Tetraploidy in mice, embryonic cell number, and the grain of the developmental map.

Tetraploid mice prepared by electrofusion develop for up to 14 days in utero. The embryos are essentially normal save that the forebrain and its associated tissues fail to develop properly. Here, we report measurements of cell counts in tissues and volume measurements of tetraploid and control embryos together with observations on the morphology of tetraploid embryos. The results show that the tetraploid embryos are about 85% normal size, but have only a little under half the number of cells of control embryos, with their nuclei being about twice the size of those of diploid cells. Close examination of sectioned material, in contrast, showed that tetraploid morphology and morphogenesis were indistinguishable from those of controls, except in forebrain-associated material. This conclusion gives some insight into an important developmental question, how fine can the developmental map be for normal cellular differentiation to proceed? As tetraploids have only about half the expected number of cells, the ability of these embryos to develop normally in all regions except the forebrain and its derivatives argues that pattern formation mechanisms can cope with the abnormally small number of cells in all regions except the forebrain. The results as a whole argue for size regulation in mammalian embryos being achieved by assaying absolute size rather than counting cell numbers.     

Flow cytometric assay for evaluation of the effects of cell density on cytotoxicity and induction of apoptosis

BACKGROUND: We used a flow cytometric assay, which allows us to perform precise measurements within a wide range of cell concentrations to study the effect of the density of cultured cells on their sensitivity to cytotoxic compounds. METHODS: To measure cytotoxic action, cells are plated in a 96-well plate at a density ranging from 700 to 100,000 cells/ml and are allowed to grow for 72 h in the presence of various concentrations of a cytotoxic agent. To quantitate the number of surviving cells, each sample is analyzed in a flow cytometer with equal acquisition time. Viable cells are identified by light scattering characteristics identical to those for untreated cells. To estimate the amount of viable, apoptotic, or necrotic (late apoptotic) cells, the samples are stained with Annexin V and propidium iodide. RESULTS: Using this method, we found that the cytotoxicity of ascorbic acid for malignant lymphoid CEM-C7 cells can be increased significantly when cell density decreases, reaching a value that is typically lower than the normal physiological concentration of ascorbic acid in blood. CONCLUSION: The flow cytometric analysis described in this study can be useful in comparing the effects of cell density on the cytotoxic action of various compounds.     

Remodeling of retinal ganglion cell dendrites in the absence of action potential activity

The dendrites of ganglion cells in the retina have an excess number of spines and branches that are normally lost during the first postnatal month of development. We investigated whether this dendritic remodeling can be prevented when the action potential activity of ganglion cells is abolished by chronic intraocular injections of tetrodotoxin (TTX) during the first 4 or 5 postnatal weeks in the cat. Dendritic tree morphologies of alpha and beta ganglion cells from TTX-treated, non-TTX-treated (contralateral eye), and normal control retinae were compared after intracellular filling with Lucifer yellow. Qualitative observations and quantitative measurements indicate that TTX treatment does not prevent the normally occurring loss of spines and dendritic branches. Indeed, the dendritic trees of both alpha and beta cells in TTX injected eyes actually have even fewer spines and branches than normal cells at equivalent ages. However, because the total dendritic lengths of these cells are also reduced after TTX blockade, spine density is indistinguishable from untreated animals at the same age. In addition, although dendritic field areas are not altered with treatment, the complexity of the dendritic trees is reduced. These observations suggest that dendritic remodeling can occur in the absence of ganglion cell action potential activity. Thus, the factors that influence the dendritic and axonal development of retinal ganglion cells must differ, because similar TTX treatment during the period of axonal remodeling does have profound effects on the final pattern of terminal arborizations.     

Remodeling of retinal ganglion cell dendrites in the absence of action potential activity.

