Linear electrical properties of passive and active currents in spherical heart cell clusters.

Impedance studies were performed on small spherical clusters of embryonic chick heart cells grown in tissue culture. Each syncytial cluster was impaled with two microelectrodes; one injected low amplitude stochastic current and the other recorded the resulting perturbation of intracellular potential. The current and potential records were digitized, decomposed into their sinusoidal components, and the frequency domain impedance of the cluster was determined. The impedance data were compared with a theory for current flow in a spherical syncytium and values were derived for parameters describing the membranes and intercellular clefts of the tissue. The clusters were spontaneously active but usually became temporarily quiescent when impaled with two electrodes. The potential stabilized at a value close to -30 mV. At this depolarized potential, active slow currents, presumably present in the cardiac action potential, contributed noticeably to the linear impedance, producing a resonant peak in the magnitude of the impedance at a frequency of 1-3 Hz. The linearized impedance functions for these currents were characterized in the presence and absence of tetrodotoxin (TTX) and D-600. TTX had no noticeable effect on the impedance but D-600 essentially abolished the active currents. Although the ionic basis of these currents is not known, frequency domain analysis appears to be a viable technique for studying slow currents in heart muscle.     

Induction of 6-thioguanine resistance in human cells treated with N-acetoxy-2-acetylaminofluorene

Induction of 6-thioguanine resistance was studied in human cells treated with the direct-acting chemical carcinogen N-acetoxy-2-acetylaminofluorene (NA-AAF). At low concentrations (2.5–7.5 μM) induction of resistant clones was linear and followed one-hit kinetics, while at 10 μM the yield of resistant clones was higher and appeared to result from the combination of one-hit and two-hit kinetics. A study of about 50 resistant clones revealed that most had reduced levels of hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity (25–85% of controls) and were able to use exogenous hypoxanthine for growth (“Type II mutants,” deMars, 1974); a few had very low HGPRT activity (1–8% of controls) and were unable to use exogenous hypoxanthine (“Type I mutants”). Use of [9-¹⁴C]NA-AAF allowed us to examine the frequency of induction of thioguanine resistance as a function of binding to DNA (μmole AAF/mole DNA-P). Calculations from these data suggest that most “hits” on the HGPRT locus do not result in detectable mutations: At three different levels of binding and induced mutation frequency, the yield was 2.5-3 detectable mutants/10 000 molecules of acetylaminofluorene bound to the HGPRT locus. These data suggest that most bound acetylaminofluorene molecules either produce no change in the primary sequence of DNA (possibly as a result of repair or correct “read through” by the DNA polymerase) or result in changes which are phenotypically undetectable.     

Effects of phospholipid and detergent on the substrate specificity of adrenal cytochrome P-450scc. Substrate binding and kinetics of cholesterol side chain oxidation

Cytochrome P-450scc was isolated from mitochondria of bovine adrenal cortex by hydrophobic chromatography on octyl Sepharose followed by affinity chromatography on cholesterol-7-(thiomethyl)carboxy-3 beta-acetate-Sepharose. The partially purified eluate from the octyl Sepharose resin was free of adrenodoxin and adrenodoxin reductase and displayed biphasic binding characteristics for cholesterol, cholesterol sulfate, and cholesterol acetate (CA). Chromatography of the octyl Sepharose eluate on CA-Sepharose removed extraneous proteins and resolved the cytochrome P-450scc into two fractions, each of which displayed monophasic binding with all three substrates. These fractions behaved identically with respect to their ability to bind substrates, their kinetic properties, and their rate of migration during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The dissociation constants of the cytochrome P-450scc.substrate complexes are 1.1, 2.6, and 1.3 microM for cholesterol, cholesterol sulfate, and cholesterol acetate, respectively. Addition of phospholipids isolated from adrenal cortex mitochondria or adrenodoxin had no effect on the equilibrium binding constants. Addition of Emulgen 913, however, decreased the binding affinities 10-20-fold. Emulgen 913 also inhibited the interaction of adrenodoxin with the cytochrome. An active side chain cleavage system was reconstituted with purified P-450 by addition of saturating amounts of adrenodoxin, adrenodoxin reductase, and NADPH-generating system. The apparent Km values for this reconstituted system of cholesterol, cholesterol sulfate, and cholesterol acetate are 1.8, 1.9, and 0.6 microM, respectively. Since the Km values of substrate oxidation are similar to the Kd values of the cytochrome P-450.substrate complexes, it seems likely that the binding of substrates, particularly when the side chain cleavage system is free of mitochondrial membranes, is not rate-limiting. Based on these results and electrophoretic data, it appears that one cytochrome P-450 present in adrenal mitochondria can oxidize cholesterol, its sulfate, and its acetate. This enzyme represented about 60% of the cytochrome P-450 present in the octyl Sepharose eluate. The factors responsible for the biphasic kinetics of oxidation by intact mitochondria and biphasic binding of sterol substrates by partially purified preparations of cytochrome P-450scc are still unknown.     

