Changes In Transmembrane Potential Research Articles

Graphene oxide in low concentrations can change mitochondrial potential, autophagy, and apoptosis paths in two strains of invertebrates with different life strategies

Nanoparticles, like graphene oxide (GO), are particles with unique physiochemical properties that enable their wide application in various areas of life. The effects of GO on individual cell organelles like mitochondria and the effects of interactions are worth investigating, as they can activate multiple cellular processes, such as autophagy or apoptosis. Mitochondrial injury plays an essential role in the majority of cell death routines. In the project, we investigated cell health status measured as mitochondrial inner membrane depolarization, autophagy, and apoptosis induction during long-term GO administration in food (0.02 μg g−1 and 0.2 μg g−1 of food). Two unique Acheta domesticus strains that differ in life strategy were used: wild-type and long-lived at three different life stages (larva, young adult, mature adult). The changes in mitochondrial trans-membrane potential were marked in the wild-type strain. The autophagy was lower in all GO-treated groups in both strains, and the apoptosis was lower in both strains in the mature adult crickets. Low GO concentrations treatment for the whole life, despite mitochondrial dysfunction, may lead to inhibition of autophagy and apoptosis by arresting the cell cycle for the duration of repair, and other repair tools are involved in the process of restoring homeostasis.

Mitochondrial Function and Resistance to Oxidative Stress in the Kidney during Pregnancy.

We demonstrated that the serum of pregnant rats increases viability of kidney epithelial cells and promotes their proliferation. The intensity of oxidative stress in the kidneys was also reduced during pregnancy, but only in rats that were not exposed to acute ischemic kidney injury. This decrease in oxidative stress was not associated with changes in transmembrane mitochondrial potential, the size of mitochondria, time of opening of mitochondrial permeability transition pore (mPTP), mitochondrial respiration rate, antioxidant activity, or nitric oxide level.

Optogenetic confirmation of transverse-tubular membrane excitability in intact cardiac myocytes.

T-tubules (TT) form a complex network of sarcolemmal membrane invaginations, essential for well-co-ordinated excitation-contraction coupling (ECC) and thus homogeneous mechanical activation of cardiomyocytes. ECC is initiated by rapid depolarization of the sarcolemmal membrane. Whether TT membrane depolarization is active (local generation of action potentials; AP) or passive (following depolarization of the outer cell surface sarcolemma; SS) has not been experimentally validated in cardiomyocytes. Based on the assessment of ion flux pathways needed for AP generation, we hypothesize that TT are excitable. We therefore explored TT excitability experimentally, using an all-optical approach to stimulate and record trans-membrane potential changes in TT that were structurally disconnected, and hence electrically insulated, from the SS membrane by transient osmotic shock. Our results establish that cardiomyocyte TT can generate AP. These AP show electrical features that differ substantially from those observed in SS, consistent with differences in the density of ion channels and transporters in the two different membrane domains. We propose that TT-generated AP represent a safety mechanism for TT AP propagation and ECC, which may be particularly relevant in pathophysiological settings where morpho-functional changes reduce the electrical connectivity between SS and TT membranes. KEY POINTS: Cardiomyocytes are characterized by a complex network of membrane invaginations (the T-tubular system) that propagate action potentials to the core of the cell, causing uniform excitation-contraction coupling across the cell. In the present study, we investigated whether the T-tubular system is able to generate action potentials autonomously, rather than following depolarization of the outer cell surface sarcolemma. For this purpose, we developed a fully optical platform to probe and manipulate the electrical dynamics of subcellular membrane domains. Our findings demonstrate that T-tubules are intrinsically excitable, revealing distinct characteristics of self-generated T-tubular action potentials. This active electrical capability would protect cells from voltage drops potentially occurring within the T-tubular network.

