Membrane potential plays various key roles in live bacterial and eukaryotic cells. So far, the effects of membrane potential on action of antimicrobial peptides (AMPs) and cell-penetrating peptides ...(CPPs) have been examined using cells and small lipid vesicles. However, due to the technical drawbacks of these experiments, the effect of membrane potential on the actions of AMPs and CPPs and the elementary processes of interactions of these peptides with cell membranes and vesicle membranes are not well understood. In this short review, we summarize the results of the effect of membrane potential on the action of an AMP, lactoferricin B (LfcinB), and a CPP, transportan 10 (TP10), in vesicle membranes revealed by the single giant unilamellar vesicle (GUV) method. Parts of the actions and their elementary steps of AMPs and CPPs interacting vesicle membranes under membrane potential are clearly revealed using the single GUV method. The experimental methods and their analysis described here can be used to elucidate the effects of membrane potential on various activities of peptides such as AMPs, CPPs, and proteins. Moreover, GUVs with membrane potential are more suitable as a model of cells or artificial cells, as well as GUVs containing small vesicles.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Lactoferricin B (LfcinB) is a cationic antimicrobial peptide, and its capacity to damage the bacterial plasma membrane is suggested to be a main factor in LfcinB's antimicrobial activity. However, ...the specific processes and mechanisms in LfcinB-induced membrane damage are unclear. In this report, using confocal laser-scanning microscopy, we examined the interaction of LfcinB with single Escherichia coli cells and spheroplasts containing the water-soluble fluorescent probe calcein in the cytoplasm. LfcinB induced rapid calcein leakage from single E. coli cells and from single spheroplasts, indicating that LfcinB interacts directly with the plasma membrane and induces its rapid permeabilization. The proton ionophore carbonyl cyanide m-chlorophenylhydrazone suppressed this leakage. Next, we used the single giant unilamellar vesicle (GUV) method to examine LfcinB's interaction with GUVs comprising polar lipid extracts of E. coli containing a water-soluble fluorescent probe, Alexa Fluor 647 hydrazide (AF647). We observed that LfcinB stochastically induces local rupture in single GUVs, causing rapid AF647 leakage; however, higher LfcinB concentrations were required for AF647 leakage from GUVs than from E. coli cells and spheroplasts. To identify the reason for this difference, we examined the effect of membrane potential on LfcinB-induced pore formation, finding that the rate of LfcinB-induced local rupture in GUVs increases greatly with increasing negative membrane potential. These results indicate that membrane potential plays an important role in LfcinB-induced local rupture of lipid bilayers and rapid permeabilization of E. coli plasma membranes. On the basis of these results, we discuss the mode of action of LfcinB's antimicrobial activity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
For antimicrobial peptides (AMPs) with antimicrobial and bactericidal activities and cell-penetrating peptides (CPPs) with activity to permeate through plasma membrane, their interactions with lipid ...bilayer region in plasma membrane play important roles in these functions. However, the elementary processes and mechanisms of their functions have not been clear. The single giant unilamellar vesicle (GUV) method has revealed the details of elementary processes of interaction of some AMPs and CPPs with lipid vesicles. In this review, we summarize the mode of action of AMPs such as magainin 2 (Mag) and CPPs such as transportan 10 (TP10), revealed by the single GUV methods, and especially we focus on the role of membrane tension in actions of Mag and TP10 and the mechanisms of their actions. First, we explain the characteristics of the single GUV method briefly. Next, we summarize the recent view on the effect of tension on physical properties of lipid bilayers and describe the role of tension in actions of Mag and TP10. Some experimental results indicate that Mag-induced pore is a stretch-activated pore. The effect of packing of transbilayer asymmetric lipid on Mag-induced pore formation is described. On the other hand, entry of fluorescent dye, carboxyfluorescein (CF)-labeled TP10 (i.e., CF-TP10), into single GUVs without pore formation is affected by tension and high concentration of cholesterol. Pre-pore model for translocation of CF-TP10 across lipid bilayer is described. The experimental methods and their analysis described here are useful for investigation of functions of the other types of AMPs, CPPs, and proteins.