Resistivity of Red Blood Cells Against High-Intensity, Short-Duration Electric Field Pulses Induced by Chelating Agents

The interaction of human red blood cells (RBCs) with diethylenetriamine-pentaacetic acid (DTPA) or its Gd-complex (Magnevist, a widely used clinical magnetic resonance contrast agent containing free DTPA ligands) led to the following, obviously interrelated phenomena. (i) Both compounds protected erythrocytes against electrohemolysis in isotonic solutions caused by a high-intensity DC electric field pulse. (ii) The inhibition of electrohemolysis was observed only when cells were electropulsed in low-conductivity solutions. (iii) The uptake of Gd-DTPA by electropulsed RBCs was relatively low. (iv) (Gd-) DTPA reduced markedly deformability of erythrocytes, as revealed by the electrodeformation experiments using high-frequency electric fields. Taken together, the results indicate that (Gd-) DTPA produce stiffer erythrocytes that are more resistant to electric field exposure. The observed effects of the chelating agents on the mechanical properties and the electropermeabilization of RBCs must have an origin in molecular changes of the bilayer or membrane-coupled cytoskeleton, which, in turn, appear to result from an alteration of the ionic equilibrium (e.g., Ca²⁺ sequestration) in the vicinity of the cell membrane. Received: 19 January 1999/Revised: 1 April 1999     

Reversible Electropermeabilization of Mammalian Cells by High-Intensity, Ultra-Short Pulses of Submicrosecond Duration

Mouse myeloma cells were electropermeabilized by single square-wave electric pulses with amplitudes of up to ∼150 kV/cm and durations of 10–100 nsec. The effects of the field intensity, pulse duration and medium conductivity on cell viability and field-induced uptake of molecules were analyzed by quantitative flow cytometry using the membrane-impermeable fluorescent dye propidium iodide as indicator molecule. Despite the extremely large field strengths, the majority of cells survived the exposure to ultra-short field pulses. The electrically induced dye uptake increased markedly with decreasing conductivity of the suspending medium. We assigned this phenomenon to the transient electrodeformation (stretching) force that assumes its maximum value if cells are suspended in low-conductivity media, i.e., if the external conductivity σ_e is smaller than that of the cytosol σ_i. The stretching force vanishes when σ_e is equal to or larger than σ_i. Due to their capability of delivering extremely large electric fields, the pulse power systems used here appear to be a promising tool for the electropermeabilization of very small cells and vesicles (including intracellular organelles, liposomes, etc.). Received: 15 May 2001/Revised: 20 July 2001     

Treatment of Cells with Detergent Activates Caspases and Induces Apoptotic Cell Death

Due to their amphiphilic properties, detergents readily disrupt cellular membranes and cause rapid cytolysis. In this study we demonstrate that treatment of cells with sublytic concentrations of detergents such as Triton X-100, Nonidet P-40, n-octylglucoside and the bile salt sodium deoxycholate induce typical signs of apoptosis including DNA fragmentation and cleavage of poly(ADP-ribose) polymerase molecules. The detergent concentration required for apoptosis was below the critical micellar concentration. Induction of apoptosis was not restricted to human cells but similarly occurred in a variety of other vertebrate cell lines. Unstimulated peripheral blood mononuclear cells were susceptible to apoptosis induction by detergent suggesting that apoptosis in this circumstance is not mediated by CD95. Cell death was not due to influx of calcium from the medium. Apoptosis was blocked and cytolysis prevented by treatment with peptide inhibitors of caspases. These findings suggest a process of apoptosis that is initiated upon nonspecific alterations at the cell membrane level. Physiologic correlates of this process still have to be defined. Received: 12 November 1999/Revised: 6 March 2000     

The Effect of Electrical Deformation Forces on the Electropermeabilization of Erythrocyte Membranes in Low- and High-Conductivity Media

