Cytolytic Research Articles

Study on immunopathogenic effect of bacterial protein antigen and the cytolytic toxin antigen of vibrio vulnificus in BALB / c Mice

Objective To investigate whether Vibrio vulnificus bacterial protein antigen and the cytolytic toxin antigen can induce the effective immune protection against Vibrio vulnificus infection.Methods BALB/c mice were immunized with bacterial cytolytic toxin antigen protein antigen of Vibrio vulnificus to evaluate its ability to stimulate immune response.The protective efficacy of immunized mice was evaluated by active immunization and intraperitoneal challenge with V.vulnificus in mice.Results The immunized mice produced higher percentage of CD19+ B lymphocytes and high level specific antibodies (titers up to 1∶25600).All immunized mice survived from lethal challenge with V.vulnificus,while only 13.33％ of mice in control group survived at the end of challenged experiment.Conclusions The bacterial protein antigen and cytolytic toxin antigen of Vibrio vulnificus are capable of inducing specific antibody response in mice to confer effective protection against lethal challenge with V.vulnificus. Key words: Vibrio vulnificus; Cytolytic toxin; Bacterial protein antigen; Active immune

Cyclosporin A, but not FK506 induces osmotic lysis of pancreas zymogen granules (ZG), intra‐acinar enzyme release and lysosome instability by activating a K+ channel

The immunosuppressant tacrolimus (FK506) has improved pancreas allograft survival compared to cyclosporin A (CsA), possibly due to reduced acute pancreatitis following ischemia‐reperfusion injury. Ion permeabilities in zymogen granules (ZG) membranes, including a KCNQ1 K+ channel, promote hormone‐stimulated enzyme secretion. We investigated whether a differential modulation of ZG and lysosomal ion permeabilities, and enzyme secretion by CsA/FK506 contributes to pancreatitis. Rat ZG and lysosomes were isolated by gradient centrifugation, ion permeabilities assayed by osmotic lysis, and single channel currents recorded in a planar lipid bilayer. Amylase release was measured in permeabilized acini and lysosomal cathepsin B release detected by immunoblotting. CsA (1–10μM), but not FK506, enhanced ZG osmotic lysis by selectively increasing K+ permeability up to 5‐fold. ZG membrane K+ channels showed ~2‐fold increased single channel open probability with CsA only. CsA selectively increased basal (~2‐fold), but not cholecystokinin‐octapeptide (1nM)‐induced amylase secretion in K+ medium only. CsA (5μM), but not FK506, increased cathepsin B release from lysosomes. CsA selectively opens the ZG K+channel and induces cathepsin B release from lysosomes, which cause increased in situ lysis of ZG and may induce or aggravate acute allograft pancreatitis. Funded by Mukoviszidose e.V. (F 04/04) and DFG TH 345/11‐1.

Utilization of vinasse for the production of polyhydroxybutyrate by Haloarcula marismortui

Vinasse, a recalcitrant waste of the ethanol industry was employed for the production of polyhydroxyalkanoate (PHA) by the extremely halophilic archaeon, Haloarcula marismortui in shake flasks. The PHA was recovered by osmotic lysis of the cells and subsequent purification by sodium hypochlorite and organic solvents. Through UV-vis spectroscopy, differential scanning calorimetry, Fourier transform infrared, and nuclear magnetic resonance spectroscopy, the PHA was found to have characteristics very similar to that of the standard polyhydroxybutyrate (PHB) from Sigma. Inhibitory effect of polyphenols contained in vinasse was assessed by a quick and reliable cup-plate agar-diffusion method. Raw vinasse (10%) was utilized leading to accumulation of 23% PHA (of cell dry weight) and following an efficacious pre-treatment process through adsorption on activated carbon, 100% pre-treated vinasse could be utilized leading to 30% accumulation of PHB by H. marismortui. Maximum specific growth rate, specific production rate, and volumetric productivity attained using 10% raw vinasse were comparable to that obtained using a previously reported nutrient deficient medium (NDM), while the values with 100% pre-treated vinasse were higher than that determined using NDM medium. This is the first report of polyhydroxybutyrate production by a halophilic microorganism utilizing vinasse.

