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排序方式: 共有249条查询结果,搜索用时 15 毫秒
1.
Mercurated nucleic acid probes, a new principle for non-radioactive in situ hybridization 总被引:3,自引:0,他引:3
This report describes the localization of specific nucleic acid sequences in interphase nuclei and metaphase chromosomes by a new hybridocytochemical method based on the use of mercurated nucleic acid probes. After the hybridization a sulfhydryl-hapten compound is reacted with the hybrids formed. A number of such ligands were synthesized and tested. A fluorescyl ligand could be used for the direct visualization of highly repetitive sequences. For indirect immunocytochemical visualization trinitrophenyl ligands were found to be more sensitive than biotinyl analogues. These ligands were applied for the detection of target sequences in metaphase chromosomes and interphase nuclei of somatic cell hybrids, human lymphoid cell lines and blood cell cultures. The sequences were in the range of high to low copy numbers. The lower limit of sensitivity is indicated by the visualization of two human unique DNA fragments (40 and 15.6 kb) in human metaphases. The method is rapid, gives consistent results and can be used for both RNA and DNA probes. Other potentials of the new principle are discussed. 相似文献
2.
C Troelstra R M Landsvater J Wiegant M van der Ploeg G Viel C H Buys J H Hoeijmakers 《Genomics》1992,12(4):745-749
We have cloned the human DNA excision repair gene ERCC6 by virtue of its ability to correct the uv sensitivity of Chinese hamster overy cell mutant UV61. This mutant is a member of complementation group 6 of the nucleotide excision repair-deficient rodent mutants. By means of in situ hybridization and Southern blot analysis of mouse x human somatic cell hybrids, the gene was localized to human chromosome 10q11-q21. An RFLP detected within the ERCC6 locus can be helpful in linkage analysis. 相似文献
3.
I. Cornelese-ten Velde J. Wiegant H. J. Tanke J. S. Ploem 《Histochemistry and cell biology》1989,92(2):153-160
Summary Reflection contrast microscopy (RCM) is a sensitive tool to detect minor amounts of precipitated diaminobenzidine (DABox) in immunoperoxidase stained specimens. One of the main issues in immunocytochemistry is the ongoing need for more sensitive and quantitative techniques. Therefore we applied RCM, using a new simple model system, to methods previously described for increased sensitivity in immunocytochemistry with bright field microscopy. Addition of imidazole was found the most sensitive method and addition of Nickel and Cobalt ions gave the most enhanced colour intensity. Variation of the enzyme reaction parameters yielded a continuous increase in reflection with time. This was then discussed in view of other model studies of peroxidase kinetics. A quantitative relationship between the amount of peroxidase and the reflection of DABox was observed, indicating that quantitative immunoperoxidase studies with RCM are feasible.In situ hybridization (ISH) was then used as a useful biological model for RCM to test the optimal conditions for DAB staining found in the model system (high concentrations of DAB and peroxidase and 2 h incubation time). There was no background staining in the model system, also after prolonged incubation time. The ISH experiments showed that the contrast (ratio) between specific signal and chromosome background did not increase in time, whereas only the use of high avPO concentrations yielded the highest contrast. 相似文献
4.
We have analyzed a total of 12 different global and local multiple
protein-sequence alignment methods. The purpose of this study is to
evaluate each method's ability to correctly identify the ordered series of
motifs found among all members of a given protein family. Four
phylogenetically distributed sets of sequences from the hemoglobin, kinase,
aspartic acid protease, and ribonuclease H protein families were used to
test the methods. The performance of all 12 methods was affected by (1) the
number of sequences in the test sets, (2) the degree of similarity among
the sequences, and (3) the number of indels required to produce a multiple
alignment. Global methods generally performed better than local methods in
the detection of motif patterns.
相似文献
5.
Roeland Van Wijk Marij Welters Jan E. M. Souren Hans Ovelgonne Fred A. C. Wiegant 《Journal of cellular physiology》1993,155(2):265-272
In this work, we demonstrate that a nonlethal dose of arsenite administered to quiescent C3H10T1/2 fibroblasts can enhance the mitogenic effect of suboptimal concentrations of serum. The mitogenic effect was dependent on the serum concentration and on the time interval between the administration of arsenite and that of serum. This suggests that mitogen sensitivity changes in time after arsenite treatment. It is shown that the concentrations of arsenite that enhance the mitogenic effect of serum also increase the mRNA levels of c-fos, HSP68, and HSP84 and induce the specific synthesis of Heat Shock Proteins (HSPs). The physiological significance of this phenomenon is most likely to counteract the long-term toxic effect of arsenite by early induction of compensation for cell loss. © 1993 Wiley-Liss, Inc. 相似文献
6.
Mariëtte P. C. van de Corput Roeland W. Dirks Wouter W. Wiegant Joop Wiegant Klaus Mühlegger A. K. Raap 《Histochemistry and cell biology》1997,108(4-5):359-364
Oestradiol has been conjugated to allylamine-dUTP with an 11-atom spacer to allow enzymatic incorporation of the label into
DNA sequences. In a comparative DNA and mRNA FISH study we have used DNA probes that were either labelled with digoxigenin,
biotin or oestradiol. Results show that oestradiol-labelled probes can detect DNA and RNA sequences in FISH equally well as
digoxigenin- and biotin-labelled probes. Further, no crossreactivity between the various hapten-specific antibodies and the
three haptens were observed. Binding of the rabbit anti-oestradiol antibody to endogenous oestrogen in various tissues was
not observed under the conditions tested. In view of the increasing demands for multi-colour DNA and mRNA FISH applications,
oestradiol is a welcome addition to the collection of haptens employed in FISH.
