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1.
In the present study, the bombesin-induced changes in cytosolic free Ca2+ ([Ca2+]i) were investigated in single Fura-2 loaded SV-40 transformed hamster β-cells (HIT). Bombesin (50–500 pM) caused frequency-modulated repetitive Ca2+ transients. The average frequency of the Ca2+ transients induced by bombesin (200 pM) was 0.58 ± 0.02 min−1 (n = 121 cells). High concentrations of bombesin (≥ 2 nM) triggered a large initial Ca2+ transient followed by a sustained plateau or by a decrease to basal levels. In Ca2+- free medium, bombesin caused only one or two Ca2+ transients and withdrawal of extracellular Ca2+ abolished the Ca2+ transients. The voltage-dependent Ca2+ channel (VDCC) blockers, verapamil (50 μM) and nifedipine (10 μM), reduced amplitude and frequency of the Ca2+ transients and stopped the Ca2+ transients in some cells. Thapsigargin caused a sustained rise in [Ca2+]i) in the presence of extracellular Ca2+ while in its absence the rise in [Ca2+]i) was transient. Verapamil (50 μM) inhibited the thapsigargin-induced increase in [Ca2+], by about 50%. Depletion of intracellular Ca2+ stores by repetitive stimulation with increasing concentrations of bombesin or thapsigargin in Ca2+-free medium caused an agonist-independent increase in [Ca2+]i) when extracellular Ca2+ was restored, which was larger than in control cells that had been incubated in Ca2+-free medium for the same period of time. This rise in [Ca2+]i and the thapsigargin-induced increase in [Ca2+]i) were only partly inhibited by VDCC-blockers. Thus, depletion of the agonist-sensitive Ca2+ pool enhances Ca2+ influx through VDCC and voltage-independent Ca2+ channels (VICC). In conclusion, the bombesin-induced Ca2+ response in single HIT cells is periodic in nature with frequency-modulated repetitive Ca2+ transients. Intracellular Ca2+ is mobilized during each Ca2+ transient, but Ca2+ influx through VDCC and VICC is required for maintaining the sustained nature of the Ca2+ response. Ca2+ influx in whole or part is activated by a capacitative Ca2+ entry mechanism. 相似文献
2.
3.
Fanny Giudicelli Anne-Laure Brabant Isabelle Grit Patricia Parnet Valérie Amarger 《PloS one》2013,8(7)
Methionine, folic acid, betaine and choline interact in the one-carbon metabolism which provides methyl groups for methylation reactions. An optimal intake of these nutrients during pregnancy is required for successful completion of fetal development and evidence is growing that they could be involved in metabolic long-term programming. However, the biological pathways involved in the action of these nutrients are still poorly known. This study investigated the interaction between methyl donors and protein content in maternal diet during the preconceptual, pregnancy and lactation periods and the consequences on the rat offspring in the short and long term. Methyl donor supplementation reduced leptin secretion in offspring, whereas insulin levels were mostly affected by protein restriction. The joint effect of protein restriction and methyl donor excess strongly impaired postnatal growth in both gender and long term weight gain in male offspring only, without affecting food intake. In addition, rats born from protein restricted and methyl donor supplemented dams gained less weight when fed a hypercaloric diet. Methylation of the leptin gene promoter in adipose tissue was increased in methyl donor supplemented groups but not affected by protein restriction only. These results suggest that maternal methyl donor supplementation may influence energy homeostasis in a gender-dependent manner, without affecting food intake. Moreover, we showed that macronutrients and micronutrients in maternal diet interact to influence the programming of the offspring. 相似文献
4.
