Aggregatation,fusion and aqueous content release from liposomes induced by lysozyme derivatives: Effect on the lytic activity

Chemically modified lysozymes, namely: N-succinyl lysozyme, glycine methyl ester of N-succinyl lysozyme and oxoindole lysozyme have been prepared. Aggregation, fusion and leakage of phospholipid vesicles induced by these derivatives have been studied in comparison with the effect of the unmodified protein. The experiments were carried out with negatively charges 9PC/ PA, 9:1) and uncharged (PC and PC/DOPE/Chol (10:5:5)) lipid vesicles of different packing. Fusion and aggregation of negatively charged phospholipid vesicles is induced by proteins positively charged at pH 7·0 involving electrostatic interactions. a similar pattern on fusion and aggregation of the least stably packed lipid vesicles points also to hydrophobic forces playing a role in the lipid-protein interaction. A conformational change of the protein involved increasing β-turns, loops and unordered structure at the expenses of β-sheet without affecting λhelix content. The conformational effect is necessary to provoke the effects studied, since one of the derivatives (N-succinyl lysozyme) neither changes conformation nor causes aggregation and fusion of vesicles. However, there is no relationship between lysozyme activity and fusion or aggregation of lipid vesicles that catalytic and fusogenci sites of, indicating lysozyme are topographically different     

Biogenesis of rat mitochondrial citrate carrier (CIC): the N-terminal presequence facilitates the solubility of the preprotein but does not act as a targeting signal

Most mitochondrial preproteins carry a cleavable N-terminal presequence that mediates targeting to mitochondria and translocation across the mitochondrial membranes. In this study, we characterized the presequence of the citrate carrier (CIC, tricarboxylate carrier) of rat liver mitochondria. The CIC presequence was found to be dispensable both for targeting to mitochondria and insertion into the inner membrane. Unlike the presequence of the related phosphate carrier, fusion of the CIC presequence to the cytosolic enzyme dihydrofolate reductase did not confer mitochondrial targeting, indicating that the CIC presequence does not act as a targeting signal. However, the presequence was required to keep the CIC in a soluble state. Mature CIC lacking the presequence was prone to aggregation. We conclude that mitochondrial presequences do not necessarily act as mediators of targeting. In the case of the CIC, the presequence appears to determine the folding state of the preprotein.     

Chitosan-reduced gold nanoparticles: a novel carrier for the preparation of spray-dried liposomes for topical delivery

Exposure of skin to various chemical and physical agents results in excessive stress to the outermost cell layer of the skin, causing different degenerative effects that can be minimized by using antioxidant formulations. The major challenge, in this regard, is to develop a formulation, which can prevent photodegradation of the actives, thus allowing a significant amount to be deposited at the site. In recent decades, liposomal formulations have been extensively employed to overcome the barrier properties of the skin and photodegradation of actives. In the present study, chitosan-reduced gold nanoparticles were investigated for its potential as a carrier to prepare liposomes by a spray-drying method. Liposomes so obtained were characterized for phospholipid recovery, diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy, particle size, zeta potential, encapsulation efficiency, and deposition of drug and gold nanoparticles in the rat skin. Further, a liposomal gel formulation was prepared using Carbopol^® 980 NF (Noveon Systems, Kochi, India) and evaluated for drug deposition in the skin. Antioxidant activity of vitamin C encapsulated in gold liposomes was determined on a human leukemia (HL-60) cell line. The use of gold nanoparticles as a carrier showed improved phospholipid recovery and thus overcomes the liposome scalability problem. DRIFT spectra confirmed the presence of phospholipid in the formulation. Liposomal gel showed improved drug deposition, as compared to control and marketed preparations. A more interesting contribution of the chitosan-reduced gold nanoparticles was an enhanced antioxidant activity seen in case of the vitamin C–loaded gold liposomal formulation. Liposomal formulation was found to be stable for 3 months at 30°C and 65% relative humidity.     