Electrical breakdown of erythrocytes induces hemoglobin release which increases markedly with decreasing conductivity of the pulse medium. This effect presumably results from the transient, conductivity-dependent deformation forces (elongation or compression) on the cell caused by Maxwell stress. The deformation force is exerted on the plasma membrane of the cell, which can be viewed as a transient dipole induced by an applied DC electric field pulse. The induced dipole arises from the free charges that accumulate at the cell interfaces via the Maxwell-Wagner polarization mechanism. The polarization response of erythrocytes to a DC field pulse was estimated from the experimental data obtained by using two complementary frequency-domain techniques. The response is very rapid, due to the highly conductive cytosol. Measurements of the electrorotation and electrodeformation spectra over a wide conductivity range yielded the information and data required for the calculation of the deformation force as a function of frequency and external conductivity and for the calculation of the transient development of the deformation forces during the application of a DC-field pulse. These calculations showed that (i) electric force precedes and accompanies membrane charging (up to the breakdown voltage) and (ii) that under low-conductivity conditions, the electric stretching force contributes significantly to the enlargement of ``electroleaks' in the plasma membrane generated by electric breakdown. Received: 12 December 1997/Revised: 13 March 1998     

In Vitro Selection of a Deoxyribozyme That Can Utilize Multiple Substrates

Deoxyribozymes that could catalyze the formation of an internucleotide phosphorothioester linkage were selected from a random sequence pool. During the course of the selection, the pool was successively challenged with five oligonucleotide substrates, each of which terminated in the same hexanucleotide sequence. Selected deoxyribozyme ligases could use all five substrates, albeit to different degrees, and appeared to form secondary structures that allow differential pairing between the deoxyribozyme and each substrate. These results suggest that early replicases may have been able to bind a variety of oligonucleotide substrates while catalyzing ligation via a common junction. Received: 12 December 2000 / Accepted: 28 June 2001     

Carotenoids Induce Apoptosis in the T-lymphoblast Cell Line Jurkat E6.1

Epidemiologically, a high-carotenoid intake via a fruit- and vegetable-rich diet is associated with a decreased risk of various forms of cancer. The mechanisms by which carotenoids exert this protective effect are controversial. In this study, we examined the potency of a range of carotenoids commonly found in human plasma to induce apoptosis in Jurkat E6.1 malignant T-lymphoblast cells. At a concentration of 20 &#119 M, the order of potency to induce apoptosis after 24 h was: &#103 -carotene > lycopene > lutein> &#103 -cryptoxanthin=zeaxanthin. Canthaxanthin failed to induce apoptosis under these conditions. &#103 -Carotene induced apoptosis in a time- and concentration-dependent manner with a lowest effective concentration of about 3 &#119 M. Pre-conditioning of &#103 -carotene for 72 h destroyed its pro-apoptotic activity almost completely, whereas degradation for 6 h or less did not, indicating that either &#103 -carotene itself and/or an early degradation product of &#103 -carotene are the death-inducing compounds. Apoptosis induced by &#103 -carotene was characterized by chromatin condensation and nuclear fragmentation, DNA degradation, PARP cleavage and caspase-3 activation. The antioxidant BO-653 inhibited the degradation of &#103 -carotene in vitro and significantly increased its cytotoxicity, indicating that a pro-oxidant effect of &#103 -carotene is unlikely to cause its pro-apoptotic activity. The induction of apoptosis in transformed cells by carotenoids may explain their protective effect against cancer formation in humans. Possible pathways for induction of apoptosis by carotenoids are discussed.     

The Chloride Conductance of Tight Junctions of Rat Ileum Can Be Increased by cAMP But Not by Carbachol

It is well known, that in mammalian small intestine, cAMP increases Cl⁻ permeability of the apical membrane of enterocytes as part of its secretory action. Paradoxically, this is usually accompanied by an increase of the transepithelial resistance. In the present study we report that in the presence of bumetanide (to block basolateral Cl⁻ uptake) cAMP always decreased the transepithelial resistance. We examined whether this decrease in resistance was due to a cAMP-dependent increase of the paracellular electrolyte permeability in addition to the increase of the Cl⁻ permeability of the apical cell membrane. We used diffusion potentials induced by serosal replacement of NaCl, and transepithelial current passage to evoke transport number effects. The results revealed that cAMP (but not carbachol) could increase the Cl⁻ permeability of the tight junctions in rat ileum. Moreover, we observed a variation in transepithelial resistance of individual tissue preparations, inversely related to the cation selectivity of the tissue, suggesting that Na⁺ permeability of the tight junctions can vary between preparations. Received: 7 September 1996/Revised: 5 November 1996     