Patterns of Root Colonization in Epacridaceous Plants Collected from Different Sites 总被引:2,自引:0,他引:2
Root colonization was studied in ten species of the Epacridaceaeat three sites in Victoria by morphological and cross-inoculationexperiments. The sites and genera chosen were Cranbourne [Epacrisimpressa Labill. andLeucopogon ericoides(Smith) R. Br.] andRye [L. parviflorus(Andrews) Lindley] on the Mornington Peninsula,and the Grampians[Astroloma conostephioides(Sond.) Benth.,A.humifusum(Cav.) R. Br.,A pinifolium(R. Br.) Benth,Brachylomadaphnoides(Smith) Benth.,E. impressa, E. impressavar.grandifloraBenth.andStyphelia adscendensR. Br.] in western Victoria. For morphologicalstudies, samples of roots from each species at each site werecleared and stained and examined microscopically. For cross-inoculationstudies, cuttings from each site were struck in potting mediuminoculated with soil from the same and other sites. The ericoidmycorrhizae in the roots of plants found at or grown in Cranbourneand Rye soils were similar. Both were significantly differentfrom the internal hyphae found in the roots of plants foundat or grown in Grampians soils, which were three times largerin diameter and formed dense coils which filled the host celland invaded adjacent epidermal cells. This suggests that morethan one fungus is involved in the relationships, that the MorningtonPeninsula sites had a different fungus from the Grampians siteand that host specificity is low. Vesicular structures werealso found commonly on plants at the Grampians site, in contrastwith other sites. Epacridaceae; root; fungus; mycorrhiza; morphology; inoculation 相似文献
5.
The wingless- and int-related proteins (Wnts) have an important role during embryonic development and limb patterning. To investigate their function during chondrocyte differentiation, we used NIH3T3 cells producing seven members of the Wnt family and secreted frizzled-related protein (sFRP-2) for co-culture experiments with the rat chondrogenic cell line pColl(II)-EGFP-5. Pilot experiments showed a negative effect of Wnt-7a on the proliferation of three rodent chondrogenic cell lines, RCJ3.1(C5.18), CFK-2, and C1. To establish a reporter system for chondrogenic differentiation we then produced a stably transfected chondrogenic cell line based on RCJ3.1(C5.18) for further experiments, which expresses green fluorescence protein (EGFP) under the collagen type II promoter (pColl(II)-EGFP-5). This cell line permits convenient observation of green fluorescence as a marker for differentiation in life cultures. The colony size of this cell line in agarose suspension cultures was reduced to 20-40% of control, when exposed to Wnt-1, 3a, 4, 7a, and 7b for 14 days. Similarly, reporter gene expression and the synthesis of cartilage-specific proteoglycans were inhibited by this group of Wnts. In contrast, pColl(II)-EGFP-5 cells exposed to Wnt-5a and Wnt-11 reached 140% of control, and reporter gene expression and proteoglycan synthesis were stimulated. The effects of Wnt-7a and Wnt-5a were additive in pColl(II)-EGFP-5 cells and some but not all Wnt effects were antagonized by the inhibition of proteoglycan sulfation with chlorate, by sFRP-2, which may modulate Wnt receptor binding, or by inhibitors of protein kinase C. These results suggest two functional Wnt subclasses that differentially regulate proliferation and chondrogenic differentiation in vitro which may have implications for cartilage differentiation in vivo. Since some, but not all Wnt effects were sensitive to inhibitors of proteoglycan synthesis or protein kinase C, multiple modes of signal transduction may be involved. 相似文献
6.
Rad51p and Rad54p,but not Rad52p,elevate gene repair in Saccharomyces cerevisiae directed by modified single-stranded oligonucleotide vectors 总被引:1,自引:0,他引:1
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Synthetic single-stranded DNA vectors have been used to correct point and frameshift mutations in episomal or chromosomal targets in the yeast Saccharomyces cerevisiae. Certain parameters, such as the length of the vector and the genetic background of the organism, have a significant impact on the process of targeted gene repair, and point mutations are corrected at a higher frequency than frameshift mutations. Genetic analyses reveal that expression levels of the recombination/repair genes RAD51, RAD52 and RAD54 can affect the frequency of gene repair. Overexpression of RAD51 enhances the frequency 4-fold for correction of an episomal target and 5-fold for correction of a chromosomal target; overexpression of RAD54 is also effective in stimulating gene repair, to the same extent as RAD51 in the chromosomal target. In sharp contrast, RAD52 gene expression serves to reduce gene repair activity in rescue experiments and in experiments where RAD52 is overexpressed in a wild-type strain. This may suggest an antagonist role for Rad52p. Consistent with this notion, the highest level of targeted repair occurs when the RAD51 gene is overexpressed in a strain of yeast deficient in RAD52 gene function. 相似文献
7.
