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61.
A E Berson C Young S L Morrison G H Fujii J Sheung B Wu J B Bolen A L Burkhardt 《Biochemical and biophysical research communications》1999,259(3):533-538
We report the molecular cloning and initial characterization of a novel fatty acid acylated serine/threonine protein kinase. The putative open reading frame is predicted to encode a 305 amino acid protein possessing a carboxy-terminal protein kinase domain and amino-terminal myristylation and palmitylation sites. The protein kinase has been accordingly denoted as the myristylated and palmitylated serine/threonine protein kinase (MPSK). Human and mouse MPSKs share approximately 93% identity at the amino acid level with complete retention of acylation sites. Radiation hybridization localized the human MPSK gene to chromosome 2q34-37. Northern analysis demonstrated that the human MPSK 1.7 kilobase mRNA is widely distributed. Epitope tagged human MPSK was found to be acylated by myristic acid at glycine residue 2 and by palmitic acid at cysteines 6 and/or 8. Palmitylation of MPSK in these experiments was found to require an intact myristylation site. While epitope tagged MPSK in immune complexes or purified human glutathione S transferase-MPSK was found to autophosphorylate at one or more threonine residues, the enzyme was not found to phosphorylate several other common exogenous substrates. Indeed, only PHAS-I was identified as an exogenous substrate which was found to be phosphorylated on threonine and serine residues. 相似文献
62.
Hierarchical analysis of variation in the mitochondrial 16S rRNA gene among Hymenoptera 总被引:5,自引:2,他引:3
Nucleotide sequences from a 434-bp region of the 16S rRNA gene were
analyzed for 65 taxa of Hymenoptera (ants, bees, wasps, parasitoid wasps,
sawflies) to examine the patterns of variation within the gene fragment and
the taxonomic levels for which it shows maximum utility in phylogeny
estimation. A hierarchical approach was adopted in the study through
comparison of levels of sequence variation among taxa at different
taxonomic levels. As previously reported for many holometabolous insects,
the 16S data reported here for Hymenoptera are highly AT-rich and exhibit
strong site-to-site variation in substitution rate. More precise estimates
of the shape parameter (alpha) of the gamma distribution and the proportion
of invariant sites were obtained in this study by employing a reference
phylogeny and utilizing maximum-likelihood estimation. The effectiveness of
this approach to recovering expected phylogenies of selected hymenopteran
taxa has been tested against the use of maximum parsimony. This study finds
that the 16S gene is most informative for phylogenetic analysis at two
different levels: among closely related species or populations, and among
tribes, subfamilies, and families. Maximization of the phylogenetic signal
extracted from the 16S gene at higher taxonomic levels may require
consideration of the base composition bias and the site-to-site rate
variation in a maximum-likelihood framework.
相似文献
63.
This report describes a convenient method for the rapid and efficient
release of N-linked oligosaccharides from low microgram amounts of
glycoproteins. A 96-well MultiScreen assay system containing a
polyvinylidene difluoride (PVDF) membrane is employed to immobilize
glycoproteins for subsequent enzymatic deglycosylation. Recombinant
tissue-type plasminogen activator (rt-PA) is used to demonstrate the
deglycosylation of 0.1-50 micrograms of a glycoprotein. This method enabled
the recovery of a sufficient amount of N-linked oligosaccharides released
enzymatically with peptide N-glycosidase F (PNGaseF) from as little as 0.5
microgram rt-PA for subsequent analysis by matrix-assisted laser
desorption/ionization time-of-flight (MALDI- TOF) mass spectrometry. The
immobilization of rt-PA to the PVDF membrane did not sterically inhibit the
PNGaseF-mediated release of oligosaccharides from rt-PA as determined by
tryptic mapping experiments. Comparison of the oligosaccharides released
from 50 micrograms of rt-PA by either the 96-well plate method or by a
standard solution digestion procedure showed no significant differences in
the profiles obtained by high-pH anion-exchange chromatography with pulsed
amperometric detection (HPAEC-PAD). Both neutral and sialylated
oligosaccharide standards spiked into wells were recovered equally as
determined by HPAEC-PAD. One advantage of this approach is that reduction
and alkylation can be performed on submicrogram amounts of glycoproteins
with easy removal of reagents prior to PNGaseF digestion. In addition, this
method allows 60 glycoprotein samples to be deglycosylated in 1 day with
MALDI-TOF or HPAEC-PAD analysis being performed on the following day.
