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101.
102.
103.
Characterization and localization of myosin in the brush border of intestinal epithelial cells 总被引:28,自引:26,他引:2
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The brush border of intestinal epithelial cells consists of a tightly packed array of microvilli, each of which contains a core of actin filaments. It has been postulated that microvillar movements are mediated by myosin interactions in the terminal web with the basal ends of these actin cores (Mooseker, M.S. 1976. J. Cell. Biol. 71:417-433). We report here that two predictions of this model are correct: (a) The brush border contains myosin, and (b) myosin is located in the terminal web. Myosin is isolated in 70 percent purity by solubilization of Triton-treated brush borders in 0.6 M KI, and separation of the components by gel filtration. Most of the remaining contaminants can be removed by precipitation of the myosin at low ionic strength. This yield is approximately 1 mg of myosin/30 mg of solubilized brush border protein. The molecule consists of three subunits with molecular weights of 200,000, 19,000, and 17,000 daltons in a 1:1:1 M ratio. At low ionic strength, the myosin forms small, bipolar filaments with dimensions of 300 X 11nm, that are similar to filaments seen previously in the terminal web of isolated brush borders. Like that of other vertebrate, nonmuscle myosins, the ATPase activity of isolated brush border myosin in 0.6 M KCI is highest with EDTA (1 μmol P(i)/mg-min; 37 degrees C), intermediate with Ca++ (0.4 μmol P(i)/mg-min), and low with Mg++ (0.01 μmol P(i)/mg-min). Actin does not stimulate the Mg-ATPase activity of the isolated enzyme. Antibodies against the rod fragment of human platelet myosin cross-react by immunodiffusion with brush border myosin. Staining of isolated mouse or chicken brush borders with rhodamine-antimyosin demonstrates that myosin is localized exclusively in the terminal web. 相似文献
104.
Metabolism of cationized lipoproteins by human fibroblasts: biochemical and morphologic correlations
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Human plasma low density lipoprotein (LDL) that had been rendered polycationic by coupling with N, N-dimethyl-1, 3-propanediamine (DMPA) was shown by electron microscopy to bind in clusters to the surface of human fibroblasts. The clusters resembled those formed by polycationic ferritin (DMPA-feritin), a visual probe that binds to anionic site on the plasma membrane. Biochemical studies with (125)I-labeled DMPA-LDL showed that the membrane-bound lipoprotein was internalized and hydrolyzed in lysosomes. The turnover time for cell bound (125)I-DMPA-LDL, i.e., the time in which the amount of (125)I-DMPA-LDL degraded was equal to the steady-state cellular content of the lipoprotein, was about 50 h. Because the DMPA-LDL gained access to fibroblasts by binding nonspecifically to anionic sites on the cell surface rather than by binding to the physiologic LDL receptor, its uptake failed to be regulated under conditions in which the uptake of native LDL was reduced by feedback suppression of the LDL receptor. As a result, unlike the case with native LDL, the DMPA-LDL accumulated progressively within the cell, and this led to a massive increase in the cellular content of both free and esterified cholesterol. Studies with (14)C-oleate showed that at least 20 percent of the accumulated cholesteryl esters represented cholesterol that had been esterified within the cell. After 4 days of incubation with 10 μg/ml of DMPA-LDL, fibroblasts had accumulated so much cholesteryl ester that neutral lipid droplets were visible at the light microscope level with Oil Red O staining. By electron microscopy, these intracellular lipid droplets were observed to lack a tripartite limiting membrane. The ability to cause the overaccumulation of cholesteryl esters within cells by using DMPA-LDL provides a model system for study of the pathologic consequences at the cellular level of massive deposition of cholesteryl ester. 相似文献
105.
106.