Molecular force transduction by ion channels – diversity and unifying principles

Cells perceive force through a variety of molecular sensors, of which the mechanosensitive ion channels are the most efficient and act the fastest. These channels apparently evolved to prevent osmotic lysis of the cell as a result of metabolite accumulation and/or external changes in osmolarity. From this simple beginning, nature developed specific mechanosensitive enzymes that allow us to hear, maintain balance, feel touch and regulate many systemic variables, such as blood pressure. For a channel to be mechanosensitive it needs to respond to mechanical stresses by changing its shape between the closed and open states. In that way, forces within the lipid bilayer or within a protein link can do work on the channel and stabilize its state. Ion channels have the highest turnover rates of all enzymes, and they can act as both sensors and effectors, providing the necessary fluxes to relieve osmotic pressure, shift the membrane potential or initiate chemical signaling. In this Commentary, we focus on the common mechanisms by which mechanical forces and the local environment can regulate membrane protein structure, and more specifically, mechanosensitive ion channels.

Lipid Raft Formation and Properties are Necessary and Sufficient to Explain the Properties of Membrane Domains in B. Burgdorferi and are Necessary for its Membrane Integrity

In eukaryotic cells liquid ordered (Lo) microdomains (rafts) rich in sphingolipids and cholesterol are believed to have an important role in many membrane functions. Borrelia burgdorferi (B.b), the bacterium that is the causative agent of Lyme disease, contains host-derived free cholesterol and large amounts of cholesteryl glycolipids. We recently found that microdomains large enough to be seen by transmission electron microscopy (TEM) form in its outer membrane (La Rocca et al (2010) Cell Host and Microbe 8, 331-342). In this study, the extent to which B.b. membrane domains have lipid raft properties was investigated. We carried out lipid substitutions in B.b. cells in which cholesterol lipids were partly substituted with various sterols having differing abilities to support ordered domain formation. TEM was used to visualize B.b. membrane domains, while in live, unfixed B.b. cells FRET and fluorescence anisotropy were used to detect domain segregation and measure overall membrane order, respectively. Both in ordinary B.b. cells and in cells after substitution with sterols that promote ordered domain formation in model membranes there was domain segregation and membrane order consistent with liquid ordered/liquid disordered domain co-existence. in contrast, after substitution with sterols that do not support ordered domain formation in model membranes there was no domain segregation and membrane order was more consistent with the liquid disordered state. Association of biotinylated lipids with B.b. domains required that they contain saturated rather than unsaturated acyl chains. Membrane integrity and growth after sterol substitution also required raft-forming sterols. Sterols not supporting ordered domain formation resulted in osmotic lysis of the organisms. These properties demonstrate that B. burgdorferi membrane domains have the properties of lipid rafts.

Characterizing osmotic lysis kinetics under microfluidic hydrodynamic focusing for erythrocyte fragility studies

The biomechanics of erythrocytes, determined by the membrane integrity and cytoskeletal structure, provides critical information on diseases such as diabetes mellitus, myocardial infarction, hypertension, and sickle cell anemia. Here we demonstrate a simple microfluidic tool for examining erythrocyte fragility based on characterizing osmotic lysis kinetics. Hydrodynamic focusing is used for generating rapid dilution of the buffer and producing lysis of erythrocytes during their flow. The lysis kinetics are tracked by monitoring the release of intracellular contents from cells via recording the light intensity of erythrocytes at various locations in the channel. Such release profile reflects sensitively the changes in erythrocyte fragility induced by chemical, heating, and glucose treatment. Our tool provides a simple approach for probing red blood cell fragility in both basic research and clinical settings.