INVESTIGATION OF THE TRANSMEMBRANE POTENTIAL OF MITOCHONDRIA AND THE GENERATION OF FREE RADICAL COMPOUNDS IN LYMPHOCYTES OF CERVICAL CANCER PATIENTS

Summary. Important indicators that reflect changes in the functional state of non-malignant cells from the tumor environment and can be used as predictors of the occurrence of complications as a result of radiation and chemo radiation therapy are the level of polarization of the mitochondrial membrane and the intensity of production of reactive forms of oxygen and nitrogen (free radical compounds, FR). Aim: To investigate changes in the mitochondrial transmembrane potential (MTP) and the intensity of FR formation in peripheral blood lymphocytes (PBL) of cervical cancer (CC) patients before the chemo radiation therapy. Object and methods: In the study used peripheral blood samples of 26 patients with СС before starting chemo radiation therapy and 29 apparently healthy individuals (AHI, control group). PBL isolation was performed on Histopaque®-1077. The level of TMP in lymphocytes was determined using the dye JC-1, the intensity of FR formation using the dye DCFH-DA. Results: It was shown that the tendency to lower TMP (by 1.46 times) and significantly lower FR formation (by 2.84 times) was observed in the PBL of patients with CC. In the examined patients with a greater degree of the size of the primary tumor and its’ invasion (T2 compared to T1) or a greater degree of tumor differentiation (G2 compared to G2/3-3), the TMP level was higher (by 1.35 and 1.47 times, respectively). In CC patients, in contrast to AHI, a significant correlation was found between the TMP level and the intensity of FR formation (r=0.473). It has been shown that in the PBL of CC patients with the stage of the tumor process T1N0M0 and the degree of the tumor differentiation G2, the level of TMP and the intensity of BP formation are significantly lower (by 1.91 times and 1.88 times, respectively) than in similar patients with uterine cancer, who has been examined before. Conclusions: The functioning of lymphocytes of patients with CC before chemo radiation therapy is already changed according to the indicators of TMP value and FR formation, which are significantly lower than in AHI lymphocytes. In contrast to the control group, a significant correlation was found between the TMP levels and BP formation in the PBL of the examined CC patients. It has been shown that in CC patients with a disease stage of T1N0M0 and a G2 tumor differentiation degree changes in the level of TMP and the FR formation are more pronounced than in a similar group of patients with uterine cancer. The obtained results are background for evaluating the changes occurring in the cells from the tumor environment after radiation therapy.

In Ukrainian

This research is aimed at the investigation the electrosurface and biochemical parameters of bacterial cells B. cereus B4368, L. plantarum, E. coli K-A, P. fluorescens B5040 under the influence of copper in ionic form and as nanoparticles in order to determine the nature and level of their toxic effect on bacteria. Copper nanoparticles synthesized in aqueous solution with NaBH4 and stabilized with dextran were used. Changes in membrane transport parameters were assessed by the value of ATPase activity; changes in transmembrane potential were assessed by the method of penetrating tetraphenylphosphonium cations (TPP+); and bacterial integrity was assessed by UV spectroscopy of cellular metabolites. A concentration-dependent inhibition of the membrane ATPase reaction and dissipation of the transmembrane potential under the action of both forms of copper was found, and the inhibitory effect in the case of the nanoparticles was on average 20 % higher than in the ionic form. As a result of heterocoagulation of dextran-stabilized copper nanoparticles and bacteria, a decrease in the negative ξ - potential of bacteria was observed, which was 40 % more effective under the action of copper nanoparticles compared to Cu2+ ions. The most significant changes in membrane parameters were observed in the range 10–60 μM of copper concentrations. With B. cereus B4368 cells taken as an example, we found a violation of the barrier function of their cell membrane under the influence of both copper preparations. In the case of copper nanoparticles, nucleic acid leakage from the bacterial cytoplasm was detected, which was confirmed by the absorption band at 260 nm. The results obtained indicate a high level of sensitivity of the studied electrosurface and biochemical parameters of bacterial cells to the effects of ionic and nanoparticle copper, which allows them to be used as indicators of the toxicity of metal nanoparticles in the development of metal-containing probiotic preparations.

Inactivation of foodborne pathogens in ground pork tenderloin using 915 MHz microwave heating depending on power level

The main purpose of this research was to investigate the effect of power level of 915 MHz microwave heating on the inactivation of foodborne pathogens in ground pork and its bactericidal mechanism. It was demonstrated that the heating rate was proportional to the power level. For instance, the heating rates observed at microwave heating powers of 2, 3, 4, and 5 kW were 1.70, 2.77, 3.35, and 4.03℃/s, respectively. The bactericidal effect of microwave heating also significantly (P < 0.05) increased with increasing power level. In particular, when ground pork was subjected to microwave heating at 5 kW, the reduction level of pathogens was>2 log units higher than at 2 kW. To determine the bactericidal mechanism of microwave heating depending on power level, changes in transmembrane potential and DNA damage were determined using fluorescence. The extent of depolarization in membrane potential of pathogens significantly (P < 0.05) increased as power level increased. There was no significant difference in degree of DNA damage at different power levels. However, the percentage of DNA damage was>86% at all power levels. The transmembrane potential assay indicates that the bacteria exhibited dramatic pore formation on the membrane at 5 kW. Through transmission electron microscopy, the electroporation effect was enhanced as power level increased. Moreover, the quality of ground pork subjected to microwave heating at 2–5 kW was determined by measuring the moisture content, cooking loss, and texture profile.