Ancient Gene Duplication and Differential Gene Flow in Plastid Lineages: The GroEL/Cpn60 Example

Cryptomonads, small biflagellate algae, contain four different genomes. In addition to the nucleus, mitochondrion, and chloroplast is a fourth DNA-containing organelle the nucleomorph. Nucleomorphs result from the successive reduction of the nucleus of an engulfed phototrophic eukaryotic endosymbiont by a secondary eukaryotic host cell. By sequencing the chloroplast genome and the nucleomorph chromosomes, we identified a groEL homologue in the genome of the chloroplast and a related cpn60 in one of the nucleomorph chromosomes. The nucleomorph-encoded Cpn60 and the chloroplast-encoded GroEL correspond in each case to one of the two divergent GroEL homologues in the cyanobacterium Synechocystis sp. PCC6803. The coexistence of divergent groEL/cpn60 genes in different genomes in one cell offers insights into gene transfer from evolving chloroplasts to cell nuclei and convergent gene evolution in chlorophyll a/b versus chlorophyll a/c/phycobilin eukaryotic lineages. Received: 24 April 1998 / Accepted: 12 June 1998     

The Sensitivity of the Human Kv1.3 (hKv1.3) Lymphocyte K+ Channel to Regulation by PKA and PKC is Partially Lost in HEK 293 Host Cells

cDNA encoding the full-length hKv1.3 lymphocyte channel and a C-terminal truncated (Δ459-523) form that lacks the putative PKA Ser⁴⁶⁸ phosphorylation site were stably transfected in human embryonic kidney (HEK) 293 cells. Immunostaining of the transfected cells revealed a distribution at the plasma membrane that was uniform in the case of the full-length channel whereas clustering was observed in the case of the truncated channel. Some staining within the cell cytoplasm was found in both instances, suggesting an active process of biosynthesis. Analyses of the K⁺ current by the patch-clamp technique in the whole cell configuration showed that depolarizing steps to 40 mV from a holding potential (HP) of −80 mV elicited an outward current of 2 to 10 nA. The current threshold was positive to −40 mV and the current amplitude increased in a voltage-dependent manner. The parameters of activation were −5.7 and −9.9 mV (slope factor) and −35 mV (half activation, V _0.5) in the case of the full-length and truncated channels, respectively. The characteristics of the inactivation were 14.2 and 24.6 mV (slope factor) and −17.3 and −39.0 mV (V _0.5) for the full-length and truncated channels, respectively. The activation time constant of the full-length channel for potentials ranging from −30 to 40 mV decreased from 18 to 12 msec whereas the inactivation time constant decreased from 6600 msec at −30 mV to 1800 msec at 40 mV. The unit current amplitude measured in cells bathing in 140 mm KCl was 1.3 ± 0.1 pA at 40 mV, the unit conductance, 34.5 pS and the zero current voltage, 0 mV. Both forms of the channels were inhibited by TEA, 4-AP, Ni²⁺ and charybdotoxin. In contrast to the native (Jurkat) lymphocyte Kv1.3 channel that is fully inhibited by PKA and PKC, the addition of TPA resulted in 34.6 ± 7.3% and 38.7 ± 9.4% inhibition of the full-length and the truncated channels, respectively. 8-BrcAMP induced a 39.4 ± 5.4% inhibition of the full-length channel but had no effect (8.6 ± 8.3%) on the truncated channel. Cell dialysis with alkaline phosphatase had no effects, suggesting that the decreased sensitivity of the transfected channels to PKA and PKC was not due to an already phosphorylated channel. Patch extract experiments suggested that the hKv1.3 channel was partially sensitive to PKA and PKC. Cotransfecting the Kvβ1.2 subunit resulted in a decrease in the value of the time constant of inactivation of the full-length channel but did not modify its sensitivity to PKA and PKC. The cotransfected Kvβ2 subunit had no effects. Our results indicate that the hKv1.3 lymphocyte channel retains its electrophysiological characteristics when transfected in the Kvβ-negative HEK 293 cell line but its sensitivity to modulation by PKA and PKC is significantly reduced. Received: 18 June 1997/Revised: 7 October 1997     

