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1.
Structure-function analysis of epidermal growth factor: site directed mutagenesis and nuclear magnetic resonance 总被引:2,自引:0,他引:2
The role of leucine-47 in determining the structure and activity of human epidermal growth factor was examined using site-directed mutagenesis. Wild type protein and four variants in which Leu47 was replaced by valine, glutamate, aspartate and alanine were produced from yeast. 1H NMR experiments demonstrated that substitution of Leu47 had little effect on the protein structure. The observed reduction in receptor binding affinity caused by the substitutions could thus be attributed to perturbation of a residue directly involved in receptor interactions. 相似文献
2.
Analysis of liver/bone/kidney alkaline phosphatase mRNA, DNA, and enzymatic activity in cultured skin fibroblasts from 14 unrelated patients with severe hypophosphatasia. 总被引:5,自引:2,他引:3
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M J Weiss K Ray M D Fallon M P Whyte K N Fedde M A Lafferty R A Mulivor H Harris 《American journal of human genetics》1989,44(5):686-694
Hypophosphatasia is a heritable disorder characterized by defective bone mineralization and a deficiency of liver/bone/kidney alkaline phosphatase (L/B/K ALP) activity in serum and tissues. Severe forms of the disease, which are generally lethal in infancy, are inherited in an autosomal recessive fashion. The gene defects that produce hypophosphatasia are poorly understood, but many are likely to occur at the L/B/K ALP locus. To investigate these gene defects, we analyzed L/B/K ALP DNA, RNA, and enzyme activity in cultured dermal fibroblasts from 14 patients with perinatal or infantile hypophosphatasia and from 12 normal individuals. Southern blot analyses of the L/B/K ALP genes from patients and controls revealed identical restriction patterns. Control fibroblast ALP activity correlated with the corresponding L/B/K ALP mRNA levels estimated by blot hybridization analysis and densitometry (r = .94, P less than .0001). In contrast, fibroblasts from the hypophosphatasia patients were deficient in ALP enzyme activity but expressed apparently full-sized L/B/K ALP mRNA at normal levels. Bone specimens from one of the patients were examined and found to be deficient in histochemical ALP but contained immunologic cross-reactive material detected by anti-human liver ALP antiserum. Our results demonstrate that the deficiency of ALP activity in fibroblasts from 14 patients with severe hypophosphatasia is not due to decreased steady-state levels of the corresponding mRNA. The presence of enzymatically inactive L/B/K ALP protein in one of these patients is consistent with a point mutation or small in-frame deletion in the coding region of L/B/K ALP gene. 相似文献
3.
Asialoglycoprotein receptor phosphorylation and receptor-mediated endocytosis in hepatoma cells. Effect of phorbol esters 总被引:6,自引:0,他引:6
The asialoglycoprotein (ASGP) receptor on Hep G2 cells undergoes constitutive recycling and ligand endocytosis in the presence of phorbol dibutyrate, at a 50% reduced rate relative to control cells (Fallon, R. J., and Schwartz, A. L. (1986) J. Biol. Chem. 261, 15081-15089). The relevance of receptor phosphorylation to these events was investigated by selective immunoprecipitation of surface receptors with polyclonal anti-human ASGP antiserum and pulse-chase labeling with [32P]orthophosphate to identify subcellular locations of initial receptor phosphorylation events as well as the eventual fate of phosphorylated receptor during recycling. The surface immunoprecipitation method recovers greater than 95% of surface ASGP receptors and only 5% or less of intracellular (brief[35S]methionine pulse-labeled) receptors. With this assay we detected low levels of ASGP receptor phosphorylation at the cell surface in control cells (0.1 mol of P/mol of R) which were rapidly (less than 1 min) stimulated 20-fold by 400 nM phorbol dibutyrate addition (1.7 mol of P/mol of R). Staurosporine, a protein kinase C inhibitor, blocks this stimulation by phorbol. Receptor phosphorylation at early time points in the presence of phorbol esters was restricted to the plasma membrane. Subsequent chase in the presence of excess unlabeled phosphate and phorbol esters lowered [32P] ATPi specific activity by 68% at 1 h. Surface immunoprecipitation during this chase period showed the phosphorylated ASGP receptors were rapidly lost from the cell surface (t1/2 = 20 min). In contrast, examination of intracellular receptor during the pulse-chase experiment in phorbol dibutyrate-treated cells showed the presence of phosphorylated pool(s) of ASGP receptors which were detectable for 6 h of chase. Since no labeled receptor can be detected at the cell surface at this time, the described intracellular phosphorylated receptors are in a non-recycling pool. 相似文献
4.
