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41.
The linear extrapolation method was used to evaluate the unfolding free energy changes (delta G degrees N-U) for phenylmethanesulfonyl chymotrypsin (PMS-Ct) at pH 6.0. The nonlinear least-squares fits of difference spectral data using urea and guanidinium chloride as denaturants gave identical values for delta G degrees N-U and delta epsilon degrees U, the latter being extinction coefficient differences between native and unfolded forms of the protein in the limit of zero concentration of denaturant. The independence of these parameters from the nature of solvent suggests strongly that they are characteristic properties of the protein alone. The delta G degrees N-U data at pH 6.0 and 4.0, which differ by more than 100-fold in stability of the protein, were incorporated into a thermodynamic cycle involving free energy changes for titration of native and unfolded PMS-Ct from pH 4.0 to 6.0. The purpose of the cycle was to test whether delta G degrees N-U obtained by use of the linear extrapolation method exhibits the characteristics required of a thermodynamic function of state. Within error, the thermodynamic cycle was found to accommodate the delta G degrees N-U quantities obtained at pH 4.0 and 6.0 for PMS-Ct. 相似文献
42.
Tomlinson MG Kurosaki T Berson AE Fujii GH Johnston JA Bolen JB 《The Journal of biological chemistry》1999,274(19):13577-13585
Bruton's tyrosine kinase (Btk) is mutated in X-linked agammaglobulinemia patients and plays an essential role in B cell receptor signal transduction. Btk is a member of the Tec family of nonreceptor protein-tyrosine kinases that includes Bmx, Itk, Tec, and Txk. Cell lines deficient for Btk are impaired in phospholipase C-gamma2 (PLCgamma2)-dependent signaling. Itk and Tec have recently been shown to reconstitute PLCgamma2-dependent signaling in Btk-deficient human cells, but it is not known whether the atypical Tec family members, Bmx and Txk, can reconstitute function. Here we reconstitute Btk-deficient DT40 B cells with Bmx and Txk to compare their function with other Tec kinases. We show that in common with Itk and Tec, Bmx reconstituted PLCgamma2-dependent responses including calcium mobilization, extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) activation, and apoptosis. Txk also restored PLCgamma2/calcium signaling but, unlike other Tec kinases, functioned in a phosphatidylinositol 3-kinase-independent manner and failed to reconstitute apoptosis. These results are consistent with a common role for Tec kinases as amplifiers of PLCgamma2-dependent signal transduction, but suggest that the pleckstrin homology domain of Tec kinases, absent in Txk, is essential for apoptosis. 相似文献
43.
Time-dependent effects of trimethylamine-N-oxide/urea on lactate dehydrogenase activity: an unexplored dimension of the adaptation paradigm.
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Given that enzymes in urea-rich cells are believed to be just as sensitive to urea effects as enzymes in non-urea-rich cells, it is argued that time-dependent inactivation of enzymes by urea could become a factor of overriding importance in the biology of urea-rich cells. Time-independent parameters (e.g. Tm, k(cat), and Km) involving protein stability and enzyme function have generally been the focus of inquiries into the efficacy of naturally occurring osmolytes like trimethylamine-N-oxide (TMAO), to offset the deleterious effects of urea on the intracellular proteins in the urea-rich cells of elasmobranchs. However, using urea concentrations found in urea-rich cells of elasmobranches, we have found time-dependent effects on lactate dehydrogenase activity which indicate that TMAO plays the important biological role of slowing urea-induced dissociation of multimeric intracellular proteins. TMAO greatly diminishes the rate of lactate dehydrogenase dissociation and affords significant protection of the enzyme against urea-induced time-dependent inactivation. The effects of TMAO on enzyme inactivation by urea adds a temporal dimension that is an important part of the biology of the adaptation paradigm. 相似文献
44.
Substitution bias, rapid saturation, and the use of mtDNA for nematode systematics 总被引:13,自引:0,他引:13
Only relatively recently have researchers turned to molecular methods for
nematode phylogeny reconstruction. Thus, we lack the extensive literature
on evolutionary patterns and phylogenetic usefulness of different DNA
regions for nematodes that exists for other taxa. Here, we examine the
usefulness of mtDNA for nematode phylogeny reconstruction and provide data
that can be used for a priori character weighting or for parameter
specification in models of sequence evolution. We estimated the
substitution pattern for the mitochondrial ND4 gene from intraspecific
comparisons in four species of parasitic nematodes from the family
Trichostrongylidae (38-50 sequences per species). The resulting pattern
suggests a strong mutational bias toward A and T, and a lower
transition/transversion ratio than is typically observed in other taxa. We
also present information on the relative rates of substitution at first,
second, and third codon positions and on relative rates of saturation of
different types of substitutions in comparisons ranging from intraspecific
to interordinal. Silent sites saturate extremely quickly, presumably owing
to the substitution bias and, perhaps, to an accelerated mutation rate.
Results emphasize the importance of using only the most closely related
sequences in order to infer patterns of substitution accurately for
nematodes or for other taxa having strongly composition-biased DNA. ND4
also shows high amino acid polymorphism at both the intra- and
interspecific levels, and in higher level comparisons, there is evidence of
saturation at variable amino acid sites. In general, we recommend using
mtDNA coding genes only for phylogenetics of relatively closely related
nematode species and, even then, using only nonsynonymous substitutions and
the more conserved mitochondrial genes (e.g., cytochrome oxidases). On the
other hand, the high substitution rate in genes such as ND4 should make
them excellent for population genetics studies, identifying cryptic
species, and resolving relationships among closely related congeners when
other markers show insufficient variation.
相似文献
45.