Branch Length Heterogeneity Leads to Nonindependent Branch Length Estimates and Can Decrease the Efficiency of Methods of Phylogenetic Inference

Branch length estimates play a central role in maximum-likelihood (ML) and minimum-evolution (ME) methods of phylogenetic inference. For various reasons, branch length estimates are not statistically independent under ML or ME. We studied the response of correlations among branch length estimates to the degree of among-branch length heterogeneity (BLH) in the model (true) tree. The frequency and magnitude of (especially negative) correlations among branch length estimates were both shown to increase as BLH increases under simulation and analytically. For ML, we used the correct model (Jukes–Cantor). For ME, we employed ordinary least-squares (OLS) branch lengths estimated under both simple p-distances and Jukes–Cantor distances, analyzed with and without an among-site rate heterogeneity parameter. The efficiency of ME and ML was also shown to decrease in response to increased BLH. We note that the shape of the true tree will in part determine BLH and represents a critical factor in the probability of recovering the correct topology. An important finding suggests that researchers cannot expect that different branches that were in fact the same length will have the same probability of being accurately reconstructed when BLH exists in the overall tree. We conclude that methods designed to minimize the interdependencies of branch length estimates (BLEs) may (1) reduce both the variance and the covariance associated with the estimates and (2) increase the efficiency of model-based optimality criteria. We speculate on possible ways to reduce the nonindependence of BLEs under OLS and ML. Received: 9 March 1999 / Accepted: 4 May 1999     

Current Intraspecific Dynamics of Sequence Evolution Differs from Long-Term Trends and Can Account for the AT-Richness of Honeybee Mitochondrial DNA

The very high AT content of hymenopteran mtDNA has warranted speculation about nucleotide substitution processes in this group. Here we investigate the pattern of honeybee, Apis mellifera, mtDNA nucleotide polymorphisms inferred from phylogeny in terms of differences between the ATPase6, COI, COII, COIII, cytochrome b, and ND2 genes and strand asymmetry in mutation rates. The observed transition/transversion ratios and the distribution of nonsynonymous substitutions between regions differed significantly. The pattern of differences between genes leading to these heterogeneities (the ATPase6 and COIII genes group apart from the rest) differed markedly from that predicted on the basis of long-term evolutionary change and may indicate differences between current and long-term dynamics of sequence evolution. Also, there is strong strand asymmetry in substitutions, which probably results in a mutability of G and C sufficiently high to account for the AT-richness of honeybee mtDNA. Received: 21 October 1998 / Accepted: 27 January 1999     

Molecular Regulation of Renal Phosphate Transport

Received: 18 April 1996/Revised: 26 June 1996     

Effect of Dimethyl Sulfides on the Induction of Apoptosis in Human Leukemia Jurkat Cells and HL-60 Cells

Organosulfur compounds have been established to possess anticancer effects. To provide a better understanding of the biological function of dimethyl sulfides, dimethyl monosulfide (Me₂S), dimethyl disulfide (Me₂S₂), dimethyl trisulfide (Me₂S₃) and dimethyl tetrasulfide (Me₂S₄) were used as experimental materials to investigate their effects on apoptosis induction in human leukemia Jurkat cells and HL-60 cells. Treatment with 20 μM dimethyl sulfides for 24 h decreased the viability of both cells. The cell viability-reducing effect of these sulfides was in the following order: Me₂S₄ ≈ Me₂S₃ > Me₂S₂ ≈ Me₂S for Jurkat cells and Me₂S₄ > Me₂S₃ > Me₂S₂ ≈ Me₂S for HL-60 cells. Me₂S₃ and Me₂S₄ significantly induced DNA fragmentation and caspase-3 activation. The addition of GSH or NAC completely suppressed the sulfide-induced apoptosis. Our results indicate that dimethyl sulfides with a larger number of sulfur atoms more strongly induced apoptosis in both human leukemia cells via ROS production and caspase-3 activation.     

