Ion channels activated by swelling of Madin Darby Canine Kidney (MDCK) cells

Summary According to previous studies hyposmotic swelling of Madin Darby Canine Kidney (MDCK) cells leads to a marked decrease of cell membrane resistance. The present study has been performed to identify the underlying ion channels using the patchclamp technique: reduction of extracellular osmolarity to 230 mmol/liter leads to a transient activation of K⁺ channels and a sustained activation of anion channels. The K⁺ channels are inwardly rectifying with a single-channel slope conductance of 56 ± 3 pS at –50 mV (cell negative) and of 29 ± 2 pS at 0 mV PD across the patch 150 mmol/liter K⁺ in pipette). The same channels are activated by an increase of intracellular calcium activity, as shown previously. The anion channels display a single-channel slope conductance of 41 ± 4 pS at –50 mV (cell negative) and of 25 ± 3 pS at 0 mV PD across the patch (150 mmol/liter Cl^– in pipette). The channel is anion selective and conducts both bicarbonate and chloride with a preference for bicarbonate. Its open probability is not affected by changing intracellular calcium from 0.1–10 mol/liter. The channels observed explain the effects of cell swelling on PD, ion selectivity and resistance of the cell membrane in MDCK cells.The authors gratefully acknowledge the valuable discussion with Drs. P. Deetjen, E. Wöll and F. Friedrich, the skilled technical assistance of G. Siber and S. David, and the excellent mechanic and electronic support by K.-H. Streicher, Ing. M. Hirsch and M. Plank. This study was supported by the Fonds zur Förderung der wissenschaftlichen Forschung, Grant No. P5813 and P6792M.     

Novel Madin Darby Canine Kidney cell clones exhibit unique phenotypes in response to morphogens

Summary Novel Madin Darby Canine Kidney cell clones were isolated. These cell clones exhibit differential responsiveness to inducers of tubule or cyst formation in collagen gel culture: hepatocyte growth factor or inducers of intracellular cAMP formation, respectively. In gel culture, clone OR93.22.D6 forms cysts and responds with morphological transformation to both hepatocyte growth factor and prostaglandin E₁, and is most typical of a previously described cell type except for its higher transepithelial electrical resistance. OR55.25.II20 forms tubules in culture, is unresponsive to hepatocyte growth factor, and forms prostaglandin-induced spherical cysts. OR55.28.V2 forms dense cell spheres under control conditions, is induced to form tubules by hepatocyte growth factor, and is unresponsive to prostaglandin. OR55.29 forms only cysts, and is the only clone to form domes in monolayer culture. Tubule formation induced by hepatocyte growth factor, in all clones except OR55.25.II20, is blocked by a neutralizing antibody. In defined medium, without hepatocyte growth factor or prostaglandin, OR55.25.II20 forms spontaneous tubules. This finding indicates that a tubulogenic serum factor is not responsible for the observed phenotype. Increasing prostaglandin concentrations lead to inhibition of tubule formation and increased cyst formation. This observation suggests that induction of intracellular cAMP formation negatively regulates tubule formation in these cells, and implies that cystogenesis may represent a “default pathway” for impaired tubulogenesis. These observations demonstrate that some facets of renal tubulogenesis may be independent of hepatocyte growth factor, and that care must be exercised when comparing biological studies utilizing different clones.     

Change of intracellular calcium of neural cells induced by extracellular ATP

Exposure of various neural cells to ATP increased intracellular Ca2+ and the production of inositol trisphosphate. The Ca2+ responses were also observed in the absence of extracellular Ca2+, suggesting that a part of Ca2+ mobilization took place from cytosolic storage. Since adenosine had no effect on intracellular Ca2+ increment, ATP appears to act through a P2-purinergic receptor. Islet-activating protein or pertussis toxin pretreatment hardly influenced the increase in intracellular Ca2+ and inositol trisphosphate production induced by ATP, suggesting that IAP-sensitive GTP-binding proteins do not play a practical role in this reaction.     