The dendrites of ganglion cells in the retina have an excess number of spines and branches that are normally lost during the first postnatal month of development. We investigated whether this dendritic remodeling can be prevented when the action potential activity of ganglion cells is abolished by chronic intraocular injections of tetrodotoxin (TTX) during the first 4 or 5 postnatal weeks in the cat. Dendritic tree morphologies of alpha and beta ganglion cells from TTX-treated, non-TTX-treated (contralateral eye), and normal control retinae were compared after intracellular filling with Lucifer yellow. Qualitative observations and quantitative measurements indicate that TTX treatment does not prevent the normally occurring loss of spines and dendritic branches. Indeed, the dendritic trees of both alpha and beta cells in TTX injected eyes actually have even fewer spines and branches than normal cells at equivalent ages. However, because the total dendritic lengths of these cells are also reduced after TTX blockade, spine density is indistinguishable from untreated animals at the same age. In addition, although dendritic field areas are not altered with treatment, the complexity of the dendritic trees is reduced. These observations suggest that dendritic remodeling can occur in the absence of ganglion cell action potential activity. Thus, the factors that influence the dendritic and axonal development of retinal ganglion cells must differ, because similar TTX treatment during the period of axonal remodeling does have profound effects on the final pattern of terminal arborizations.     

Influence of the bilayer composition on the binding and membrane disrupting effect of Polybia-MP1, an antimicrobial mastoparan peptide with leukemic T-lymphocyte cell selectivity

This study shows that MP-1, a peptide from the venom of the Polybia paulista wasp, is more toxic to human leukemic T-lymphocytes than to human primary lymphocytes. By using model membranes and electrophysiology measurements to investigate the molecular mechanisms underlying this selective action, the porelike activity of MP-1 was identified with several bilayer compositions. The highest average conductance was found in bilayers formed by phosphatidylcholine or a mixture of phosphatidylcholine and phosphatidylserine (70:30). The presence of cholesterol or cardiolipin substantially decreases the MP-1 pore activity, suggesting that the membrane fluidity influences the mechanism of selective toxicity. The determination of partition coefficients from the anisotropy of Trp indicated higher coefficients for the anionic bilayers. The partition coefficients were found to be 1 order of magnitude smaller when the bilayers contain cholesterol or a mixture of cholesterol and sphingomyelin. The blue shift fluorescence, anisotropy values, and Stern-Volmer constants are indications of a deeper penetration of MP-1 into anionic bilayers than into zwitterionic bilayers. Our results indicate that MP-1 prefers to target leukemic cell membranes, and its toxicity is probably related to the induction of necrosis and not to DNA fragmentation. This mode of action can be interpreted considering a number of bilayer properties like fluidity, lipid charge, and domain formation. Cholesterol-containing bilayers are less fluid and less charged and have a tendency to form domains. In comparison to healthy cells, leukemic T-lymphocyte membranes are deprived of this lipid, resulting in decreased peptide binding and lower conductance. We showed that the higher content of anionic lipids increases the level of binding of the peptide to bilayers. Additionally, the absence of cholesterol resulted in enhanced pore activity. These findings may drive the selective toxicity of MP-1 to Jurkat cells.     

Relation of insulin receptors to insulin resistance.

The status of insulin-receptor interactions in a variety of insulin-resistant states is reviewed. Utilizing large adipocytes from adult rats and small fat cells from young rats, we have conducted a series of in vitro experiments in an attempt to determine the cellular alteration(s) responsible for the insulin resistance associated with obesity. Stimulation of glucose oxidation by insulin is reduced in large cells. Studies using a mimicker of insulin action, spermine, as well as measurements of 125I-insulin binding to large and small cells indicate that receptor number and affinity are not responsible for hormone resistance. Furthermore, when rapid and direct measurements of sugar uptake were made, insulin stimulation was virtually identical in both cell types. These findings indicate that large adipocytes have an efficient insulin-responsive D-glucose transport system and suggest that the apparent hormone resistance may be due to alterations in intracellular glucose metabolism. It has been proposed that altered insulin-receptor interaction underlies the insulin resistance of human obesity. We have investigated this particular aspect of insulin action by 125I-insulin binding studies. Similar numbers of insulin receptors per cell and affinity for insulin were observed in adipocytes obtained from normal weight subjects and morbidly obese patients. Thus, the initial step in insulin action is unaltered in human obesity.     

