(mitochondria-to-cytoplasm gradients) to be made and suggests that the trans-mitochondrial membrane protonmotive force in the intact cell cannot be greater than -143 mV.Assessment of the electrical potential and ion gradients across subcellular compartments in the intact cell is an important goal that has not been pursued directly; isolation of subcellular compartments and subsequent in vitro studies have been the predominant approach, although possible redistribution of ionic species during isolation raises doubts with respect to the validity of this procedure. In this communication, we present a quantitative approach to the evaluation of the transmembrane electrical potential and pH gradients across a subcellular compartment in an intact cell. The requisite information for such evaluations includes: (i) relative volumes of the subcellular compartment and the cell; (ii) a known (measured) value of trans-plasma membrane electrical potential and pH gradient; and (iii) measured total concentrations of ions and weak acids/bases that distribute passively in the cell and in the compartment. These distributions give estimates of the cytosol-to-extracellular space gradient plus the compartment-tocytosol gradients.We have used this approach to evaluate the total trans-mitochondrial protonmotive force (the electrical plus pH gradients across the mitochondrial membrane) in mouse neuroblastoma Hanks' medium (3) containing 10 mM Hepes (pH 7.4) and 0.05% bovine serum albumin and finally resuspended in the same medium at 25-30 mg wet weight/ml. These suspensions were passed through a wire mesh (20-to 40-,m pore size diameter) in order to produce suspension homogeneity. Suspensions treated in this manner showed no clumps (no more than three cells grouped together) under a microscope. The dry weights of the cell suspension and the medium were determined for each preparation.For microelectrode measurements, cells were grown in Nunclon plastic petri dishes for 2-3 days as described above. Measurements of membrane potential were made while most of the cells were still in the exponential growth phase.Microelectrode Measurements. The medium in the petri dish was poured off and replaced by Hanks' medium containing 10 mM Hepes (pH 7.4) and 0.05% bovine serum albumin. The dish was placed on an inverted high-power microscope with a warmed stage (20-25°C). The electrode assembly was advanced by a remote stepping-motor hydraulic system. The microscope and the electrode carrier were contained within an electrically shielded cage and a humidified chamber. Lighting was via a fiber optic, heat-filtered high intensity source (Leitz, West Germany). After the lid of the petri dish was removed, the electrode was inserted under direct vision into randomly selected cells adhering to the floor of the dish. Only those Abbreviation: TPMP, triphenylmethyl phosphonium ion. Thomas (5). Some electrodes were ground to a chisel point on an optically flat grinding machine to allow easier penetration of the cell membrane.Isotope Distribution Measurement...

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Suspension cultures of L-929 fibroblasts grown to densities of 6 to 10 X 1 0 6 cellslml through daily centrifugation and resuspension i n fresh media, have been maintained for periods up to five months without change in viability or cell size. DNA synthesis and mitosis in these cultures is limited to 5% of the cells per day, a fraction very nearly equal to the fraction of cells rendered nonviable, most likely during the manipulations associated with medium renewal. The kinetics of the flow of cells into the S and M periods following (a) renewal of the medium and (b) dilution of the high density cultures, suggest that the large majority of the cells are i n a Go or early GI phase, resuming growth readily i n response to decreased cell density. This is further indicated by the sequence of the marked shifts occurring i n the cell volume distribution spectrum of the high density cultures after dilution. Long term, steady state regulation of growth with retention of intact viability was thus demonstrated i n the case of a long established aneuploid cell line. The fact that this occurs in suspension but not in attached cultures, supports the concept that impairment of growth control in such cells affects predominantly regulatory mechanisms located at the cell surface rather than those concerned with intracellular synthesis and metabolism.

Sulfur mustard (SM; 2,2(')-dichloroethyl sulfide) is a percutaneous alkylating agent first used as a chemical weapon at Ypres, Belgium, in World War I. Despite its well-documented history, the primary lesions effecting dermal-epidermal separation and latent onset of incapacitating blisters remain poorly understood. By immunofluorescent imaging of human epidermal keratinocytes (HEK) and epidermal tissues exposed to SM (400 microM for 5 min), we have amassed unequivocal evidence that SM disrupts adhesion complex molecules, which are also disrupted by epidermolysis bullosa-type blistering diseases of the skin. Images of keratin 14 (K14) in control cells showed tentlike filament networks linking the HEK's basolateral anchoring sites to the dorsal surface of its nuclei. Images from 6-h postexposure profiles revealed early disruption (

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