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The effect of membrane potential on plasma membrane damage generated by antimicrobial peptides (AMPs) is an important, yet poorly characterized, process. Here, we studied the effect of membrane ...potential (φm) on pore formation by magainin 2 (Mag) in single giant unilamellar vesicles (GUVs) composed of dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) membranes. Various membrane potentials in GUVs containing gramicidin A were generated as a result of K+ concentration gradients. First, we examined Mag-generated membrane permeation of the water-soluble fluorescent probe calcein in single DOPG/DOPC-GUVs in the presence of membrane potential. The results indicate that the rate constant (kp) of Mag-induced pore formation increased with increasing negative membrane potentials. Analysis of the rim intensity of single GUVs interacting with low concentrations of a fluorescent probe, carboxyfluorescein-labeled Mag (CF-Mag), using confocal laser scanning microscopy (CLSM) shows that the concentration of CF-Mag in the membrane greatly increased with negative membrane potentials. This indicates that the binding constant of CF-Mag to the membrane increased with more negative membrane potentials. To elucidate the location of Mag in a GUV with φm during Mag-induced pore formation, we examined the interaction of Mag and a low concentration of a CF-Mag mixture with single GUVs containing the water-soluble fluorescent probe AF647 using CLSM. The data indicate that CF-Mag locates in the external leaflet of single GUVs until just before pore formation. Based on these data, we conclude that the increase in the surface concentration of Mag is one of the primary causes of the increase in kp with negative membrane potential.
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•Magainin 2 (Mag) caused leakage of calcein from GUVs under membrane potential, φm.•Rate constant of Mag-induced pore formation (kp) increased as ∣φm∣ increased.•Binding constant of CF-Mag to GUV membrane increased with an increase in ∣φm∣.•Under φm, Mag locates in outer leaflet of GUV until just before pore formation.•Increase in Mag surface concentration is one of main causes of the increase in kp.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Cell-penetrating peptides (CPPs) can translocate across plasma membranes to enter the cytosol of eukaryotic cells without decreasing cell viability. We revealed the mechanism underlying this ...translocation by examining the effect of membrane potential, φm, on the entry of a CPP, transportan 10 (TP10), into the lumen of single giant unilamellar vesicles (GUVs). For this purpose, we used the single GUV method to detect the entry of carboxyfluorescein (CF)-labeled TP10 (CF-TP10) into the lumen of single GUVs. First, we used various K+ concentration differences to apply different negative membrane potentials on single GUVs containing gramicidin A in their membrane and confirmed these potentials using the φm-sensitive fluorescent probe 3,3′-dihexyloxacarbocyanine iodine. The fluorescence intensity of the GUV membranes (i.e., the rim intensity) due to 3,3′-dihexyloxacarbocyanine iodine increased with |φm| up to 118 mV, and its dependence on |φm| less than 28 mV agreed with a theoretical estimation (i.e., the dye concentration in the inner leaflet of a GUV is larger than that in the outer leaflet according to the Boltzmann distribution). We then examined the effect of φm on the entry of CF-TP10 into GUVs using single GUVs containing small GUVs or large unilamellar vesicles inside the mother GUV lumen. We found that CF-TP10 entered the GUV lumen without pore formation and the rate of entry of CF-TP10 into the GUV lumen, Ventry, increased with an increase in |φm|. The rim intensity due to CF-TP10 increased with an increase in |φm|, indicating that the CF-TP10 concentration in the inner leaflet of the GUV increased with |φm|. These results indicate that the φm-induced elevation in Ventry can be explained by the increase in CF-TP10 concentration in the inner leaflet with |φm|. We discuss the mechanism underlying this effect of membrane potential based on the pre-pore model of the translocation of CF-TP10 across a GUV membrane.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The entry of cell-penetrating peptides (CPPs) into live cells and lipid vesicles has been monitored using probe (e.g., fluorescent dye)-labeled CPPs. However, probe labeling may alter the interaction ...of CPPs with membranes. We have developed a new method to detect the entry of nonlabeled CPPs into the lumens of single giant unilamellar vesicles (GUVs) without pore formation in the GUV membrane. The GUVs contain large unilamellar vesicles (LUVs) whose lumens contain a high (self-quenching) concentration of the fluorescent dye calcein. If the CPPs enter the GUV lumen and interact with these LUVs to induce calcein leakage, the fluorescence intensity (FI) due to calcein in the GUV lumen increases. The lipid compositions of the LUVs and GUVs allow leakage from LUVs but not from the GUVs. We applied this method to detect the entry of transportan 10 (TP10) into single GUVs comprising dioleoylphosphatidylglycerol and dioleoylphosphatidylcholine and examined the interaction of low concentrations of nonlabeled TP10 with single GUVs whose lumens contain Alexa Fluor 647 hydrazide (AF647) and the LUVs mentioned above. The FI of the GUV lumen due to calcein increased continuously with time without leakage of AF647, indicating that TP10 entered the GUV without pore formation in the GUV membrane. The lumen intensity due to calcein increased with TP10 concentration, indicating that the rate of entry of TP10 into the GUV lumen increased. We estimated the minimum TP10 concentration in a GUV lumen detected by this method. We discuss the entry of nonlabeled TP10 and the characteristics of this method.
