The effect of the cryoprotectants,dimethylsulfoxide and glycerol on water transport in the human red blood cell

The response of human red blood cells to the cryoprotective agents, DMSO and glycerol, has been investigated using a pulsed NMR method. The experimentally determined parameters are: (1) the intracellular transverse relaxation time, T_2a; (2) the mean residence time of intracellular water, τ_a, which is effectively a reciprocal measure of the rate of water transport across the red blood cell membrane; and (3) the activation energy for this process. The quantitative data indicate that the observed effects are colligative rather than species-specific in origin.     

From a cytotoxic agent to the discovery of a novel antimalarial agent

A novel cytotoxin 3,5-bis(4-chlorobenzylidene)-1-[4-{2-(4-morpholinyl)ethoxy}phenyl-carbonyl]-4-piperidone hydrochloride 2 demonstrated potent antimalarial properties with IC₅₀ values of 0.60 and 1.97 μM against the drug sensitive D6 strain and the C235 drug-resistant strain of Plasmodium falciparum. This compound concentrates in red blood cells, lowers glutathione concentrations in erythrocytes and permeates across CACO-2 cells. These data reveal 2 to be a promising lead compound in the quest for novel antimalarial agents.     

Erythrocyte membrane glycerophospholipid organization is normal in multiple sclerosis

The phospholipid composition of erythrocyte membranes from patients with multiple sclerosis (MS) was found to be normal, in agreement with previous reports. The transbilayer asymmetry of the glycerophospholipids in MS red cells was probed using bee venom phospholipase A₂ and was also found not to be significantly different from normal. Abnormal membrane glycerophospholipid organisation is therefore not involved in the increased red cell size, osmotic fragility, and electrophoretic mobility associated with MS.     

The Electrophoretic Velocity of Human Red Cells, of Their Ghosts and Mechanically Produced Fragments, and of Certain Lipid Complexes

Ghosts prepared in CO₂-saturated water from unwashed human red cells can be fragmented mechanically, but ghosts from thrice washed cells cannot. If the ghosts are prepared by freezing and thawing, this difference is not observed. The electrophoretic velocity varies also with the way in which the ghosts are prepared. The pH-mobility dependence of washed red cells flatten off to a plateau at pH 9, and the electrophoretic velocity is zero at about pH 2. Ghosts prepared by freezing and thawing have almost the same pH-mobility dependence, but if the ghosts are prepared in CO₂-saturated hyptonic saline, the mobility at pH 9.4 is 0.75 times that of washed cells. Fragments of ghosts of unwashed red cells have a smaller mobility than that of the red cells. Trypsin reduces the mobility of washed red cells and of ghosts. Sols of lipid complexes (lecithin, cephalin, and lipositol), at varying pH's, have a mobility 1.2 times that of the washed red cell. The pH-mobility relation is otherwise similar. These complexes can be coated with dextran and trypsin.     

TRH-receptor mobility and function in intact and cholesterol-depleted plasma membrane of HEK293 cells stably expressing TRH-R-eGFP

Here we investigated the effect of disruption of plasma membrane integrity by cholesterol depletion on thyrotropin-releasing hormone receptor (TRH-R) surface mobility in HEK293 cells stably expressing TRH-R-eGFP fusion protein (VTGP cells). Detailed analysis by fluorescence recovery after photobleaching (FRAP) in bleached spots of different sizes indicated that cholesterol depletion did not result in statistically significant alteration of mobile fraction of receptor molecules (M_f). The apparent diffusion coefficient (D_app) was decreased, but this decrease was detectable only under the special conditions of screening and calculation of FRAP data. Analysis of mobility of receptor molecules by raster image correlation spectroscopy (RICS) did not indicate any significant difference between control and cholesterol-depleted cells. Results of our FRAP and RICS experiments may be collectively interpreted in terms of a “membrane fence” model which regards the plasma membrane of living cells as compartmentalized plane where lateral diffusion of membrane proteins is limited to restricted areas by cytoskeleton constraints. Hydrophobic interior of plasma membrane, studied by steady-state and time-resolved fluorescence anisotropy of hydrophobic membrane probe DPH, became substantially more “fluid” and chaotically organized in cholesterol-depleted cells. Decrease of cholesterol level impaired the functional coupling between the receptor and the cognate G proteins of G_q/G₁₁ family.In conclusion: the presence of an unaltered level of cholesterol in the plasma membrane represents an obligatory condition for an optimum functioning of TRH-R signaling cascade. The decreased order and increased fluidity of hydrophobic membrane interior suggest an important role of this membrane area in TRH-R–G_q/G₁₁α protein coupling.     

