UV-A irradiation induces a decrease in the mitochondrial respiratory activity of human NCTC 2544 keratinocytes

UV-A irradiation caused a dose-dependent decrease in cellular oxygen consumption (56%) and ATP content (65%) in human NCTC 2544 keratinocytes, one hour after treatment. This effect was partially reversed by maintaining the irradiated cells in normal culture conditions for 24h. Using malate/glutamate or succinate as substrates for mitochondrial electron transport, the oxygen uptake of digitoninpermeabilised cells was greatly inhibited following UV-A exposure. These results strongly suggest that UV-A irradiation affects the state 3 respiration of the mitochondria. However, under identical conditions, UV-A exposure did not reduce the mitochondrial transmembrane potential. The antioxidant, vitamin E inhibited UV-A-induced lipid peroxidation, but did not significantly prevent the UV-A-mediated changes in cellular respiration nor the decrease in ATP content, suggesting that these effects were not the result of UV-A dependent lipid peroxidation. UV-A irradiation also led to an increase in MnSOD gene expression 24 hours after treatment, indicating that the mitochondrial protection system was enhanced in response to UV-A treatment. These findings provide evidence that impairment of mitochondrial respiratory activity is one of the early results of UV-A irradiation for light doses much lower than the minimal erythemal dose.     

Mitochondrial ryanodine‐sensitive Ca2+ channels of rat liver

To examine ryanodine‐sensitive Ca²⁺ channels in mitochondria of rat hepatocytes and their role in energy state of the cells via investigation of the ryanodine effect on mitochondrial membrane potential. Oxygen consumption was measured by polarography using the Clark electrode. The substrates of oxidation such as pyruvate (5mM), α‐ketoglutarate (5mM), or succinate (5mM) were used. Oxidative phosphorylation was stimulated by the addition of adenosine diphosphate (200nM). Mitochondrial membrane potential was measured using a voltage‐sensitive fluorescent probe tetramethylrhodamine‐methyl‐ester (0.1μM) and was analyzed by a flow cytometer. To evaluate the intact mitochondria, we used carbonil cyanide m‐chlorophenyl hydrazone (CCCP, 10μM). Changes in the ionized calcium concentration in rat liver mitochondria were measured using a fluorescent probe Fluo‐4 AM. Effect of ryanodine on oxygen consumption of rat liver mitochondria depends on the oxidation substrate and the incubation time. Oxidation of pyruvate in the presence of ryanodine (0.05μM) decreased the membrane potential of rat liver mitochondria by 38.4%. At higher concentrations, ryanodine (0.1μM or 1μM) led to decrease of membrane potential by 51.7% and 42.8%, respectively. In contrast, oxidation of α‐ketoglutarate in the presence of ryanodine (0.05μM) increased mitochondrial membrane potential by 16.8%. However, at higher concentrations, ryanodine (0.1μM or 1μM) triggered a decreasing of membrane potential by 42.5% and 31.0%, respectively. Therefore, ryanodine at various concentrations (0.05μM, 0.1μM, or 1μM) causes differential effects on Ca²⁺ concentration in the mitochondria matrix under oxidation of pyruvate or α‐ketoglutarate. The data suggest the presence of ryanodine receptors in mitochondrial membrane of rat hepatocytes. Their inhibition with higher concentrations of ryanodine leads to decreasing of intra‐mitochondrial Ca²⁺ concentration and affecting the energy state of mictochondria in hepatocytes.     

Design,synthesis and biological activity of cell‐penetrating peptide‐modified octreotide analogs

Four novel octreotide analogs with cell‐penetrating peptides (CPPs) at the N‐terminus or C‐terminus were synthesized by a stepwise Fmoc solid‐phase synthesis strategy. The synthesized peptides were analyzed and characterized using reverse phase HPLC and MALDI‐TOF mass spectrometry. The antiproliferative activity of the analogs was tested in vitro on human gastric (SGC‐7901) and hepatocellular cancer (BEL7402) cell lines using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. Interestingly, these analogs showed a higher anticancer activities than the parent octreotide except CMTPT03 analog. The results demonstrate that the designed octreotide analogs enhance their anticancer activity after linking together the CPPs to octreotide at the N‐terminus, and are potential molecules for future use in cancer therapy and drug targeting. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Organelle‐specific probe JC‐1 identifies membrane potential differences in the mitochondrial function of bovine sperm

