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71.
Kinetic characterization of Escherichia coli outer membrane phospholipase A using mixed detergent-lipid micelles 总被引:3,自引:0,他引:3
The substrate specificity of Escherichia coli outer membrane phospholipase A was analyzed in mixed micelles of lipid with deoxycholate or Triton X-100. Diglycerides, monoglycerides, and Tweens 40 and 85 in Triton X-100 are hydrolyzed at rates comparable to those of phospholipids and lysophospholipids. p-Nitrophenyl esters of fatty acids with different chain lengths and triglycerides are not hydrolyzed. The minimal substrate characteristics consist of a long acyl chain esterified to a more or less hydrophilic headgroup as is the case for the substrate monopalmitoylglycol. Binding occurs via the hydrocarbon chain of the substrate; diacyl compounds are bound three to five times better than monoacyl compounds. When acting on lecithins, phospholipase A1 activity is six times higher than phospholipase A2 activity or 1-acyl lysophospholipase activity. Activity on the 2-acyl lyso compound is about two times less than that on the 1-acyl lysophospholipid. The enzyme therefore has a clear preference for the primary ester bond of phospholipids. In contrast to phospholipase A1 activity, phospholipase A2 activity is stereospecific. Only the L isomer of a lecithin analogue in which the primary acyl chain was replaced by an alkyl ether group is hydrolyzed. The D isomer of this analogue is a competitive inhibitor, bound with the same affinity as the L isomer. On these ether analogues the enzyme shows the same preference for the primary acyl chain as with the natural diester phospholipids. Despite its broad specificity, the enzyme will initially act as a phospholipase A1 in the E. coli envelope where it is embedded in phospholipids. 相似文献
72.
The chemical synthesis of a phosphatidylglucose 总被引:1,自引:0,他引:1
73.
Expression of porcine pancreatic phospholipase A2. Generation of active enzyme by sequence-specific cleavage of a hybrid protein from Escherichia coli. 总被引:1,自引:0,他引:1 下载免费PDF全文
P de Geus C J van den Bergh O Kuipers H M Verheij W P Hoekstra G H de Haas 《Nucleic acids research》1987,15(9):3743-3759
The cDNA coding for the porcine pancreatic prophospholipase A2 (proPLA) has been cloned and expressed in E. coli. Expression of proPLA could only be obtained in the form of intracellular aggregates after fusing the 15 kDa proPLA to a large (greater than or equal to 45 kDa) bacterial peptide. The fusion protein was readily purified from cell lysates, and specifically cleaved. Cleavage of the fusion protein was achieved with either hydroxylamine (at Asn/Gly sequences in the denatured protein), or trypsin (between the pro- and the mature PLA in the renatured fusion protein). The former method releases a proPLA-like enzyme, while the latter directly yields PLA. Renaturation of the fusion protein was made possible by the use of a recently reported new S-sulphonation method. The released (pro)PLA was purified (yields of 2-3 mg/ltr of culture medium), and showed identical properties compared to native (pro)PLA. 相似文献
74.
Tempo and mode of concerted evolution in the L1 repeat family of mice 总被引:10,自引:0,他引:10
Martin SL; Voliva CF; Hardies SC; Edgell MH; Hutchison CA d 《Molecular biology and evolution》1985,2(2):127-140
A 300-bp DNA sequence has been determined for 30 (10 from each of three
species of mice) random isolates of a subset of the long interspersed
repeat family L1. From these data we conclude that members of the L1 family
are evolving in concert at the DNA sequence level in Mus domesticus, Mus
caroli, and Mus platythrix. The mechanism responsible for this phenomenon
may be either duplicative transposition, gene conversion, or a combination
of the two. The amount of intraspecies divergence averages 4.4%, although
between species base substitutions accumulate at the rate of approximately
0.85%/Myr to a maximum divergence of 9.1% between M. platythrix and both M.
domesticus and M. caroli. Parsimony analysis reveals that the M. platythrix
L1 family has evolved into a distinct clade in the 10-12 Myr since M.
platythrix last shared a common ancestor with M. domesticus and M. caroli.