Effects of selegiline,a monoamine oxidase B inhibitor,on differentiation of P19 embryonal carcinoma stem cells,into neuron-like cells

Selegiline, the irreversible inhibitor of monoamine oxidase B (MAO-B), is currently used to treat Parkinson’s disease. However, the mechanism of action of selegiline is complex and cannot be explained solely by its MAO-B inhibitory action. It stimulates gene expression, as well as expression of a number of mRNAs or proteins in nerve and glial cells. Direct neuroprotective and anti-apoptotic actions of selegiline have previously been observed in vitro. Previous studies showed that selegiline can induce neuronal phenotype in cultured bone marrow stem cells and embryonic stem cells. Embryonal carcinoma (EC) cells are developmentaly pluripotene cells which can be differentiated into all cell types under the appropriate conditions. The present study was carried out to examine the effects of selegiline on undifferentiated P19 EC cells. The results showed that selegiline treatment had a dramatic effect on neuronal morphology. It induced the differentiation of EC cells into neuron-like cells in a concentration-dependent manner. The peak response was in a dose of selegiline significantly lower than required for MAO-B inhibition. The differentiated cells were immunoreactive for neuron-specific proteins, synaptophysin, and β-III tubulin. Stem cell therapy has been considered as an ideal option for the treatment of neurodegenerative diseases. Generation of neurons from stem cells could serve as a source for potential cell therapy. This study suggests the potential use of combined selegiline and stem cell therapy to improve deficits in neurodegenerative diseases.     

Effects of hyperthermia, irradiation, and cytotoxic drugs on fluorescein isothiocyanate staining intensity for flow cytofluorometry

Measurement of fluorescein isothiocyanate (FITC) staining intensity of cultured lymphoblastoid cells following hyperthermia showed large increases without concomitant increases in nuclear protein. Similar measurements of cells following incubation with cytotoxic drugs showed fluorescent intensity increases that exceeded the increases in nuclear protein that were due to the cell cycle blocking action of the drug. The reverse, however, was true for cells following irradiation. In contrast, FITC staining intensity and nuclear protein measurements of cells proceeding through the cell cycle after removal of the cycle blocking agent showed nearly parallel changes, although there were reproducible minor differences, especially following blocking with hydroxyurea. These results suggest that FITC staining intensity is a function not only of nuclear protein content but also of stain access to the reaction sites of the protein constituents of the chromatin. Thus, it is possible that FITC staining may be used as a probe of changes in chromatin structure following experimental manipulation of cells in vitro or treatment of tumors in vivo.     

Mechanisms of Pathogenesis, Infective Dose and Virulence in Human Parasites

The number of pathogens that are required to infect a host, termed infective dose, varies dramatically across pathogen species. It has recently been predicted that infective dose will depend upon the mode of action of the molecules that pathogens use to facilitate their infection. Specifically, pathogens which use locally acting molecules will require a lower infective dose than pathogens that use distantly acting molecules. Furthermore, it has also been predicted that pathogens with distantly acting immune modulators may be more virulent because they have a large number of cells in the inoculums, which will cause more harm to host cells. We formally test these predictions for the first time using data on 43 different human pathogens from a range of taxonomic groups with diverse life-histories. We found that pathogens using local action do have lower infective doses, but are not less virulent than those using distant action. Instead, we found that virulence was negatively correlated with infective dose, and higher in pathogens infecting wounded skin, compared with those ingested or inhaled. More generally, our results show that broad-scale comparative analyses can explain variation in parasite traits such as infective dose and virulence, whilst highlighting the importance of mechanistic details.     

Evidence for a calcium-dependent outward conductance in endocrine cells of the cockroach, Diploptera punctata

Abstract The cell membranes of the corpora allata of the cockroach Diploptera punctata contain voltage-dependent calcium channels. Depolarizing current injection into cells of the corpora allata in the presence of the calcium channel blockers, cadmium, cobalt or verapamil allows the production of multiple action potentials, as does treatment with the intracellular calcium chelator, BAPTA/AM. These results suggest that calcium currents are involved both in decreasing the excitability and in activating an outward current in cells of the corpora allata. Electrophysiological measurements also suggest a concomitant reduction in outward conductance following the multiple action potentials produced in the presence of the channel blockers or BAPTA/ AM. We hypothesize that the calcium current may play an important role in the regulation of intracellular calcium concentration and Juvenile Hormone biosynthesis.     

