Preliminary results on the non-thermal effects of 200-350 GHz radiation on the growth rate of S. cerevisiae cells in microcolonies.

We report preliminary results from studies of biological effects induced by non-thermal levels of non-ionizing electromagnetic radiation. Exponentially growing Saccharomyces cerevisiae yeast cells grown on dry media were exposed to electromagnetic fields in the 200-350 GHz frequency range at low power density to observe possible non-thermal effects on the microcolony growth. Exposure to the electromagnetic field was conducted over 2.5 h. The data from exposure and control experiments were grouped into either large-, medium- or small-sized microcolonies to assist in the accurate assessment of growth. The three groups showed significant differences in growth between exposed and control microcolonies. A statistically significant enhanced growth rate was observed at 341 GHz. Growth rate was assessed every 30 min via time-lapse photography. Possible interaction mechanisms are discussed, taking into account Frohlich's hypothesis.

Use of the statistical properties of the wavelet-transform coefficients for optimization of integration time in Fourier transform spectrometry.

We show that an analysis of the mean and variance of discrete wavelet coefficients of coaveraged time-domain interferograms can be used as a specification for determining when to stop coaveraging. We also show that, if a prediction model built in the wavelet domain is used to determine the composition of unknown samples, a stopping criterion for the coaveraging process can be developed with respect to the uncertainty tolerated in the prediction.

Furosemide stimulates K transport in HCD57 erythroid cells.

We examined the influence of serum and furosemide on K movement and cell volume in HCD57 cells, a murine erythroleukemia cell line, which require erythropoietin (EPO) for survival. We found that maintenance of cell volume depends on the concentration of serum in the culture medium. In isotonic medium containing 20% serum, HCD57 cells maintain their steady-state volume. In contrast, the cells shrink progressively as medium serum content is reduced. In serum-free medium, raising external K to 75 mm prevents cell shrinkage and a further increase in K to 145 mm results in swelling, revealing a role for K permeability in the regulation of cell volume. Of particular interest has been a serendipitous finding with furosemide. Below an external K concentration of 2.1 +/- 0.3 mm in medium containing 2% serum, furosemide inhibits K uptake, probably stemming from its well known inhibitory action on KCl cotransport. However, above that K concentration, furosemide stimulates K uptake in a dose-dependent manner. Moreover, furosemide potentiates cell shrinkage induced by serum withdrawal. These findings suggest that the transport machinery mediating cellular shrinkage, once primed by serum depletion, becomes receptive to a second stimulus.

Design of access control methods for protecting the confidentiality of patient information in networked systems.

Public awareness of the potential for violation of personal privacy in clinical information systems is increasing. Much of this increase can be attributed to the popularity and publicity of the World Wide Web. Nightly news reports of intruder break-ins and flaws in Internet software security have stimulated public interest in the security of clinical information systems available over the web. As part of the development of systems designed to provide clinical narratives to physicians over the Internet, we are exploring designs that provide additional protection and security to these systems. Specifically, we are developing and testing automated access control measures based on provider-patient relationships for controlling access to personally identifiable patient information.

Potentiation of regulatory volume decrease by P2U purinoceptors in HSG-PA cells.

HSG-PA human salivary gland duct cells exhibit progressively increased regulatory volume decrease (RVD) in response to decreased medium osmolarity. The P2U purinoceptor agonist UTP causes a potentiation of RVD, the extent of which is most pronounced in 220 mosM medium and is least apparent in 180 mosM medium. We examined the underlying mechanisms for this effect. Exposure of HSG-PA cells to UTP promotes Ca2+ mobilization, hyperpolarization, and net K+ efflux, suggesting the participation of Ca(2+)-activated K+ channels in RVD. To delineate the anion counterpart of K+ movement during RVD, cell swelling in the presence of gramicidin, which abolishes the membrane potential, was measured. In response to a sudden dilution in hypotonic media, gramicidin-treated cells swelled immediately, followed by a "secondary swelling" in 180 but not in 220 mosM medium. The results suggest that in 180 mosM cells perform spontaneous RVD mediated by increased anion conductance. In 220 mosM medium in which RVD is minimal, the increase in anion conductance is marginal. In our model of RVD in which cells were challenged by UTP, the ensuing hyperpolarization provides the driving force for net Cl- efflux, which is confirmed by tracer flux studies during purinoceptor-activated RVD. Thus RVD, which has long been regarded as a self-sufficient cellular program, appears to be subject to extracellular control in HSG-PA cells through receptor-mediated processes.