Hierarchical recruitment by AMPA but not staurosporine of pro-apoptotic mitochondrial signaling in cultured cortical neurons: evidence for caspase-dependent/independent cross-talk

Excitotoxicity mediated via the ( S )-α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) subtype of receptor for l -glutamate contributes to various neuropathologies involving acute brain injury and chronic degenerative disorders. In this study, AMPA-induced neuronal injury and staurosporine (STS)-mediated apoptosis were compared in primary neuronal cultures of murine cerebral cortex by analyzing indices up- and downstream of mitochondrial activation. AMPA-mediated apoptosis involved induction of Bax, loss of mitochondrial transmembrane potential (ΔΨ_m), early release of cytochrome c (cyt c ), and more delayed release of second mitochondrial activator of caspases (SMAC), Omi, and apoptosis-inducing factor (AIF) with early calpain and minor late activation of caspase 3. STS-induced apoptosis was characterized by a number of differences, a more rapid time course, non-involvement of ΔΨ_m, and relatively early recruitment of SMAC and caspase 3. The AMPA-induced rise in intracellular calcium appeared insufficient to evoke ΔΨ_m as release of cyt c preceded mitochondrial depolarization, which was followed by the cytosolic translocation of SMAC, Omi, and AIF. Bax translocation preceded cyt c release for both stimuli inferring its involvement in apoptotic induction. Inclusion of the broad spectrum caspase inhibitor zVAD-fmk reduced the AMPA-induced release of cyt c , SMAC, and AIF, while only affecting the redistribution of Omi and AIF in the STS-treated neurons. Only AIF release was affected by a calpain inhibitor (calpastatin) which exerted relatively minor effects on the progression of cellular injury. AMPA-mediated release of apoptogenic proteins was more hierarchical relative to STS with its calpain activation and caspase-dependent AIF redistribution arguing for a model with cross-talk between caspase-dependent/independent apoptosis.     

The octarepeat region of prion protein, but not the TM1 domain, is important for the antioxidant effect of prion protein

The cellular prion protein (PrP(c)) plays a crucial role in the pathogenesis of prion diseases, but its physiological function is far from understood. Several candidate functions have been proposed including binding and internalization of metal ions, a superoxide dismutase-like activity, regulation of cellular antioxidant activities, and signal transduction. The transmembrane (TM1) region of PrP(c) (residues 110-135) is particularly interesting because of its very high evolutionary conservation. We investigated a possible role of TM1 in the antioxidant defense, by assessing the impact of overexpressing wt-PrP or deletion mutants in N(2)A mouse neuroblastoma cells on intracellular reactive oxygen species (ROS) levels. Under conditions of oxidative stress, intracellular ROS levels were significantly lowered in cells overexpressing either wild-type PrP(c) (wt-PrP) or a deletion mutant affecting TM1 (Delta8TM1-PrP), but, as expected, not in cultures overexpressing a deletion mutant lacking the octapeptide region (Deltaocta-PrP). Overexpression of wt-PrP, Delta8TM1-PrP, or Deltaocta-PrP did not affect basal ROS levels. Interestingly, the mitochondrial membrane potential was significantly lowered in Deltaocta-PrP-transfected cultures in the absence of oxidative stress. We conclude that the protective effect of PrP(c) against oxidative stress involves the octarepeat region but not the TM1 domain nor the high-affinity copper binding site described for human residues His96/His111.     

Secretion of endothelin converting enzyme-1a: The hydrophobic signal anchor domain alone is not sufficient to promote membrane localization

Endothelin converting enzyme-1 (ECE-1) is a type II membrane protein that is important for the proteolytic activation of big endothelin-1 to endothelin-1. Although the highly conserved zinc-binding motif is known to be located in the extracellular domain, the role(s) of the N-terminal and membrane-spanning signal anchor domains in the biosynthesis and function of ECE-1 isoforms, ECE-1a, ECE-1b, and ECE-1c, remain undetermined. In this study, we provide evidence that the deletion of the cytoplasmic N-terminal tail (residues 1-55) of ECE-1a results in the cleavage of a potential signal peptide located in the signal anchor domain leading to the partial secretion of the recombinant enzyme into the media. However, the truncation of N-terminal and/or signal anchor domain does not affect the activity of ECE-1a. Therefore, our results demonstrate that the hydrophobic signal anchor domain alone is not sufficient for the membrane anchoring of ECE-1a and that the N-terminal domain of ECE-1a is important for membrane targeting as well as the intracellular localization of the enzyme.     