The morphological basis of intracellular measurements in the cockroach tactile spine neuron

Summary The femoral tactile spine of the cockroach (Periplaneta americana) contains a single sensory neuron, which adapts rapidly and completely to step deformations of the spine. Techniques for stable intracellular recording from the tactile spine neuron have recently been established, allowing electrophysiological investigation of mechanotransduction and adaptation in this sensory neuron. However, intracellular recordings from the neuron produce a wide range of action potential heights and thresholds, raising the possibility that some penetrations are in adjacent, but closely coupled supporting glial cells. This problem is exacerbated because the cell cannot be visualized during penetration.Systematic measurements of action potential heights and thresholds were made in tactile spine cells, together with identification of some penetrated cells by intracellular injection of Lucifer Yellow. All stained cells were clearly sensory neurons, although their action potentials amplitudes varied from 9 mV to 80 mV. Smaller action potentials were broader than larger action potentials, and the changes in height and shape could be explained by a simple cable conduction model using measured morphological and electrical parameters. The model could also account for the observed relationship between action potential height and threshold.These results indicate that reliable recording from the tactile spine neuron is possible, but that variability in the positions of the penetration or the spike initiating zone cause an apparently wide range of electrophysiological measurements.     

The role of the BCG dose and the mouse strain in the inhibition of development of neoplasms susceptible and nonsusceptible to the action of natural cytotoxic cells

The susceptibility of Ehrlich Ascites Carcinoma (EAC) cells to the action of natural cytotoxic cells of DBA/2 and Balb/c mice in vitro was established. Leukaemia L 1210 cells proved insensitive to the in vitro action of natural cytotoxic cells of DBA/2 mice, but not to those of Balb/c ones. BCG, one of the inductors of cytotoxic NK cells, when administered to DBA/2 or Balb/c mice before introduction of EAC cells inhibited the growth of this tumour but did not retard the development of leukaemia L 1210 in DBA/2 mice. The change in the number of peritoneal exsudate cells (PEC) in DBA/2 mice after intraperitoneal injection of BCG was demonstrated to be dependent on the dose and the time elapsed after bacilli introduction. The antitumour action of BCG does not depend on changes in the number of PEC caused by the bacilli. Both large (3.0 mg) and small (0.02 mg) doses of BCG inhibit the development of EAC in Balb/c mice ("sensitive" to BCG), notwithstanding the time of administration of the bacilli. In DBA/2 mice ("resistant" to BCG) development of EAC can be inhibited only by the large dose of BCG since small one is sometimes ineffective.     

Calcium ion uptake, somatotropin release, and fine structure of somatotrophs in cultures of the rat anterior pituitary upon the action of an oligopeptide (Boc-Gln-Leu-Lysinal)

Cultured cells from the anterior pituitary glands of adult rats were treated with the tripeptide aldehyde proteinase inhibitor, Boc-Gln-Leu-Lys-H. The oligopeptide had a profound releasing effect on growth hormone, whereas the prolactin release remained unchanged at 10(-3) mol/l drug concentration after an incubation for 2 h. In the presence of the oligopeptide a time- and dose-dependent calcium influx into cultured cells has been shown which was proved to be almost completely antagonized with magnesium ions but not with Nifedipine. In addition, radioactive calcium ions could be detected in a number of cells by light microscopic autoradiography when cultures were treated with Boc-Gln-Leu-Lys-H for short periods. The selective Gel action of the oligopeptide on growth hormone producing cells has been demonstrated also in fine structural investigations: multigranular and single exocytotic profiles have been observed. Accordingly, we have postulated that Boc-Gln-Leu-Lysinal mimics the effects of the known ionophores. Its mode of action needs, however, further studies especially on isolated somatotrophs.     

Chalones and the Control of Normal, Regenerative, and Neoplastic Growth of the Skin

SYNOPSIS Chalones,inhibitors of cell dmsion have been isolatedand studied from a number of mammalian tissues, most notably,the epidermis The epidermal rhalone is a glycoprotein It exhibitsconsiderable, but not complete specificity The epidermal chalone decreases mitotic activity by inhibitingcells in the G 2 phase of the cell cycle from entering mitosis,and probably also by inhibiting ceils in the G 1 phase of thecell cycle from entering mitosis To inhibit cells in G 2 fromentering mitosis the chilone requnes adrenalin, and for maximalactivity hydrocortisone It is not known if idrenalin and hydrocortisoneare required for chalone inhibition of cells in G 1 In addition to inhibiting cell division in normal epidermalcells the epidermal chalone can inhibit cell division in regeneratingepidermal cells induced to proliferate by chemical damage Thephase of the cell cycle in which the chalone inhibits legeneratingepidermal cells from entering mitosis is not known Epidermal tumors contain a decreased amount of chalone Mitosisin epidermal tumors is inhibited by treatment with epidermalchalone Tumor cells are inhibitedfrom entering mitosis fromeither the G 1 or G 2 phases of the cell cycle Chalones are said to inhibit mitosis by a negative feedbackmechanism However, experiments which presumably result in adecrease in chalone concentration do not result in an increasein mitotic activity It is suggested that if chalones are physiological controllers of cell division they do not act by a simplenegative feedback mechanism but require the action of a substanceto decrease their concentration     