Membrane polarization at the excitation threshold induced by external electric fields in cardiomyocytes of rats at different developmental stages.

External electric fields (E), used for cardiac pacing and defibrillation/cardioversion, induce a spatially variable change in cardiomyocyte transmembrane potential (ΔVm) that depends on cell geometry and E orientation. This study investigates E-induced ΔVm in cardiomyocytes from rats at different ages, which show marked size/geometry variation. Using a tridimensional numerical electromagnetic model recently proposed (NM3D), it was possible: (a) to evaluate the suitability of the simpler, prolate spheroid analytical model (PSAM) to calculate amplitude and location of ΔVm maximum (ΔVmax) for E = 1V.cm-1; and (b) to estimate the ΔVmax required for excitation (ΔVT) from experimentally determined threshold E values (ET). Ventricular myocytes were isolated from neonatal, weaning, adult, and aging Wistar rats. NM3D was constructed as the extruded 2D microscopy cell image, while measured minor and major cell dimensions were used for PSAM. Acceptable ΔVm estimates can be obtained with PSAM from paralelepidal cells for small θ. ET, but not ΔVT, was higher for neonate cells. ΔVT was significantly greater in the cell from older animals, which indicate lower responsiveness to E associated with aging, rather than with altered cell geometry/dimensions. ΔVT might be used as a non-invasive indicator of cell excitability as it is little affected by cell geometry/size.

Time-Dependent Features of Mass Transfer and Transmembrane Potential in Erythrocytes During Equilibration in Cryoprotective Solutions

On the basis of the developed physical and mathematical model of mass transfer, which takes into account the transmembrane transfer of non-electrolytes, basic ions and the associated changes in the transmembrane potential, the redistribution of osmotically active substances during equilibration of erythrocytes in cryoprotective solutions was investigated. Time parameters of changes in concentrations of osmotically active substances inside and outside cells, as well as transmembrane electric potential, were calculated. It is shown that during the exposure of human erythrocytes to 1M solutions of glycerol, 1,2-propanediol (1,2-PD) and dimethylsulfoxide (DMSO), the sign of their transmembrane electric potential changes three times, and in solutions of ethylene glycol (EG) and of acetamide (AA) – once. The analysis of the obtained results showed that the most acceptable for further cryopreservation from the point of view of erythrocytes reaching a state close to equilibrium in a 1M solution of glycerol was their equilibration for 5.5 min, and in solutions of DMSO, AA, EG and 1,2-PD with the same concentration – 1 min. At the same time, the cells remain somewhat dehydrated (by 5.5–7.5%), and the concentrations of cryoprotectants inside erythrocytes change insignificantly during longer exposure. The indicated degree of dehydration does not affect cell viability, but reduces the likelihood of intracellular ice formation during subsequent freezing.

Pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine Sulfonamides as Novel Potential Anticancer Agents: Apoptosis, Oxidative Stress, and Cell Cycle Analysis.

The current study continues the evaluation of the anticancer potential of three de novo synthesized pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine sulfonamides-MM129, MM130, and MM131-against human cancer cells of HeLa, HCT 116, PC-3, and BxPC-3 lines. The pro-apoptotic activity of the investigated sulfonamides was shown by observations of changes in the mitochondrial transmembrane potential of the tested cells, externalization of phosphatidylserine on the cellular membrane surface, and cell morphology in microscopic imaging. The computational studies have shown that MM129 exhibited the lowest binding energy values when docked against CDK enzymes. In addition, the highest stability was shown for complexes formed between MM129 and CDK5/8 enzymes. All examined compounds induced cell cycle arrest in the G0/G1 phase in the BxPC-3 and PC-3 cells and simultaneously caused the accumulation of cells in the S phase in the HCT 116 cells. In addition, the increase in the subG1 fraction was observed in PC-3 and HeLa cells. The application of a fluorescent H2DCFDA probe revealed the high pro-oxidative properties of the tested triazine derivatives, especially MM131. In conclusion, the obtained results suggest that MM129, MM130, and MM131 exhibited strong pro-apoptotic properties towards investigated cells, mainly against the HeLa and HCT 116 cell lines, and high pro-oxidative potential as well. Moreover, it is suggested that the anticancer activity of the tested compounds may be associated with their ability to inhibit CDK enzymes activities.