Effects of extracellular nucleotides on electrical properties of subconfluent Madin Darby canine kidney cells

ATP and ADP but not AMP lead to sustained hyperpolarization of Madin Darby canine kidney (MDCK) cells. The present study has been performed to test for an influence of other nucleotides on the potential difference across the cell membrane (PD) in subconfluent MDCK cells. PD has been continuously monitored with conventional microelectrodes during rapid exchange of extracellular fluid. Application of 1 mumol/1 UTP leads to a rapid (less than 2 s) hyperpolarization of the cell membrane by -17.0 +/- 0.4 mV (from -50.1 +/- 0.6 mV), a reduction of cell membrane resistance and an increase of the sensitivity of PD to alterations of extracellular potassium. The concentration needed for half maximal effect of UTP is approximately equal to 0.2 mumol/1. ITP is similarly effective, whereas UDP, GTP and GDP are less effective. Up to 1 mmol/1 UMP, GMP, TTP or CTP do not significantly alter PD. In calcium-free extracellular fluid the hyperpolarizing effect of UTP is blunted (-11.6 +/- 2.3 mV) and only transient. In conclusion, UTP similar to purine triphosphates hyperpolarizes MDCK cells by increasing the potassium conductance. The activation of potassium channels requires calcium, which is apparently recruited from both intra- and extracellular sources.     

High Yield Production of Influenza Virus in Madin Darby Canine Kidney (MDCK) Cells with Stable Knockdown of IRF7

Influenza is a serious public health problem that causes a contagious respiratory disease. Vaccination is the most effective strategy to reduce transmission and prevent influenza. In recent years, cell-based vaccines have been developed with continuous cell lines such as Madin-Darby canine kidney (MDCK) and Vero. However, wild-type influenza and egg-based vaccine seed viruses will not grow efficiently in these cell lines. Therefore, improvement of virus growth is strongly required for development of vaccine seed viruses and cell-based influenza vaccine production. The aim of our research is to develop novel MDCK cells supporting highly efficient propagation of influenza virus in order to expand the capacity of vaccine production. In this study, we screened a human siRNA library that involves 78 target molecules relating to three major type I interferon (IFN) pathways to identify genes that when knocked down by siRNA lead to enhanced production of influenza virus A/Puerto Rico/8/1934 in A549 cells. The siRNAs targeting 23 candidate genes were selected to undergo a second screening pass in MDCK cells. We examined the effects of knockdown of target genes on the viral production using newly designed siRNAs based on sequence analyses. Knockdown of the expression of a canine gene corresponding to human IRF7 by siRNA increased the efficiency of viral production in MDCK cells through an unknown process that includes the mechanisms other than inhibition of IFN-α/β induction. Furthermore, the viral yield greatly increased in MDCK cells stably transduced with the lentiviral vector for expression of short hairpin RNA against IRF7 compared with that in control MDCK cells. Therefore, we propose that modified MDCK cells with lower expression level of IRF7 could be useful not only for increasing the capacity of vaccine production but also facilitating the process of seed virus isolation from clinical specimens for manufacturing of vaccines.     

Mobilization of intracellular calcium by extracellular ATP and by calcium ionophores in the Ehrlich ascites-tumour cell

We have studied the changes of the intracellular free calcium concentration ([Ca2+]i) effected by external ATP, which induces formation of inositol trisphosphate, and by the divalent cation ionophores ionomycin and A23187. Both, ATP (40 microM) and ionophores (1-80 mumol/l cells ionomycin; 20-400 mumol/l cells A23187), produced a transient rise of [Ca2+]i which reached its maximum within 15-30 s and declined near resting values (about 200 nM) within 1-3 min. When the [Ca2+]i peak surpassed 500 nM a transient cell shrinkage due to simultaneous activation of Ca2+-dependent K+ and Cl- channels was also observed. The cell response was similar in medium containing 1 mM Ca2+ and in Ca2+-free medium, suggesting that the Ca mobilized to the cytosol comes preferently from the intracellular stores. Treatment with low doses of ionophore (1 mumol/l cells for ionomycin; 20 mumol/l cells for A23187) depressed the response to a subsequent treatment, either with ionophore or with ATP. Treatment with ATP did also inhibit the subsequent response to ionophore, but in this case the inhibition was dependent on time, the stronger the shorter the interval between both treatments. This result suggests that the permeabilization of Ca stores by ATP is transient and that Ca can be taken up again by the intracellular stores. Refill was most efficient when Ca2+ was present in the incubation medium. Addition of either ATP or ionomycin (1-25 mumol/l cells) to cells incubated in medium containing 1 mM Ca2+ decreased drastically the total cell Ca content during the following 3 min of incubation. In the case of ATP the total cell levels of Ca returned to the initial values after 7-15 min, whereas in the case of the ionophore they remained decreased during the whole incubation period. These results indicate that Ca released from the intracellular stores by either ATP or ionophores is quickly extruded by active mechanisms located at the plasma membrane. They also suggest that, under the conditions studied here, with 1 mM Ca2+ outside, the Ca-mobilizing effect of ionophores is stronger in endomembranes than in the plasma membrane.     

