Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:EXPT01586 (G418)
 2,237 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phosphorylase kinase is a multimeric enzyme of composition (alpha, beta, gamma, delta)4 whose catalytic activity resides in the gamma-subunit. As an approach to understand further its regulation, a cDNA for the gamma-subunit of phosphorylase kinase (gamma PhK) has been cloned into a mammalian expression vector behind the mouse metallothionein-1 promoter. NIH 3T3 cells were co-transfected with this construct (pEV gamma PhK) and pSV2neo, G418-resistant clones were selected, and several were found to have stably incorporated the gamma-subunit cDNA into their genomic DNA. Phosphorylase kinase activity was clearly present in extracts from cultures of pEV gamma PhK-transformed cells and increased several-fold after 24 h of incubation with Zn2+, whereas it was undetectable in the parent 3T3 cells. A significant, but variable, proportion (15-70%) of the activity was Ca2+-dependent. We conclude that the phosphorylase kinase activity expressed by the cells transformed with pEV gamma PhK is due to free gamma-subunit and gamma-subunit associated with cellular calmodulin, which replaces the delta-subunit normally associated with the gamma-subunit in the holoenzyme.
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PMID:Expression of a cDNA for the catalytic subunit of skeletal-muscle phosphorylase kinase in transfected 3T3 cells. 248 39

Human papillomaviruses (HPVs) are known as etiologic agents of various diseases in regions of the human epithelium. Specific type HPV16 is most frequently found in association with human squamous cell carcinoma. To examine biological activity of HPV type 16 in human cells, primary foreskin epidermal cells and dermal fibroblasts were transfected by recombinant viral DNA containing neo gene with Ca2+-phosphate precipitation. Epidermal cells were maintained in 0.5% Chelex-treated fetal calf serum, low calcium medium supplemented with bovine pituitary extracts and hormone mix. Fibroblasts were cultured in DMEM plus 10% fetal calf serum. The transfected cells were then selected with G418-resistant phenotype. These cells were propagated to maintain in culture and subsequently became stable lines carrying HPV16 genomes, while mock transfected control cells died off at approximately 40-50 population doublings (PD) in a parallel experiment. We have established two independently immortalized human epidermal cell lines (PHK16-I and II) which harbor different copies of HPV16 genome and express HPV16 specific mRNA. Although younger populations of PHK16 lines were fairly sensitive to high Ca2+-level to be differentiating keratinocytes, progressive changes of the cellular phenotype were demonstrated in terms of changes in Ca2+-response and anchorage independent growth during over 300 PD. Altered Ca2+-regulation of growth and differentiation appeared to be common reliable phenotype associated with stable transformation of skin epidermal cells. In contrast, none of the HPV16-transfected fibroblast line immortalized but simply showed extended life span up to 100 PD in average. The result suggested that this biological activity of HPV16 could be reflected in HPV-tropism related to epithelial transformation. We then studied correlations between HPV16 gene expression and the regulation of growth and differentiation of PHK lines during the progressive transformation. Northern blot analysis of RNA from cells in earlier passages demonstrated that down-regulation of HPV16 E6/E7 transcription was associated with keratinocyte differentiation induced by added 1.0mM calcium. The p97 promoter for HPV 16 early genes covering E6/E7 was specifically responsible for this Ca2+-regulation. The eventual loss of Ca2+-regulation could be implicated in a process of progressive transformation of HPV16-epidermal cell system.
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PMID:[HPV16 participates in progressive transformation of normal epidermal cells]. 253 84