.
The Arabidopsis NPR1 disease resistance protein is a novel cofactor that confers redox regulation of DNA binding activity to the basic domain/leucine zipper transcription factor TGA1 总被引:12,自引:0,他引:12
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Després C Chubak C Rochon A Clark R Bethune T Desveaux D Fobert PR 《The Plant cell》2003,15(9):2181-2191
104.
A novel in-vitro system for the simultaneous exposure of bladder smooth muscle cells to mechanical strain and sustained hydrostatic pressure 总被引:2,自引:0,他引:2
Haberstroh KM Kaefer M DePaola N Frommer SA Bizios R 《Journal of biomechanical engineering》2002,124(2):208-213
The novel hydrostrain system was designed in an effort to establish and maintain conditions that simulate the in-vivo mechanical environment of the bladder. In this laboratory system, ovine bladder smooth muscle cells on flexible, 10-cm-dia silastic membranes were exposed simultaneously to hydrostatic pressure (40 cm H2O, a pressure level currently associated with bladder pathologies) and mechanical strains (up to 25 percent) under standard cell culture conditions for 7 h. Under these conditions, Heparin Binding-Epidermal Growth Factor and Collagen Type III mRNA expression were significantly increased (p<0.01 and 0.1, respectively); however, no changes were observed in Collagen Type I mRNA expression. Decreases in the Collagen Type I:Type III ratio following simultaneous exposure of bladder smooth muscle cells to pathological levels of hydrostatic pressure and mechanical strain in vitro are in agreement with clinically observed increases in Collagen Type III with concomitant decreased human bladder compliance. The results of the present study, therefore, provide cellular/molecular level information relevant to bladder pathology that could have significant implications in the field of clinical urology. 相似文献
105.
High affinity mimotope of the polysaccharide capsule of Cryptococcus neoformans identified from an evolutionary phage peptide library 总被引:6,自引:0,他引:6
Beenhouwer DO May RJ Valadon P Scharff MD 《Journal of immunology (Baltimore, Md. : 1950)》2002,169(12):6992-6999
Cryptococcus neoformans causes a life-threatening meningoencephalitis in a significant percentage of AIDS patients. Mice immunized with a glycoconjugate vaccine composed of the glucuronoxylomannan (GXM) component of the cryptococcal capsular polysaccharide conjugated to tetanus toxoid (TT) produce Abs that, based on the epitope recognized, can be either protective or nonprotective. Since nonprotective Abs block the efficacy of protective Abs, we are interested in developing a vaccine that would focus the immune response specifically to protective epitopes. Previously, we screened a phage display library with 2H1, a protective anti-GXM mAb, and isolated PA1, a representative peptide that had a K(d) of 295 nM for 2H1. Mice immunized with PA1 conjugated to keyhole limpet hemocyanin developed high anti-peptide (1/13,000), but low anti-GXM (maximum, 1/200) titers. We now report our efforts to improve this vaccine by screening a sublibrary with six random amino acids added to either end of the PA1 motif to identify higher affinity peptides. P206.1, a peptide isolated from this sublibrary, had 80-fold higher affinity for 2H1 (K(d) = 3.7 nM) than PA1. P206.1 bound protective, but not nonprotective, anti-GXM Abs. Mice immunized with P206.1 conjugated to various carriers did not mount an Ab response to GXM despite developing high anti-peptide titers. However, mice primed with GXM-TT and boosted with P206.1-TT developed significant anti-GXM titers (maximum, 1/180,000). This latter immunization scheme focused the immune response on protective epitopes, since only 2-5% of these titers were directed against nonprotective de-O-acetylated GXM epitopes compared with 20-60% in animals primed and boosted with GXM-TT. 相似文献
106.
Rena G Woods YL Prescott AR Peggie M Unterman TG Williams MR Cohen P 《The EMBO journal》2002,21(9):2263-2271
107.
Elizabeth A. Leermakers John M. Jakicic Jackie Viteri Rena R. Wing 《Obesity (Silver Spring, Md.)》1998,6(5):346-352
Objective : Weight gain occurs frequently in men aged 25–40. This study compared the effectiveness of a clinic-based and a home-based intervention with a no-treatment control group in preventing this weight gain. Research Methods and Procedures : Men (n = 67)—aged 25 to 40, sedentary, with a body mass index of 22 to 30, recruited from the University of Pittsburgh—were randomly assigned to 4-month treatments focused on increasing aerobic exercise and reducing fat intake through a clinic-based (CB) or a home-based (HB) program, or to a de-layed-treatment control group. Subjects were reassessed at 4 months. Results : Adherence and outcome did not differ significantly between the CB and HB programs, except that CB subjects recorded their food intake more frequently, and a greater number of CB subjects achieved a total of 120 miles of exercise over the 4 months. Subjects in the two intervention conditions combined lost significantly more weight (-1.6 ± 2.5 kg) than control subjects, who gained 0.2 ±1.9 kg (p<0.01); this effect of treatment was seen primarily in men with a body mass index of 27 to 30 (-2.7 kg for CB and HB combined vs. +1.5 kg for control). Treated subjects also had somewhat greater improvements in body composition, aerobic fitness, and weekly energy expenditure than controls, although these differences did not reach significance. Discussions: Both CB and HB intervention show promise in preventing weight gain in young men, especially in those who are slightly overweight. Larger studies, using more representative samples of young men, appear warranted. 相似文献
108.