Apoptotic effect of Vibrio vulnificus cytolysin on A549 human lung adenocarcinoma cells

Vibrio vulnificus cytolysin (VVC) has a very strong cytotoxic effect on various types of mammalian cells. However, the inhibitory effect of VVC on the proliferation of human lung cancer cells has scarcely been reported. This study aimed to analyze the effects of recombinant VVC (rVVC) on the A549 human lung adenocarcinoma cell line and to investigate the underlying molecular mechanisms governing these effects. This study showed that rVVC inhibited the proliferation of A549 cells in a concentration- and time-dependent manner (as measured by the MTT assay). rVVC failed to induce the release of intracellular lactate dehydrogenase (LDH) from the target cells suggesting that osmotic lysis may not contribute to its cytolysin-induced cytotoxicity. Treatment of A549 cells with an IC50 (concentration of drug that inhibits cell growth by 50%) of rVVC resulted in morphological changes and blebbing typical of apoptosis. Annexin-V FITC analysis by FCM indicated that rVVC-induced apoptosis in A549 cells occurs in a dose-dependent manner. A DNA fragmentation assay was utilized to investigate the apoptosis induced by rVVC. The pro-apoptotic activity of rVVC was attributed to its ability to modulate, in a concerted manner, the expression of Bcl-2 and Bax proteins, which were down- and up-regulated, respectively. Caspase-9 and -3 were subsequently activated, however caspase-8 was not. These results prove that rVVC effectively induces programmed cell death and suggests that rVVC-induced apoptosis in the A549 cell line is mediated by the regulation of Bcl-2 protein expression and the activation of caspase-9 and -3.

Karlodinium veneficum—The little dinoflagellate with a big bite

For decades, high densities of the dinoflagellate Karlodinium veneficum have been associated with aquatic faunal mortalities worldwide. This small (<8–12 μm) athecate phytoplankton, common in coastal aquatic ecosystems, has a mixed nutritional mode, relying on both photosynthesis and phagotrophy for growth (mixotrophy). It is frequently present in relatively low cell abundance (10 2–10 3 cells mL −1), but is capable of forming intense blooms of 10 4–10 5 cells mL −1 that are often associated with fish kills. A suite of toxic compounds (karlotoxins) have been characterized, both in the laboratory and in the field, with hemolytic, ichthyotoxic, and cytotoxic properties. These toxins have been shown to generate pores in membranes with desmethyl sterols and increase the ionic permeability resulting in membrane depolarization, disruption of motor functions, osmotic cell swelling and lysis. The biological raison d’etre for karlotoxin production appears to be prey capture but grazing deterrence is an additional advantage. Strain variation in types of karlotoxins and toxin cell quotas is extensive. Since its initial description in 1956 by Dorothy Ballentine toxic and nontoxic strains are common. Despite numerous name changes it is now clearly recognized as a cosmopolitan species with extensive ecosystem impacts.

Prestin in HEK cells is an obligate tetramer

The unusual membrane motor protein prestin is essential for mammalian hearing and for the survival of cochlear outer hair cells. While prestin has been demonstrated to be a homooligomer, by Western blot and FRET analyses, the stoichiometry of self association is unclear. Prestin, coupled to the enhanced green fluorescent protein, was synthesized and membrane targeted in human embryonic kidney cells by plasmid transfection. Fragments of membrane containing immobilized fluorescent molecules were isolated by osmotic lysis. Diffraction-limited fluorescent spots consistent in size with single molecules were observed. Under continuous excitation, the spots bleached to background in sequential and approximately equal-amplitude steps. The average step count to background levels was 2.7. A binomial model of prestin oligomerization indicated that prestin was most likely a tetramer, and that a fraction of the green fluorescent protein molecules was dark. As a positive control, the same procedure was applied to cells transfected with plasmids coding for the human cyclic nucleotide-gated ion channel A3 subunit (again coupled to the enhanced green fluorescent protein), which is an obligate tetramer. The average step count for this molecule was also 2.7. This result implies that in cell membranes prestin oligomerizes to a tetramer.

