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Co-culture conditions are well established in which Schwann cells (SCs) derived from immature or adult rats proliferate and form myelin in response to contact with sensory axons. In a companion article, we report that populations of adult-derived human Schwann cells (HASCs) fail to function under these co-culture conditions. Furthermore, we report progressive atrophy of neurons in co-cultures containing populations of either human fibroblasts. Two factors that might account for the insufficiency of the co-culture system to support HASC differentiation are the failure of many HASCs to proliferate and the influence of contaminating fibroblasts. To minimize fibroblast contamination of neuron-HASC co-cultures, we used fluorescence-activated cell sorting to highly purify HASC populations (to more than 99.8%). To stimulate expansion of the HASC population, a mitogenic mixture of heregulin (HRGβ1 amino acid residues 177-244; 10 nM), cholera toxin (100 ng/mL), and forskolin (1 μM) was used. When these purified and expanded HASCs were co-cultured with embryo-derived rat sensory neurons, neuronal shrinkage did not occur and after 4 to 6 weeks some myelin segments were seen in living co-cultures. This myelin was positively identified as human by immunostaining with a monoclonal antibody specific to the human peripheral myelin protein P0 (antibody 592). Although this is the first reported observation of myelination by HASCs in tissue culture, it should be noted that myelination occurred more slowly and in much less abundance than in comparable cultures containing adult rat-derived SCs. We anticipate that further refinements of the HASC co-culture system that enhance myelin formation will provide insights into important aspects of human SC biology and provide new opportunities for studies of human peripheral neuropathies. © 1995 John Wiley & Sons, Inc. 相似文献
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An established keratinocyte line (XB), derived from a mouse teratoma, terminally differentiates in suspension culture in a manner similar to human epidermal keratinocytes. When surface-grown XB cells are placed in suspension culture, they lose colony forming ability very rapidly; within three days the loss is virtually complete. Measurement of the ability of the suspended cells to synthesize protein and RNA show that they begin to lose both after 12 hours, the rate of uridine and glycine incorporation falling nearly to zero in about 36 hours. The cells then become insoluble in ionic detergents, owing to the formation of disulfide-stabilized keratin filaments, and digest their nuclei. The total RNA content of the cells (a measure of ribosomes) begins to drop sharply about 12 hours after the cells are placed in suspension culture, and most RNA is eliminated by 24 hours. This process is independent of the presence of serum in the medium. DNA also begins to disappear from the cells, but this process is slower than ribosomal destruction and is strongly affected by the presence of serum. After seven days in the absence of serum, half the DNA still remains, and nearly all the nuclei are still visible, whereas during the same period in the presence of serum all visible nuclei and all DNA disappear. In contrast to the destructive process that takes place in the keratinocytes, 3T3 cells are much more stable in suspension culture. They show a reversible decline in their rate of amino acid incorporation, but no decline in their rate of uridine incorporation, and they undergo little loss in colony forming efficiency for several days. They retain most of their RNA and nuclei with full DNA content. The destructive process in suspended XB cells seems to be a model for the cell death that takes place in terminal differentiation of the keratinocyte. 相似文献
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Pseudomonas fluorescens strains are known to produce a wide range of secondary metabolites including phenazines, siderophores, pyoluteorin, and 2,4
diacetylphloroglucinol (DAPG). DAPG is of particular interest because of its antifungal properties and because its production
is associated with inhibition of phytopathogenic fungi in natural disease-suppressive soils. This trait has been exploited
to develop strains of P. fluorescens that have potential application as biocontrol agents. Although the biochemistry, genetics and regulation of DAPG production
have been well-studied, relatively little is known about how DAPG inhibits fungal growth and how fungi respond to DAPG. Employing
a yeast model and a combination of phenotypic assays, molecular genetics and molecular physiological probes, we established
that inhibition of fungal growth is caused by impairment of mitochondrial function. The effect of DAPG on yeast is largely
fungistatic but DAPG also induces the formation of petite cells. Expression of the multidrug export proteins Pdr5p and Snq2p
is increased by DAPG-treatment but this appears to be a secondary effect of mitochondrial damage as no role in enhancing DAPG-tolerance
was identified for either Pdr5p or Snq2p. 相似文献
440.
R L Morrissey D T Zolock D D Bikle R N Empson T J Bucci 《Biochimica et biophysica acta》1978,538(1):23-33
The dynamics of intestinal response in rachitic chicks to 1alpha,25-dihydroxycholecalciferol were evaluated by various biochemical parameters. The following observations were made: 1. The earliest detected intestinal response to 1alpha,25-dihydroxycholecalciferol was increased in vitro calcium uptake and in vivo calcium transport, occurring by 2 h and 2.5 h respectively. 2. Increased RNA polymerase activity was observed by 4 h after 1alpha,25-dihydroxycholecalciferol treatment. 3. Calcium binding protein was detected by 5 h, but could not be detected 2.5 h after 1alpha,25-dihydroxycholecalciferol treatment. 4. Increased alkaline phosphatase activity and in vitro accumulation of inorganic phosphate were first demonstrable 6 h after 1alpha,25-dihydroxycholecalciferol treatment. 5. In vivo duodenal calcium accumulation in the mucosa was elevated after 5 h, peaked at 6.5 h, and then began to decrease at 9 h. In vitro duodenal calcium accumulation was elevated at 2 h, peaked at 12 h, and decreased to control level by 18 h. Our data emphasize the lack of correlation between the appearance of calcium binding protein or increased alkaline phosphatase activity and the transport rate of calcium across the duodenum after treatment with 1alpha,25-dihydroxycholecalciferol. The data suggest a correlation between duodenal calcium accumulation and the appearance of calcium binding protein or increased alkaline phosphatase activity. 相似文献