James C. Bartholomew

Feeding acetyl-l-carnitine and lipoic acid to old rats significantly improves metabolic function while decreasing oxidative stress

Mitochondrial-supported bioenergetics decline and oxidative stress increases during aging. To address whether the dietary addition of acetyl-l-carnitine [ALCAR, 1.5% (wt/vol) in the drinking water] and/or (R)-α-lipoic acid [LA, 0.5% (wt/wt) in the chow] improved these endpoints, young (2–4 mo) and old (24–28 mo) F344 rats were supplemented for up to 1 mo before death and hepatocyte isolation. ALCAR+LA partially reversed the age-related decline in average mitochondrial membrane potential and significantly increased (P = 0.02) hepatocellular O2 consumption, indicating that mitochondrial-supported cellular metabolism was markedly improved by this feeding regimen. ALCAR+LA also increased ambulatory activity in both young and old rats; moreover, the improvement was significantly greater (P = 0.03) in old versus young animals and also greater when compared with old rats fed ALCAR or LA alone. To determine whether ALCAR+LA also affected indices of oxidative stress, ascorbic acid and markers of lipid peroxidation (malondialdehyde) were monitored. The hepatocellular ascorbate level markedly declined with age (P = 0.003) but was restored to the level seen in young rats when ALCAR+LA was given. The level of malondialdehyde, which was significantly higher (P = 0.0001) in old versus young rats, also declined after ALCAR+LA supplementation and was not significantly different from that of young unsupplemented rats. Feeding ALCAR in combination with LA increased metabolism and lowered oxidative stress more than either compound alone.

Regulation of CYP1A1 by indolo [3, 2-b] carbazole in murine hepatoma cells

To determine the basis for unexpected differences in CYP1A1 inducing potencies and efficacies for the diet-derived indole derivative, indolo[3,2-b]carbazole (ICZ) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), we conducted a systematic analysis of events involved in the induced expression of CYP1A1 in murine hepatoma-derived cell lines (Hepa-1). In contrast to the effects of TCDD, induction kinetics and CYP1A1 mRNA half-life were dependent on ICZ concentration, and the response from low doses of inducer was transient due to rapid clearance of ICZ. TCDD and ICZ produced the same maximum response (i.e. equal efficacies) from a TCDD-responsive CAT reporter construct in Hepa-1 cells. When measured by the immediate responses associated with CYP1A1 expression, including cellular uptake of inducer, receptor transformation and binding to DRE (gel mobility shift assay), initiation of transcription (nuclear run-on assay), and short-term accumulation of mRNA (Northern blot assay), ICZ also exhibited an efficacy equal to that of TCDD and a potency that corresponds to its receptor affinity. ICZ is a potent and selective noncompetitive inhibitor of ethoxyresorufin O-deethylase activity (K = 1.5 nM). Taken together these results indicate that ICZ is a bifunctional modulator of CYP1A1 expression with intrinsic efficacy equal to that of TCDD.

Characterization and Separation of Plant Protoplasts Via Flow Cytometry and Cell Sorting

Summary Large populations of protoplasts from five species: Euphorbia lathyris, Nicotiana glauca, N. langsdorfii, Petunia parodii and P. inflata (albino) have been characterized by flow cytometry on the basis of laser light scatter and chlorophyll fluorescence or exogenously-added stains, fluorescein isothiocyanate (FITC) and rhodamine isothiocyanate (RITC). Stain concentrations were 2.2 to 3.6 μl FITC and 7.2 μl RITC stock solutions per ml enzyme-protoplast solution using 5 mg FITC or RITC per ml absolute ethanol or acetone. Optimum conditions for FITC and RITC staining was found to be at pH 7.5 or greater and with acetone as the stain solvent Stained E. lathyris mesophyll protoplasts produced callus and regenerated shoots, indicating non-toxicity of the fluorochromes. E. lathyris protoplasts fused from two differentially-stained populations produced unique histograms when compared to mixed, but unfused populations. Further analysis and sorting of mesophyll protoplasts from these species was done with a Becton Dickinson Fluorescence-Activated Cell Sorter (FACS IV). Euphorbia and Petunia protoplasts were sorted according to various combinations of parameters: light scatter and fluorescence from chlorophyll, FITC, RITC or both stains. Up to 2000 intact protoplasts were sorted and recovered within 1 h. Sorting can be done under sterile conditions to allow culturing of the collected protoplasts.