Calcium is Necessary for Motility in the Diatom Amphora coffeaeformis

The marine diatom Amphora coffeaeformis required Ca²⁺ and bicarbonate for motility. Movement was inhibited by the Ca²⁺-blocking agents ruthenium red and α-isopropyl-α-[(N-methyl-N-homoveratryl)-α- aminopropyl]-3,4,5-trimethoxy phenyl acetonitrile and the metabolic energy uncoupler, carbonyl cyanide 3-chlorophenylhydrazone. 3-(3′,4-Dichlorophenyl)-1,1-Dimethyl urea was without effect on cells at a concentration that prevented O₂ production in the light. Although Sr²⁺ could replace Ca²⁺ in the attachment of cells to glass, it did not substitute for Ca²⁺ in motility.     

Movements of individual BKCa channels in live cell membrane monitored by site-specific labeling using quantum dots

The movements of BK_Ca channels were investigated in live cells using quantum dots (QDs). The extracellular N-terminus was metabolically tagged with biotin, labeled with streptavidin-conjugated QDs and then monitored using real-time time-lapse imaging in COS-7 cells and cultured neurons. By tracking hundreds of channels, we were able to determine the characteristics of channel movements quantitatively. Channels in COS-7 cells exhibited a confined diffusion in an area of 1.915 μm², with an initial diffusion coefficient of 0.033 μm²/s. In neurons, the channel movements were more heterogeneous and highly dependent on subcellular location. While the channels in soma diffused slowly without clear confinement, axodendritic channels showed more rapid and pseudo-one-dimensional movements. Intriguingly, the channel movement in somata was drastically increased by the neuronal β4 subunit, in contrast to the channels in the axodendritic area where the mobility were significantly decreased. Thus, our results demonstrate that the membrane mobility of BK_Ca channels can be greatly influenced by the expression system used, subunit composition, and subcellular location. This QD-based, single-molecule tracking technique can be utilized to investigate the cellular mechanisms that determine the mobility as well as the localization of various membrane proteins in live cells.     

Haemolytic and cytotoxic action of latarcin Ltc2a

Activity and action mechanisms of latarcin 2a (Ltc2a), an antimicrobial peptide from the venom of the spider Lachesana tarabaevi (Zodariidae), were studied in vitro on human cells. Cytotoxicity of Ltc2a for erythrocytes (EC₅₀ = 3.4 μM), leukocytes (EC₅₀ = 19.5 μM) and erythroleukemia K562 cells (EC₅₀ = 3.3 μM) has been found to be primary related to plasma membrane destabilization. Using fluorescently labeled Ltc2a, three common features are found for erythrocytes and K562 cells: pronounced inhomogeneity of cellular response to Ltc2a; complex multistage character of Ltc2a-cell interactions; a positive feedback between Ltc2a binding to plasma membrane and development of toxic effects. Discocyte - echinocyte - spherocyte - ghost is a sequence of Ltc2a-induced transformations of erythrocytes that are accompanied by multistage enhancement of Ltc2a membrane binding, formation of small (ca. 2.0 nm) membrane pores, osmotic imbalance development and reorganization of the pores into large (ca. 13 nm) membrane openings that are preserved in ghosts. Ltc2a induces membrane blebbing and swelling of K562 cells followed by cell death. Cytotoxic action occurs through formation of membrane pores (ca. 3.7 nm) which show greater permeability for anionic than cationic molecules. The pore formation is accompanied with self-assisted Ltc2a internalization and accumulation in mitochondria, mitochondrion inactivation and apoptosis-independent phosphatidylserine externalization.     

Inhibition of K+-sensitive p-nitrophenylphosphatase activity on rhesus-positive red cells after incubation with IgG-anti-rhesus-D

The incubation of ghosts derived from human Rhesus-positive red cells with IgG-anti-Rhesus-D inhibited the K⁺-sensitive p-nitrophenylphosphatase activity. This enzyme has a partial function in the (Na⁺ + K⁺)-ATPase system related to the phosphorylation step, which is important for active potassium transport through the red cell membrane. The specificity of the impairment by the antigen-antibody reaction in the Rhesus-D system was proved by the following controls. Ghosts obtained from Rhesus-negative red cells incubated by IgG-anti-Rhesus-D and those of Rhesus-positive red cells treated with non-immune serum did not show any reduction of the K⁺-p-nitrophenylphosphatase activity. The ghost preparation with lanthanum carried out after hypotonic hemolysis of the washed red cells in 2 mM LaCl₃ at pH 6 was the most suitable procedure to explore this topic in comparison to other techniques for preparing ghosts of red cells.     