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IJS, KILJ, NUK, PNG, UL, UM
The mechanism of entry of cell-penetrating peptides (CPPs) into the cytosol of various cells has been studied by examining the interaction of CPPs with lipid bilayers and their entry into lipid ...vesicle lumens using various methods. Here we describe a single giant unilamellar vesicle (GUV) method to study CPPs. In this new method, we use GUVs containing small GUVs in the mother GUV lumen or GUVs containing large unilamellar vesicles (LUVs) in the GUV lumen and investigate the interaction of fluorescent probe-labeled CPPs with single GUVs in real time using confocal laser scanning microscopy. This method can detect CPPs in the GUV lumen with high sensitivity, allowing immediate measurement of the time course of entry of CPPs into the vesicle lumen. This method allows simultaneous measurement of the entry of CPPs and of CPP-induced pore formation, allowing the relationship between the two events to be determined. One can also simultaneously measure the entry of CPPs and the CPP concentration in the GUV membrane. The rate of entry of CPPs into a single GUV lumen can be estimated by obtaining the fraction of GUVs into which CPPs entered before a specific time t without pore formation among all examined GUVs (i.e., the fraction of entry) and the lumen intensity due to LUVs with bound CPPs. This method is therefore useful for elucidating the mechanism of entry of CPPs into lipid vesicles.
Obesity is associated with metabolic dysfunction and over nutrition. Increased body mass index and obesity are strongly amalgamated with changes in the physiological function of adipose tissue, ...leading to altered secretion of adipocytokines, inflammatory mediators release as well as chronic inflammation and insulin resistance. The purposes of this study were to review the evidence of how obesity and inflammation may lead to insulin resistance and cancer.
Recent findings suggested that increased level of inflammatory mediators in obesity, plays an introductory and cabalistic role in the development of different types of inflammatory disorders including type 2 diabetes mellitus. Link between elevated body mass index and type 2 diabetes mellitus (T2DM). Several of the factors−such as increased levels of leptin, plasminogen activator inhibitor-1, decreased levels of adiponectin, insulin resistance, chronic inflammation etc. consequently result in carcinogenesis and carcinogenic progression too.
This review summarizes how cytokine production in adipose tissue of obese subject creates a chronic inflammatory environment that favors tumor cell motility and invasion to enhance the metastatic potential of tumor cells. High levels of cytokine in the circulation of affected individuals have been associated with a significantly worse outcome. This article also reconnoiters the mechanisms that link obesity to numerous disorders such as inflammation, diabetes, cancers and most specifically combine these processes in a single image. Understanding these mechanisms may assist to understand the consequences of obesity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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•A new method of continuous detection of entry of peptides into single vesicles has been developed.•CF-TP10 entered the GUV lumen without leakage of AF647, i.e., without pore ...formation.•The time course of CF-TP10 concentration in the GUV lumen was monitored continuously.•The rate of entry of CF-TP10 into GUV lumen increased with CFTP10 concentration.•The presence of LUVs in the GUV lumen increased the rate of entry CF-TP10 into single GUVs.