Na,K-ATPase expression and cell volume during hypertonic stress in human renal cells.

Primary cultures of human renal cortex cells were incubated in hypertonic medium and low K+ medium to determine the effect on Na,K-ATPase alpha and beta subunit expression, cell water, and intracellular ions. Cells exhibited functional characteristics of proximal tubules based on PTH stimulation of cAMP and the presence of Na(+)-dependent phosphate transport. When either NaCl or sucrose was added to increase medium osmolality to 500 mOsm/kg, beta subunit mRNA increased relative to control between 2.4 and 3.2-fold by six hours, and was still near twofold higher after 24 hours, while alpha subunit mRNA increased to about 1.5 times control by six hours. In low K+ medium, only beta mRNA increased. Hypertonic incubation increased Na,K-ATPase activity by 39% to 66% after 24 hours. Cell water was 70% of control at one hour, but increased to 90% of control by 24 hours. Only about 40% of the volume regulatory increase depended on accumulation of Na+ and K+. These results demonstrate that primary cultures of human proximal tubule cells can respond to hypertonic stress by induction of Na,K-ATPase.

Regulation of Na(+)-K(+)-ATPase expression in cultured renal cells by incubation in hypertonic medium.

To determine whether alterations in cell volume affect Na(+)-K(+)-adenosinetriphosphatase (ATPase) expression, a subclone of the Madin-Darby canine kidney (MDCK) cell line was incubated in anisotonic serum-free medium and alpha- and beta-subunit mRNA, Na(+)-K(+)-ATPase activity, and active K+ transport were measured. In medium adjusted to 500 mosmol/kgH2O by adding NaCl, the alpha-subunit mRNA concentration was 2.93 +/- 0.14 (SE) times control and beta-mRNA was 1.93 +/- 0.27 times control. When sucrose was added to increase osmolality, alpha-subunit mRNA increased to 1.85 +/- 0.18 times control. Na(+)-K(+)-ATPase activity of homogenates from cells incubated in 500 mosmol/kgH2O medium for 24 h increased to 2.62 +/- 0.52 times control when NaCl was added and 2.31 +/- 0.34 times control when sucrose was added. Active K+ transport increased between 60 and 90% after cells were incubated in 450 mosmol/kgH2O medium with either NaCl or sucrose added. Stimulation of Na(+)-K(+)-ATPase expression in renal cells facing hypertonic stress may represent a long-term mechanism that allows cells to maintain cation gradients in a hypertonic environment.

Synthesis and translocation of Na(+)-K(+)-ATPase alpha- and beta-subunits to plasma membrane in MDCK cells.

Synthesis and translocation of Na(+)-K(+)-ATPase alpha-catalytic and beta-glycoprotein subunits from intracellular membranes to the plasma membrane were studied in Madin-Darby canine kidney cells (MDCK-T) by combining the methods of pulse-chase labeling, subcellular fractionation on sorbitol gradients, and immunoprecipitation. Immunoprecipitation from homogenates revealed that radioactive methionine incorporated into beta-subunit was equal to that incorporated into alpha-subunit after 15 min of labeling. Because the ratio of total methionines in alpha- vs. beta-subunit is approximately 5:1, these results suggest that beta-subunit is synthesized in molar excess over alpha-subunit. Half of the newly synthesized beta-subunit, likely unassembled units, were degraded by 60 min after labeling, while alpha-subunits were stable through 120 min after synthesis, suggesting alpha may be limiting for alpha beta-assembly. By 120 min the ratio of counts incorporated into alpha vs. beta approached 5, which is predicted by a 1:1 ratio of alpha to beta. The sorbitol gradient resolved two major membrane samples: a mixture of endoplasmic reticulum and Golgi populations and a plasma membrane-enriched sample. Immature beta (beta i) could not be detected in the plasma membrane-enriched samples at levels greater than could be attributed to cross-contamination by intracellular membranes. Mature beta (beta m) became detectable after 30 min, and conversion of beta i to beta m was 90% complete at 120 min. A peak of labeled alpha-subunit appeared in the plasma membrane-enriched sample at 60 min, coincident with the appearance of labeled beta m-subunit in this sample, suggesting movement as alpha beta-heterodimers.

Patterns of mRNA expression during early cell growth differ in kidney epithelial cells destined to undergo compensatory hypertrophy versus regenerative hyperplasia.