Enhancement of divalent anion transport across the human red blood cell membrane by the water-soluble dansyl chloride derivative 2-(N-piperidine)ethylamine-1-naphtyl-5-sulfonylchloride (PENS-Cl)

Sulfate transport across the red cell membrane is enhanced by the newly synthesised, water-soluble and nonpenetrating dansyl chloride derivative 2-(N-piperidine)ethylamine-1-napththyl-5-sulfonylchloride (PENS-Cl). The transport is only enhanced if the potentiating agent 2-(4-aminophenyl-3-sulfonic acid)-6-methylbenzothiazol-7-sulfonic acid (APMB)is present during incubation with PENS-Cl. The enhanced flux is reduced by the anion-transport inhibitor 4,4′-diisothiocyanatodihydrostilbene-2,2′-disulfonate (H₂ DIDS) to about the same low level as in untreated controls. In contrast to dansyl chloride, PENS-Cl does not increase cation leakage from the red cells. The effects of PENS-Cl on sulfate transport resemble those produced by dansyl chloride. However, it can be shown that PENS-Cl only reacts with one subset of sites that are modified by dansyl chloride and involved in bringing about the enhancement of sulfate transport. This subset does not include the sites accessible to dansyl chloride in the absence of APMB. It comprises only a fraction of the sites exposed to dansyl chloride in the presence of APMB. Very little labelling of proteins of the red cell membrane can be seen after exposure of ghosts to the PENS-Cl, while dansyl chloride labels all major proteins.     

Characterization of a Red Beet Protein Homologous to the Essential 36-Kilodalton Subunit of the Yeast V-Type ATPase

V-type proton-translocating ATPases (V-ATPases) (EC 3.6.1.3) are electrogenic proton pumps involved in acidification of endomembrane compartments in all eukaryotic cells. V-ATPases from various species consist of 8 to 12 polypeptide subunits arranged into an integral membrane proton pore sector (V₀) and a peripherally associated catalytic sector (V₁). Several V-ATPase subunits are functionally and structurally conserved among all species examined. In yeast, a 36-kD peripheral subunit encoded by the yeast (Saccharomyces cerevisiae) VMA6 gene (Vma6p) is required for stable assembly of the V₀ sector as well as for V₁ attachment. Vma6p has been characterized as a nonintegrally associated V₀ subunit. A high degree of sequence similarity among Vma6p homologs from animal and fungal species suggests that this subunit has a conserved role in V-ATPase function. We have characterized a novel Vma6p homolog from red beet (Beta vulgaris) tonoplast membranes. A 44-kD polypeptide cofractionated with V-ATPase upon gel-filtration chromatography of detergent-solubilized tonoplast membranes and was specifically cross-reactive with anti-Vma6p polyclonal antibodies. The 44-kD polypeptide was dissociated from isolated tonoplast preparations by mild chaotropic agents and thus appeared to be nonintegrally associated with the membrane. The putative 44-kD homolog appears to be structurally similar to yeast Vma6p and occupies a similar position within the holoenzyme complex.     

The State of Water in Human and Dog Red Cell Membranes

The apparent activation energy for the water diffusion permeability coefficient, P_d, across the red cell membrane has been found to be 4.9 ± 0.3 kcal/mole in the dog and 6.0 ± 0.2 kcal/mole in the human being over the temperature range, 7° to 37°C. The apparent activation energy for the hydraulic conductivity, L_p, in dog red cells has been found to be 3.7 ± 0.4 kcal/mole and in human red cells, 3.3 ± 0.4 kcal/mole over the same temperature range. The product of L_p and the bulk viscosity of water, η, was independent of temperature for both dog and man which indicates that the geometry of the red cell membrane is not temperature-sensitive over our experimental temperature range in either species. In the case of the dog, the apparent activation energy for diffusion is the same as that for self-diffusion of water, 4.6–4.8 kcal/mole, which indicates that the process of water diffusion across the dog red cell membrane is the same as that in free solution. The slightly, but significantly, higher activation energy for water diffusion in human red cells is consonant with water-membrane interaction in the narrower equivalent pores characteristic of these cells. The observation that the apparent activation energy for hydraulic conductivity is less than that for water diffusion across the red cell membrane is characteristic of viscous flow and suggests that the flow of water across the membranes of these red cells under an osmotic pressure gradient is a viscous process.     