Regulatory volume increase (RVI) and apoptotic volume decrease (AVD) in U937 cells in hypertonic medium

Changes in intracellular water, K+ and Na+ of U937 cells incubated in hyperosmolar medium supplemented with 200 mM sucrose have been studied. Cells were stained with acrydine orange, ethydium bromide, APOPercentage dye, which marks the phosphatidyl serine distribution on the plasma membrane; and FLICA polycaspase fluorescent dye. It was found that cell shrinkage produced by direct osmotic effect induced both a regulatory volume increase and apoptotic volume decrease. The regulatory volume increase dominated at the early stage, whereas apoptotic volume decrease prevailed at the later stage. Therefore, U937 cells were capable of triggering apoptosis and apoptotic volume decrease, despite the unimpaired regulatory volume increase response, and the current opinion that the dysfunction of the regulatory volume increase is a prerequisite for apoptosis and apoptotic volume decrease (Subramanyam et al., 2010) should be revised. It is concluded that the apoptotic volume decrease plays a significant role in preventing osmotic lysis in apoptotic cells, rather than in initiating apoptosis.

Heavy chain-only antibodies and tetravalent bispecific antibody neutralizing Staphylococcus aureus leukotoxins

Panton-Valentine leukocidin (PVL) is a pore-forming toxin associated with current outbreaks of community-associated methicillin-resistant strains and implicated directly in the pathophysiology of Staphylococcus aureus-related diseases. Humanized heavy chain-only antibodies (HCAb) were generated against S. aureus PVL from immunized transgenic mice to neutralize toxin activity. The active form of PVL consists of the two components, LukS-PV and LukF-PV, which induce osmotic lysis following pore formation in host defense cells. One anti-LukS-PV HCAb, three anti-LukF-PV HCAbs with affinities in the nanomolar range, and one engineered tetravalent bispecific HCAb were tested in vitro and in vivo, and all prevented toxin binding and pore formation. Anti-LukS-PV HCAb also binds to γ-hemolysin C (HlgC) and inhibits HlgC/HlgB pore formation. Experiments in vivo in a toxin-induced rabbit endophthalmitis model showed that these HCAbs inhibit inflammatory reactions and tissue destruction, with the tetravalent bispecific HCAb performing best. Our findings show the therapeutic potential of HCAbs, and in particular, bispecific antibodies.

Molecular mechanism of pyroptosis and inflammasome activation

Background Pyroptosis is a caspase 1-dependent programmed cell death. It features rapid plasma-membrane rupture and release of proinflammatory intracellular contents and induces a special programmed cell death, intermediate between apoptosis and necrosis,named pyroptosis. Objective This article reviews the molecular mechanism of pyroptosis and inflammasome activation to explore the possible future research and its prospect of clinical application. Content Pyroptosis is a conserved cell death process mediating inflammation and immune responses. Various pathological stimuli could trigger formation and activation of a multiprotein complex called the inflammasome, leading to the activation of caspase 1. A combination of downstream processes such as plasma-membrane pore formation, release of multiple activated inflammatory cytokines and DNA cleavage mediated by caspase 1, would eventually result in cell osmotic lysis and induce pyroptosis. Trend Exploring the molecular mechanism of pyroptosis and elucidate how inflammasome activates caspase 1 within pyroptosis will offer us some new targets to prevent and cure complicated immune diseases such as sepsis and inflammatory bowel disease. Key words: Programmed cell death; Pyroptosis; Inflammasome; Caspase-1

Effect of BMAP-28 Antimicrobial Peptides on Leishmania major Promastigote and Amastigote Growth: Role of Leishmanolysin in Parasite Survival