Leucine in cultured cells: Its metabolism and use as a marker for protein turnover

Abstract The metabolism of leucine by normal and virally transformed mouse fibroblasts in culture has been investigated. Cultures pulsed with [U-14C]leucine were studied for the distribution of radioactivity in the free amino acid pool, cellular protein, lipid and CO2. Less than 1% of the label taken up was found in lipid and no radioactivity was recovered as CO2 when cultures were pulsed in growth medium. That these cells retain the capacity to oxidize leucine was shown by experiments in which cultures pulsed in a balanced salt solution (Krebs-Ringer bicarbonate) showed linear evolution of CO2. Leucine proved to be a desirable marker for protein turnover studies due to its minimal metabolism when labeling was performed in growth medium. The appearance of radioactive leucine in the medium of cells previously labeled with [14C]leucine was shown to be directly correlated with loss of radioactivity in cellular protein. A class of rapidly degrading proteins and a second group of proteins with a slower rate of turnover could be distinguished when cells were labeled with [14C] and [3H]leucine for varying lengths of time. Density inhibited cultures degraded both these classes of proteins about twice as rapidly as did growing cells.

The use of fluorescence correlation spectroscopy to probe chromatin in the cell nucleus

All systems in thermodynamic equilibrium are subject to spontaneous fluctuations from equilibrium. For very small system, the fluctuations can be made apparent, and can be used to study the behavior of the system without introducing any external perturbations. The mean squared amplitude of these fluctuations contains information about the absolute size of the system. The characteristic time of the fluctuation autocorrelation function contains kinetic information. In the experiments reported here, these concepts are applied to the binding equilibrium between ethidium bromide and DNA, a system where the fluorescence properties of the dye greatly enhance the effect of spontaneous fluctuations in the binding equilibrium. Preliminary experiments employ well-characterized DNA preparations, including calif thymus DNa, SV40 DNA, and calf thymus nucleohistone particles. Additional measurements are described which have been made in small regions of individual nuclei, isolated from green monkey kidney cells, observing as few as 5000 dye molecules. The data indicate that the strength of dye binding increases in nuclei isolated from cells which have been stimulated to enter the cell growth cycle. The viscosity of nuclear material is inferred to be between one and two orders of magnitude greater than that of water, and it decreases as the cells leave the resting state and enter the cell growth cycle. Washing the nuclei also lowers the viscosity. These experiments demonstrate that fluorescence correlation spectroscopy can provide information at the subnuclear level that is otherwise unavailable.

Analysis of Expression of Human Mammary Epithelial Antigens in Normal and Malignant Breast Cells at the Single Cell Level by Flow Cytofluorimetry

The expression of cell-type specific antigens on the surface of human mammary epithelial cells (HME antigens) is analyzed at the single cell level by flow cytofluorimetry. Methods are described to deal with two technical difficulties that have hampered the quantitation of surface antigens on cells from primary cultures by flow cytofluorimetry, namely, cell clumping and the presence of several cell types. By staining the cells simultaneously with two different fluorescent markers: the cell surface HME antigens with fluorescein-tagged antibodies (green fluorescence) and the cell DNA with propidium iodide (red fluorescence), it was possible to determine the expression of HME antigens per unit DNA, analyze their expression on breast cells in mixed populations, and analyze their expression in different phases of the cell cycle. The HME antigens were diminished in malignant breast epithelial cells as compared to their normal counterpart.

Fatty acid requirements and temperature dependence of monooxygenase activity in rat liver microsomes

The effect of variation in the microsomal membrane fatty acid composition on Arrhenius plot phase transition temperatures for p-nitroanisole O-demethylation and benzo[a]pyrene hydroxylation has been investigated. In liver microsomes from normal-dieted rats, p-nitroanisole O-demethylase activity has a break temperature at 24 degrees C, while that of benzo[a]pyrene hydroxylase occurs at 29 degrees C indicating that these two enzymes may exist in different patches of membrane. The microsomal membrane fatty acid composition was altered by starving rats for 48 h and then refeeding them a fat-free diet for 4 or 5 days. In microsomes having diet-altered fatty acid compositions, benzo[a]pyrene hydroxylase has a break temperature at 33 degrees C, a value higher than that observed in normal-dieted rats. This observation correlates with the increase in saturation observed in the diet-altered fatty acid composition and thus may correspond to a phase transition roughly dependent on the fatty acid melting point. Induced and basal levels of cytochrome P-450 and P-448 in animals having different microsomal fatty acid composition are reported. Phenobarbital-induced levels of p-nitroanisole O-demethylase in normal microsomes were six times higher than those in microsomes having diet-altered composition, whereas 3-methylcholanthrene-induced levels of benzo[a]pyrene hydroxylase were similar regardless of diet. The low level of p-nitroanisole O-demethylase activity in membranes with altered fatty acid compositions suggests that a particular type(s) of fatty acid was not present in sufficient quantity to permit the induction of maximal enzyme activity. Since the induced benzo[a]pyrene hydroxylase activity was the same regardless of diet, there was presumably sufficient quantities of the appropriate fatty acids present in the membrane for induction of this activity. Therefore, particular fatty acids may be necessary for the induction of maximal activity of particular enzymes in the mixed function monooxygenase system.

