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71.
72.
A procedure is described for the isolation of highly purified heavy-chain immunoglobulin mRNAs from a variety of mouse plasmacytomas (IgA, IgG, and IgM producers). The use of fresh tissue and the rapid isolation and direct extraction of membrane-bound polyribosomes were found to be essential in obtaining large quantities of undegraded heavy-chain mRNAs. The individual mRNAs were purified by two cycles of oligo(dT)-cellulose chromatography, sodium dodecyl sulfate--sucrose gradient centrifugation, and electrophoresis on 98% formamide containing polyacrylamide gels. When added to a cell-free protein-synthesizing system from wheat germ, the MPC-11 gamma2b and H2020 alpha heavy-chain mRNAs efficiently directed the synthesis of a predominant product of 55 000 molecular weight, while the synthesis of a 70 000 dalton protein in addition to other lower molecular weight polypeptides were observed with MOPC 3741 mu mRNA. All of these proteins were immunoprecipitable with class-specific heavy-chain antisera, and in the case of the gamma2b in vitro products good correspondence in a comparative trypsin--chymotrypsin fingerpring with in vivo labeled gamma2b heavy chain was observed. The gamma2b and a alpha heavy-chain mRNAs possessed a chain length of approximately 1800 nucleotides and the mu mRNA a size of approximately 2150 nucleotides when examined under stringent denaturation conditions. The purities of the alpha, gamma2b, and mu mRNAs were estimated to be 60--80%, 50--70%, and 50--83%, respectively, on the basis of their hybridization rates with cDNA probes in comparison to mRNA standards of known complexity. Heavy-chain mRNAs of the same class isolated from different mouse strains (Balb/C or NZB) display no detectable sequence differences in cross hybridization experiments, even though the cDNA--mRNA hybrids are submitted to stringent S1 nuclease digestion. These results indicate that allotypic determinants represent only a minor fraction of the heavy-chain constant region sequence in the mouse. 相似文献
73.
A switch region inversion contributes to the aberrant rearrangement of a mu immunoglobulin heavy chain gene in MPC-11 cells. 总被引:4,自引:2,他引:2 下载免费PDF全文
We describe the unique features of an aberrantly rearranged mu immunoglobulin heavy chain gene isolated from MPC-11 cells (a gamma 2b producing Balb/c plasmacytoma). A novel rearrangement has occurred 1.5 Kb 5' of the MPC-11 mu gene (denoted 18b mu) resulting in the deletion of the majority of the repetitive switch region (S mu) and 5' flanking DNA including the Joining (JH) sequences. The remainder (275 bp) of the S mu repeat has undergone a complete sequence inversion. DNA sequences 5' of the inverted S mu sequence do not resemble Variable (VH), Diversity (D), JH or their conserved flanking sequences. A DNA sequence localized 5' of the inverted S mu sequence, (p18b mu-1.4) detects a small family of homologous sequences in Balb/c DNA. The 18b mu-1.4 like sequences lack homology to S mu, exhibit flanking sequence polymorphisms in 5 out of 6 inbred mouse strains and undergo partial or complete deletion in 5 out of 10 plasmacytomas tested. Two 18b mu-1.4 homologous sequences display a higher copy number in C57Bl/6, AL/N and CAL9 mouse strains. 相似文献
74.
