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J R Wrathall

Publications and source records attributed to J R Wrathall.

58 records · Page 4Linked to original sources
PubMed1981

Cultures enriched in Schwann-like cells from dissociated nerve segments of the adult cat.

Methods for producing Schwann cell-rich cultures from nerve segments of the adult cat were investigated. Efficient dissociation was achieved with a combination of collagenase and trypsin. Differential attachment of elements within the resulting suspension allowed production of Schwann cell-rich cultures derived by outgrowth from slowly attaching segments of degenerating myelin sheath tube containing Schwann-like cells. Rapid trypsinization during subculture further increased the proportion of Schwann-like cells in these cultures to 90% or more.

Animals↗
PubMed1975

Suppression of melanoma cell tyrosinase activity and tumorigenicity after incorporation of bromouracil for one or two cell divisions.

We have studied the kinetics of suppression of tyrosinase activity and tumorigenicity in unsynchronized B16 mouse melanoma cells (clone B559) exposed to 5-bromodeoxyuridine (BrdU, 3 mug/ml) for one or two cell divisions, then cultured in BrdU-free medium (RM) for five or six days. Bromouracil replaced about 23% of thymine residues after 24 hours (1 cell division) and almost 40% after 48 hours (2 cell divisions) in the presence of BrdU. Upon subsequent growth in RM the extent of replacement declined in a manner consistent with dilution by new DNA synthesis, reaching 5-10% substitution by day 7 of these experiments. Tyrosinase activity was significantly reduced after treatment with BrdU for 24 or 48 hours but continued to decline after the cultures were changed to RM, approaching undetectable levels on day 7. The time course of reduction was similar to that previously determined in cells grown continuously for seven days in the presence of BrdU. Therefore, suppression of tyrosinase activity can result from incorporation of BrdU during a single cell cycle, but requires about seven days for full manifestation of the effect. Tumorigenicity decreased to 55% after 24 hours and to 15% after 48 hours with BrdU but rapidly reversed to approach that of untreated melanoma cells when subsequently grown in RM for 5-6 days. The effects of BrdU on total RNA or protein synthesis, or on plating efficiency appeared insufficient to account for the degree of suppression observed. Our results indicate that substitution by bromouracil into either strand of DNA loci controlling tyrosinase activity or tumorigenic potential may be sufficient for suppression. In addition, they demonstrate that such brief treatment with BrdU may be used to probe the regulation of differentiated function and tumorigenicity in these melanoma cells.

Animals↗
PubMed1973

Suppression of pigmentation in mouse melanoma cells by 5-bromodeoxyuridine: effects on tyrosinase activity and melanosome formation.

Low concentrations (1-3 microg/ml) of 5-bromodeoxyuridine (BrdU) reversibly suppress pigmentation in a highly pigmented clone (B(5)59) of cultured B16 mouse melanoma cells. We have found that unpigmented cells (clone C(3)471), derived by long-term culture of B(5)59 cells in 1 microg of BrdU/ml, were completely amelanotic with no biochemically or cytochemically detectable tyrosinase activity or ultrastructural evidence of premelanosomes. The process by which pigmentation is suppressed was studied in B(5)59 cells during a 7-day period of growth with BrdU (3 microg/ml). Assays of tyrosinase activity showed that activity was reduced after 1 day and decreased progressively, approaching zero by 7 days. A quantitatively minor part of this reduction was directly attributable to the appearance of a dialyzable inhibitor of tyrosinase activity. Acrylamide gel electrophoresis revealed two bands of activity corresponding in Rx values to the T(1) and T(2) forms of soluble tyrosinase. Both were progressively reduced during growth with BrdU but one form (T(1)) was consistently affected earlier than the other (T(2)). Ultrastructural-cytochemical studies also showed an early effect on the localization of tyrosinase reaction product. At day 3, reaction product was no longer present in Golgi saccules and Golgi-associated smooth surfaced tubules, but was still seen within premelanosomes, compound melanosomes, and occasional Golgi-associated vesicles. By 7 days tyrosinase reaction product was usually not demonstrable. The number of premelanosomes was progressively decreased during growth with BrdU. Premelanosomes became concentrated in the juxtanuclear region and at day 3 many were contained within abnormally large and numerous compound melanosomes. Premelanosomes and compound melanosomes were rarely seen at 7 days, by which time the cultures were nearly amelanotic. The coordinated suppression of melanogenesis by BrdU may provide a useful model in which to study the normal regulation of this process.

Animals↗