The mitochondrial probe 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethylbenzimidazolyl‐carbocyanine iodide (JC‐1) not only identifies mitochondria exhibiting low membrane potentials by the emission of green fluorescence (range, 510–520 nm) but also differentiates these from mitochondria exhibiting relatively high membrane potentials. This discrimination occurs because JC‐1 forms aggregates at high membrane potentials. These J‐aggregates emit a bright red‐orange fluorescence at 590 nm. In this study, JC‐1 was combined with the classical dead cell stain, propidium iodide (PI), to identify a spectrum of functional sperm along with degenerate sperm. Flow cytometric analysis of bull sperm showed that the aggregate:monomer ratio differed among bulls before cryopreservation (P < 0.001) but not afterwards (P > 0.05). The effects of stain equilibration time, sperm concentration, and live:dead ratios were examined. The addition of SYBR‐14 to the JC‐1 and PI combination enhanced the distinction between the red PI‐stained and red‐orange JC‐1–stained populations. This discrimination between J‐aggregates and the PI‐stained sperm was affected by sperm concentration. These studies show that JC‐1 can be useful in monitoring mitochondrial function in bovine sperm. Mol. Reprod. Dev. 53:222–229, 1999. © 1999 Wiley‐Liss, Inc.     

Reactive oxygen species‐provoked mitochondria‐dependent cell death during ageing of elm (Ulmus pumila L.) seeds

Previous studies have shown that controlled deterioration treatment (CDT) induces programmed cell death in elm (Ulmus pumila L.) seeds, which undergo certain fundamental processes that are comparable to apoptosis in animals. In this study, the essential characteristics of mitochondrial physiology in elm seeds during CDT were identified by cellular ultrastructural analysis, whole‐body optical imaging, Western blotting and semi‐quantitative RT–PCR. The alteration in mitochondrial morphology was an early event during CDT, as indicated by progressive dynamic mitochondrial changes and rupture of the mitochondrial outer membrane; loss of mitochondrial transmembrane potential (Δψ_m) ensued, and mitochondrial ATP levels decreased. The mitochondrial permeability transition pore inhibitor cyclosporine A effectively suppressed these changes during ageing. The in situ localization of production of reactive oxygen species (ROS), and evaluation of the expression of voltage‐dependent anion‐selective channel and cyclophilin D indicated that the levels of mitochondrial permeability transition pore components were positively correlated with ROS production, leading to an imbalance of the cellular redox potential and ultimately to programmed cell death. Pre‐incubation with ascorbic acid slowed loss of mitochondrial Δψ_m, and decreased the effect of CDT on seed viability. However, there were no significant changes in multiple antioxidant elements or chaperones in the mitochondria during early stages of ageing. Our results indicate that CDT induces dynamic changes in mitochondrial physiology via increased ROS production, ultimately resulting in an irreversible loss of seed viability.     

Potentials for prolonging shelf‐life of platelets by near infrared low‐level light

Platelets are uniquely stored at room temperature, during which they gradually loss their quality owing to deteriorating functions of mitochondria over time. Given the well‐documented beneficial effect of near infrared low‐level light (LLL) on mitochondrial functions, we explored a potential for LLL to protect mitochondrial function and extend the shelf‐life of platelets beyond the current 5 days. We found that exposure of a platelet‐containing storage bag to 830 nm light‐emitting diode (LED) light at 0.5 J/cm² prior to storage could significantly retain a pH value and viability of the platelets stored for 8 days with improved quality compared to those stored similarly for 5 days in controls. The LLL inhibited reactive oxygen species (ROS) and lactate production, while sustaining ATP synthesis and mitochondrial membrane potential and morphology in the stored platelets. It also sustained aggregation capacity and in vivo survival of stored platelets, concomitant with no significant activation, as suggested by similar CD62p expression and enhanced agonist‐induced aggregation and recovery following infusion in the presence compared to absence of LLL treatment. This simple, additive‐free, cost‐effective, noninvasive approach can be readily incorporated into the current platelet storage system to potentially improve quality of stored platelets.      