BackgroundProtozoan parasites, such as Leishmania, still pose an enormous public health problem in many countries throughout the world. Current measures are outdated and have some associated drug resistance, prompting the search into novel therapies. Several innovative approaches are under investigation, including the utilization of host defence peptides (HDPs) as emerging anti-parasitic therapies. HDPs are characterised by their small size, amphipathic nature and cationicity, which induce permeabilization of cell membranes, whilst modulating the immune response of the host. Recently, members of the cathelicidin family of HDPs have demonstrated significant antimicrobial activities against various parasites including Leishmania. The cathelicidin bovine myeloid antimicrobial peptide 28 (BMAP-28) has broad antimicrobial activities and confers protection in animal models of bacterial infection or sepsis. We tested the effectiveness of the use of BMAP-28 and two of its isomers the D-amino acid form (D-BMAP-28) and the retro-inverso form (RI-BMAP-28), as anti-leishmanial agents against the promastigote and amastigote intracellular Leishmania major lifecycle stages.Methodology/Principal FindingsAn MTS viability assay was utilized to show the potent antiparasitic activity of BMAP-28 and its protease resistant isomers against L. major promastigotes in vitro. Cell membrane permeability assays, caspase 3/7, Tunel assays and morphologic studies suggested that this was a late stage apoptotic cell death with early osmotic cell lysis caused by the antimicrobial peptides. Furthermore, BMAP-28 and its isomers demonstrated anti-leishmanial activities against intracellular amastigotes within a macrophage infection model.Conclusions/SignificanceInterestingly, D-BMAP-28 appears to be the most potent antiparasitic of the three isomers against wild type L. major promastigotes and amastigotes. These exciting results suggest that BMAP-28 and its protease resistant isomers have significant therapeutic potential as novel anti-leishmanials.

Enhancement of Cytotoxicity against IL-15 Up-regulate NKG2D Expression on Cytokine-induced Killer Cells

Objective To analyse the effects of IL-15 on the expression of NKG2D and the cytotoxicity of cytokine-induced killer(CIK) cells against human esophagus carcinoma cell EC9706 in vitro.Methods Peripheral blood mononuclear cells were isolated from healthy donors then divided into two groups: the control group(cells were cultured in the presence of IFN-γ,anti-CD3 antibody and IL-2) and IL-15 group(cells were cultured in the presence of IFN-γ,anti-CD3 antibody,IL-2 and 10 ng/ml IL-15).Phenotypic characteristics of CIK cells and the expression of NKG2D were analyzed by flow cytometry.After 14 days culture,cytotoxicity of CIK cells against EC9706 cells was measured by using a standard LDH releasing assay.Effector cells were added to target cells at E∶T radios of 20∶1,30∶1.In blocking experiments,NKG2D monoclonal antibody was added to CIK cells for 15 min before plating at 30∶1 E∶T ratio.Results NKG2D molecules were significantly up-regulated during the culture period on CD3+ cells(the CIK cells population) and the CD56+cells.The differences of NKG2D expressions were significant between IL-15 group and the control group(P0.05).Cytotoxicity of CIK cells treated by the IL-15 group was higher than that of the control group,both at 20∶1 E∶T ratios and at 30∶1 E∶T ratios(P0.05).When the NKG2D molecules on CIK cell ular membrance were blocked by the NKG2D monoclonal antibody,the cytolytic activity in the control group cells and IL-15 group cells was significantly inhibited.Conclusion IL-15 up-regulated the expression of NKG2D on CIK cells,which enhanced the NKG2D mediated cytotoxicity against EC9706 cells.CIK cells played a role through the NKG2D molecules.

Unfolding Distinguishes the Vibrio cholerae Cytolysin Precursor from the Mature Form of the Toxin

Vibrio cholerae cytolysin (VCC) is a potent cytolytic toxin that induces colloid osmotic lysis of its target eukaryotic cells by forming transmembrane oligomeric β-barrel channels. VCC is secreted by the bacteria as an inactive precursor (Pro-VCC) and is subsequently activated by proteolytic removal of an N-terminal "Pro-domain", thus generating the active form of the toxin (Mature-VCC). Earlier studies have indicated an intramolecular chaperone-like role of the Pro-domain favoring efficient secretion of the toxin from the periplasm into the extracellular space. However, the exact role of the Pro-domain in the VCC structure--function mechanism remains unclear. Here, we have compared the Pro-VCC and Mature-VCC molecules in terms of their structural and conformational properties. We have studied unfolding of the two variants of the VCC molecule in response to an array of denaturing conditions, including low-pH, chemical denaturant and heat-induced unfolding. Pro-VCC shows a more profound tendency to unfold in response to such denaturing conditions compared to Mature-VCC. Biophysical characterization of the isolated Pro-domain further suggests that the increased unfolding propensity of Pro-VCC does not arise because of an increased level of unfolding of the Pro-region itself. Altogether, our results imply that a natively folded architecture of the Pro-VCC molecule with sufficient structural and conformational plasticity presumably allows it to adopt a suitable configuration that is possibly required for its efficient secretion and subsequent proteolytic maturation under physiological conditions.