The effect of size of red cells on the kinetics of their oxygen uptake

Using a double-beam stopped-flow apparatus estimations were made of the velocity constant for the initial uptake of oxygen by fully reduced erythrocytes (k''_c). Mammalian cells were studied with volumes varying from 20 µ³ (goat) to 90 µ³ (man), as were bullfrog cells (680 µ³). Measurements were made under physiological conditions of pH, P _CO2, and temperature. In man k''_c was 80 mM ^-1 sec^-1 and in other species smaller cells generally had a greater value for k''_c than did the larger cells. In the goat it was 1.8 times as great as the human value; in the bullfrog it was only one-fifth as great. These differences could not be accounted for by interspecific differences in hemoglobin kinetics. The differences probably represent a true effect of size conferring some biological advantage on the species with the smaller cells. The cell membrane offered resistance to oxygen passage. Using the usual red cell model of an infinite sheet of reduced hemoglobin, membrane permeability appeared to differ among mammals. If, as is likely, the effective cell halfthickness differs among mammals, actual membrane permeability differences may be less. A method for measurement of oxygen saturation of dilute cell suspensions is also described.     

Membrane mobility agent alters the consequences of lectin-cell interaction in a malignant cell membrane

The membrane mobility agent 2-(2-methoxyethoxy)-ethyl 8-(cis-2-n-octylcyclopropyl)-octanoate promotes cap formation from wheat germ agglutinin-receptor combinations at the expense of agglutination in membranes of malignant mastocytoma cells.     

A new variant glyoxalase I allele that is readily detectable in stimulated lymphocytes and lymphoblastoid cell lines but not in circulating lymphocytes or erythrocytes

We describe an allele of the human glyoxalase GLO locus that encodes an enzymatically inactive form of the protein, which would not have been detected if only circulating erythrocytes and lymphocytes had been studied. The new allele is named GLO*3 and its protein product, GLO 3. Circulating blood cells of GLO*2/GLO*3 heterozygotes have just one electrophoretic band that migrates as the normal 2-2 dimer. Lymphoblastoid cell lines and phytohemagglutinin-stimulated lymphocytes from the same individuals have two electrophoretic bands, one with the mobility of the 2-2 dimer and one with the mobility of the 2-1 dimer that is present in GLO*2/GLO*1 heterozygotes, but a band with the mobility of the 1-1 dimer is not present. Therefore, the GLO*3 allele encodes a monomer that has the electrophoretic mobility of GLO 1 but is enzymatically inactive unless it is combined with normal monomers in 2-3 and 1-3 heterodimers. The failure to detect the GLO 3 protein in red cells and unstimulated lymphocytes is attributed to a relatively great instability or small rate of production in those cells. Consistent with this interpretation is the reduction of GLO activity in red cells of GLO*2/GLO*3 and GLO*1/GLO*3 heterozygotes to 65% or less of that in normal homozygotes and heterozygotes, while the activity of GLO*3 heterozygous lymphoblastoid cells is about 80% of normal. In contrast, the GLO activity of lymphoblastoid cells that had one copy of the GLO locus deleted by γ-irradiation was 50%–60% of normal. Our observations indicate that certain kinds of mutant alleles of the GLO locus, and perhaps other loci, may not be detected in electrophoretic surveys on circulating blood cells only. The segregation of alleles that are not expressed in circulating red and white blood cells could confuse attempts to determine parentage, as they might have in the family described here. The observations also demonstrate the feasibility of mapping human genes by using ionizing radiation to create partial chromosome deletions in cultured cells.     