Design of NK‐2‐derived peptides with improved activity against equine sarcoid cells

Equine sarcoid is a topically accessible model for the evaluation of anticancer peptides acting by physical membrane disruption avoiding the complexity of a systemic application. We aim at evaluating and improving natural peptides for host defence as lead structures, where we focus on the cationic and amphipathic peptide NK‐2. Cytotoxicity tests, fluorescence microscopy and a chip‐based biosensor, which enabled real‐time monitoring of cell metabolism, were applied. Cancer cell killing was dynamic with an initial phase of increased cellular respiration, followed by membrane destruction. NK‐2 was substantially improved and shortened. Novel peptides exhibited a fivefold improved activity against sarcoid cells, while haemolysis remained almost unaltered. Similar Zeta potential and similar amount of surface phosphatidylserine of sarcoid and normal skin cells are responsible for a lack of selectivity between these two cell types. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Inhibition of catechol-O-methyltransferase by natural pentacyclic triterpenes: structure–activity relationships and kinetic mechanism

Inhibitors of COMT are clinically used for the treatment of Parkinson’s disease. Here, we report the first natural pentacyclic triterpenoid-type COMT inhibitors and their structure-activity relationships and inhibition mechanism. The most potent compounds were found to be oleanic acid, betulinic acid and celastrol with IC₅₀ values of 3.89–5.07 μM, that acted as mixed (uncompetitive plus non-competitive) inhibitors of COMT, representing a new skeleton of COMT inhibitor. Molecular docking suggested that they can specifically recognise and bind with the unique hydrophobic residues surrounding the catechol pocket. Furthermore, oleanic acid and betulinic acid proved to be less disruptive of mitochondrial membrane potential (MMP) compared to tolcapone, thus reducing the risk of liver toxicity. These findings could be used to produce an ideal lead compound and to guide synthetic efforts in generating related derivatives for further preclinical testing.     

Ratio‐dependent significance thresholds in reciprocal 15N‐labeling experiments as a robust tool in detection of candidate proteins responding to biological treatment

Metabolic labeling of plant tissues with ¹⁵N has become widely used in plant proteomics. Here, we describe a robust experimental design and data analysis workflow implementing two parallel biological replicate experiments with reciprocal labeling and series of 1:1 control mixtures. Thereby, we are able to unambiguously distinguish (i) inherent biological variation between cultures and (ii) specific responses to a biological treatment. The data analysis workflow is based on first determining the variation between cultures based on ¹⁵N/¹⁴N ratios in independent 1:1 mixtures before biological treatment is applied. In a second step, ratio‐dependent SD is used to define p‐values for significant deviation of protein ratios in the biological experiment from the distribution of protein ratios in the 1:1 mixture. This approach allows defining those proteins showing significant biological response superimposed on the biological variation before treatment. The proposed workflow was applied to a series of experiments, in which changes in composition of detergent resistant membrane domains was analyzed in response to sucrose resupply after carbon starvation. Especially in experiments involving cell culture treatment (starvation) prior to the actual biological stimulus of interest (resupply), a clear distinction between culture to culture variations and biological response is of utmost importance.     

Stimulatory effect of regucalcin on mitochondrial ATP‐dependent calcium uptake activity in rat kidney cortex

