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1.
2.
Isolation and characterization of different molecular and chromatographic forms of heparin-binding growth factor 1 from bovine brain 总被引:4,自引:0,他引:4
Bovine brain heparin-binding growth factor 1 (HBGF-1), a single polypeptide (Mr 17,400) with an amino-terminal acetylalanine and three cysteines within the sequence, isolates in multiple truncated and chromatographic forms. The relative yields of the various forms of HBGF-1 depend upon the methods used for purification. Extraction of brain tissue at neutral pH in the presence of protease inhibitors yielded intact acetylala-HBGF-1 and Asn21-HBGF-1 in a ratio of 2.3 to 1. Omission of the protease inhibitors during extraction markedly reduced the yield of acetylala-HBGF-1 and generated predominantly a mixture of Asn21-HBGF-1 and Phe15-HBGF-1. Acetylala-HBGF-1 and Asn21-HBGF-1 can be separated by cation-exchange chromatography prior to further purification. Isolated acetylala-HBGF-1 and Asn21-HBGF-1 distributed into three chromatographic peaks each on reverse-phase high-performance chromatography. Reduction of samples with dithiothreitol prior to reverse-phase chromatography reduced the three peaks of each molecular species into a single peak. Exposure of a single chromatographic peak of HBGF-1 to pH 8 in the absence of a reducing agent generated two or more additional chromatographic peaks upon subsequent chromatography. Although each chromatographic form of different molecular species of HBGF-1 exhibited potent mitogenic activity, reduction of HBGF-1 forms prior to reverse-phase chromatography appeared to increase the specific mitogenic activity of both purified molecular forms. 相似文献
3.
Xiao-yan Ding Wallace L. McKeehan Jianming Xu Horst Grunz 《Development genes and evolution》1992,201(6):334-339
Summary Mesoderm formation is a result of cell-cell interactions between the vegetal and animal hemisphere and is thought to be mediated by inducing peptide growth factors including members of the FGF and TGF superfamilies. Our immunochemical study analyses the distribution of FGF receptors coded by the human flg gene during embryogenesis of Xenopus laevis. Immunostaining was detected in the dorsal and ventral ectoderm and also in the marginal zone of early cleavage, blastula and gastrula stages. Signals were very strong in the mid and late blastula (stage 8 and 9) and declined slightly in the early gastrula (stage 10). A dramatic decrease was observed up to the late gastrula (stage 11+). In stage 13 embryos, immunostaining was only found in cells around the blastopore. Isolated ectoderm cultured in vitro showed a similar temporal expression and decrease of the signal as the normal embryos. These results indicate that receptor expression is independent of the interaction of the animal cells with the vegetal part of the embryo. Of interest is the fact that the signal cannot only be found at or near the cell surface but also within the cell. This suggests the presence of an intracellular isoform of the receptor resulting from the endogenous expression of splice variants and the internalization of transmembrane receptor. Taken together our results suggest that the loss of competence (for bFGF around stage 10) is not directly correlated with the presence of receptors. The possible roles of heparan sulphate glucosaminoglycans (low affinity receptors) and control mechanisms in the intracellular signalling pathway downstream of the receptor level should be taken into consideration. 相似文献
4.
Hiroyoshi Hoshi Mikio Kan Wallace L. McKeehan 《In vitro cellular & developmental biology. Plant》1987,23(10):723-732
Summary Hepatocytes were isolated from human fetal liver in order to analyze the direct effects of growth factors and hormones on
human hepatocyte proliferation and function. Mechanical fragmentation and then dissociation of fetal liver tissue with a collagenase/dispase
mixture resulted in high yield and viability of hepatocytes. Hepatocytes were selected in arginine-free, ornithine-supplemented
medium and defined by morphology, albumin production and ornithine uptake into cellular protein. A screen of over twenty growth
factors, hormones, mitogenic agents and crude organ and cell extracts for effect on the stimulation of hepatocyte growth revealed
that EGF, insulin, dexamethasone, and factors concentrated in bovine neural extract and hepatoma cell-conditioned medium supported
attachment, maintenance and growth of hepatocytes on a collagen-coated substratum. The population of cells selected and defined
as differentiated hepatocytes had a proliferative potential of about 4 cumulative population doublings. EGF and insulin synergistically
stimulated DNA synthesis in the absence of other hormones and growth factors. Although neural extracts enhanced hepatocyte
number, no effect on DNA synthesis of neural extracts or purified heparin-binding growth factors from neural extracts could
be demonstrated in the absence or presence of defined hormones, hepatoma-conditioned medium or serum. Hepatoma cell-conditioned
medium had the largest impact on both hepatocyte cell number and DNA synthesis under all conditions. Dialyzed serum protein
(1 mg/ml) at 10 times higher protein concentration had a similar effect to hepatoma cell-conditioned medium (100 μg/ml). The
results suggest that hepatoma cell conditioned medium may be a concentrated and less complicated source than serum for purification
and characterization of additional normal hepatocyte growth factors.