The importance of G0 in the site of action of interferon in the cell cycle

Abstract It has been suggested that the ‘metabolic event’ controlling the transition of quiescent cells into the proliferating phase is a possible target for the growth inhibitory action of interferon [1]. Direct evidence of this mechanism of action has been hindered by the lack of techniques for the quantification of quiescent cells in a tumor population. The present report applies a recently available technique [2] to investigate more closely the site of interferon action in the G1 part of the cell cycle, and to emphasize that a G0 population is a necessary but not sufficient determinant of a cell lines response to growth inhibition by interferon.

Aryl hydrocarbon hydroxylase induction in mouse liver cells--relationship to position in the cell cycle.

The inducibility of aryl hydrocarbon hydroxylase (AHH) by benzo[a]-pyrene (BaP) has been studied in synchronously grown cultures of mouse liver cells. These cells (NMuLi cl 8) have low basal levels of AHH which can be induced greater than 100-fold by BaP. Cells were synchronized in G1(G0) by serum starvation and in S by release from serum starvation in combination with excess thymidine. When released from G1(G0) by replating at lower cell density in fresh medium with 20% serum, cells began entering S with a lag of 12 h. Addition of BaP (1 microgram/ml) 8 h before serum stimulation, at the time of stimulation or 7.5 h after stimulation all gave similar induction kinetics: the AHH activity peaked as the cells began entering S regardless of when the BaP was added. Cells blocked in various parts of S by excess thymidine were inducible for AHH activity as efficiently as cells moving through S and into G2. These results indicate that the inducibility of AHH is greater when cells are actively proliferating and may be a contributing factor to why growing cells are more sensitive to mutagenesis and transformation than quiescent cells.

Biochemical basis for the acquisition of resistance to benzo[a]pyrene in clones of mouse liver cells in culture☆

In a Namru mouse liver epithelial cell strain designated NMuLi, aryl hydrocarbon hydroxylase (AHH) activity peaked at 12 h post-induction with 1 microgram/ml of benzo(a)pyrene (BaP) in both confluent and growing cells. Maximal levels of AHH activity were reached on day two post-plating. This induced activity was inhibited in vitro 78% by gassing the incubation mixture with carbon monoxide for 15 s, and inhibited 93% by addition of 40 microgram/ml of 7,8 benzoflavone(BF). Induced AHH levels were higher in epithelial clones that were sensitive to the toxicity of BaP than in resistant clones. The survival fraction of clones from NMuLi and of subclones derived from a sensitive clone of NMuLi after BaP treatment was a negative exponential function of the maximal induced AHH activity in the clones. One of the clones, NMuLi cl 8, was extremely susceptible to the toxic effects of BaP, the +/-(trans)-7alpha, 8beta-dihydroxy-7,8-dihydro-BaP(7,8-diol), and the (+/-)-7alpha, 8beta-dihydroxy-9beta, 10beta-epoxy-7,8,9,10-tetrahydro-BaP (diol-epoxide), known metabolites of BaP. The toxicity of BaP and the 7,8 diol to this clone was inhibited by BF, suggesting that these cells possessed an enzyme activity inhibitable by BF that could epoxidize BaP to the 7,8 oxide and then epoxidize the resultant 7,8 diol to the diol-epoxide. Another clone derived from NMuLi, clone 7, was relatively resistant to the toxic effects of BaP and the 7,8-diol, but still extremely susceptible to the toxic effects of the diol-epoxide. The slight toxicity to BaP in this clone was inhibited by BF, but the toxicity of the 7,8-diol to this clone was not inhibited by BF. A typical cytochrome P450 inhibitor, metyrapone, had no effect on the toxicity of BaP, the 7,8-diol, or the diol-epoxide to either clone 7 or clone 8. The results suggest that these liver cells possess two enzymes that play some role in polycyclic hydrocarbon-induced toxicity. Enzyme A, a BaP-inducible enzyme that is inhibitable by BF, efficiently metabolizes BaP to the 7,8-diol and the 7,8-diol to the diol-epoxide. It is responsible for most of the hydrocarbon toxicity. Enzyme B is not inhibitable by BF and metabolizes the 7,8-diol less efficiently to the diol-epoxide or efficiently to other, less toxic products.