The effect of regucalcin, which is a regulatory protein of Ca²⁺ signaling, on Ca²⁺‐ATPase activity in isolated rat renal cortex mitochondria was investigated. The presence of regucalcin (50, 100, and 250 nM) in the enzyme reaction mixture led to a significant increase in Ca²⁺‐ATPase activity. Regucalcin significantly stimulated ATP‐dependent ⁴⁵Ca²⁺ uptake by the mitochondria. Ruthenium red (10⁻⁶ M) or lanthunum chloride (10⁻⁶ M), an inhibitor of mitochondrial Ca²⁺ uptake, markedly inhibited regucalcin (100 nM)‐increased mitochondrial Ca²⁺‐ATPase activity and ⁴⁵Ca²⁺ uptake. The effect of regucalcin (100 nM) in elevating Ca²⁺‐ATPase activity was completely prevented by the presence of digitonin (10⁻²%), a solubilizing reagent of membranous lipids, vanadate, an inhibitor of phosphorylation of ATPase, or dithiothreitol (50 mM), a protecting reagent of the sulfhydryl (SH) group of the enzyme. The activating effect of regucalcin (100 nM) on Ca²⁺‐ATPase activity was not further enhanced by calmodulin (0.30 μM) or dibutyryl cyclic AMP (10⁻⁴ M), which could increase Ca²⁺‐ATPase activity. Trifluoperazine (TFP; 50 μM), an antagonist of calmodulin, significantly decreased Ca²⁺‐ATPase activity. The activating effect of regucalcin on the enzyme was also seen in the presence of TFP, indicating that regucalcin's effect is not involved in mitochondrial calmodulin. The present study demonstrates that regucalcin can stimulate Ca²⁺‐pump activity in rat renal cortex mitochondria, and that the protein may act on an active site (SH group) related to phosphorylation of mitochondrial Ca²⁺‐ATPase. J. Cell. Biochem. 80:285–292, 2000. © 2000 Wiley‐Liss, Inc.     

Ion channel activity of transmembrane segment 6 of Escherichia coli proton‐dependent manganese transporter

Synthetic peptides corresponding to the sixth transmembrane segment (TMS6) of secondary‐active transporter MntH (Proton‐dependent Manganese Transporter) from Escherichia coli and its two mutations in the functionally important conserved histidine residue were used as a model for structure–function study of MntH. The secondary structure of the peptides was estimated in different environments using circular dichroism spectroscopy. These peptides interacted with and adopted helical conformations in lipid membranes. Electrophysiological experiments demonstrated that TMS6 was able to form multi‐state ion channels in model biological membranes. Electrophysiological properties of these weakly cation‐selective ion channels were strongly dependent on the surrounding pH. Manganese ion, as a physiological substrate of MntH, enhanced the conductivity of TMS6 channels, influenced the transition between closed and open states, and affected the peptide conformations. Moreover, functional properties of peptides carrying two different mutations of His²¹¹ were analogous to in vivo functional characteristics of Nramp/MntH proteins mutated at homologous residues. Hence, a single functionally important TMS can retain some of the functional properties of the full‐length protein. These findings could contribute to understanding the structure–function relationship at the molecular level. However it remains unclear to what extent the peptide‐specific channel activity represents a functional aspect of the full‐length membrane carrier protein. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 718–726, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Synthesis,biological activity and conformational analysis of head‐to‐tail cyclic analogues of LL37 and histatin 5

LL37 and histatin 5 are antimicrobial peptides. LL37 exhibits killing activity against a broad spectrum of pathogens, whereas histatin 5 is primarily an antifungal agent. Head‐to‐tail cyclization of histatin 5 did not affect its antimicrobial and haemolytic activity. The cyclic LL37 exhibits identical antifungal and haemolytic activity as does LL37. Its antimicrobial activity varied in one dilution depending on the kind of bacteria. The structure of cyclic peptides was studied by circular dichroism spectroscopy. Both peptides undergo a conformational change leading to stabilisation of their α‐helical structure in the presence of negatively charged sodium dodecyl sulfate micelles. However, with cyclic histatin 5, the presence of Zn²⁺ ions is also necessary to fuse the peptide to the micelle. The specific action of the Zn²⁺ ions is attributed to the presence of a zinc‐binding motif, His‐Glu‐X‐X‐His. It has been speculated that this zinc complexing may be related to the well‐established anticandidal activity. In the case of cyclic LL37, also the presence of a zwitterionic dodecylphosphocholine micelle induces formation of the helical structure. A microwave‐assisted procedure for the cleavage of a peptide from the 2‐chlorotrityl chloride resin was, for the first time, successfully used to obtain protected peptide fragments that can be applied to the preparation of head‐to‐tail cyclopeptides or to condensation of peptidic fragments. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Dammarane triterpenes targeting α-synuclein: biological activity and evaluation of binding sites by molecular docking