Transmembrane potential, an indicator in situ reporting cellular senescence and stress response in plant tissues

BackgroundPlant cells usually sustain a stable membrane potential due to influx and/or efflux of charged ions across plasma membrane. With the growth and development of plants, different tissues and cells undergo systemic or local programmed decline. Whether the membrane potential of plasma membrane could report senescence signal of plant tissues and cells is unclear.ResultsWe applied a maneuverable transmembrane potential (TMP) detection method with patch-clamp setup to examine the senescence signal of leaf tissue cells in situ over the whole life cycle in Arabidopsis thaliana. The data showed that the TMPs of plant tissues and cells were varied at different growth stages, and the change of TMP was higher at the vegetative growth stage than at the reproductive stage of plant growth. The distinct change of TMP was detectable between the normal and the senescent tissues and cells in several plant species. Moreover, diverse abiotic stimuli, such as heat stress, hyperpolarized the TMP in a short time, followed by depolarized membrane potential with the senescence occurring. We further examined the TMP of plant chloroplasts, which also indicates the senescence signal in organelles.ConclusionsThis convenient TMP detection method can report the senescence signal of plant tissues and cells, and can also indicate the potential of plant tolerance to environmental stress.

Cycloartane triterpenoid from Euphorbia macrostegia modulates ER stress signaling pathways to induce apoptosis in MDA-MB231 and MCF-7 breast cancer cell lines.

Unfolded protein response (UPR) is involved in breast cancer (BC) progression and drug resistance. Many natural products (NPs) could modulate UPR and used for therapeutic purposes. Herein, we aimed to investigate the molecular mechanism of Cycloart-23E-ene-3β, 25-diol (Cycloart-E25), cytotoxicity, as a NP extracted from Euphorbia macrostegia and focused on endoplasmic-reticulum stress (ERS) and UPR signaling pathways. Reactive oxygen species (ROS) were probed by DCFDA fluorescence dye. Apoptosis was assayed by annexin V/propidium iodide (PI), immunoblotting of anti- and proapoptotic, Bcl-2 and Bax proteins, and mitochondrial transmembrane potential (ΔΨm) changes. Thioflavin T (ThT) staining and immunoblotting of UPR signaling components (CHOP, PERK, ATF6, BiP, and XBP1) were recruited for the assessment of ERS. Our results indicated that Cycloart-E25 noticeably increases ROS levels in both MB-231 MDA-MB-231 and MCF-7 cell lines, p>0.05. Flow cytometry assessments revealed an increase in the cell population undergoing apoptosis. Also, the Bax/Bcl-2 ratio increased in a dose-dependent manner following Cycloart-E25 treatment, significantly, p>0.05. Mitochondrial involvement could be deduced by significant decreases in ΔΨm, p>0.05. Cycloart-E25 potently induces protein aggregation and upregulated CHOP, PERK, ATF6, BiP, and XBP1 factors in both MDA-MB-231 MB-231 and MCF-7 cell lines, indicating the involvement of ERS in Cycloart-E25-mediated apoptosis. In conclusion, Cycloart-E25 increased the accumulation of misfolded proteins and upregulated UPR components. Therefore, induction of ERS may be involved in the trigger of apoptosis in BC cell lines. Cycloart-E25 induced apoptosis in breast cancer cell lines through ERS. More assessments are needed to confirm its in vivo anti-tumoral effects.

Frequency responses for induced neural transmembrane potential by electromagnetic waves (1 kHz to 1 GHz)

Abstract Many biophysical effects of electromagnetic radiation are interpreted based on the induced voltage on cellular membranes. It is very instructive to study wideband frequency responses showing how an impinging electromagnetic wave carrying a certain time waveform translates into a time-dependent change in the cell-membrane potentials in any desired tissue. A direct numerical solution of this problem with realistic models for the body and cells results in meshcells of nanometer dimensions, which is unaffordable for almost any computing machine. In this paper, we exploit a multiscale method with serial frequency responses to arrive at the final frequency response for the induced transmembrane potential changes in cerebral cells induced by electromagnetic waves incident on the body. The results show a bandpass characteristic; a frequency window of approximately 10 kHz to 100 MHz as the most sensitive frequency band for neuronal membrane sensing of external electromagnetic fields.

