Ross Shiman

Quantitative assay of melanin in melanoma cells in culture and in tumors

Abstract A simple method has been developed for the quantitative fluorimetric determination of melanin. In these studies, we have used synthetic melanin prepared from l -dopa by mushroom tyrosinase and natural melanin present in B16 mouse melanoma cells grown in culture or as a solid tumor. In the assay procedure, the melanin is oxidatively degraded into soluble products by heating in an alkaline, dilute hydrogen peroxide solution under standard conditions. The resulting solution has a characteristic fluorescence which is quantitatively related to the melanin initially present. The assay is very sensitive and can be applied directly to tissues or cell cultures, without prior isolation of the melanin.

Deoxythymidine Kinase from Rabbit Kidney Cells Infected with Herpes Simplex Virus Types 1 and 2

Deoxythymidine kinase (TdR kinase) from rabbit kidney cells infected in vitro with herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) was further investigated. Enzyme activity induced by HSV-2 was more heat-labile and more sensitive to inhibition by deoxythymidine nucleotides than HSV-1 induced enzyme activity in both crude extracts and partially purified preparations. The thermostability of TdR kinase activity from cells coinfected with HSV-1 and HSV-2 was intermediate between enzymes induced separately. The difference in thermostability between the two enzyme activities was also observed in extracts from 1-β-D-arabinofuranosylcytosine (ara-C) treated infected cells. HSV-1 and HSV-2 induced enzymes also differed in their response to substrate concentration. Gel filtration with Sephadex G-100 revealed the presence of monomer, dimer and aggregated forms of HSV-1 induced enzyme. The molecular weights of the monomer and dimer were estimated to be 58,000 and 97,000, respectively. Such multiple forms were not observed with HSV-2; the molecular weight of the HSV-2 induced enzyme was estimated to be 45,000 to 60,000, depending upon the eluting buffers. HSV-1 induced TdR kinase activity showed a different electrophoretic activity pattern in polyacrylamide gel electrophoresis than did the enzyme induced by HSV-2.

[76] Tyrosine hydroxylase (bovine adrenal glands)

Publisher Summary This chapter discusses the assay, purification, and properties of tyrosine hydroxylase. When tyrosine is hydroxylated, the 3-hydrogen is replaced by a hydroxyl group. If 3, 5-ditritio-L-tyrosine is used, the displaced tritium exchanges with the water, and 1 mole of THO is found per mole of dihydroxyphenylalanine formed. The specific activity of the tritiated water isolated from the reaction mixture gives a measure of the extent of the reaction. All purification procedures are carried out at 0–3°. One unit of tyrosine hydroxylase is the amount that catalyzes the formation of 1 micromole of product per minute under the standard assay conditions. Specific activity is expressed as units per milligram of protein. The enzyme is more stable in the presence of L-tyrosine, 0.3 M sucrose, or carboxylic-acid buffers. Hydrogen peroxide at low levels causes an irreversible loss in the activity of the enzyme. The pH optimum for the enzyme-catalyzed reaction is pH 6.2.

Correlation of phenylalanine hydroxylase activity with cell density in cultured hepatoma cells

Abstract We have found that the specific activity of phenylalanine hydroxylase varies over at least a forty-fold range during the growth cycle of Reuber hepatoma (H4) cells growing in monolayer culture. The variation has three phases: (1) a very rapid drop in specific activity upon subculturing to a low cell density; (2) a region of low specific activity and (3) after confluency, a rise to a high specific activity. All the results indicate that the cell density in the culture dish is primarily responsible for this fall and rise in activity. Neither conditioning of the growth medium, the rate of cell division, nor enzyme leakage from the cells appear to play a major role in the changes observed. Lactic dehydrogenase specific activity was determined in all experiments; a much smaller, but still cell density-dependent variation was observed for this enzyme.

Susceptibilities of 540 anaerobic gram-negative bacilli to amoxicillin, amoxicillin-BRL 42715, amoxicillin-clavulanate, temafloxacin, and clindamycin.

Agar dilution MIC testing of amoxicillin, amoxicillin-BRL 42715, amoxicillin-clavulanate, temafloxacin, and clindamycin against 496 beta-lactamase-producing anaerobic gram-negative rods revealed MICs for 90% of the strains tested of 256.0 (amoxicillin), 2.0 (amoxicillin-BRL 42715 and amoxicillin-clavulanate), and 4.0 (temafloxacin and clindamycin) microgram/ml. Amoxicillin, temafloxacin, and clindamycin inhibited all 44 beta-lactamase-negative strains (MICs for 90% of the strains tested, less than or equal to 2.0 micrograms/ml). BRL 42715 will not be developed, but temafloxacin merits clinical evaluation.

Cell density dependent regulation of phenylalanine hydroxylase activity in hepatoma cells: Evidence for both an active and inactive enzyme form☆

Abstract The cell density dependent regulation of phenylalanine hydroxylase activity in Reuber hepatoma (H4) cells growing in monolayer culture has been examined in detail. We found that 48 h or more after subculture phenylalanine hydroxylase activity in the cells is an exponential function of cell density (cells/cm2). No discontinuity in the relationship is seen with the formation of a confluent monolayer. A rapid loss or a rapid gain in enzyme activity in the cells is observed after diluting or concentrating the cell cultures. The two processes appear qualitatively different. The loss in activity is a first order process which starts at the time of subculture with the rate of loss dependent on the density of subculture. The gain in activity begins 6–8 h after subculture to a higher density; it can be blocked by cycloheximide and has a maximum rate of increase that is about 10% of the maximum rate of loss of activity. Using immunochemical procedures, we found the same amount of phenylalanine hydroxylase associated antigen in Reuber cells from low density as from high density cultures, over a range of phenylalanine hydroxylase specific activities from 0.2 to 4.2. After concentrating cells to a higher density, no increase in enzyme antigen was observed, despite a several-fold increase in enzyme activity and a requirement for protein synthesis during the process. These observations imply the presence of an active and inactive phenylalanine hydroxylase with the relative amounts of each determined by the cell density. The effects of db-cAMP are discussed. Evidence is presented here that the hydrocortisone stimulation of phenylalanine hydroxylase activity works through a different mechanism than the cell density dependent process.

Hydrocortisone induction of phenylalanine hydroxylase isozymes in cultured hepatoma cells

Abstract The hydrocortisone stimulation of phenylalanine hydroxylase activity in Reuber H4 hepatoma cells is shown to be associated with an alteration in phenylalanine hydroxylase isozyme composition. Three forms of phenylalanine hydroxylase were identified in H4 cells which have been treated with hydrocortisone; however, only one of these forms appears to be present prior to glucocorticoid treatment. The relative amounts, as well as the total amount, of the three forms and their chromatographic behavior on hydroxylapatite are nearly identical to the three phenylalanine hydroxylase isozymes found in adult rat liver. The hydroxylase isozyme composition in 2 day old rats is similar to that found in adult rats and in H4 cells treated with hydrocortisone.