Brandenburg K Garidel P Schromm AB Andrä J Kramer A Egmond M Wiese A 《European biophysics journal : EBJ》2005,34(1):28-41
Outer-membrane proteases T (OmpT) are important defence molecules of Gram-negative bacteria such as Escherichia coli found in particular in clinical isolates. We studied the interaction of OmpT with the membrane-forming lipids phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) from the inner leaflet and lipopolysaccharide (LPS) from the outer leaflet of the outer membrane. These investigations comprise functional aspects of the protein–lipid interaction mimicking the outer-membrane system as well as the bioactivity of LPS:OmpT complexes in the infected host after release from the bacterial surface. The molecular interaction of the lipids PE, PG, and LPS with OmpT was investigated by analysing molecular groups in the lipids originating from the apolar region (methylene groups), the interface region (ester), and the polar region (phosphates), and by analysing the acyl-chain melting-phase behaviour of the lipids. The activity of OmpT and LPS:OmpT complexes was investigated in biological test systems (human mononuclear cells and Limulus amoebocyte lysate assay) and with phospholipid model membranes. The results show a strong influence of OmpT on the mobility of the lipids leading to a considerable fluidization of the acyl chains of the phospholipids as well as LPS, and a rigidification of the phospholipid, but not LPS head groups. From this, a dominant role of the protein on the function of the outer membrane can be deduced. OmpT released from the outer membrane still contains slight contaminations of LPS, but its strong cytokine-inducing ability in mononuclear cells, which does not depend on the Toll-like receptors 2 and 4, indicates an LPS-independent mechanism of cell activation. This might be of general importance for infections induced by Gram-negative bacteria. 相似文献
75.
Multispectral fluorescence lifetime imaging system for intravascular diagnostics with ultrasound guidance: in vivo validation in swine arteries 下载免费PDF全文
Julien Bec Dinglong M. Ma Diego R. Yankelevich Jing Liu William T. Ferrier Jeffrey Southard Laura Marcu 《Journal of biophotonics》2014,7(5):281-285
Fluorescence lifetime technique has demonstrated potential for analysis of atherosclerotic lesions and for complementing existing intravascular imaging modalities such as intravascular ultrasound (IVUS) in identifying lesions at high risk of rupture. This study presents a multimodal catheter system integrating a 40 MHz commercial IVUS and fluorescence lifetime imaging (FLIm) using fast helical motion scanning (400 rpm, 0.75 mm/s), able to acquire in vivo in pulsatile blood flow the autofluorescence emission of arterial vessels with high precision (5.08 ± 0.26 ns mean average lifetime over 13 scans). Co‐registered FLIm and IVUS data allowed 3D visualization of both biochemical and morphological vessel properties. Current study supports the development of clinically compatible intravascular diagnostic system integrating FLIm and demonstrates, to our knowledge, the first in vivo intravascular application of a fluorescence lifetime imaging technique. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim) 相似文献
76.
Alexandre Bergeron Andra Brezai Rami Shukr Louis Villeneuve Bruce G. Allen Wasay M. S. Qureshi Kathryn E. Hentges Angelino Calderone 《Journal of cellular physiology》2021,236(2):1281-1294
Cardiomyocyte migration represents a requisite event of cardiogenesis and the regenerative response of the injured adult zebrafish and neonatal rodent heart. The present study tested the hypothesis that the appearance of the intermediate filament protein nestin in neonatal rat ventricular cardiomyocytes (NNVMs) was associated in part with the acquisition of a migratory phenotype. The cotreatment of NNVMs with phorbol 12,13‐dibutyrate (PDBu) and the p38α/β mitogen‐activated protein kinase inhibitor SB203580 led to the de novo synthesis of nestin. The intermediate filament protein was subsequently reorganized into a filamentous pattern and redistributed to the leading edge of elongated membrane protrusions translating to significant lengthening of NNVMs. PDBu/SB203580 treatment concomitantly promoted the reorganization of nonmuscle myosin IIB (NMIIB) located predominantly at the periphery of the plasma membrane of NNVMs to a filamentous phenotype extending to the leading edge of elongated membrane protrusions. Coimmunoprecipitation assay revealed a physical interaction between NMIIB and nestin after PDBu/SB203580 treatment of NNVMs. In wild‐type and heterozygous NMIIB embryonic hearts at E11.5–E14.5 days, nestin immunoreactivity was identified in a subpopulation of cardiomyocytes elongating perpendicular to the compact myocardium, at the leading edge of nascent trabeculae and during thickening of the compact myocardium. In mutant embryonic hearts lacking NMIIB protein expression, trabeculae formation was reduced, the compact myocardium significantly thinner and nestin immunoreactivity undetectable in cardiomyocytes at E14.5 days. These data suggest that NMIIB and nestin may act in a coordinated fashion to facilitate the acquisition of a migratory phenotype in neonatal and embryonic cardiomyocytes. 相似文献
77.