Resveratrol Enhances the Anti-Cancer Effects of Cis-Platinum on Human Cervical Cancer Cell Lines by Activating the SIRT3 Relative Anti-Oxidative Pathway.

Background: Cervical cancer exerts considerable mortality in the world. The combinations of chemotherapy with cis-platinum were the first-line treatment in late-stage cervical cancer but may cause severe adverse effects. Resveratrol (RES, 3,5,4′-trihydroxy-trans-stilbene) is a phytoalexin, and it showed anti-cancer effects but with low toxicity and side effects. Herein, we examined the anti-cancer effects of cis-platinum combined with RES in human cervical cancer cell lines. Methods: The antiproliferative effect was examined by cell counting and short-term MTT assay. Cell apoptosis was detected. The cell cycle distribution was determined by flow cytometry. Intracellular reactive oxygen species and mitochondrial transmembrane potential change were observed and calculated by confocal microscopy. The Si-RNA interference of SIRT3 in cancer cells was performed. Protein expression was checked by Western blotting. Results: RES inhibited the growth of SiHa cell lines, and it significantly enhanced the cis-platinum-induced cell apoptosis and cell cycle arresting in 48 h. The activation of the SIRT3 relative anti-oxidative pathway was proved to be the reason for the enhanced anti-cancer effects of cis-platinum and RES combination. Si-RNA interference of SIRT3 compromised the anti-cancer effect of cis-platinum and RES combination. Furthermore, the silencing of SIRT3 RNA inhibited the expression of the anti-oxidant enzyme (MnSOD, GPx, SOD-1, and CAT) and decreased the generation of H2O2 in the cis-platinum and RES combination group. Conclusion: RES enhances the anti-cancer effects of cis-platinum on SiHa cells by activating the SIRT3 relative anti-oxidative pathway. RES may act as a potential synergistic agent and be useful in the treatment of cervical cancer.

The influence of venetoclax, used alone or in combination with cladribine (2-CdA), on CLL cells apoptosis in vitro: Preliminary results

Venetoclax (VEN), a highly selective BCL-2 inhibitor, is successfully used in the treatment of chronic lymphocytic leukemia (CLL). The purine analogue - cladribine (2-CdA) - is also administered to CLL patients, especially as a part of chemoimmunotherapy. To compare the effects of the VEN+2-CdA regimen with that of the 2 drugs used alone on the apoptosis of CLL lymphocytes in vitro. Mononuclear cells were collected from 103 previously untreated CLL patients. They were incubated with VEN (40 nM) or/and 2-CdA (16 μM) for 48 h. Cytotoxicity, overall apoptosis, mitochondrial transmembrane potential changes (ΔΨm), and expression of selected apoptosis-involved proteins were measured. The cytotoxicity, overall apoptosis, caspase-3 or caspase-9 expression, and ΔΨm were significantly higher after VEN+2-CdA addition compared to both drugs used alone, with a very strong synergistic effect observed. The percentage of BCL-2-positive cells decreased after VEN and VEN+2-CdA addition compared to controls. The TP53-expressing cells increased under the influence of all tested regimens. The VEN+2-CdA increased the expression of BIM, BAX and NOXA compared to either controls or VEN or 2-CdA alone. Similar increases in PUMA expression were observed after VEN, 2-CdA and VEN+2-CdA addition. The FAS-associated death-domain protein (FADD) expression was significantly higher after 2-CdA and 2-CdA+VEN addition as compared to control. Our results confirm the involvement of both VEN and 2-CdA in the intrinsic apoptotic pathway. They also demonstrate that these agents have a synergistic effect on CLL cells in vitro. Further studies are needed to assess the influence of VEN+2-CdA on the expression of apoptosis-involved genes.