One-Dimensional Mathematical Model of the Atrioventricular Node Including Atrio-Nodal, Nodal, and Nodal-His Cells

Mathematical models are a repository of knowledge as well as research and teaching tools. Although action potential models have been developed for most regions of the heart, there is no model for the atrioventricular node (AVN). We have developed action potential models for single atrio-nodal, nodal, and nodal-His cells. The models have the same action potential shapes and refractoriness as observed in experiments. Using these models, together with models for the sinoatrial node (SAN) and atrial muscle, we have developed a one-dimensional (1D) multicellular model including the SAN and AVN. The multicellular model has slow and fast pathways into the AVN and using it we have analyzed the rich behavior of the AVN. Under normal conditions, action potentials were initiated in the SAN center and then propagated through the atrium and AVN. The relationship between the AVN conduction time and the timing of a premature stimulus (conduction curve) is consistent with experimental data. After premature stimulation, atrioventricular nodal reentry could occur. After slow pathway ablation or block of the L-type Ca²⁺ current, atrioventricular nodal reentry was abolished. During atrial fibrillation, the AVN limited the number of action potentials transmitted to the ventricle. In the absence of SAN pacemaking, the inferior nodal extension acted as the pacemaker. In conclusion, we have developed what we believe is the first detailed mathematical model of the AVN and it shows the typical physiological and pathophysiological characteristics of the tissue. The model can be used as a tool to analyze the complex structure and behavior of the AVN.     

Interleukin-4 enhances anti-IgM stimulation of B cells by improving cell viability and by increasing the sensitivity of B cells to the anti-IgM signal

The lymphokine IL-4 is a potent enhancer of anti-IgM-induced B cell proliferation. Although the mechanism of this enhancement is not known, a commonly held view suggests that IL-4 acts together with anti-IgM as a costimulating factor for the activation of a subpopulation of B cells. To evaluate this hypothesis we examined the effect of IL-4 on the proportion of B cells stimulated to divide by different doses of anti-IgM using flow cytometry in combination with measurements of tritiated-thymidine incorporation. The results suggest a novel and surprisingly simple model for the mode of action of IL-4. Our analysis revealed that at high saturating anti-IgM concentrations, the proportion of live B cells which enter into S phase of the cell cycle is the same (approximately 65%) for cells cultured with or without IL-4. Cultures containing IL-4, however, exhibit a twofold increase in thymidine uptake over cultures without IL-4. This increase can be explained completely by the ability of IL-4 to enhance the viability of small dense B cells over the first 24 hr from approximately 50 to 90% of the starting cell number. Normalizing the maximum response levels obtained with and without IL-4 reveals that B cells incubated with IL-4 exhibit a 10-fold decrease in the concentration of anti-IgM required to stimulate the half-maximum proliferation level. Furthermore, evaluation of the number of cells in S phase by flow cytometry and analysis of the kinetics of cell proliferation revealed that the increased response effected by IL-4 at lower anti-IgM concentrations was due to a greater number of proliferating B cells rather than the same number of cells undergoing a faster division rate. We also found a highly nonlinear relationship between B cell number and proliferative response, implying a requirement for an additional, cell cooperation-mediated, activating signal for maximum B cell proliferation. IL-4 enhanced proliferation by the same proportion at all cell concentrations indicating that it does not replace or alter this requirement for cell cooperation. Taken together these results suggest that anti-IgM in combination with a second unidentified cell-cooperation-dependent signal leads to proliferation of up to 65% of small resting B cells. IL-4 does not provide an essential activation signal but serves to raise the sensitivity of B cells to the anti-IgM-generated signal.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of the mitogenic response of an epidermal growth factor-dependent keratinocyte cell line by dexamethasone, insulin, and transforming growth factor-beta