Parkinson''s disease (PD) is a neurodegenerative disorder that affects adult people whose treatment is palliative. Thus, we decided to test three dammarane triterpenes 1, 1a, 1b, and we determined that 1 and 1a inhibit β-aggregation through thioflavine T rather than 1b. Since compound 1 was most active, we determined the interaction between α-synuclein and 1 at 50 µM (Kd) through microscale thermophoresis. Also, we observed differences in height and diameter of aggregates, and α-synuclein remains unfolded in the presence of 1. Also, aggregates treated with 1 do not provoke neurites'' retraction in N2a cells previously induced by retinoic acid. Finally, we studied the potential sites of interaction between 1 with α-synuclein fibrils using molecular modelling. Docking experiments suggest that 1 preferably interact with the site 2 of α-synuclein through hydrogen bonds with residues Y39 and T44.     

Control of RAB7 activity and localization through the retromer‐TBC1D5 complex enables RAB7‐dependent mitophagy

Retromer is an endosomal multi‐protein complex that organizes the endocytic recycling of a vast range of integral membrane proteins. Here, we establish an additional retromer function in controlling the activity and localization of the late endosomal small GTPase RAB7. Surprisingly, we found that RAB7 not only decorates late endosomes or lysosomes, but is also present on the endoplasmic reticulum, trans‐Golgi network, and mitochondrial membranes, a localization that is maintained by retromer and the retromer‐associated RAB7‐specific GAP TBC1D5. In the absence of either TBC1D5 or retromer, RAB7 activity state and localization are no longer controlled and hyperactivated RAB7 expands over the entire lysosomal domain. This lysosomal accumulation of hyperactivated RAB7 results in a striking loss of RAB7 mobility and overall depletion of the inactive RAB7 pool on endomembranes. Functionally, we establish that this control of RAB7 activity is not required for the recycling of retromer‐dependent cargoes, but instead enables the correct sorting of the autophagy related transmembrane protein ATG9a and autophagosome formation around damaged mitochondria during Parkin‐mediated mitophagy.     

An experimental test of density‐dependent selection on temperament traits of activity,boldness and sociability

Temperament traits are seen in many animal species, and recent evolutionary models predict that they could be maintained by heterogeneous selection. We tested this prediction by examining density‐dependent selection in juvenile common lizards Zootoca vivipara scored for activity, boldness and sociability at birth and at the age of 1 year. We measured three key life‐history traits (juvenile survival, body growth rate and reproduction) and quantified selection in experimental populations at five density levels ranging from low to high values. We observed consistent individual differences for all behaviours on the short term, but only for activity and one boldness measure across the first year of life. At low density, growth selection favoured more sociable lizards, whereas viability selection favoured less active individuals. A significant negative correlational selection on activity and boldness existed for body growth rate irrespective of density. Thus, behavioural traits were characterized by limited ontogenic consistency, and natural selection was heterogeneous between density treatments and fitness traits. This confirms that density‐dependent selection plays an important role in the maintenance of individual differences in exploration‐activity and sociability.     

Use of a Novel Cell Adhesion Method and Digital Measurement to Show Stimulus‐dependent Variation in Somatic and Oral Ciliary Beat Frequency in Paramecium

When Paramecium encounters positive stimuli, the membrane hyperpolarizes and ciliary beat frequency increases. We adapted an established immobilization protocol using a biological adhesive and a novel digital analysis system to quantify beat frequency in immobilized Paramecium. Cells showed low mortality and demonstrated beat frequencies consistent with previous studies. Chemoattractant molecules, reduction in external potassium, and posterior stimulation all increased somatic beat frequency. In all cases, the oral groove cilia maintained a higher beat frequency than mid‐body cilia, but only oral cilia from cells stimulated with chemoattactants showed an increase from basal levels.     