The parsimony tree also provides a means to derive the average half-life of
L1 sequences in the genome. The rates of gain and loss of individual copies
of L1 were estimated to be approximately equal, such that approximately
one-half of them turn over every 3.3 Myr.
相似文献
75.
Genetics and biochemistry of the peptidoglycan-associated proteins b and c of Escherichia coli K12. 总被引:17,自引:0,他引:17
Cornelis Verhoef Ben Lugtenberg Ria van Boxtel Pieter de Graaff Hubertus Verheij 《Molecular & general genetics : MGG》1979,169(2):137-146
Summary To collect information on synthesis and regulation of the peptidoglycan-associated pore-forming outer membrane proteins b and c, mutants resistant to phages Mel and TuIa were analyzed. Genetic analysis showed three linkage groups, corresponding with the genes tolF (phenotype b-c+), meo A (phenotype b+c-) and ompB (phenotypes b-c-, b- c+, b++ c- and b++ c±). It has recently been described that also a b+ c- phenotype can occur in the latter linkage group [Chai, T., Foulds, J., J. Bacteriol. 130, 781–786 (1977)]. Among ompB (b- c+)/meoA (b+ c-) double mutants strains were found with the b+ c- phenotype, showing that ompB is not the structural gene for protein b. Studies on purified proteins b and c showed profound differences between the two proteins with respect to the electrophoretic mobility of fragments obtained by treatment with cyanogen bromide, trypsin and chymotrypsin. The amino acid in position three of the amino-termini of proteins b and c, isolated from isogenic strains, were identified as isoleucine and valine respectively. Both the genetic and biochemical results are consistent with a model recently published [Ichihara, S., Mizushima, S., J. Biochem. (Japan) 83, 1095–1100 (1978)] which predicts that tolF and meoA are the structural genes for the proteins b and c respectively and that ompB is a regulatory gene whose product regulates the levels of both proteins. 相似文献
76.
Hof D Cheung K de Rooij DJ van den Hoogen FH Pruijn GJ van Venrooij WJ Raats JM 《Arthritis research & therapy》2005,7(2):R302-R309
Modifications occurring on autoantigens during cell death have been proposed to have a role in the initiation of autoimmune
diseases. Patients suffering from mixed connective tissue disease (MCTD) produce autoantibodies directed to U1 small nuclear
ribonucleoprotein (snRNP), and antibodies against a 70 kDa protein component, the U1-70K (70K) protein, are the most prominent.
During apoptosis, 70K is cleaved by caspase-3 to a 40 kDa product, which remains associated with the complex. Autoantibodies
preferentially recognizing the apoptotic form of 70K have been described previously, and an apoptosis-specific epitope on
70K has been identified. This study shows that 29 of 53 (54%) MCTD sera preferentially recognize the apoptotic form of 70K
over intact 70K. Moreover, we show that antibodies directed to an apoptosis-specific epitope on 70K are more specifically
associated with MCTD than other anti-70K antibodies, suggesting that apoptotic 70K is a better antigen for the detection of
these antibodies in MCTD patients. Longitudinal analysis of 12 MCTD patients showed in several patients that early sera are
relatively enriched with antibodies recognizing an apoptosis-specific epitope, and that the levels of these apoptosis-specific
antibodies decrease in time. These findings indicate that the early detection of apoptotic 70K is of considerable interest
for anti-U1 snRNP-positive patients. 相似文献
77.