Characterization of a serine protease-mediated cell death program activated in human leukemia cells

Tightly controlled proteolysis is a defining feature of apoptosis and caspases are critical in this regard. Significant roles for non-caspase proteases in cell death have been highlighted. Staurosporine causes a rapid induction of apoptosis in virtually all mammalian cell types. Numerous studies demonstrate that staurosporine can activate cell death under caspase-inhibiting circumstances. The aim of this study was to investigate the proteolytic mechanisms responsible for cell death under these conditions. To that end, we show that inhibitors of serine proteases can delay cell death in one such system. Furthermore, through profiling of proteolytic activation, we demonstrate, for the first time, that staurosporine activates a chymotrypsin-like serine protease-dependent cell death in HL-60 cells independently, but in parallel with the caspase controlled systems. Features of the serine protease-mediated system include cell shrinkage and apoptotic morphology, regulation of caspase-3, altered nuclear morphology, generation of an endonuclease and DNA degradation. We also demonstrate a staurosporine-induced activation of a putative 16 kDa chymotrypsin-like protein during apoptosis.     

Apoptosis of human leukemic HL—60 cells induced to differentiate by treatment with RA or DMSO

In present study,we studied the effect of all-trans retinoic acid(ATRA)and dimethylsulfoxide(DMSO)on the induction of apoptosis in HL-60 cell line.Based on morphological changes by Hochest 33342 staining and identification of internuclesomal NDA celeavage by gel electrophoresis,we observed aberrant nuclear chromatin condensation and ladder-like pattern of DNA degradation. Using Flow Cytometric method.We found sub-G1 peak in RA-treated HL-60 cells starting 5 to 6d after the initiation of the treatment However,Such an obvious apoptotic peak was not identified in DMSO-differentiated cells.Combining the research accomplished before.our study approves further that apoptosis could be a common mode of death of terminally differentiated HL-60 cells.     

Protection from Cell Death by mcl-1 Is Mediated by Membrane Hyperpolarization Induced By K+ Channel Activation

Mcl-1, a member of the Bcl-2 family, has been identified as an inhibitor of apoptosis induced by anticancer agents and radiation in myeloblastic leukemia cells. The molecular mechanism underlying this phenomenon, however, is not yet understood. In the present study, we report that hyperpolarization of the membrane potential is required for prevention of mcl-1 mediated cell death in murine myeloblastic FDC-P1 cells. In cells transfected with mcl-1, the membrane potential, measured by the whole-cell patch clamp, was hyperpolarized more than −30 mV compared with control cells. The membrane potential was repolarized by increased extracellular K⁺ concentration (56 mV per 10-fold change in K⁺ concentration). Using the cell-attached patch-clamp technique, K⁺ channel activity was 1.7 times higher in mcl-1 transfected cells (NP _o = 22.7 ± 3.3%) than control cells (NP _o = 13.2 ± 1.9%). Viabilities of control and mcl-1 transfected cells after treatment with the cytotoxin etoposide (20 μg/ml), were 37.9 ± 3.9% and 78.2 ± 2.0%, respectively. Suppression of K⁺ channel activity by 4-aminopyridine (4-AP) before etoposide treatment significantly reduced the viability of mcl-1 transfected cells to 49.0 ± 4.6%. These results indicate that as part of the prevention of cell death, mcl-1 causes a hyperpolarization of membrane potential through activation of K⁺ channel activity. Received: 30 March 1999/Revised: 20 July 1999     

The Complete Mitochondrial DNA Sequence of the Domestic Sheep (Ovis aries) and Comparison with the Other Major Ovine Haplotype

The complete mitochondrial DNA (mtDNA) molecule of the domestic sheep, Ovis aries, was sequenced, together with part of the mtDNA of a specimen representing the other major O. aries haplotype group. The length of the complete ovine mtDNA presented is 16,616 nucleotides (nt). This length is not absolute, however, due to heteroplasmy caused by the occurrence of different numbers of a 75-nt-long tandem repeat in the control region. The sequence data were included in analyses of intraspecific ovine molecular differences, molecular comparisons with bovine mtDNAs, and phylogenetic analyses based on complete mtDNAs. The comparisons with bovine mtDNAs were based on the central domains of the ovine control regions, representing both major ovine haplotype groups, and the corresponding domains of Bos taurus and B. indicus. The comparisons showed that the difference between the bovids was 1.4 times greater than the intraspecific ovine difference. These findings suggest that the strains of wild sheep from which domestic sheep originated were more closely related than were the B. primigenius subspecies which gave rise to B. indicus and B. taurus cattle. Datings based on complete mtDNAs suggest that the bovine and ovine lineages diverged about 30 million years before present. This dating is considerably earlier than that proposed previously. Received: 5 September 1997 / Accepted: 5 May 1998     