Mechanisms of miconazole-induced rise in cytoplasmic calcium concentrations in Madin Darby canine kidney (MDCK) cells

The effect of miconazole on intracellular calcium levels ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells was studied using fura-2 as the Ca2+ indicator. Miconazole increased [Ca2+]i dose-dependently at concentrations of 5-100 microM. The [Ca2+]i transient consisted of an initial rise, a gradual decay and an elevated plateau (220 s after addition of the drug). Removal of extracellular Ca2+ partly reduced the miconazole response. Mn2+ quench of fura-2 fluorescence confirmed that miconazole induced Ca2+ influx. The miconazole-sensitive intracellular Ca2+ store overlapped with that sensitive to thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ pump, because 20 microM miconazole depleted the thapsigargin (1 microM)-sensitive store, and conversely, thapsigargin abolished miconazole-induced internal Ca2+ release. Miconazole (20-50 microM) partly inhibited the capacitative Ca2+ entry induced by 1 microM thapsigargin, measured by depleting intracellular Ca2+ store in Ca(2+)-free medium followed by addition of 10 mM CaCl2. Miconazole induced capacitative Ca2+ entry on its own. Pretreatment with 0.1 mM La3+ partly inhibited 20 microM miconazole-induced Mn2+ quench of fura-2 fluorescence and [Ca2+]i rise, suggesting that miconazole induced Ca2+ influx via two pathways separable by 0.1 mM La3+. Miconazole-induced internal Ca2+ release was not altered when the cytosolic level of inositol 1,4,5-trisphosphate (IP3) was substantially inhibited by the phospholipase C inhibitor U73122.     

Kinetics of intracellular calcium release by inositol 1,4,5-trisphosphate and extracellular ATP in porcine cultured aortic endothelial cells.

Quantitative, time-resolved measurements have been made of intracellular Ca ion release by inositol 1,4,5-trisphosphate (InsP3) and extracellular ATP in porcine aortic endothelial cells in tissue culture. Intracellular free [Ca] was detected with the calcium dye fluo-3 and InsP3 released intracellularly by photolysis of 'caged' InsP3 in whole-cell voltage-clamped aortic endothelial cells. A rise of [Ca] was recorded at InsP3 concentrations greater than 0.2 microM. The timecourse at low InsP3 concentrations comprised a delay of mean 300 ms (range 266-330 ms), a peak in 2-3 s before declining with a half-time of 5-10 s. The delay and time-to-peak decreased with increasing concentrations of InsP3 over the range 0.2-5 microM. At very high concentrations of InsP3 (> 5 microM), the delay in the Ca response was short, always less than 20 ms. The results are consistent with a direct binding and gating action of InsP3 on the Ca channel of the cellular store. Following InsP3 action there is a refractoriness of the InsP3 Ca release process which recovers with a timecourse of half-time about 30 s. A comparison can be made between the timecourse of InsP3 and extracellular ATP actions. High concentrations of ATP (500 microM) acted with a delay of mean 1.8 s (range 1.2-2.5 s), whereas even moderate concentrations of InsP3 acted much more quickly, suggesting that there are slow coupling steps before or during the production of InsP3 in response to extracellular ATP. Both ATP and InsP3 evoked an increase in membrane conductance to K+, probably via Ca.     