The tale of tail-anchored proteins: coming from the cytosol and looking for a membrane

A group of integral membrane proteins, known as C-tail anchored, is defined by the presence of a cytosolic NH2-terminal domain that is anchored to the phospholipid bilayer by a single segment of hydrophobic amino acids close to the COOH terminus. The mode of insertion into membranes of these proteins, many of which play key roles in fundamental intracellular processes, is obligatorily posttranslational, is highly specific, and may be subject to regulatory processes that modulate the protein's function. Although recent work has elucidated structural features in the tail region that determine selection of the correct target membrane, the molecular machinery involved in interpreting this information, and in modulating tail-anchored protein localization, has not been identified yet.     

Mitochondrial membrane depolarization and the selective death of dopaminergic neurons by rotenone: protective effect of coenzyme Q10

Chronic exposure to the pesticide rotenone induces a selective degeneration of nigrostriatal dopaminergic neurons and reproduces the features of Parkinson's disease in experimental animals. This action is thought to be relevant to its inhibition of the mitochondrial complex I, but the precise mechanism of this suppression in selective neuronal death is still elusive. Here we investigate the mechanism of dopaminergic neuronal death mediated by rotenone in primary rat mesencephalic neurons. Low concentrations of rotenone (5-10 nM) induce the selective death of dopaminergic neurons without significant toxic effects on other mesencephalic cells. This cell death was coincident with apoptotic events including capsase-3 activation, DNA fragmentation, and mitochondrial membrane depolarization. Pretreatment with coenzyme Q10, the electron transporter in the mitochondrial respiratory chain, remarkably reduced apoptosis as well as the mitochondrial depolarization induced by rotenone, but other free radical scavengers such as N-acetylcysteine, glutathione, and vitamin C did not. Furthermore, the selective neurotoxicity of rotenone was mimicked by the mitochondrial protonophore carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), a cyanide analog that effectively collapses a mitochondrial membrane potential. These data suggest that mitochondrial depolarization may play a crucial role in rotenone-induced selective apoptosis in rat primary dopaminergic neurons.     

The effect of liposomes on thermotropic membrane phase transitions of bovine spermatozoa and oocytes: implications for reducing chilling sensitivity

We have examined the effects of combinations between egg-phosphatidylcholine (EPC) or dipalmitoylphosphatidylcholine (DPPC) liposomes with either bovine spermatozoa or oocytes on cellular chilling sensitivity, lipid phase transition temperature (T(m)), and the ability of the oocytes to develop to the blastocyst stage. Spermatozoa and oocytes were exposed to EPC and DPPC liposomes at various temperatures (spermatozoa: 4, 12, 16, and 25 degrees C; oocytes: 4, 16, and 32 degrees C). The membrane integrity of the spermatozoa-control group decreased significantly following exposure to 16 or 12 degrees C, compared to ambient temperature (25 degrees C). In contrast, the EPC-sperm group had a greater resistance to chilling at each temperature and showed a decline in membrane integrity only at the lowest temperatures investigated. However, the DPPC-sperm group was injured significantly at all temperatures tested. Similar to the sperm, oocytes from the control group that were exposed to 16 degrees C were injured more severely than oocytes that were electrofused with EPC or DPPC liposomes. The membrane integrity of the oocytes at 16 degrees C that were electrofused with either EPC or DPPC liposomes was approximately the same as the control group held at 32 degrees C (normalized to 100%), compared to 46% in the control group at 16 degrees C (P<0.01). The transition temperatures of the sperm and oocyte membranes revealed different T(m) for the different liposome treatments. All groups had a significantly higher cleavage rate, as well as increased blastocyst formation when oocytes were exposed to temperatures above or below their T(m). We suggest that the T(m) of spermatozoa or oocytes can be changed by spontaneous association or electrofusion of liposomes with cellular membranes and, consequently, the chilling sensitivity can be altered. The resulting possibility is that embryo development after cryopreservation could be improved with such a method.     