Alison M. Michie Graham Rena Margaret M. Harnett Miles D. Houslay 《Cell biochemistry and biophysics》1998,28(2-3):161-185
We have previously shown that the major cAMP phosphodiesterase (PDE) isoforms present in murine thymocytes are the cGMP-stimulated
PDE activity (PDE-2) and the cAMP-specific PDE activity (PDE-4), and that these isoforms are differentially regulated following
ligation of the TCR (Michie, A. M., Lobban, M. D., Mueller, T., Harnett, M. M., and Houslay, M. D. [1996]Cell. Signalling
8, 97–110). We show here that the anti-CD3-stimulated elevation in PDE-4 activity in murine thymocytes is dependent on protein
tyrosine kinase and protein kinase C (PKC)-mediated signals as the TCR-coupled increase in PDE-4 activity can be abrogated
by both the tyrosine kinase inhibitor, genistein, and the PKC selective inhibitors chelerythrine and staurosporine. Moreover,
the PKC-activating phorobol ester, phorbol-12-myristate, 13-acetate (PMA) caused an increase in PDE-4 activity, similar to
that observed in cells challenged with anti-CD3 monoclonal antibodies and which was not additive with cochallenge using anti-CD3
antibodies. Both the PMA-and the anti-CD3 antibody-mediated increases in PDE-4 activity were blocked by treatment with either
cycloheximide or actinomycin D. Despite the upregulation of PDE-4 activity consequent to TCR ligation, intracellular cAMP
levels increased on challenge of thymocytes with anti-CD3 antibody, indicating that adenylate cyclase activity was also increased
by TCR ligation. It is suggested that the anti-CD3-mediated increase in PDE-4 activity was owing to a rapid PKC-dependent
induction of PDE-4 activity following crosslinking of the TCR complex. This identifies “crosstalk” occurring between the PKA
and PKC signaling pathways initiated by ligation of the antigen receptor in murine thymocytes. That both adenylate cyclase
and PDE-4 activities were increased may indicate the presence of compartmentalized cAMP responses present in these cells. 相似文献
109.
The mitochondrial DNA (mtDNA) size of the terrestrial gastropod Albinaria turrita was determined by restriction enzyme mapping and found to be approximately 14.5 kb. Its partial gene content and organization were examined by sequencing three cloned segments representing about one-fourth of the mtDNA molecule. Complete sequences of cytochrome c oxidase subunit II (COII), and ATPase subunit 8 (ATPase8), as well as partial sequences of cytochrome c oxidase subunit I (COI), NADH dehydrogenase subunit 6 (ND6), and the large ribosomal RNA (IrRNA) genes were determined. Nine putative tRNA genes were also identified by their ability to conform to typical mitochondrial tRNA secondary structures. An 82-nt sequence resembles a noncoding region of the bivalve Mytilus edulis, even though it might contain a tenth tRNA gene with an unusual 5-nt overlap with another tRNA gene. The genetic code of Albinaria turrita appears to be the same as that of Drosophila and Mytilus edulis. The structures of COI and COII are conservative, but those of ATPase8 and ND6 are diversified. The sequenced portion of thelrRNA gene (1,079 nt) is characterized by conspicuous deletions in the 5 and 3 ends; this gene represents the smallest coelomate IrRNA gene so far known. Sequence comparisons of the identified genes indicate that there is greater difference between Albinaria and Mytilus than between Albinaria and Drosophila. An evolutionary analysis, based on COII sequences, suggests a possible nonmonophyletic origin of molluskan mtDNA. This is supported also by the absence of the ATPase8 gene in the mtDNA of Mytilus and nematodes, while this gene is present in the mtDNA of Albinaria and Cepaea nemoralis and in all other known coelomate metazoan mtDNAs. 相似文献
110.
Antonius L. J. J. Bronckers Rena N. D'Souza William T. Butler Donacian M. Lyaruu Simon van Dijk Steffen Gay Joseph H. M. Wöltgens 《Cell and tissue research》1993,272(2):237-247
A non-collagenous protein, extracted from rat incisor dentin, is a dentin sialoprotein (DSP). We examined immunohistochemically the developmental appearance and tissue distribution of DSP in 1 to 3-day-old rat molar and incisor tooth germs. The earliest staining for DSP was observed in newly differentiated odontoblasts. In more advanced stages, immunostaining for DSP gradually increased in pre-dentin, odontoblasts and dentin, and appeared in many cells of the dental papilla. In early stages of development before the breakdown of the dental basement membrane, pre-ameloblasts were also positive for DSP. This staining disappeared from the ameloblast cell body soon after deposition of the first layer of mineralized dentin. Radiolabelling of tooth matrix proteins with 14C-serine in vitro followed by immunoprecipitation and fluorography confirmed that DSP was synthesized by tooth-forming cells. The immunolocalization for DSP was different from that of either collagen type-I, osteocalcin or the amelogenins. Whereas collagen type-I and osteocalcin were restricted to the mesenchymal dental tissues, the amelogenins were detectable in both epithelial and mesenchymal dental cells and tissues at the epithelio-mesenchymal interface at early stages of development, prior to the onset of dentin mineralization. We conclude that DSP is expressed in and secreted by odontoblasts and some dental papilla cells from early stages of dentinogenesis onwards, i.e. later than type-I collagen, but before deposition of the first layer of mineralized dentin. In pre-mineralizing stages, some of the matrix proteins may be endocytosed from the pre-dentin by both cell types involved in the epithelio-mesenchymal interaction. 相似文献