A type III secretion system (T3SS) in pathogenic Yersinia
species functions to translocate Yop effectors, which modulate cytokine
production and regulate cell death in macrophages. Distinct pathways of
T3SS-dependent cell death and caspase-1 activation occur in
Yersinia-infected macrophages. One pathway of cell death
and caspase-1 activation in macrophages requires the effector YopJ. YopJ is an
acetyltransferase that inactivates MAPK kinases and IKKβ to cause
TLR4-dependent apoptosis in naïve macrophages. A YopJ isoform in Y.
pestis KIM (YopJKIM) has two amino acid substitutions,
F177L and K206E, not present in YopJ proteins of Y.
pseudotuberculosis and Y. pestis CO92. As compared
to other YopJ isoforms, YopJKIM causes increased apoptosis, caspase-1
activation, and secretion of IL-1β in Yersinia-infected
macrophages. The molecular basis for increased apoptosis and activation of
caspase-1 by YopJKIM in Yersinia-infected
macrophages was studied. Site directed mutagenesis showed that the F177L and
K206E substitutions in YopJKIM were important for enhanced apoptosis,
caspase-1 activation, and IL-1β secretion. As compared to
YopJCO92, YopJKIM displayed an enhanced capacity to
inhibit phosphorylation of IκB-α in macrophages and to bind IKKβ in
vitro. YopJKIM also showed a moderately increased ability to inhibit
phosphorylation of MAPKs. Increased caspase-1 cleavage and IL-1β secretion
occurred in IKKβ-deficient macrophages infected with Y.
pestis expressing YopJCO92, confirming that the
NF-κB pathway can negatively regulate inflammasome activation.
K+ efflux, NLRP3 and ASC were important for secretion of
IL-1β in response to Y. pestis KIM infection as shown using
macrophages lacking inflammasome components or by the addition of exogenous KCl.
These data show that caspase-1 is activated in naïve macrophages in
response to infection with a pathogen that inhibits IKKβ and MAPK kinases
and induces TLR4-dependent apoptosis. This pro-inflammatory form of apoptosis
may represent an early innate immune response to highly virulent pathogens such
as Y. pestis KIM that have evolved an enhanced ability to
inhibit host signaling pathways. 相似文献
78.
79.
80.
Jurij Dolen?ek Andra? Sto?er Ma?a Skelin Klemen Evan W. Miller Marjan Slak Rupnik 《PloS one》2013,8(12)
Oscillatory electrical activity is regarded as a hallmark of the pancreatic beta cell glucose-dependent excitability pattern. Electrophysiologically recorded membrane potential oscillations in beta cells are associated with in-phase oscillatory cytosolic calcium activity ([Ca2+]i) measured with fluorescent probes. Recent high spatial and temporal resolution confocal imaging revealed that glucose stimulation of beta cells in intact islets within acute tissue slices produces a [Ca2+]i change with initial transient phase followed by a plateau phase with highly synchronized [Ca2+]i oscillations. Here, we aimed to correlate the plateau [Ca2+]i oscillations with the oscillations of membrane potential using patch-clamp and for the first time high resolution voltage-sensitive dye based confocal imaging. Our results demonstrated that the glucose-evoked membrane potential oscillations spread over the islet in a wave-like manner, their durations and wave velocities being comparable to the ones for [Ca2+]i oscillations and waves. High temporal resolution simultaneous records of membrane potential and [Ca2+]i confirmed tight but nevertheless limited coupling of the two processes, with membrane depolarization preceding the [Ca2+]i increase. The potassium channel blocker tetraethylammonium increased the velocity at which oscillations advanced over the islet by several-fold while, at the same time, emphasized differences in kinetics of the membrane potential and the [Ca2+]i. The combination of both imaging techniques provides a powerful tool that will help us attain deeper knowledge of the beta cell network. 相似文献