相似文献
64.
A plasmid library of Acinetobacter calcoaceticus HindIII fragments was
constructed, and clones that complemented an Escherichia coli pabA mutant
were selected. Plasmids containing a 3.9-kb fragment of A. calcoaceticus
DNA that also complemented E. coli trpD and trpC-(trpF+) mutants were
obtained. We infer that complementation of E. coli pabA mutants was the
result of the expression of the amphibolic anthranilate-
synthase/p-aminobenzoate-synthase glutamine-amidotransferase gene and that
the plasmid insert carried the entire trpGDC gene cluster. In E. coli
minicells, the plasmid insert directed the synthesis of polypeptides of
44,000, 33,000, and 20,000 daltons, molecular masses that are consistent
with the reported molecular masses of phosphoribosylanthranilate
transferase, indoleglycerol-phosphate synthase, and anthranilate-synthase
component II, respectively. A 3,105- bp nucleotide sequence was determined.
Comparison of the A. calcoaceticus trpGDC sequences with other known trp
gene sequences has allowed insight into (1) the evolution of the amphibolic
trpG gene, (2) varied strategies for coordinate expression of trp genes,
and (3) mechanisms of gene fusions in the trp operon.
相似文献
65.
Differential scanning calorimetry of bovine rhodopsin in rod-outer-segment disk membranes. 总被引:1,自引:0,他引:1
S M Khan W Bolen P A Hargrave M M Santoro J H McDowell 《European journal of biochemistry》1991,200(1):53-59
Rhodopsin-containing retinal rod disk membranes from cattle have been examined by differential scanning calorimetry. Under conditions of 67 mM phosphate pH 7.0, unbleached rod outer segment disk membranes gave a single major endotherm with a temperature of denaturation (Tm) of 71.9 +/- 0.4 degrees C and a thermal unfolding calorimetric enthalpy change (delta Hcal) of 700 +/- 17 kJ/mol rhodopsin. Bleached rod outer segment disk membranes (membranes that had lost their absorbance at 498 nm after exposure to orange light) gave a single major endotherm with a Tm of 55.9 +/- 0.3 degrees C and a delta Hcal of 520 +/- 17 kJ/mol opsin. Neither bleached nor unbleached rod outer segment disk membranes gave endotherms upon thermal rescans. When thermal stability is examined over the pH range of 4-9, the major endotherms of both bleached and unbleached rod outer segment disk membranes were found to show maximum stability at pH 6.1. The observed delta Hcal values for bleached and unbleached rod outer segment disk membranes exhibit membrane concentration dependences which plateau at protein concentrations beyond 1.5 mg/mL. For partially bleached samples of rod outer segment disk membranes, the calorimetric enthalpy change for opsin appears to be somewhat dependent on the degree of bleaching, indicating intramembrane nearest neighbor interactions which affect the unfolding of opsin. Delta Hcal and Tm are particularly useful for assessing stability and testing for completeness of regeneration of rhodopsin from opsin. Other factors such as sample preparation and the presence of low concentrations of ethanol also affect the delta Hcal values while the Tm values remain fairly constant. This shows that the delta Hcal is a sensitive parameter for monitoring environmental changes of rhodopsin and opsin. 相似文献
66.