An increase in cell size and protein content is characteristic of cells undergoing hypertrophy and of replicating cells prior to DNA synthesis. Cell enlargement in the two situations could be regulated by similar early events with an interruption of the cell cycle occurring in hypertrophy, or the two processes could be uncoupled. In vivo models were used to compare hypertrophy induced by unilateral nephrectomy and hyperplasia induced by folic acid injection in rabbit renal cortical cells. Within 48 hr, cell volume increased in both groups but the number of cells in the cell cycle and DNA synthesis was increased only after folic acid. Patterns of mRNA expression of the following three groups of cell cycle-dependent genes were analyzed: (i) protooncogenes (c-fos, c-myc, and c-Ha-ras), (ii) structural protein genes (vimentin and beta-actin), and (iii) transport protein genes (Na+, K+-ATPase, ADP-ATP translocase, and calcyclin). mRNAs for all genes, except calcyclin and c-Ha-ras, were detected in controls. Folic acid generally induced rapid, transient increases in mRNA levels, but after unilateral nephrectomy, expression of most mRNAs showed a gradual, progressive increase. These data indicate that gene expression in the early stages of cell enlargement differs in cells destined to undergo proliferation vs. hypertrophy. The term "sustained message amplification" is proposed to describe the hypertrophied cell.

Pretranslational regulation of Na-K-ATPase in cultured canine kidney cells by low K+.

Long-term upregulation of the sodium pump [Na-K-adenosine triphosphatase (Na-K-ATPase)] entails an increase in the number of enzyme molecules. We incubated Madin-Darby canine kidney (MDCK) cells in low K+ medium and studied the time course and magnitude of change in the relative abundance of the two Na-K-ATPase subunits (alpha and beta), in the synthesis rate of the subunits, and in the relative abundance of alpha- and beta-mRNA. When cells were incubated in medium containing 0.25 mM K+, intracellular Na+ increased from 25.2 +/- 0.9 (SE) mmol/l cell H2O to 69.8 +/- 9.6 at 4 h and 132 +/- 6 at 16 h. Cell K+ fell from 146 +/- 4 mmol/l cell H2O to 105 +/- 9 at 4 h and 42.3 +/- 4.7 at 16 h. The relative abundance of Na-K-ATPase subunits, measured with immunoblots of cell homogenates, increased such that after 24 h alpha was 1.71 +/- 0.33 and beta was 1.67 +/- 0.22 times control. After 8 h of K+ depletion, alpha-synthesis rate, measured by immunoprecipitation of pulse-labeled cells, increased to 2.30 +/- 0.50 and beta increased to 2.07 +/- 0.42 times control. The alpha- and beta-subunit mRNA abundance, measured by hybridizing alpha- and beta-cDNA probes to total RNA, increased within 30 min to 1.93 +/- 0.24 and 2.29 +/- 0.64 times control, respectively. We conclude that regulatory adjustments of Na-K-ATPase abundance involve an increase in translation after a rapid and coordinate increase in the concentrations of alpha- and beta-subunit mRNA.

Phosphate transport in Ehrlich ascites tumor cells: inhibition by H+.

The effect of changes in extracellular pH (pHo) and intracellular pH (pHi) on Na+-dependent and Na+-independent inorganic phosphate (Pi) transport in Ehrlich cells was investigated. In the presence of Na+, acutely reducing pHo from 7.30 to 5.50 results first in a transient (approximately 7 min) stimulation of Pi transport. The enhanced rate of transport is a saturable function of the extracellular [H+]; the Ks equals 2.3 X 10(-6) M (pHo 6.68). However, Pi transport is progressively inhibited as pHi falls below 6.50. The effect of pHi on Pi transport measured at various intracellular [Na+] suggests that inhibition develops as a consequence of H+ interaction with an intracellular Na+ site(s) on the Na+-dependent carrier. At pHo 7.4, about 15% of the steady state Pi flux persists in the absence of Na+. However, when pHo is reduced, transport is stimulated to the same extent and with the same time course and kinetic characteristics as in the presence of Na+. Thus, H+ stimulated Pi transport does not require Na+, raising the possibility that the Na+-independent component is mediated by the anion (Cl-) exchanger.

H+ transport and the regulation of intracellular pH in Ehrlich ascites tumor cells.