VDAC electronics: 2. A new,anaerobic mechanism of generation of the membrane potentials in mitochondria

Mitochondrial hexokinase (HK) and creatine kinase (CK) known to form complexes with a voltage dependent anion channel (VDAC) have been reported to increase cell death resistance under hypoxia/anoxia. In this work we propose a new, non-Mitchell mechanism of generation of the inner and outer membrane potentials at anaerobic conditions. The driving force is provided by the Gibbs free energy of the HK and CK reactions associated with the VDAC–HK and the ANT (adenine nucleotide translocator)–CK–VDAC complexes, respectively, both functioning as voltage generators. In the absence of oxygen, the cytosolic creatine phosphate can be directly used by the ANT–CK–VDAC contact sites to produce ATP from ADP in the mitochondrial matrix. After that, ATP released through the fraction of unbound ANTs in exchange for ADP is used in the mitochondrial intermembrane space by the outer membrane VDAC–HK electrogenic complexes to convert cytosolic glucose into glucose-6-phosphate. A simple computational model based on the application of Ohm's law to an equivalent electrical circuit showed a possibility of generation of the inner membrane potential up to − 160 mV, under certain conditions, and of relatively high outer membrane potential without wasting of ATP that normally leads to cell death. The calculated membrane potentials depended on the restriction of ATP/ADP diffusion in narrow cristae and through the cristae junctions. We suggest that high inner membrane potential and calcium extrusion from the mitochondrial intermembrane space by generated positive outer membrane potential prevent mitochondrial permeability transition, thus allowing the maintenance of mitochondrial integrity and cell survival in the absence of oxygen.     

Interaction of liposomes with homologous series of fluorescent berberine derivatives: New cationic probes for measuring membrane potential

The interaction of liposomes with a series of fluorescent berberine derivatives having different alkyl chain lengths has been investigated. The hydrophobicity of the binding site on the phospholipid membrane increases and mobility decreases with the length of the alkyl chain. If lauryl sulphate micelles are used to bind berberines, the hydrophobicity of the binding site is the same for all derivatives. The dye series represents a model with constant charge and growing lipophilicity. Both electrostatic forces and lipophilicity play an important role in binding. By virtue of the excellent sensitivity of the dyes to medium polarity, berberines prove to be suitable probes for measuring membrane potential, but only in cases when a negative charge is generated in the liposomal interior. The fluorescence response is a linear function of the potential magnitude.     

Effect of oleate on the apparent Km of monoamine oxidase and the amount of membrane-bound hexokinase in isolated rat hepatocytes: Further evidence for the controlling role of the surface charge in hexokinase binding

Brief incubation of isolated rat hepatocytes in the presence of the oleate-bovine serum albumin complex resulted in a release to the cytosol of a portion of hexokinase (EC 2.7.1.1) normally bound to intracellular membranes. This was correlated with an increase of the negative surface potential of the outer mitochondrial membrane, as measured in situ by determining changes of K_m of monoamine oxidase (EC 1.4.3.4). It is suggested that non-esterified fatty acids produce a partial release of bound hexokinase in the liver cell by changing the surface charge of intracellular membranes.     

Interaction of phosphonium ions with Arthrobacter globiformis: evidence for the operation of an efflux system

Abstract Substrate-induced efflux of methyltriphenylphosphonium ion (MTP) from Arthrobacter globiformis has been demonstrated using organisms depleted of endogenous reserves by starvation. Further evidence for the operation of a mediated efflux system was provided by studies on the effect of analogues of MTP on MTP accumulation.     