Effects of thymoquinone on liver miRNAs and oxidative stress in Ehrlich acid mouse solid tumor model
I Meral M Pala F Akbas S Ustunova C Yildiz MH Demirel 《Biotechnic & histochemistry》2018,93(4):301-308
We investigated the effects of thymoquinone (TQ) on the expression of liver microRNAs (miRNAs), liver histopathology and oxidative stress in Ehrlich acid solid tumor model induced mice. We used 24 male BALB/c mice divided randomly into three groups. Control (C) group mice were injected intraperitoneally (i.p.) with 0.5 ml saline for four weeks. Tumor (T) group mice were injected i.p. with 0.5 ml saline for four weeks, then Ehrlich acid tumor cells were injected subcutaneously into the neck to induce solid tumor formation. TQ (T + Tq) group mice injected i.p. with 10 mg/kg TQ for four weeks, then Ehrlich acid tumor cells were injected subcutaneously into the neck of the mice in this group to induce solid tumor formation. At the end of the study, liver from all groups were removed for histopathological and miRNAs analysis, and oxidative stress measurement. We found that the expression of miR-206b-3p was up-regulated and the oxidative stress and necrosis increased in the liver tissue of mice with Ehrlich acid solid tumor. TQ application decreased the oxidative stress, prevented necrosis, increased regeneration and down-regulated the expression of miR-206b-3p in the liver tissue. 相似文献
78.
The outer membrane phospholipase A (OMPLA) of Enterobacteriaceae has been proposed to span the membrane 14 times as antiparallel amphipathic beta-strands, thereby exposing seven loops to the cell surface. We have employed the epitope insertion method to probe the topology of OMPLA of Salmonella typhimurium. First, missense mutations were introduced at various positions in the pldA gene, encoding OMPLA, to create unique BamHI sites. These BamHI sites were subsequently used to insert linkers, encoding a 16-amino-acid B-cell epitope. Proper assembly of all mutant proteins was revealed by their heat modifiability in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The accessibility of the inserted epitopes was assessed. Immunofluorescence analysis of intact cells with antibodies against the inserted epitope showed that three of seven predicted loops are indeed cell surface exposed. Trypsin accessibility experiments verified the cell surface exposure of two additional loops and provided support for the proposed periplasmic localization of three predicted turns. For two other predicted exposed loops, the results were not conclusive. These results support to a large extent the proposed topology model of OMPLA. Furthermore, the observation that the substitutions Glu66Pro and Glu247Gly virtually abolished enzymatic activity indicates that these residues might play a major role in catalysis. 相似文献
79.
R. van der Heijden E. R. Verheij J. Schripsema A. Baerheim Svendsen R. Verpoorte P. A. A. Harkes 《Plant cell reports》1988,7(1):51-54
Treatment of suspension cultures of some Tabernaemontana species (Apocynaceae) with elicitors (e.g. cellulase, Candida albicans) result in a rapid de novo production of antimicrobial active triterpenes. The triterpenes are identified as ursene carboxylic acid derivatives. These triterpenes are not produced by an elicited cell suspension culture of Catharanthus roseus, another Apocynaceae. 相似文献
80.
In phospholipase A2 from Naja melanoleuca snake venom all four lysines were converted into the epsilon-amidinated derivatives without reaction of the alpha-amino group. The amidinated phospholipase (AMPA) showed high enzymatic activity. Starting from AMPA, chemical modification reactions were carried out at the alpha-amino function. This group was blocked with a tert-butyloxycarbonyl or a phenylthiocarbamyl group. Furthermore the polypeptide chain was shortened by one residue by removing the N-terminal asparagine, resulting in the formation of des-Asn1-AMPA. The native enzyme was shortened by eight residues by cyanogen bromide cleavage at the single methionine residue. Although all modified proteins show a reduced affinity for monomeric lipids, they are easily saturated with micellar substrate analogs. Whereas the removal of the N-terminal octapeptide abolished all enzymatic activity the other modified enzymes possess a low (1%), but measurable enzymatic activity. It is concluded that chemical modifications in the N-terminal region give rise to a distortion of the active site, thus reducing the activity of the lipid-bound enzyme. 相似文献