Hydroxyl ion movements across the human erythrocyte membrane. Measurement of rapid pH changes in red cell suspensions

A stopped flow rapid reaction apparatus capable of following changes of ±0.02 pH unit in 0.1 ml of solution in less than 0.005 sec has been developed, utilizing a commercially available pH-sensitive glass electrode. Using this instrument, extracellular pH at 37°C was followed from less than 0.025 sec to 300 sec after mixing equal volumes of the following CO₂-free solutions: (A) normal human red cells, washed three times and resuspended in 150 mM NaCl at pH 7.2 with a hematocrit of 18%; and, (B) 150 mM NaCl adjusted with HCl or NaOH to pH 2.1 to pH 10.3. A minimum of 2 ml of mixture had to flow through the electrode chamber to ensure complete washout. The mixing process produced a step change in the pH of the extracellular fluid, after which exchanges across the red cell membrane and buffering by intracellular hemoglobin caused it to return toward pH 7.2 with an approximately exponential time course. Under the assumption that pH changes after mixing represent exchanges of hydroxyl for chloride ions across the cell membrane, hydroxyl ion permeabilities (P _OH ^- in cm/sec) were calculated and found to vary from 2 x 10^-4 at pH 9 to 4 x 10^-1 at pH 4 according to the empirical relationship P _OH ^- = 170 exp (-1.51 pH). The form of the dependence of P _OH ^- on extracellular pH does not appear compatible with a simple fixed charge theory of membrane permselectivity.     

Membrane mobility agents : V. Genetic variability in the fusibility of hen red cells

The membrane mobility agent, A₂C, promotes fusion of hen red cells from several strains of Leghorn hens with high efficiency. Cells from Cornish hens are also easily fused by the same procedure, but cells from Rhode Island Red hens fuse more slowly and to a lesser extent under the same conditions. Cells from several strains of White Rock hens do not fuse when treated in the same way. A potentially useful new parameter of the genetic control of the behavior of membrane (and/or cytoskeletal structure) is thus indicated.     

A spectral shift in cytochrome a induced by calcium ions

Ca²⁺ induces a red shift in the absorption spectrum of ferrocytochrome a when added to uncoupled mitochondria, sub-mitochondrial particles or isolated cytochrome aa₃. The shift is identical within experimental error to the previously reported energy-linked shift in intact mitochondria (Wikström, M. K. F. (1972), Biochim. Biophys. Acta 283, 385–390). One mol of calcium produces the shift in one mol of cytochrome a, the K_D being approx. 20–30 μM. The calcium-induced shift is readily reversed by chelating agents such as EDTA, ethyleneglycol-bis-(μ-aminoethyl ether)N,N′-tetraacetic acid (EGTA) and ATP and is insensitive to uncoupling agents and inhibitors of calcium transport (La³⁺ and ruthenium red). It is shown that the binding site for calcium that is responsible for the spectral shift is located on the outside of the permeability barrier of the mitochondrial cristae membrane.It is proposed that calcium simulates the energy-linked shift in cytochrome a by binding to a site of cytochrome aa₃ that is occupied by protons in energized mitochondria and that is located at the external surface of the mitochondrial membrane.     

Cell specificity and molecular mechanism of antibacterial and antitumor activities of carboxyl-terminal RWL-tagged antimicrobial peptides

Antimicrobial peptides (AMPs) constitute a diverse class of naturally occurring or synthetic antimicrobial molecules that have potential for use in the treatment of drug-resistant infections. Several undesirable properties of AMPs, however, may ultimately hinder their development as antimicrobial agents. Thus, new synthetic strategies, including primarily the de novo design of AMPs, urgently need to be developed. In this study, a series of peptides, H-(RWL) _n (n = 1, 2, 3, 4, or 5), were designed. H represents GLRPKYS from the C-terminal sequence of AvBD-4. Our results showed that these RWL-tagged peptides can kill not only bacteria but also human hepatocellular carcinoma HepG2 cells. However, the peptide tagged with two repeats of RWL (GW13) showed less affinity to human embryonic lung fibroblast MRC-5 cells or human red blood cells (hRBCs) than HepG2 cells. These results demonstrated that GW13, with high amphiphilicity, exerted great selectivity toward bacteria and cancer cells, sparing host mammalian cells. The mechanism of action against bacteria was elucidated through combined studies of scanning electron microscopy (SEM) and fluorescence assays, showing that the peptide possessed membrane-lytic activities against microbial cells. The fluorescence assays illustrated that GW13 induced apoptosis in HepG2 cells. The cell morphology of HepG2 cells, observed by SEM, further illustrated that GW13 causes cell death by damaging the cell membrane. Our results indicate that GW13 has considerable potential for future development as an antimicrobial and antitumor agent.