Quenching of tryptophan fluorescence by brominated phospholipid 总被引:7,自引:0,他引:7
Bromolipids [1-palmitoyl-2-(dibromostearoyl)phosphatidylcholine] with bromines at the 4,5-, 6,7-, 9,10-, 11,12-, and 15,16-positions were used to examine the fluorescence quenching of a synthetic, membrane-spanning peptide (Lys2-Gly-Leu8-Trp-Leu8-Lys-Ala-amide) incorporated into both small and large unilamellar vesicles. The peptide-lipid vesicles were analyzed to show that at least 75% of the peptide was in a transbilayer configuration, placing the single tryptophan in its predicted place in the center of the bilayer. Quenching profiles of the peptide in bromolipid showed maximal (90%) quenching by the 15,16-bromolipid, indicating that the bromolipids can accurately locate the position of a tryptophan in the bilayer. The quenching by the other bromolipids decreased with an r6 dependence and an apparent R0 of 9 A. In addition, indole in methanolic solution was subjected to quenching by a variety of mono- and dibrominated hydrocarbons. The quenching was analyzed, by using a modified Stern-Volmer equation, and found to be greatly dependent upon the number and positioning of the bromines. Monobromobutanes were found to have a quenching efficiency of only 7% while dibromobutanes, with bromines on adjacent carbon atoms, had efficiencies of over 80%. In addition, the dibromobutanes exhibited significant "static" quenching whereas the monobrominated butanes did not. These data suggest that the bromolipids are more appropriately defined as short-range quenchers rather than strictly contact quenchers. 相似文献
67.
A test of the linear extrapolation of unfolding free energy changes over an extended denaturant concentration range. 总被引:6,自引:0,他引:6
Guanidine hydrochloride (GdnHCl) and thermally induced unfolding measurements on the oxidized form of Escherichia coli thioredoxin at pH 7 were combined for the purpose of assessing the functional dependence of unfolding free energy changes on denaturant concentration over an extended GdnHCl concentration range. Conventional analysis of GdnHCl unfolding exhibits a linear plot of unfolding delta G vs [GdnHCl] in the transition zone. In order to extend unfolding delta G measurements outside of that narrow concentration range, thermal unfolding measurements were performed using differential scanning calorimetry (DSC) in the presence of low to moderate concentrations of GdnHCl. The unfolding delta G values from the DSC measurements were corrected to 25 degrees C using the Gibbs-Helmholtz equation and mapped onto the delta G vs [GdnHCl] plot. The dependence of unfolding delta G on [GdnHCl] was found to be linear over the full denaturant concentration range, provided that the chloride ion concentration was kept at a threshold of greater than or equal to 1.5 M. In the DSC experiments performed in the presence of GdnHCl, chloride concentrations were maintained at 1.5 M by addition of appropriate amounts of NaCl. The linear extrapolation method (LEM) gives an unfolding free energy change in the absence of denaturant (delta G degrees N-U) in excellent agreement with the delta G determined by DSC measurement in 1.5 M NaCl. The various methods give a consensus unfolding delta G value of 8.0 kcal/mol at 25 degrees C in the absence of denaturant.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
68.
Large-scale production of polyoma middle T antigen by using genetically engineered tumors. 总被引:4,自引:0,他引:4 下载免费PDF全文
D R Kaplan B Bockus T M Roberts J Bolen M Israel B S Schaffhausen 《Molecular and cellular biology》1985,5(7):1795-1799
A recombinant plasmid containing a metallothionein promoter-polyoma middle T cDNA fusion was constructed and used to transfect NIH 3T3 cells. Transformed cells expressing middle T were injected into nude mice. Within 3 weeks, each mouse produced tumors containing middle T equivalent to that in 250 to 1,000 100-mm dishes of polyomavirus-infected cells. This middle T, partially purified by immunoaffinity chromatography, retained activity as measured by its ability to be phosphorylated in vitro. The combined approach of fusing strong promoters to genes of interest and utilizing nude mice to grow large quantities of cells expressing the gene provides a quick, inexpensive alternative to other expression systems. 相似文献
69.
S. A. Morse T. A. Mietzner G. Bolen A. Le Faou G. Schoolnik 《Antonie van Leeuwenhoek》1987,53(6):465-469
The major iron-regulated protein (MIRP) was purified, from both Neisseria gonorrhoeae and N. meningitidis by selective extraction with cetyltrimethylammonium bromide followed by ion-exchange and moleculair-seive chromatography. Solutions of the purified proteins had a characteristic pink color. The overall amino acid composition of these proteins was similar, although differences were noted in the number of serine, threonine, and lysine residues. Nevertheless, the N-terminal amino acid sequence was identical through 47 residues for both the meningococcal and gonococcal MIRP. Plasma emission spectrophotometry revealed that the meningococcal 37K protein contained ca. 1 mole Fe/mole protein. 相似文献
70.