Accepted 20 June 1997 相似文献
7.
alpha-Endorphin and gamma-endorphin, two closely related peptides of the pro-opiomelanocortin family with characteristic biological activities, were purified to homogeneity from single human pituitary glands and chemically identified. Isolation of the peptides was based on size fractionation by Sephadex G-75 chromatography followed by two HPLC steps using reverse-phase and paired-ion reverse-phase systems and was monitored by radioimmunoassay. During the isolation procedure alpha- and gamma-endorphin-sized material behaved chromatographically and immunologically indistinguishably from synthetic alpha- and gamma-endorphin. The amino acid composition and NH2-terminus of isolated peptides demonstrated their identity as authentic alpha-endorphin and gamma-endorphin. Acetylated forms were absent. In addition, evidence is provided that large forms with alpha- and gamma-endorphin immunoreactivity detected during gel filtration are human lipotropin-(1-74) and -(1-75), respectively. The data substantiate that alpha-endorphin and gamma-endorphin exist as endogenous peptides in the human pituitary gland. 相似文献
8.
ACTH-induced inhibition of endogenous rat brain protein phosphorylation in vitro: Structure activity
Henk Zwiers Victor Marius Wiegant Peter Schotman Willem Hendrik Gispen 《Neurochemical research》1978,3(4):455-463
ACTH1–24 inhibits the endogenous phosphorylation in vitro of distinct SPM protein bands. Using N-terminal fragments of ACTH, the structure-activity requirements for this effect were studied. A rather complex interaction of the ACTH fragments with endogenous SPM phosphorylation was observed. The effects were not only dependent on the primary structure of the peptide used, but also on the protein band studied and the ATP/SPM ratio used in the incubation system. ACTH1–24 did not interfere with the ATP-hydrolyzing activity of the SPM preparation, nor did it influence the endogenous phosphatase activity. Therefore, a direct interaction of ACTH with SPM protein kinase(s) is likely to be responsible for its effect on phosphorylation. 相似文献
9.
Fred A. Opmeer J. Peter H. Burbach Victor M. Wiegant Jan M. van Ree 《Life sciences》1982,31(4):323-328
The influence of chronic morphine exposure on the biotransformation of β-endorphin (βE) was investigated using the myenteric plexus-longitudinal muscle of guinea-pig ileum. A membrane preparation was incubated with βE and the degradation of βE as well as the accumulation of several βE fragments in the incubation medium were followed with time. The levels of peptides were determined by specific radioimmunoassays after separation by high-pressure liquid chromatography. It was found that exposure to morphine did not affect the disappearance of βE, but altered the time course of accumulation of βE fragments. In fact, the accumulation of γ-endorphin, α-endorphin and des-tyrosine1-α-endorphin was enhanced, while that of des-tyrosine1-γ-endorphin was not changed. Additionally, the disappearance of γ-endorphin appeared to be stimulated by morphine exposure. These data provide evidence that the fragmentation of βE is changed by chronic morphine exposure in such a way that the turn-over of γ-endorphin is increased. 相似文献
10.
In vitro stimulation of alkaline phosphatase activity in immature embryonic chick pelvic cartilage by adenosine 下载免费PDF全文
Cyclic AMP content in embryonic chick pelvic cartilage increases significantly as the embryo ages from 8 to 10 d. This in ovo elevation in cyclic AMP content precedes maximal cartilage alkaline phosphatase activity by some 24 h. We studied whether this temporal relationship may be causally related, using an in vitro organ culture. Incubation of pelvic cartilage from 9- and 10-d embryos in medium containing monobutyryl cyclic AMP (BtcAMP) resulted in significant increases in alkaline phosphatase activity (220 and 66 percent, respectively) as compared to that of cartilages incubated in medium alone. This stimulation was both concentration- and time-dependent with maximal response at 0.5 mM BtcAMP and 4-h incubation, respectively. Similar incubations of cartilage in medium containing 1-methyl-3-isobutyl xanthine (MIX), 0.25 mM, also resulted in increased alkaline phosphatase activity (114 percent). However, pelvic cartilage from 11-d embryos incubated in medium containing BtcAMP or MIX showed no increase in alkaline phosphatase activity. We postulated that developmental age was the factor responsible for this difference in response and that immature cartilage (that with little or no alkaline phosphatase activity) would respond to BtcAMP whereas mature cartilage (that with significant alkaline phosphatase activity) would not. This was tested by incubating end sections of 11-d cartilage, which have little alkaline phosphatase activity, and center sections, which have significantly alkaline phosphatase activity, with both BtcAMP and MIX. Alkaline phosphatase activity in end sections (immature cartilage) was stimulated by BtcAMP and MIX, whereas it was not stimulated in the center sections. Actinomycin D and cycloheximide inhibited BtcAMP and MIX stimulation of alkaline phosphatase activity. Thus, the in vitro data suggest that cyclic AMP is a mediator for the stimulation of alkaline phosphatase activity in embryonic cartilage. 相似文献