PMHC/scFv fusion protein mediates killing of tumor cells by viral specific cytotoxic T cells

Objective To study whether the HBC-A2/scFv fusion protein mediates killing of tumor cells by viral specific cytotoxic T cells. Methods The fusion protein was attached to the CD71-expressing, HLA class Ⅰ negative tumor cells. And then, cytolysis by viral peptide-specific CTLs which were generated by co-culture of peripheral blood lymphocytes from HLA-A2 positive donors with inactivated T2 cells pulsed with the viral peptide were tested by lactate dehydrogenase (LDH) releasing. Results The fusion protein can attach the active viral peptide/HLA-A2 complex to K562, HepG2 and U937 cells through binding of CD71 scFv to CD71 (37.30% ±8.25%, 27.20% ±3.88%, 21.80% ±6.49% ) and mediate cytotoxicity of viral peptide-specific CTLs against those cells in vitro ( K562: 42.08% ± 1.14% vs 8.07%± 1.39%; HepG2: 49.72% ± 1.59% vs 12.46% ± 1.26%; U937: 39.72% ± 3.26% vs 7.13% ±1.48% ). Conclusion This viral peptide/HLA-A2 complex targeted by CD71 scFv is able to redirect viral peptide-specific T-cell mediated immune responses against tumor cells. Key words: Viral peptide/HLA-A2 complex; CD71 single chain antibody; Fusion protein; Tumor cell

Water: A Simple Solution for Tumor Spillage

Although often proposed as a means to reduce the harmful consequences of tumor spill, water lavage has yet to be systematically evaluated in relevant in vitro and in vivo models. This study evaluates the mechanisms and utility of a single water lavage to improve the sequelae of tumor spill during laparotomy. Murine colorectal tumor cell susceptibility to water-induced osmotic lysis was characterized in vitro. A reproducible model of tumor spill was established to recapitulate water or saline lavage during laparotomy. Analyses of tumor volumes calculated from noninvasive imaging were performed. The tumor volumes and survival of mice treated with water, normal saline, or sham laparotomy were assessed. Significant osmotic lysis of cultured murine colorectal cancer cells was observed after a brief exposure to water. Compared to saline or sham laparotomy, water lavage demonstrated superior clinical outcomes with a decrease in tumor burden and concomitant improvement in survival. The use of water lavage during oncologic surgeries to reduce the sequelae of tumor spill is justified and strongly supported by our study. Data from our study raise several concerns regarding the mechanisms and efficacy of saline lavage. Clinically, the use of water lavage during laparotomy would be anticipated to reduce peritoneal disease burden with minimal toxicity or cost.