A versatile chondrogenic rat calvaria cell line R-tTA-24 that permits tetracycline-regulated gene expression 总被引:1,自引:0,他引:1
Bergwitz C Wendlandt T Pötter E Glomb I Gras K von zur Mühlen A Brabant G 《Histochemistry and cell biology》2000,113(2):145-150
The clonal rat calvaria cell line RCJ3.1C5.18 (RCJ) undergoes chondrogenic differentiation after long-term culture post confluence.
To allow flexible genetic manipulation, a tetracycline-regulated gene expression system was established in this cell line.
Treatment with tetracycline in operational doses does not affect the differentiation of RCJ cells with respect to the markers
tested. After stable transfection with pUHD15.1 containing the tetracycline transactivator (tTA) in the presence of pTK-hyg
for hygromycin selection, 28 clones were isolated and characterized for alcian blue staining of cartilage-specific proteoglycans
and for collagen type II expression. Clone R-tTA-24 was selected on the basis of phenotype and displayed tetracycline-dependent
downregulation of luciferase activity (tet-OFF system) by two orders of magnitude (57–149-fold) after stable transfection
with the reporter gene pBI-EGFP/luc. The novel, chondrogenic cell line R-tTA-24 may be stably transfected with various genes
of interest for tetracycline- regulated gene expression using neomycin selection and may be a valuable tool to study the process
of chondrogenic differentiation in vitro.
Accepted: 30 November 1999 相似文献
8.
Andréia S Lessa Bruno D Paredes Juliana V Dias Adriana B Carvalho Luiz Fernando Quintanilha Christina M Takiya Bernardo R Tura Guilherme FM Rezende Antonio C Campos de Carvalho Célia MC Resende Regina CS Goldenberg 《BMC veterinary research》2010,6(1):1-10
Background
Atypical scrapie was first identified in Norwegian sheep in 1998 and has subsequently been identified in many countries. Retrospective studies have identified cases predating the initial identification of this form of scrapie, and epidemiological studies have indicated that it does not conform to the behaviour of an infectious disease, giving rise to the hypothesis that it represents spontaneous disease. However, atypical scrapie isolates have been shown to be infectious experimentally, through intracerebral inoculation in transgenic mice and sheep. The first successful challenge of a sheep with 'field' atypical scrapie from an homologous donor sheep was reported in 2007.Results
This study demonstrates that atypical scrapie has distinct clinical, pathological and biochemical characteristics which are maintained on transmission and sub-passage, and which are distinct from other strains of transmissible spongiform encephalopathies in the same host genotype.Conclusions
Atypical scrapie is consistently transmissible within AHQ homozygous sheep, and the disease phenotype is preserved on sub-passage. 相似文献9.
van Beers JJ Raijmakers R Alexander LE Stammen-Vogelzangs J Lokate AM Heck AJ Schasfoort RB Pruijn GJ 《Arthritis research & therapy》2010,12(6):R219
Introduction
Rheumatoid arthritis (RA) frequently involves the loss of tolerance to citrullinated antigens, which may play a role in pathogenicity. Citrullinated fibrinogen is commonly found in inflamed synovial tissue and is a frequent target of autoantibodies in RA patients. To obtain insight into the B-cell response to citrullinated fibrinogen in RA, its autoepitopes were systematically mapped using a new methodology. 相似文献10.