Double-activation of mitochondrial permeability transition pore opening via calcium overload and reactive oxygen species for cancer therapy

BackgroundCalcium ions (Ca2+) participates in various intracellular signal cascades and especially plays a key role in pathways relevant to cancer cells. Mitochondrial metabolism stimulated by calcium overload can trigger the opening of the mitochondrial permeability transition pore (MPTP), which leads to cancer cell death.MethodsHerein, a mitochondrial pathway for tumour growth inhibition was built via the double-activation of MPTP channel. Fe2+ doped covalent organic frameworks (COF) was synthesised and applied as template to grow CaCO3 shell. Then O2 was storaged into Fe2+ doped COF, forming O2-FeCOF@CaCO3 nanocomposite. After modification with folic acid (FA), O2-FeCOF@CaCO3@FA (OFCCF) can target breast cancer cells and realize PDT/Ca2+ overload synergistic treatment.ResultsCOF can induce the production of 1O2 under 650 nm irradiation for photodynamic therapy (PDT). Low pH and hypoxia in tumour microenvironment (TME) can activate the nanocomposite to release oxygen and Ca2+. The released O2 can alleviate hypoxia in TME, thus enhancing the efficiency of COF-mediated PDT. Abundant Ca2+ were released and accumulated in cancer cells, resulting in Ca2+ overload. Notably, the reactive oxygen species (ROS) and Ca2+ overload ensure the sustained opening of MPTP, which leads to the change of mitochondria transmembrane potential, the release of cytochrome c (Cyt c) and the activation of caspases 3 for cancer cell apoptosis.ConclusionThis multifunctional nanosystem with TME responded abilities provided a novel strategy for innovative clinical cancer therapy.Graphical

Label-Free Imaging of Membrane Potentials by Intramembrane Field Modulation, Assessed by Second Harmonic Generation Microscopy.

Optical interrogation of cellular electrical activity has proven itself essential for understanding cellular function and communication in complex networks. Voltage-sensitive dyes are important tools for assessing excitability but these highly lipophilic sensors may affect cellular function. Label-free techniques offer a major advantage as they eliminate the need for these external probes. In this work, it is shown that endogenous second-harmonic generation (SHG) from live cells is highly sensitive to changes in transmembrane potential (TMP). Simultaneous electrophysiological control of a living human embryonic kidney (HEK293T) cell, through a whole-cell voltage-clamp reveals a linear relation between the SHG intensity and membrane voltage. The results suggest that due to the high ionic strengths and fast optical response of biofluids, membrane hydration is not the main contributor to the observed field sensitivity. A conceptual framework is further provided that indicates that the SHG voltage sensitivity reflects the electric field within the biological asymmetric lipid bilayer owing to a nonzero tensor. Changing the TMP without surface modifications such as electrolyte screening offers high optical sensitivity to membrane voltage (≈40% per 100mV), indicating the power of SHG for label-free read-out. These results hold promise for the design of a non-invasive label-free read-out tool for electrogenic cells.

Detailed Characterization of Antipathogenic Properties of Human Milk N-Glycome, against Staphylococcus aureus, Indicating Its Targeting on Cell Surface Proteins.

Human milk N-glycome was previously identified to have strong antipathogenic activities. This study is aimed to characterize the detailed antibacterial properties and the potential function mechanism of human milk N-glycome against Staphylococcus aureus. A serials of traditional antibacterial assays showed that human milk N-glycome possessed both bacteriostatic and bactericidal activities, which was further confirmed by the cell structure disruption including the change of transmembrane potential and leakage of intracellular contents. The results of the bacterial surface zeta potential and hydrophobicity, bacterial binding assay, gel shift assay, and fluorescence spectra and the different synergistic effects of human milk N-glycome combined with different antibiotics indicated that the bacterial surface proteins could be the targets of human milk N-glycome. Moreover, human milk N-glycome also showed antibiofilm activity. In conclusion, human milk N-glycome exhibited good potential for acting as an antibacterial substance against S. aureus and the antibacterial mechanism was a cell surface targeting action.

Protective effects of FK 506 against haemorrhagic shock-induced microvascular hyperpermeability.