Phylogeny of the Drosophila saltans species group based on combined analysis of nuclear and mitochondrial DNA sequences 总被引:2,自引:0,他引:2
Nucleotide sequences from two nuclear loci, alcohol dehydrogenase and
internal transcribed spacer-1 of the nuclear ribosomal DNA repeats, and two
mitochondrial genes, cytochrome oxidase I and cytochrome oxidase II, were
determined from nine species in the Drosophila saltans species group. The
partition homogeneity test and partitioned Bremer support were used to
measure incongruence between phylogenetic hypotheses generated from
individual partitions. Individual loci were generally congruent with each
other and consistent with the previously proposed morphological hypothesis,
although they differed in level of resolution. Since extreme conflict
between partitions did not exist, the data were combined and analyzed
simultaneously. The total evidence method gave a more resolved and highly
supported phylogeny, as indicated by bootstrap proportions and decay
indices, than did any of the individual analyses. The cordata and elliptica
subgroups, considered to have diverged early in the history of the D.
saltans group, were sister taxa to the remainder of the saltans group. The
sturtevanti subgroup, represented by D. milleri and D. sturtevanti,
occupies an intermediate position in this phylogeny. The saltans and
parasaltans subgroups are sister clades and occupy the most recently
derived portion of the phylogeny. As with previous morphological studies,
phylogenetic relationships within the saltans subgroup were not
satisfactorily resolved by the molecular data.
相似文献
46.
Granulocyte macrophage-colony stimulating factor stimulates both association and activation of phosphoinositide 3OH-kinase and src-related tyrosine kinase(s) in human myeloid derived cells. 总被引:19,自引:1,他引:18
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S Corey A Eguinoa K Puyana-Theall J B Bolen L Cantley F Mollinedo T R Jackson P T Hawkins L R Stephens 《The EMBO journal》1993,12(7):2681-2690
The signalling pathways used by the GM-CSF receptor are currently unknown. Here we show that in human myeloid derived cells GM-CSF can stimulate; (i) the accumulation of PtdIns(3,4,5)P3; (ii) increases in p53/p56lyn and p62c-yes directed protein tyrosine kinase activities in anti-lyn and anti-c-yes antibody directed immunoprecipitates, respectively and; (iii) increases in phosphoinositide 3OH-kinase activity in antiphosphotyrosine, anti-p53/p56lyn and anti-p62c-yes antibody directed immunoprecipitates. These results suggest that GM-CSF can stimulate formation of protein tyrosine kinase co-ordinated signalling complexes, that contain p53/p56lyn, p62c-yes and an activated PtdInsP2 directed phosphoinositide 3OH-kinase, which can drive the accumulation of the putative second-messenger PtdIns(3,4,5)P3. 相似文献
47.
Transfer of bacteria/yeast shuttle plasmids from Escherichia coli into the yeast species Kluyveromyces lactis, Pichia angusta (Hansenula polymorpha), and Pachysolen tannophilus has been accomplished, presumably through inter-kingdom conjugal transfer. Plasmid pEK2 was transferred into a K. lactis mutant to complement trp auxotrophy, while plasmid YEp13 was mobilized into and complemented P. angusta and P. tannophilus Leu- auxotrophs. Plasmid DNA in the recipient strains was detected by transformation of E. coli with crude yeast cell extracts. Freely replicating plasmids without detectable alterations as well as plasmids with rearrangements were recovered from yeast transconjugants. 相似文献
48.
Nucleotide sequence comparisons were used to investigate the evolution of P
transposable elements and the possibility that horizontal transfer has
played a role in their occurrence in natural populations of Drosophila and
other Diptera. The phylogeny of P elements was examined using published
sequences from eight dipteran taxa and a new, partial sequence from
Scaptomyza elmoi. The results from a number of different analyses are
highly consistent and reveal a P-element phylogeny that contradicts the
phylogeny of the species. At least three instances of horizontal transfer
are necessary to explain this incongruence, but other explanations cannot
be ruled out at this time.
相似文献
49.
Demonstration of D-xylose reductase and D-xylitol dehydrogenase in Pachysolen tannophilus 总被引:2,自引:0,他引:2
Summary The yeast, Pachysolen tannophilus, can utilize the pentose D-xylose with accumulation of significant quantities of ethanol. Cell extracts of the organism contain NADPH-linked D-xylose reductase (aldose reductase EC 1.1.1.21) and NAD-dependent D-xylitol dehydrogenase (D-xylulose reductase EC 1.1.1.9). D-Xylose was required for induction of both the D-xylitol dehydrogenase and the D-xylose reductase. Neither enzyme was found in glucose grown cell-free extracts. 相似文献
50.
Detection and quantitation of Newcastle disease virus proteins in infected chicken embryo cells.
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A technique was analyzed by which Newcastle disease virus (NDV) proteins could be quantitatively detected in the presence of chicken embryo cellular proteins in NDV-infected cells. The technique involved removal of electropho-proteins from a sodium dodecyl sulfate-polyacrylamide-agarose gel matrix by chemical cleavage of the acrylamide gel cross-linker. The proteins were subsequently transferred and covalently bound to diazobenzyloxymethyl paper. By incubating the paper with unlabeled antisera and 125I-labeled Staphylococcus aureus protein A, the specificity of the antisera and the sensitivity of this method of quantitative antigen detection were tested. The results demonstrated that as little as 1 ng of an individual NDV protein could be detected. Furthermore, this technique can simultaneously quantitate the synthesis of multiple NDV proteins under experimental conditions in which immunofluorescence, hemadsorption, and plaque assays failed to show virus protein synthesis or the formation of virus progeny. 相似文献