Father's physique influences mate preferences but not the actual choice of male somatotype in heterosexual women and homosexual men

Body constitution plays an important role in human mate choice. Cross-cultural research reports that women on average prefer men with muscular physique. It is still unclear, however, what mechanisms influence the inter-individual variation in mate preferences and choices of partner's physique. In this study, we tested the mechanisms of an imprinting-like effect (similarity between father and an ideal and actual partner) and of homogamy (similarity between self and an ideal and actual partner) for male physique in heterosexual women and homosexual men. To assess the variation in male physique, we employed somatotype paradigm which characterizes body constitution using three components: endomorphic (heavy-set), mesomorphic (muscular), and ectomorphic (lean). In total, 149 homosexual men and 769 heterosexual women from the Czech Republic indicated the somatotype of their father, ideal and actual partner, and in homosexual men also their own somatotype. In line with previous research, the somatotype most preferred by both men and women was the mesomorphic, followed by the ectomorphic and the endomorphic one. Women's preferences for an ideal partner somatotype weakly correlated with their fathers' somatotype, especially in women who reported a positive relationship with their fathers during childhood. Among homosexual men, we found imprinting-like preferences only for the ectomorphic somatotype component and no significant association with the quality of their relationships with their fathers. We also found no significant relationship between the fathers' and actual partners' somatotype in either heterosexual women or homosexual men. Our research indicates that fathers have a rather weak influence on mate preference for somatotypes and no influence on actual mate choice.     

Plasmodium falciparum: one-step growth in a semi-defined medium and the stimulatory effect of human seric lipoproteins and liposomes

The ring stages of Plasmodium falciparum within red blood cells cultured with complete medium stop growing when transferred to a basic medium containing RPMI plus fatty acid-free bovine serum albumin and dialyzable factors from human serum. Growth and multiplication can be partially restored by the addition of lipoprotein fractions prepared from human serum. No specificity was observed with subclasses of lipoproteins. Synthetic liposomes containing lecithin, oleic acid, and cholesterol mimic the effect of lipoproteins.     

Embedding calix[4]resorcinarenes in liposomes: Experimental and computational investigation of the effect of resorcinarene inclusion on liposome properties and stability

Two calix[4]resorcinarenes, which differ in the length of the alkyl chain on the methylene bridge between the aromatic rings, have been embedded in unilamellar liposomes prepared from 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine in three host/guest ratios, following two different procedures. The effect of the insertion of the guests has been evaluated through the measurements of the viscosity and the kinetic stability of the liposomal systems by means of the fluorescent probes pyrene and 5(6)-carboxyfluorescein. The presence of the guests reduces the viscosity of the liposomes, suggesting a modification of the bilayer structure. However, this does not affect liposome stability. A calix[4]resorcinarene cavitand with a more rigid conformation compared to the parent resorcinarene, has been also synthetized and embedded in liposomes. The free energy of the insertion of the substrates in the lipid bilayer has been evaluated through Molecular Dynamics simulations.     

Autotrophic CO2 fixation by Desulfovibrio baarsii: Demonstration of enzyme activities characteristic for the acetyl-CoA pathway

Abstract The uptake of a membrane potential (Δψ) probe by strain D27 of Saccharomyces cerevisiae has been shown to have properties consistent with entry occurring by means of a non-mediated process rather than the thiamine transporter. Experiments in which the mitochondrial membrane was selectively depolarised indicated that the probe used partitioned into mitochondria thus limiting its usefulness for quantitative measurements of Δψ across the cytoplasmic membrane.     

A549/DDP细胞的抗凋亡特性与其pHi和［Ca2+］i变化相关

以临床用药剂量(30 μmol/L)的顺铂处理敏感的人肺腺癌细胞A549和抗顺铂的A549/DDP细胞,在相同条件下培养.对培养不同时间的两株细胞DNA琼脂糖凝胶电泳分析的结果表明，前者在培养12 h后即呈现DNA梯子，而后者在培养48 h后却未见凋亡特征.用流式细胞计检测其凋亡峰也获得相似的结果.当测定与凋亡相关的生物化学和生物物理变化时发现对顺铂敏感的A549细胞的线粒体膜电势显著降低，胞内更加酸化且胞内钙离子浓度升高；而抗顺铂药物的A549/DDP细胞的线粒体膜电势和pH值却维持在相对较高的水平，而其胞内钙离子浓度随培养时间的延长下降.这些结果表明，抗顺铂的人肺腺癌A549/DDP细胞的抗凋亡特性与其胞内的相对碱化和较高的线粒体膜电势,以及胞内钙离子浓度的降低相关.这些特性可能参与了A549/DDP的顺铂耐药性的调节.