Greenwood IA Ledoux J Sanguinetti A Perrino BA Leblanc N 《The Journal of biological chemistry》2004,279(37):38830-38837
Activation of Ca(2+)-dependent Cl(-) currents (I(Cl(Ca))) increases membrane excitability in vascular smooth muscle cells. Previous studies showed that Ca(2+)-dependent phosphorylation suppresses I(Cl(Ca)) in pulmonary artery myocytes, and the aim of the present study was to determine the role of the Ca(2+)-dependent phosphatase calcineurin on chloride channel activity. Immunocytochemical and Western blot studies with isoform-specific antibodies revealed that the alpha and beta forms of the CaN catalytic subunit are expressed in PA cells but that only the alpha variant translocated to the cell periphery upon a rise in intracellular [Ca(2+)]. I(Cl(Ca)) evoked by pipette solutions containing a [Ca(2+)] set at 500 nm was considerably larger when the pipette solution included constitutively active CaN containing the alpha catalytic isoform. This stimulatory effect was lost by boiling the enzyme or by the inclusion of a specific CaN inhibitory peptide and was not shared by the inclusion of the beta form of the catalytic subunit. In the absence of constitutively active CaN, cyclosporin A, an inhibitor of CaN, suppressed I(Cl(Ca)) evoked by 500 nm Ca(2+) when the current amplitude was relatively large but was ineffective in cells with smaller currents. In perforated patch recordings, cyclosporin A consistently inhibited I(Cl(Ca)) evoked as a consequence of Ca(2+) influx through voltage-dependent calcium channels. These novel data show that in PA myocytes activation of I(Cl(Ca)) is enhanced by Ca(2+)-dependent dephosphorylation and that the regulation of this conductance is highly isoform-specific. 相似文献
107.
M. Essig S. Ledoux P.-L. Tharaux C. Chatziantoniou J.-C. Dussaule 《Cell biology and toxicology》1996,12(4-6):271-274
Various in vitro preparations have been utilized to study the cellular activity of vasoactive agents on renal cortical microvessels and on mesangial cells. The receptors and the transduction pathways of bradykinin and atrial natriuretic factor were characterized on cultured cortical vascular smooth muscle cells from the rabbit kidney. A preparation of afferent arterioles that had been freshly isolated from the rat kidney was used to study the NO-dependent regulation of renin release. The influence of endothelin and angiotensin II on mesangial cell proliferation was evaluated, using cocultures of human endothelial and mesangial cells. These appropriate in vitro preparations have provided new insights on renal vascular endocrinology. However, extrapolation of in vitro data to in vivo physiology must be cautious because the phenotype of vascular cells often changes in culture conditions.Abbreviations ANF
atrial natriuretic factor
- BK
bradykinin
- CNP
C-type natriuretic peptide
- ET-1
endothelin-1
- HUVEC
human umbilical vein endothelial cells
- IBMX
isobutylmethylxanthine
- NEP
neutral endopeptidase
- PKA
protein kinase A
- RCVSMC
renal cortical vascular smoothmuscle cells 相似文献
108.
Patterns of differentiation and hybridization in North American wolflike canids, revealed by analysis of microsatellite loci 总被引:8,自引:0,他引:8
Roy MS; Geffen E; Smith D; Ostrander EA; Wayne RK 《Molecular biology and evolution》1994,11(4):553-570
Genetic divergence and gene flow among closely related populations are
difficult to measure because mutation rates of most nuclear loci are so low
that new mutations have not had sufficient time to appear and become fixed.
Microsatellite loci are repeat arrays of simple sequences that have high
mutation rates and are abundant in the eukaryotic genome. Large population
samples can be screened for variation by using the polymerase chain
reaction and polyacrylamide gel electrophoresis to separate alleles. We
analyzed 10 microsatellite loci to quantify genetic differentiation and
hybridization in three species of North American wolflike canids. We
expected to find a pattern of genetic differentiation by distance to exist
among wolflike canid populations, because of the finite dispersal distances
of individuals. Moreover, we predicted that, because wolflike canids are
highly mobile, hybrid zones may be more extensive and show substantial
changes in allele frequency, relative to nonhybridizing populations. We
demonstrate that wolves and coyotes do not show a pattern of genetic
differentiation by distance. Genetic subdivision in coyotes, as measured by
theta and Gst, is not significantly different from zero, reflecting
persistent gene flow among newly established populations. However, gray
wolves show significant subdivision that may be either due to drift in past
Ice Age refugia populations or a result of other causes. Finally, in areas
where gray wolves and coyotes hybridize, allele frequencies of gray wolves
are affected, but those of coyotes are not. Past hybridization between the
two species in the south-central United States may account for the origin
of the red wolf.
相似文献
109.
110.
Changgui Shi MD Vaskar Das PhD Xin Li MD PhD Ranjan Kc PhD Sujun Qiu MD InSug O‐Sullivan PhD Richard L. Ripper CVT Jeffrey S. Kroin PhD Fackson Mwale PhD Atiyayein A. Wallace MS Bingqian Zhu MSN Lan Zhao PhD Andre J. van Wijnen PhD Mingliang Ji MD PhD Jun Lu MD PhD Gina Votta‐Velis MD PhD Wen Yuan MD Hee‐Jeong Im PhD 《Journal of cellular physiology》2018,233(10):6589-6602