Radioimmunoassay for Central Nervous System Myelin-Specific Proteolipid Protein

A double-antibody radioimmunoassay (RIA) has been developed with antisera to purified rat brain myelin proteolipid protein (PLP). The addition of Triton X-100 allowed antibody-antigen interaction and immune precipitation in the presence of sodium dodecyl sulfate (SDS). The RIA will accurately measure 8-80 ng of PLP in buffer or human serum. The RIA is highly specific for myelin PLP and does not cross-react with material in tissues (heart, kidney, muscle, testicle, and intestine) other than the central nervous system. The antibodies to rat myelin PLP cross-react with PLP from bovine brain homogenate or myelin. Myelin PLP was found to account for 55 and 52% of total myelin protein from bovine and rat brain, respectively. Furthermore, there is a higher concentration of PLP in white than in gray matter corresponding to the degree of myelination. Unlike myelin basic protein, myelin PLP was undetectable in both bovine and rat peripheral nervous system.     

Contribution of chloride to the membrane input conductance of the ventricle: The effect of ouabain

The purpose of this study was twofold: 1) to determine the contribution of chloride to the membrane input conductance (g_i) of the guinea pig ventricle during rest and activity, and; 2) to evaluate the influence of a non-toxic dose of ouabain on the chloride input conductance (g_i(Cl)). Standard electrophysiologic and myographic techniques were used to correlate changes in g_i with changes in the characteristics of the myocardial action potential and isometric contraction. During the plateau of the action potential, g_i was 80% greater than at rest. The influence of Cl^? on the conductance properties of the ventricle was evaluated by replacing 100% of the Cl^? in the bathing medium with a membrane impermeant anion (either isehionate or gluconate). Cl^? removal decreased g_i at rest and during the action potential to the same extent. To evaluate the influence of ouabain on the electromechanical properties of the isolated ventricle, the highest dose that produced signs of neither electrical nor mechanical toxicity within 90 minutes of exposure was chosen (1.2 × 10^?6M). No significant change in g_i, either at rest or during activity accompanied the initial increase in contractility and decrease in duration of the action potential produced by ouabain. However, as these effects of ouabain continued to increase over a 45 minute period, g_i increased during the plateau phase of the action potential. Cl^? removal from the medium bathing muscles treated with ouabain demonstrated that part of the increase in g_i during the plateau is due to an increase in g_i (Cl). These results suggest that: 1) g_i (Cl) makes a significant contribution to g_i both at rest and during activity, and; 2) the increase in g_i produced by ouabain during the plateau is, at least in part, due to an induced electrogenesis of the Cl^? channel.     

Chewing stick usage in Southern Ghana

Results are presented from a survey in which a sample of 887 people living in southern Ghana were questioned as to the chewing sticks they use, reasons for choice, and whether sticks are collected or bought. It appears that four kinds of sticks account for more than 85% of the total usage. Differences were recorded in preferred species and in diversity of species used, reason for choice and source of supply, according to age, sex, ethnic origin, size of settlement and educational background.     

NUCLEAR MEMBRANES OF CULTURED MAMMALIAN CELLS IN THE PERIOD PRECEDING DNA SYNTHESIS

When kidney cells are cultured directly from the rabbit, the nuclear membranes undergo a change that can be measured as an increase in electrophoretic mobility. The change appears to begin immediately upon culture and is maximal 2 hours later, after which the mobility remains constant at the elevated level. Actinomycin D and p-fluorophenylalanine, but not EDTA or ionizing radiation, suppress the increase in nuclear electrophoretic mobility. With synchronously growing L cells, no change was detected in nuclei from cells taken during various parts of the division cycle.