Undifferentiated HL60 cells respond to extracellular ATP and UTP by stimulating phospholipase C activation and exocytosis

We have recently characterised the presence of a Ca2(+)-mobilising receptor for ATP which stimulates exocytosis in differentiated HL60 cells. Here we demonstrate that the undifferentiated HL60 cells also respond to extracellular ATP by stimulating an increase in inositol phosphates and exocytosis. Of the nucleotides (ATP, UTP, ITP, ATP gamma S, AppNHp, XTP, CTP, GTP, 8-Br-ATP and GTP gamma S) that were active in stimulating inositol phosphate formation, only UTP, ATP, ITP, ATP gamma S and AppNHp were active in stimulating secretion. On differentiation, the extent of secretion due to the purinergic agonists ATP, ITP, ATP gamma S and AppNHp remained unchanged whilst secretion due to UTP, a pyrimidine, was substantially increased. These results indicate that the effect of ATP and UTP may be mediated via separate purinergic and pyrimidinergic receptors, respectively.     

Enhancement of differentiation induction of mouse myelomonocytic leukemic cells by extracellular ATP

We examined the effect of ATP on the terminal differentiation of mouse myelomonocytic leukemic M1 cells to macrophages. Although ATP alone did not induce M1 cell differentiation, addition of ATP with the differentiation inducer, interleukin 6 (IL-6), enhanced the induction of differentiation by IL-6 about twofold. Comparison among several adenine nucleotides revealed that the order of effectiveness on differentiation enhancement was ATP > ADP > AMP ≥ adenosine. Using reactive blue 2, a P2 receptor antagonist, we confirmed that the effect of ATP on the stimulation of differentiation was mediated through the P2 purinergic receptor. Examination of cytosolic [Ca2+] elevation by ATP and comparison of potency of differentiation enhancement among several ATP analogs demonstrated that the effect of differentiation enhancement was mediated through P2y purinergic receptors expressed on M1 cell surface. Within 3 h of exposure, ATP alone slightly increased the expression of differentiation-related immediate early response genes, c-myc and JunB, and ATP also enhanced the IL-6–induced expression of these genes. Induction of JunB expression by ATP analogs correlated with their potency of differentiation enhancement, which suggested that induction of JunB by ATP is one of signaling pathways involved in the exertion of its differentiation-enhancing effect. © 1994 wiley-Liss, Inc.     

Induction of intracellular ATP synthesis by extracellular ferricyanide in human red blood cells

Summary Human red blood cells rapidly convert extracellular ferricyanide into extracellular ferrocyanide. The reaction is enhanced by the addition of a substrate, adenosine. This increase of the rate of reaction is abolished by iodoacetate. The results indicate there is a flow of electrons across the membrane of metabolizing red blood cells. The reduction of extracellular ferricyanide is accompanied by the formation of intracellular ATP. The effect of an uncoupler and of inhibitors of oxidative phosphorylation on this reaction was studied under conditions where the natural rate of ATP synthesis was slightly reduced by 10^–4 moles/liter iodoacetate. ATP formation was found to be inhibited by DNP, cyanide, and, to a lesser extent, by azide. Amytal is ineffective. Ferrocyanide enhances ATP breakdown. The action of DNP requires the presence of the cell membrane. It can probably not be related to a stimulation of the membrane ATPase of Laris and Letchworth, nor can it be explained on the basis of Mitchell's chemiosmotic hypothesis by effects on the passive permeability of the erythrocyte membrane to H⁺ or alkali ions. In contrast to methylene blue and other oxidants, ferricyanide does not stimulate oxygen consumption in adult red blood cells.     

2-O-methyl PAF as a Ca2+ mobilizer in Madin Darby canine kidney cells

In Madin-Darby canine kidney (MDCK) cells, the effect of 2-O-methyl PAF, an inactive analogue of platelet activating factor (PAF), on intracellular Ca2+ concentration ([Ca2+]i) was measured by using the Ca2+-sensitive fluorescent dye fura-2. 2-O-methyl PAF (> or = 15 microM) caused a rapid rise of [Ca2+]i in a concentration-dependent manner. 2-O-methyl PAF-induced [Ca2+]i rise was partly reduced by removal of extracellular Ca2+. 2-O-methyl PAF-induced extracellular Ca2+ influx was also suggested by Mn2+ influx-induced fura-2 fluorescence quench. The 2-O-methyl PAF-induced Ca2+ influx was blocked by nifedipine, verapamil and diltiazem. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic [Ca2+]i rise, after which 2-O-methyl PAF failed to increase [Ca2+]i; also, pretreatment with 2-O-methyl PAF depleted thapsigargin-sensitive Ca2+ stores. U73122, an inhibitor of phospholipase C, abolished ATP (but not 2-O-methyl PAF)-induced [Ca2+]i rise. These findings suggest that 2-O-methyl PAF evokes a rapid increase in [Ca2+]i in renal tubular cells by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release.     