High-Resolution Structure of the Vibrio Cholerae Cytolysin Heptamer

Vibrio cholerae cytolysin (VCC) is a β-pore forming toxin (β-PFT) that helps the devastating cholera pathogen survive within and colonize the human digestive tract. After being secreted as a water-soluble monomer (∼80 kDa), VCC is cleaved by proteases to form the mature toxin (∼65 kDa). Like many PFTs, mature VCC protomers self-assemble on cell membranes forming a transmembrane channel or pore, which causes damage or death by osmotic lysis. This may play a role in protecting the pathogen from immune cells while causing damage to the intestinal epithelium. Lack of high-resolution structures of assembled pore-forming toxins has limited our understanding of toxin assembly and pore formation.We solved the 2.9 A structure of detergent-solubilized VCC by X-ray diffraction from toxin assembled on cholesterol/asolectin liposomes. This structure, along with our previous structure of the VCC monomer, reveals details of domain rearrangements that occur during pore formation. Comparison of the monomer and the heptamer structures provides insight into why this toxin requires specific lipids and cholesterol in the cell membrane for assembly and pore formation. The charge distribution within the channel helps to explain the anionic rectification behavior observed in planar lipid recordings. This is the first example of a β-PFT for which soluble pro-toxin and assembled pore structures have been solved to high resolution and may provide a basis for therapeutic strategies that target or utilize the VCC toxin.

Mechanism of Melibiose/Cation Symport of the Melibiose Permease of Salmonella typhimurium

The MelB permease of Salmonella typhimurium (MelB-ST) catalyzes the coupled symport of melibiose and Na(+), Li(+), or H(+). In right-side-out membrane vesicles, melibiose efflux is inhibited by an inwardly directed gradient of Na(+) or Li(+) and stimulated by equimolar concentrations of internal and external Na(+) or Li(+). Melibiose exchange is faster than efflux in the presence of H(+) or Na(+) and stimulated by an inwardly directed Na(+) gradient. Thus, sugar is released from MelB-ST externally prior to the release of cation in agreement with current models proposed for MelB of Escherichia coli (MelB-EC) and LacY. Although Li(+) stimulates efflux, and an outwardly directed Li(+) gradient increases exchange, it is striking that internal and external Li(+) with no gradient inhibits exchange. Furthermore, Trp → dansyl FRET measurements with a fluorescent sugar (2'-(N-dansyl)aminoalkyl-1-thio-β-D-galactopyranoside) demonstrate that MelB-ST, in the presence of Na(+) or Li(+), exhibits (app)K(d) values of ∼1 mM for melibiose. Na(+) and Li(+) compete for a common binding pocket with activation constants for FRET of ∼1 mM, whereas Rb(+) or Cs(+) exhibits little or no effect. Taken together, the findings indicate that MelB-ST utilizes H(+) in addition to Na(+) and Li(+). FRET studies also show symmetrical emission maximum at ∼500 nm with MelB-ST in the presence of 2'-(N-dansyl)aminoalkyl-1-thio-β-D-galactopyranoside and Na(+), Li(+), or H(+), which implies a relatively homogeneous distribution of conformers of MelB-ST ternary complexes in the membrane.

Mechanism of Polymer-Induced Hemolysis: Nanosized Pore Formation and Osmotic Lysis

Hemolysis induced by antimicrobial polymers was examined to gain an understanding of the mechanism of polymer toxicity to human cells. A series of cationic amphiphilic methacrylate random copolymers containing primary ammonium groups as the cationic functionality and either butyl or methyl groups as hydrophobic side chains have been prepared by radical copolymerization. Polymers with 0-47 mol % methyl groups in the side chains, relative to the total number of monomeric units, showed antimicrobial activity but no hemolysis. The polymers with 65 mol % methyl groups or 27 mol % butyl groups displayed both antimicrobial and hemolytic activity. These polymers induced leakage of the fluorescent dye calcein trapped in human red blood cells (RBCs), exhibiting the same dose-response curves as for hemoglobin leakage. The percentage of disappeared RBCs after hemolysis increased in direct proportion to the hemolysis percentage, indicating complete release of hemoglobin from fractions of RBCs (all-or-none leakage) rather than partial release from all cells (graded leakage). An osmoprotection assay using poly(ethylene glycol)s (PEGs) as osmolytes indicated that the PEGs with MW > 600 provided protection against hemolysis while low molecular weight PEGs and sucrose had no significant effect on the hemolytic activity of polymers. Accordingly, we propose the mechanism of polymer-induced hemolysis is that the polymers produce nanosized pores in the cell membranes of RBCs, causing an influx of small solutes into the cells and leading to colloid-osmotic lysis.