Microvascular hyperpermeability, the excessive leakage of fluid and proteins from the intravascular space to the interstitium, is a devastating clinical concern in haemorrhagic shock (HS), sepsis, burn and so forth. Previous studies have shown that HS-induced microvascular hyperpermeability is associated with activation of the mitochondria-mediated 'intrinsic' apoptotic signalling cascade and caspase-3 mediated disruption of the endothelial cell barrier. In this study, our objective was to test if FK506, an immunomodulator that is also known to protect mitochondria, would protect barrier functions and decrease vascular hyperpermeability following HS by acting on this pathway. FK506 (25µM) was given 10 minutes before the shock period in a rat model of HS. The HS model was a non-traumatic/fixed pressure model of hypovolemic shock developed by withdrawing blood to reduce the mean arterial pressure to 40mm Hg for 60minutes. The mesenteric post-capillary venules were monitored for changes in permeability using intravital microscopic imaging. The changes in mitochondrial transmembrane potential (MTP) were determined using the cationic dye 5,5',6,6' tetrachoro-1,1',3,3' tetraethyl benzimidazolyl carbocyanine iodide (JC-1), that was superfused on the mesenteric vasculature followed by intravital imaging. The mesenteric caspase-3 activity was measured fluorometrically. Haemorrhagic shock induced a significant increase in hyperpermeability compared to the sham-control group and FK506 treatment decreased HS-induced hyperpermeability significantly (P<.05). FK506 dampened HS-induced loss of MTP and elevation of caspase-3 activity significantly (P<.05). FK506 has protective effects against HS-induced microvascular hyperpermeability. The maintenance of the MTP and protection against caspase-3 mediated endothelial cell barrier disruption are possible mechanisms by which FK506 attenuates HS-induced hyperpermeability. FK506, currently used in clinical settings as an immunomodulator, needs to be explored further for its therapeutic usefulness against HS-induced vascular hyperpermeability and associated complications.

In Vitro and In Vivo Anti-Candida Activity and Structural Analysis of Killer Peptide (KP)-Derivatives.

The previously described decapeptide AKVTMTCSAS (killer peptide, KP), derived from the variable region of a recombinant yeast killer toxin-like anti-idiotypic antibody, proved to exert a variety of antimicrobial, antiviral, and immunomodulatory activities. It also showed a peculiar self-assembly ability, likely responsible for the therapeutic effect in animal models of systemic and mucosal candidiasis. The present study analyzed the biological and structural properties of peptides derived from KP by substitution or deletion of the first residue, leaving unchanged the remaining amino acids. The investigated peptides proved to exert differential in vitro and/or in vivo anti-Candida activity without showing toxic effects on mammalian cells. The change of the first residue in KP amino acidic sequence affected the conformation of the resulting peptides in solution, as assessed by circular dichroism spectroscopy. KP-derivatives, except one, were able to induce apoptosis in yeast cells, like KP itself. ROS production and changes in mitochondrial transmembrane potential were also observed. Confocal and transmission electron microscopy studies allowed to establish that selected peptides could penetrate within C. albicans cells and cause gross morphological alterations. Overall, the physical and chemical properties of the first residue were found to be important for peptide conformation, candidacidal activity and possible mechanism of action. Small antimicrobial peptides could be exploited for the development of a new generation of antifungal drugs, given their relative low cost and ease of production as well as the possibility of devising novel delivery systems.

Retraction Note to: NPC-16, a novel naphthalimide-polyamine conjugate, induced apoptosis and autophagy in human hepatoma HepG2 cells and Bel-7402 cells.

The antitumor effects and molecular mechanism of NPC-16, a novel naphthalimide–polyamine conjugate, were evaluated in HepG2 cells and Bel-7402 cells. Apoptosis and necrosis were evaluated by Annexin V-FITC detection kit, and autophagy by acridine orange and Lyso-Tracker Red staining. The change of mitochondrial transmembrane potential was measured using rhodamine 123 staining. The protein expression of Beclin 1, LC3 II and mTOR, p70S6 K, 14-3-3, caspase, and Bcl-2 family members was detected by immunofluorescence assays and Western Blot. Here, we elucidated the nature of cellular response of HepG2 cells and Bel-7402 cells to NPC-16 at IC50. NPC-16 induced caspase-dependent apoptosis via the mitochondrial pathway and death receptor pathway in Bel-7402 cells. Differently, NPC-16 triggered HepG2 cells both apoptosis and autophagy, further autophagy facilitated cellular apoptosis. Furthermore, mTOR signal pathway was involved in NPC-16-mediated autophagy in HepG2 cells. Thus, NPC-16 may be useful as a potential template for investigation the molecular mechanism of naphthalimide–polyamine conjugate against hepatocellular carcinoma.