Mechanisms by which extracellular ATP and UTP stimulate the release of prostacyclin from bovine pulmonary artery endothelial cells.

Extracellular ATP and UTP caused increases in the concentration of cytoplasmic free calcium ([Ca2+]i) and the intracellular level of inositol 1,4,5-trisphosphate (IP3), a second messenger for calcium mobilization, prior to the release of prostacyclin (PGI2) from cultured bovine pulmonary artery endothelial (BPAE) cells. The agonist specificity and dose-dependence were similar for nucleotide-mediated increases in IP3 levels, [Ca2+]i and PGI2 release. An increase in [Ca2+]; and PGI2 release was observed after addition of ionomycin, a calcium ionophore, to BPAE cells incubated in a calcium-free medium. The addition of ATP to the ionomycin-treated cells caused no further increase in [Ca2+]i or PGI2 release. The inability of ATP to cause an increase in [Ca2+]i or PGI2 release in ionomycin-treated cells was apparently due to the ionomycin-dependent depletion of intracellular calcium stores since the subsequent addition of extracellular calcium caused a significant increase in both [Ca2+]i and PGI2 release. Introduction of BAPTA, a calcium buffer, into BPAE cells inhibited ATP-mediated increases in [Ca2+]i and PGI2 release, further evidence that PGI2 release is dependent upon an increase in [Ca2+]i. The increase in [Ca2+]i elicited by ATP apparently caused the activation of a calmodulin-dependent phospholipase A2 since trifluoperazine, an inhibitor of calmodulin, and quinacrine, an inhibitor of phospholipase A2, prevented the stimulation of PGI2 release by ATP. Furthermore, ATP caused the specific hydrolysis of [14C]arachidonyl-labeled phosphatidylcholine and the generation of free arachidonic acid, the rate-limiting substrate for PGI2 synthesis, prior to the release of PGI2 from BPAE cells. These findings suggest that the increase in PGI2 release elicited by ATP and UTP is at least partially dependent upon a phospholipase C-mediated increase in [Ca2+]i and the subsequent activation of a phosphatidylcholine-specific phospholipase A2. ATP analogs modified in the adenine base or phosphate moiety caused PGI2 release with a rank order of agonist potency of adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) greater than 2-methylthioATP (2-MeSATP) greater than ATP, whereas alpha, beta methyleneATP and beta, gamma methyleneATP had no effect on PGI2 release.     

Novel effects of clotrimazole on Ca2+ signaling in Madin Darby canine kidney cells

The effect of clotrimazole on Ca2+ signaling in Madin Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca2+ indicator. Clotrimazole (1-30 microM) induced a concentration-dependent [Ca2+]i increase. The [Ca2+]i increase comprised an initial rise and a slow decay. External Ca2+ removal partly inhibited the Ca2+ signals by reducing both the initial rise and the decay phase, indicating that clotrimazole triggered both Ca2+ influx and Ca2+ release. Pretreatment with 30 microM clotrimazole in Ca2+-free medium abolished the Ca2+ release induced by thapsigargin (1 microM), an endoplasmic reticulum Ca2+ pump inhibitor, and conversely, pretreatment with thapsigargin prevented clotrimazole from releasing more Ca2+. This suggests that the thapsigargin-sensitive Ca2+ store is the source of clotrimazole-induced Ca2+ release. Clotrimazole (10 microM) triggered Mn2+ quench of fura-2 fluorescence which was partly inhibited by 1 mM La3+. Addition of 3 mM Ca2+ induced a [Ca2+]i increase after preincubation with 10 microM clotrimazole in Ca2+-free medium, indicating that clotrimazole activated capacitative Ca2+ entry. However, 10 and 30 microM clotrimazole inhibited 1 microM thapsigargin-induced capacitative Ca2+ entry by 21% and 74%, respectively. Pretreatment with 40 microM aristolochic acid to inhibit phospholipase A2 reduced 30 microM clotrimazole-induced Ca2+ release by 51%, but inhibiting phospholipase C with 2 microM U73122 had little effect. This implies that clotrimazole induces Ca2+ release in an IP3-independent manner, which could be modulated by phospholipase A2-coupled events.     