Variations in yeast plasma‐membrane lipid composition affect killing activity of three families of insect antifungal peptides

Naturally occurring antimicrobial peptides and their synthetic analogues are promising candidates for new antifungal drugs. We focused on three groups of peptides isolated from the venom of bees and their synthetic analogues (lasioglossins, halictines and hylanines), which all rapidly permeabilised the plasma membrane. We compared peptides' potency against six pathogenic Candida species (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei and C. dubliniensis) and the non‐pathogenic model yeast Saccharomyces cerevisiae. Their activity was independent of the presence of the multidrug‐resistant pumps of C. glabrata but was influenced by the lipid composition of cell plasma membranes. Although the direct interaction of the peptides with ergosterol was negligible in comparison with amphotericin B, the diminished ergosterol content after terbinafine pretreatment resulted in an increased resistance of C. glabrata to the peptides. The tested peptides strongly interacted with phosphatidylglycerol, phosphatidic acid and cardiolipin and partly with phosphatidylinositol and phosphatidylethanolamine. The interactions between predominantly anionic phospholipids and cationic peptides indicated a mainly electrostatic binding of peptides to the membranes. The results obtained also pointed to a considerable role of the components of lipid rafts (composed from sphingolipids and ergosterol) in the interaction of yeast cells with the peptides.     

In vitro assessment of the growth and plasma membrane H+‐ATPase inhibitory activity of ebselen and structurally related selenium‐ and sulfur‐containing compounds in Candida albicans

Ebselen (EB, compound 1) is an investigational organoselenium compound that reduces fungal growth, in part, through inhibition of the fungal plasma membrane H⁺‐ATPase (Pma1p). In the present study, the growth inhibitory activity of EB and of five structural analogs was assessed in a fluconazole (FLU)‐resistant strain of Candida albicans (S2). While none of the compounds were more effective than EB at inhibiting fungal growth (IC₅₀ ～ 18 μM), two compounds, compounds 5 and 6, were similar in potency. Medium acidification assays performed with S2 yeast cells revealed that compounds 4 and 6, but not compounds 2, 3, or 5, exerted an inhibitory activity comparable to EB (IC₅₀ ～ 14 μM). Using a partially purified Pma1p preparation obtained from S2 yeast cells, EB and all the analogs demonstrated a similar inhibitory activity. Taken together, these results indicate that EB analogs are worth exploring further for use as growth inhibitors of FLU‐resistant fungi.     

Expression of anti‐apoptosis genes alters lactate metabolism of Chinese Hamster Ovary cells in culture

In an effort to develop robust Chinese Hamster Ovary host cell lines, a variety of anti‐apoptotic genes were over‐expressed, either singly or in combination, followed by screening of transfectants for improved cell growth, extended longevity, reduced caspase 3/7 activity, and enhanced mitochondrial membrane potential (MMP). Two particular cell lines, one containing two anti‐apoptotic genes, E1B‐19K and Aven (EA167), and another containing three, E1B‐19K, Aven, and a mutant of XIAP (EAX197), exhibited a reduction in caspase 3 activity of at least 60% and a 170% enhancement in mitochondrial membrane potential compared to controls when treated with staurosporine. In batch cell growth experiments, the peak viable cell densities and viabilities were higher resulting in a 186% increase in integrated viable cell densities. Analyses of metabolite utilization and formation of waste products indicated that the apoptotic resistant cell lines depleted all the lactate when grown in commercially available CD‐CHO medium while significant levels (>1.8 g/L) accumulated in the host cell lines. When the lactate level was replenished daily in the apoptotic resistant cell lines, the cell lines consumed lactate and the culture longevity was extended up to four additional days compared to control cell lines. Furthermore, the anti‐apoptosis cell lines also accumulated lower levels of ammonia. The ability of the apoptotic resistant cell lines to consume lactate was exploited by cultivating them in a “high” glucose medium containing 15 g/L (60 mM glucose) in which apoptotic resistant cell lines exhibited lower maximum lactate (1.8 g/L) compared to control cell lines which accumulated concentrations of lactate (2.2 g/L) that appeared to be deleterious for growth. The shaker flask titer of a therapeutic antibody product expressed in an apoptotic resistant cell line in “high” glucose medium reached 690 mg/L compared to 390 mg/L for a cell line derived from a control host cell line. These results represent to our knowledge the first example in the literature in which manipulation of the apoptosis pathway has altered the nutrient consumption profile of mammalian cells in culture; findings that underscore the interdependence of the apoptotic cellular machinery and metabolism and provide greater flexibility to mammalian bioreactor process development. Biotechnol. Bioeng. 2009;103: 592–608. © 2009 Wiley Periodicals, Inc.