Experimental cell research
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Fibroblasts derived from the papillary and reticular dermis of human skin and human keratinocytes show differences in their abilities to contract floating three-dimensional gels constructed from type I collagen. Reticular fibroblasts produce greater gel contraction than papillary fibroblasts. When equal numbers of papillary and reticular fibroblasts are mixed in the gels, papillary fibroblasts consistently inhibit gel contraction by reticular fibroblasts indicating interaction between these cell types in the contraction process. ⋯ In contrast, keratinocyte contraction of collagen gels occurs in a serum-free environment. Polyclonal, affinity-purified antibodies to human plasma fibronectin at high concentrations do not inhibit gel contraction by keratinocytes, making unlikely the possibility that fibronectin synthesized by the keratinocyte is a significant factor in the gel contraction process. We are currently examining the possibilities either that keratinocytes are synthesizing other adhesion proteins or that receptors on the cell surface can interact directly with the collagen fiber.
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Hydroxyurea-resistant S49 T-lymphoma cells have increased ribonucleotide reductase activity and deoxyribonucleoside triphosphate pools when compared with wild-type cultures. If ribonucleotide reductase inhibition is the mechanism by which deoxyadenosine is cytotoxic, then hydroxyurea (HU)-resistant S49 cells might be more resistant to deoxyadenosine toxicity when adenosine deaminase is inhibited than wild-type cells. Five S49 cell lines resistant to varying concentrations of HU were compared with wild-type cells by measuring CDP reductase activity, deoxyribonucleoside triphosphate pools, and deoxyadenosine toxicity. ⋯ The S49 cells arrested in G1 phase when exposed to dAdo, although hydroxyurea-resistant cells required higher dAdo concentrations to elicit G1-phase arrest than wild-type cells. Deoxycytidine prevented dAdo-induced G1 arrest in all cell types. In summary, these data support the hypothesis that deoxyadenosine-induced dATP accumulation results in inhibition of ribonucleotide reductase and that this may be the mechanism for both cell cycle arrest and cytotoxicity in S49 T-lymphoma cells.
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[14C]Lactose, introduced into the cytosol of isolated rat hepatocytes by means of electropermeabilization, is sequestered autophagically in the same way as the established sequestration probe, [14C]sucrose. However, unlike the inert sucrose molecule, lactose is rapidly hydrolysed in the lysosomes, and can therefore be used to probe the last step of the autophagic pathway (i.e. fusion with the lysosome). ⋯ If autophagosome-lysosome fusion is blocked with vinblastine (Kovács et al., Exp cell res 137 (1982) 191), [14C]lactose will accumulate continuously as a function of the sequestration rate, and reach a high level in the pre-lysosomal vacuoles. Density gradient analysis, using chloroquine (CLQ) to alter lysosomal density, suggests that these organelles have a broad density distribution (1.08-1.13 g/ml), thus differing significantly from the distribution of lysosomes.