The priming effect of extracellular UTP on human neutrophils: Role of calcium released from thapsigargin-sensitive intracellular stores

P2Y₂ receptors, which are equally responsive to ATP and UTP, can trigger intracellular signaling events, such as intracellular calcium mobilization and mitogen-activated protein (MAP) kinase phosphorylation in polymorphonuclear leukocytes (PMN). Moreover, extracellular nucleotides have been shown to prime chemoattractant-induced superoxide production. The aim of our study was to investigate the mechanism responsible for the priming effect of extracellular nucleotides on reactive oxygen species (ROS) production induced in human neutrophils by two different chemoattractants: formyl-methionyl-leucyl-phenylalanine (fMLP) and interleukin-8 (IL-8). Nucleotide-induced priming of ROS production was concentration- and time-dependent. When UTP was added to neutrophil suspensions prior to chemoattractant, the increase of the response reached the maximum at 1 min of pre-incubation with the nucleotide. UTP potentiated the phosphorylation of p44/42 and p38 MAP kinases induced by chemoattractants, however the P2 receptor-mediated potentiation of ROS production was still detectable in the presence of a SB203580 or U0126, supporting the view that MAP kinases do not play a major role in regulating the nucleotide-induced effect. In the presence of thapsigargin, an inhibitor of the ubiquitous sarco-endoplasmic reticulum Ca²⁺-ATPases in mammalian cells, the effect of fMLP was not affected, but UTP-induced priming was abolished, suggesting that the release of calcium from thapsigargin-sensitive intracellular stores is essential for nucleotide-induced priming in human neutrophils.     

Carbonic anhydrase activity in Madin Darby canine kidney cells. Evidence for intercalated cell properties

Madin Darby canine kidney (MDCK) renal epithelial cell cultures have been investigated with respect to their potency to express carbonic anhydrase activity using histochemical methods. Acetazolamide inhibitable carbonic anhydrase activity could be detected in the cytoplasmic compartment as well as in the apical membrane of cells when grown on solid culture supports. Cells forming domes in MDCK monolayers exhibit the highest histochemically detectable enzyme activity. The attempt to subculture clonal cell lines from MDCK monolayer cultures resulted in the establishment of 5 clones, slightly different with respect to size and shape of cells and their potency to form domes. Scanning electron microscopy ensured the identification of one clone (1A4), which distinctly differed from the others with respect to the apical membrane architecture. Co-localization of peanut agglutinin and carbonic anhydrase activity at the plasma membrane always revealed a combined occurrence of enzyme reactivity and lectin binding in the apical membrane domain. Both, lectin binding and carbonic anhydrase activity were distinctly more intense in plasma membrane regions equipped with microvilli. From the results it is concluded that MDCK cells in tissue culture retained properties of intercalated cells of the nephron collecting duct segment.     

Alterations of intracellular calcium concentration in mice neuroblastoma cells by electrical field and UVA

By using a FURA2 ratio imaging method, the intracellular free calcium concentration was investigated in cultured mice neuroblastoma cells under the influence of an amplitude-modulated (AM) field (5 kHz sine wave AM 16 Hz sinusoidal 800 V/m and 80 V/m), as well as of electric field pulses (300-ms unipolar pulses of 1000 V/m and 800 V/m, 5 pulses during 10 s and 50 pulses during 100 s). An increase in free intracellular calcium was found in about 50% of cells after field application, whereas in control experiments only about 20% of the cells showed similar increases. However, this effect depended on the amount of UV irradiation used for excitation of FURA2 fluorescence. Experiments with 1/30 to former total illumination no longer demonstrated an increase in control cells or in cells treated with AM fields. The number of cells showing calcium increase after the application of pulsed fields was reduced significantly. Therefore, the UV light itself, applied as double flashes for the fluorescence measurement, activates the cellular calcium regulation. These findings offer a possible explanation for the low reproducibility of field effects found in different laboratories, in which investigations were performed with different equipment using different intensities of UV excitation. Bioelectromagnetics 18:595–597, 1997. © 1997 Wiley-Liss, Inc.