The intracellular pH (pHi) of Ehrlich ascites tumor cells, both in the steady state and under conditions of acid loading or recovery from acid loading, was investigated by measuring the transmembrane flux of H+ equivalents and correlating this with changes in the distribution ratio of dimethyloxazolidine-2,4-dione (DMO). The pHi of cells placed in an acidic medium (pHo below 7.15) decreases and reaches a steady-state value that is more alkaline than the outside. For example when pHo is acutely reduced to 5.5, pHi falls exponentially from 7.20 +/- 0.06 to 6.29 +/- 0.04 with a halftime of 5.92 +/- 1.37 min, suggesting a rapid influx of H+. The unidirectional influx of H+ exhibits saturation kinetics with respect to extracellular [H+]; the maximal flux is 15.8 +/- 0.05 mmol/(kg dry wt X min) and Km is 0.74 +/- 0.09 X 10(-6) M. Steady-state cells with pHi above 6.8 continuously extrude H+ by a process that is not dependent on ATP but is inhibited by anaerobiosis. Acid-loaded cells (pHi 6.3) when returned to pHo 7.3 medium respond by transporting H+, resulting in a rapid rise in pHi. The halftime for this process is 1.09 +/- 0.22 min. The H+ efflux measured under similar conditions increases as the intracellular acid load increases. An ATP-independent as well as an ATP-dependent efflux contributes to the restoration of pHi to its steady-state value.

Evidence for monovalent phosphate transport in Ehrlich ascites tumor cells.

In an effort to determine whether the Na+-dependent Pi transport system of Ehrlich ascites tumor cells exhibits specificity for H2PO4- or HPO4(-2), Pi fluxes were determined by measuring 32Pi-Pi self-exchange. Three experimental approaches were employed. First, the effect of pH on steady-state Pi transport at 0.5 and 5 mM was studied. Second, the relationship between Pi transport and Pi concentration (0.25-9.2 mM) at pH 5.6 and 7.9 was determined. Third, the dependence of Pi transport on [H2PO4-] (0.05-4.2 mM) at constant [HPO4(-2)] (0.5 mM), and the converse, [HPO4(-2)] (0.06-4.5 mM) at constant [H2PO4-] (0.5 mM), was evaluated. Ks (apparent half-saturation constant) and Jmax (maximal transport rate) were calculated by two methods: weighted linear regression (WLR) and a nonparametric procedure. The dependence of Pi flux on pH indicates that optimum transport occurs at pH 6.9. Pi transport decreases as pH is reduced when extracellular Pi is either 0.5 or 5 mM. However, at pH 7.9, Pi flux is reduced only in 0.5 mM Pi. At pH 5.6, H2PO4- comprises 93% of the total Pi present, and the calculated Ks is 0.055 +/- 0.026 mM (WLR). This is the same as the Ks determined from the initial phase of the flux vs. [H2PO4-] relationship (0.056 +/- 0.020 mM). However, at pH 7.9 (where 94% of Pi is HPO4(-2)), the measured Ks is 0.58 +/- 0.11 mM (WLR), which is ten times higher than at pH 5.6. This value is also five times greater than the Ks calculated from the flux vs. [HPO4(-20)] curve (0.106 +/- 0.16 mM). Kinetic parameters calculated by the nonparametric method, though somewhat different, gave similar relative results. Taken together, these results support two conclusions: (1) H2PO4- is the substrate for the Na+-dependent Pi transport system of the Ehrlich cell, and (2) H+ can inhibit Pi transport.

Phosphate concentration and transport in Ehrlich ascites tumor cells: effect of sodium.

The effects of extracellular Pi and Na+ on cellular Pi concentration and transport were studied. Steady-state Pi exchange flux was measured by 32P uptake in the presence and absence of Na+. Model experiments were also conducted to assess the possibility that hydrolysis of organic phosphate esters contributes to the chemically measured intracellular Pi concentration of Ehrlich ascites tumor cells. The results of these experiments indicate that hydrolysis of labile organic phosphate esters does not contribute to the measured intracellular pool of Pi. The Pi transport system exhibits an apparent Ks of 0.115 mM Pi and a maximal flux of 1.73 mmole min-1 (kg dry wt)-1. When incubated in a phosphate-buffered choline chloride medium (5 mM Pi) the intracellular Pi and the Pi influx fall by 65 and 88%, respectively. At 5 mM extracellular Pi, the Na+-dependent component of Pi transport fits Michaelis-Menten kinetics with the maximal flux equal to 2.46 mmole min-1 (kg dry wt)-1 and an apparent Ks of 35.4 mM Na+. In addition, a Na+-independent component of Pi transport, comprising about 12% of the total Pi flux, was identified. The data support the hypothesis that a Pi transport system, dependent on Na+, plays a principal role in the maintenance of intracellular Pi concentration.