This work was supported by NIH grant DK35310.
Editor’s statement Many investigators have struggled with the special problems associated with culture of differentiated hepatocytes.
In this paper attention is given to the specific growth factor requirements for fetal human hepatocytes. The observation that
factors from hepatoma conditioned medium or neural extracts enhanced the growth of the cells may indicate that additional
growth factors are to be identified that are important in the survival and proliferation of hepatocytes, and may also indicate
that the malignant transformation of these cells may involve the production of autocrine growth stimulators. 相似文献
5.
Different hormonal requirements for androgen-independent growth of normal and tumor epithelial cells from rat prostate 总被引:4,自引:0,他引:4
Wallace L. McKeehan Pamela S. Adams Danna Fast 《In vitro cellular & developmental biology. Plant》1987,23(2):147-152
Summary The proliferation of isolated normal prostate epithelial cells from rat and man is androgen-independent and requires cholera
toxin, insulin, dexamethasone, epidermal growth factor (EGF) and one or more polypeptide factors that are concentrated in
bovine neural tissue. The active agents in the neural tissue extract are heparin-binding polypeptides (prostatropins), the
predominant form of which has a molecular weight of 17400 and an acetylalanine at the aminoterminus. Prostatropins supported
a half-maximal increase in normal prostate epithelial cell number at 50 picomolar. The proliferation of primary and serially-cultured
epithelial cells from androgen-responsive Dunning R3327 rat prostate tumors was also androgen-independent, but exhibited dramatic
alterations in response to hormones that stimulated normal cell proliferation. At low cell density, androgen-independent growth
of isolated tumor-derived epithelial cells was independent on cholera toxin, was stimulated by dexamethasone, required insulin
andeither EGFor prostatropin. The presence of either EGF or prostatropin masked the response to the other factor. In the absence of EGF,
purified prostatropins supported a half-maximal increase in tumor cell number at 7 picomolar. Endogenous production of EGF-like
and prostatropin-like factors or both was suggested by the reduced requirement for EGF and prostatropin at high prostate tumor
cell density. These results suggest that anti-hormonal therapies against prostate tumor growth should be based on intervention
with the activity of insulin (or insulin-like factors) or simultaneous intervention with both EGF and prostatropin (or their
homologues).
This work was supported by NIH grants CA 37589 and HL 33847, and grant 1718 from the Council for Tobacco Research.
Editor’s Statement This paper is the first report of the comparison of the hormone requirements of primary cultures of normal
and tumor prostate epithelial cells from the same system. 相似文献
6.
7.
8.
Yuhsi Matuq Pamela S. Adams Nozomu Nishi Hidetaro Yasumitsu John W. Crabb Robert J. Matusik Wallace L. McKeehan 《In vitro cellular & developmental biology. Plant》1989,25(6):581-584
Summary Rat prostate extracts contain an abundant 20–22 kilodalton heparin-binding protein with near identical chromatographic properties,
but only 0.2–1% of the mitogenic activity, of bovine brain heparin-binding growth factor-1 (acidic fibroblast growth factor).
Amino terminal amino acid sequence (met-met-thr-asp-lys-asn-leu-lys-lys-lys-ile-glu-gly-asn-trp-arg-thr-val-tyr-leu-ala-ala-ser-?-val-glu-lys-ile-asn-glu-gly-ser-pro)
and immunochemical analysis revealed that the protein is identical to the androgen-dependent protein “probasin”.
This work was supported in part by NCI grant CA37589 (W. L. M., J. W. C.) and the Medical Research Council of Canada (R. J.
M.). 相似文献
9.
Jinzhao Hou Fen Wang Wallace L. McKeehan 《In vitro cellular & developmental biology. Animal》1994,30(2):111-114
Summary The human hepatoblastoma cell line, HepG2, exhibits an array of stable properties in culture that have made it a popular cell
culture model for studies on regulation of liver-specific gene expression and properties of hepatoma cells. In contrast to
other hepatoma cell lines, HepG2 cells overexpress a characteristic detergent-extractable, wheat germ lectin-binding protein
with apparent molecular mass of 130 kDa. Using an antibody to screen a phage expression library of HepG2 complementary DNA
(cDNA), we identified and cloned a 4734 base pair cDNA which codes for a 130-kDa leucine-rich protein (lrp130) when expressed
in transfected cells. The deduced sequence of lrp130 exhibits sequences weakly homologous to the consensus sequence for the
ATP binding site in ATP-dependent kinases and the protein kinase C phosphorylation site of the epidermal growth factor receptor.
Consistent with the higher levels of expression of lrp130 antigen, Northern hybridization analysis indicated that HepG2 cells
express high levels of the major 4.8 kilobase lrp130 mRNA relative to other hepatoma cells. Although currently of unknown
function, lrp130 may be of utility as a marker for liver cell lineages represented by the HepG2 cell line. 相似文献
10.