The stimulation of DNA synthesis by epidermal growth factor (EGF) has been studied for a cell line having properties useful for investigating the mechanism of action of EGF in epithelial cell populations. These studies employ a mouse keratinocyte cell line (MK), isolated by Weissman and Aaronson (1983), which is stringently dependent on exogenous EGF for growth in serum containing medium. The studies reported here characterize the compliment of EGR receptors present on the surface of MK cells and demonstrate the regulatory influence of other hormones on the capacity of EGF to stimulate DNA synthesis. Up-regulated MK cells contain approximately 22,000 EGF receptors per cell, but when the cells are grown in the presence of EGF the receptor number is reduced to about 4,000. It is estimated that only a small number of high-affinity receptors (less than 500) are required for EGF-dependent cell proliferation. In contrast to its action in fibroblastic cells, dexamethasone is a strong inhibitor of EGF-stimulated DNA synthesis of MK cells. Insulin at high concentrations, or insulin-like growth factors I or II (IGF-I, IGF-II) at physiological concentrations, synergistically enhance the EGF response. Interestingly, insulin or IGF-I or II are also able to reverse most of the dexamethasone inhibition of DNA synthesis. Transforming growth factor-beta (TGF-beta) inhibits, in reversible manner, the EGF stimulation of DNA synthesis and this inhibition is not overcome by insulin. TGF-beta receptors have been measured in MK cells and Scatchard analysis indicates approximately 20,000 receptors per cell. None of the modulatory hormones (insulin, dexamethasone, TGF-beta) significantly altered 125I-EGF binding characteristics in MK cells, suggesting a point of action distal to 125I-EGF binding.     

Effect of Trophic Status on the Culturability and Activity of Bacteria from a Range of Lakes in the English Lake District

The bacterioplankton from a number of lakes that differed in nutrient status in the English Lake District was examined with a number of techniques for enumeration and activity assessment. Natural water samples showed a clear correlation between total counts and trophic status. Esterase activity measurements with Chemchrome B were able to distinguish high- and low-nutrient-status lakes, whereas tetrazolium salt (5-cyano-2,3-ditoyltetrazolium chloride) reduction, the direct viable count-cell elongation assay, and culturability measurements could not. Tetrazolium salt reduction and esterase activity measurements labeled a significant number of cells from water of all nutrient levels, whereas the direct viable count-cell elongation method was of use only in oligotrophic waters. Size fractionation of samples showed that the culturable cells were retained by the larger filters, especially in nutrient-rich waters. Esterase activity measurements also favored the larger cells. The differences observed between assays using water that differed in trophic status raise questions about the use of these tests as a definitive measure of viability.     

Effect of trophic status on the culturability and activity of bacteria from a range of lakes in the English Lake District

The bacterioplankton from a number of lakes that differed in nutrient status in the English Lake District was examined with a number of techniques for enumeration and activity assessment. Natural water samples showed a clear correlation between total counts and trophic status. Esterase activity measurements with Chemchrome B were able to distinguish high- and low-nutrient-status lakes, whereas tetrazolium salt (5-cyano-2,3-ditoyltetrazolium chloride) reduction, the direct viable count-cell elongation assay, and culturability measurements could not. Tetrazolium salt reduction and esterase activity measurements labeled a significant number of cells from water of all nutrient levels, whereas the direct viable count-cell elongation method was of use only in oligotrophic waters. Size fractionation of samples showed that the culturable cells were retained by the larger filters, especially in nutrient-rich waters. Esterase activity measurements also favored the larger cells. The differences observed between assays using water that differed in trophic status raise questions about the use of these tests as a definitive measure of viability.     

Investigation of the effects of continuous-wave,pulse- and amplitude-modulated microwaves on single excitable cells of chara corallina

Single internodal excitable cells of Chara corallina were exposed to CW, pulse-modulated and sinusoidally modulated S-band microwave fields in a temperature-controled waveguide exposure chamber. All electrical measurements were made external to the waveguide (ie, under no impressed microwave field). The dependent variables measured before, during, and after exposure to the S-band microwave fields included: resting potential, amplitude of the action potential, rise and decay time of the action potential, conduction velocity, and excitability. Cells maintained at 22 ± 0.1 °C during exposure showed no consistent or statistically significant microwave-dependent alterations in any of the dependent variables.