Initial limb budding is independent of apical ectodermal ridge activity; evidence from a limbless mutant 总被引:1,自引:0,他引:1
Outgrowth of normal chick limb bud mesoderm is dependent on the presence of a specialized epithelium called the apical ectodermal ridge. This ectodermal ridge is induced by the mesoderm at about the time of limb bud formation. The limbless mutation in the chick affects apical ectodermal ridge formation in the limb buds of homozygotes. The initial formation of the limb bud appears to be unaffected by the mutation but no ridge develops and further outgrowth, which is normally dependent on the ridge, does not take place. As a result, limbless chicks develop without limbs. In the present study, which utilized a pre-limb-bud recombinant technique, limbless mesoderm induced an apical ectodermal ridge in grafted normal flank ectoderm. However, at stages when normal flank ectoderm is capable of responding to ridge induction, limbless flank ectoderm did not form a ridge or promote outgrowth of a limb in response to normal presumptive wing bud mesoderm. We conclude from this that the limbless mutation affects the ability of the ectoderm to form a ridge. In addition, because the limbless ectoderm has no morphological ridge and no apparent ridge activity (i.e. it does not stabilize limb elements in stage-18 limb bud mesoderm), the limbless mutant demonstrates that the initial formation of the limb bud is independent of apical ectodermal ridge activity. 相似文献
5.
6.
In this experimental study, venous end-to-end and end-to-side microvascular anastomoses in similar and diameter-discrepant vessels were compared. In 50 rats, end-to-end microvascular repair of the divided epigastric vein and end-to-side repair of the epigastric vein into the femoral vein showed 5-day patency rates of 75 and 88 percent, respectively. These data are not statistically different. In 20 rats, microvascular repair of end epigastric to end femoral veins (size discrepant) and end epigastric to side femoral veins showed 5-day patency rates of 50 and 85 percent, respectively. These data are statistically different (p less than 0.05). We conclude from these experimental data that end-to-side venous repairs may be useful in lowering the anastomosis thrombosis rate seen when size-discrepant veins are repaired. 相似文献
7.
This paper describes a combined technique for gross skeletal staining and Feulgen staining of avian embryonic limbs. The gross skeletal stain uses Victoria blue B, and the Feulgen stain is done en bloc before the skeletal stain is applied. The method has been useful in determining the cellular origins of supernumerary structures arising from experiments in which quail wing mesoderm is grafted into chick wing buds. 相似文献
8.
An Aedes albopictus dihydrofolate reductase gene was used to construct two chimeric DNA vectors that functioned as dominant selectable markers in transfected, wild type mosquito cells. Stably transformed clones were recovered after 10–15 days in the presence of selective medium containing 1 μM methotrexate. The transformed clones contained an estimated 100–500 copies of transfected DNA per nucleus. Combined data from Southern blots and in situ hybridization to metaphase chromosomes indicated that transfected DNA was likely integrated into chromosomes both as repeated structures and as randomly integrated single copy molecules, with minimal rearrangement of coding sequences. Transfected DNA was stably maintained under selective conditions, but in some cases was lost when cells were maintained for prolonged periods in the absence of methotrexate. These observations provide a general framework for further development of stable gene transfer systems for mosquito cells in culture. 相似文献
9.
10.
To test for the presence of polarizing mesoderm in an amphibian, Xenopus laevis hindlimb bud tips were rotated 180° on the proximodistal axis and returned to the stump. Supernumerary outgrowths were induced in the preaxial stump and preaxial tip tissues, and the most postaxial digit always formed next to the grafted postaxial tissue. The occurrence of polarized supernumerary outgrowths indicated that the posterior limb border contained a polarizing zone. When the limb tip was cut at varying known lengths from the body wall, rotated, and grafted to the limb stump, the incidence of twinning along the proximodistal axis permitted insight into the distribution of the polarizing zone along the posterior border. The location of polarizing tissues was found to be similar to that in the chick wing bud at comparable stages. To confirm the posterior border stump influence on the rotated preaxial limb tip tissues, 180° tip rotations were made at the proximodistal level with the highest incidence of twinning. In these cases, the adjacent stump posterior border tissues (polarizing zone) were removed, leaving a substantial amount of the deeper postaxial stump tissue, however. The frequency of twinning from tip tissues was greatly reduced in these larvae compared to those with rotated limb tips on intact stumps. Cytological examination of supernumerary outgrowths resulting from grafts of two-nucleolate tips onto one-nucleolate stumps confirmed the preaxial source of the supernumerary outgrowths. 相似文献