Cell signaling pathways mediating epidermal growth factor stimulation of Na:K:2Cl cotransport activity in rabbit corneal epithelial cells

We characterized the signaling and ion transport pathways that mediate epidermal growth factor receptor physiological control in SV40-immortalized rabbit corneal epithelial cells (tRCEC). Our evaluation employed single-cell fluorescence imaging to measure the intracellular [Na⁺]_i in these cells loaded with the Na⁺ sensitive dye, SBFI. EGF (1 to 5 ng/ml) transiently increased [Na⁺]_i from 10 mm to as much as 35 mm after 25 min, which was followed by a decline towards its control value. These increases waned at higher EGF concentrations up to 50 ng/ml. Both inhibition of EGF receptor-linked tyrosine kinase activity (50 μm RG-13022) and cPLA₂ activity (10 μm AACOCF₃) obviated EGF-induced increases in [Na⁺]_i. In contrast, PGE₂ (10 μg/ml) and cAMP (2 mm) increased [Na⁺]_i by 25 mm. Inhibition of NKCC activity through exposure to either Cl-free Ringers or 300 μm furosemide in NaCl Ringers eliminated EGF-induced increases in [Na⁺]_i. Similarly, EGF failed to increase [Na⁺]_i following inhibition of: 1) PKA activity (10 μm H-89); 2) Erk1/2 (15 μm PD98059) or 3) p38 (15 μm SB203580) activity. Stimulation protein kinase C activity (0.1 μm PMA) transiently increased [Na⁺]_i followed by a decline towards its baseline value. EGF-induced increases in [Na⁺]_i were unaltered by inhibition of K⁺ conductance (100 μm 4-AP). Taken together, EGF stimulates Erk1/2; p38 and cPLA₂ activity. Their stimulation increases PGE₂ and cAMP levels resulting in PKA and NKCC activation. Received: 18 December 2000/Revised: 24 May 2001     

Calcium-dependent Enhancement of Depletion-activated Calcium Current in Jurkat T Lymphocytes

We have obtained evidence that the Ca²⁺-selective current activated by Ca²⁺ store depletion (Ca²⁺ release-activated Ca²⁺ current; I _crac) in Jurkat T lymphocytes is augmented in a time-dependent manner by Ca²⁺ itself. Whole cell patch clamp experiments employed high cytosolic Ca²⁺-buffering conditions to passively deplete Ca²⁺ stores. Rapidly switching to nominally Ca²⁺-free extracellular buffer instantaneously reduced I _crac measured at −100 mV to leak current level. Unexpectedly, readmission of 2 mm Ca²⁺ instantaneously restored only 38 ± 5% (mean ±sem; n = 9) of the full I _crac amplitude. The remainder reappeared in a monotonic time-dependent manner over 10 to 20 sec. Rapid vs. slow intracellular Ca²⁺ chelators did not alter this process, and inorganic I _crac blockers did not regenerate it, arguing against an intracellular site of action. The effect was specific to Ca²⁺: introduction of the permeant ions, Ba²⁺ or Sr²⁺, failed to invoke time-dependent I _crac reappearance. Moreover, equimolar substitution of Ba²⁺ for Ca²⁺ initially produced Ba²⁺ current of similar magnitude to the full Ca²⁺ current, but the Ba²⁺ current decayed monotonically to <50% of its initial amplitude in <20 sec. Conversely, return to Ca²⁺ produced a time-dependent increase in I _crac to its larger Ca²⁺ permeation level. Thus Ca²⁺ appears to selectively promote a reversible transition of I _crac that results in larger current flux, and at least partially explains the selectivity of this current for Ca²⁺ over other divalent ions. Received: 30 August 1995/Revised: 7 November 1995