Entry of cell-penetrating peptides (CPPs) into living cells by translocating across plasma membranes is an important physiological phenomenon. To elucidate the mechanism of the translocation of CPPs across lipid bilayers, it is essential to reveal its elementary processes. For this purpose, here, we have developed a new method for the continuous, quantitative detection of the entry of CPPs into giant unilamellar vesicles (GUVs), where we investigate the interaction of fluorescent probe-labeled CPPs with single GUVs containing large unilamellar vesicles (LUVs) and fluorescent probes in their lumens using confocal microscopy. Using this method, we investigated the interaction of carboxyfluorescein (CF)-labeled transportan 10 (CF-TP10) with single GUVs comprised of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) containing LUVs of the same membrane and Alexa Fluor 647 hydrazide (AF647) in their lumens. At low concentrations of CF-TP10, first the fluorescence intensity (FI) of the GUV membrane increased with time, and then after some lag time the FI of the GUV lumen due to CF-TP10 increased continuously with time without leakage of AF647. At higher concentrations of CF-TP10, after the FI of the GUV lumen due to CF-TP10 increased significantly, leakage of AF647 started. These results indicate that CF-TP10 entered the GUV lumen by translocating across the GUV membrane and then bound to the LUVs there without pore formation and that CF-TP10 concentration in the lumen increased with time. The rate of entry of CF-TP10 into GUV lumen increased with CF-TP10 concentration. We discussed the kinetics of entry of CF-TP10 into single GUVs.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
The study was conducted to evaluate the in vitro antimicrobial activity, cytotoxic, and membrane stabilization activities, and in vivo antiemetic and antipyretic potentials of ethanolic extract, ...n-hexane and ethyl acetate soluble fractions of Spilanthes paniculata leaves for the first time widely used in the traditional treatments in Bangladesh.
In antipyretic activity assay, a significant reduction (P < 0.05) was observed in the temperature in the mice tested. At dose 400 mg/kg-body weight, the n-hexane soluble fraction showed the effect (36.7 ± 0.63°C ) as like as the standard (dose 150 mg/kg-body weight) after 5 h of administration. Extracts showed significant (P < 0.001) potential when tested for the antiemetic activity compared to the standard, metoclopramide. At dose 50 mg/kg-body weight, the standard showed 67.23% inhibition, whereas n-hexane and ethyl acetate soluble fractions showed 37.53% and 24.93% inhibition of emesis respectively at dose 400 mg/kg-body weight. In antimicrobial activity assay, the n-hexane soluble fraction (400 μg/disc) showed salient activity against the tested organisms. It exerts highest activity against Salmonella typhi (16.9 mm zone of inhibition); besides, crude, and ethyl acetate extracts showed resistance to Bacillus cereus and Bacillus subtilis, and Vibrio cholera respectively. All the extracts were tested for lysis of the erythrocytes. At the concentration of 1mg/ml, ethanol extract, and n-hexane and ethyl acetate soluble fractions significantly inhibited hypotonic solution induced lysis of the human red blood cell (HRBC) (27.406 ± 3.57, 46.034 ± 3.251, and 30.72 ± 5.679% respectively); where standard drug acetylsalicylic acid (concentration 0.1 mg/ml) showed 77.276 ± 0.321% inhibition. In case of heat induced HRBC hemolysis, the plant extracts also showed significant activity (34.21 ± 4.72, 21.81 ± 3.08, and 27.62 ± 8.79% inhibition respectively). In the brine shrimp lethality bioassay, the n-hexane fraction showed potent (LC50 value 48.978 μg/ml) activity, whereas ethyl acetate fraction showed mild (LC50 value 216.77 μg/ml) cytotoxic activity.
Our results showed that the n-hexane extract has better effects than the other in all trials. In the context, it can be said that the leaves of S. paniculata possess remarkable pharmacological effects, and justify its folkloric use as antimicrobial, antipyretic, anti-inflammatory, and antiemetic agent. Therefore, further research may be suggested to find possible mode of action of the plant part.
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