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The surface activity of potassium heptylene decyl succinate (PHDS) at water-air interface was studied. The obtained results showed a very good correlation between the surface activity of PHDS and concentration of surfactant solutions and temperature of the system. The phase behaviour and structure of aggregates in PHDS)/water binary system were investigated. The phase diagram of the PHDS/water binary system was obtained depending on the concentration of components and temperature of the system. It was established that due to the double hydrocarbon chain of the PHDS molecule, the lamellar liquid crystalline phase exists across a wide range of the phase diagram. It was shown that upon the addition of water, the cross-sectional area of the PHDS molecules in lamellar liquid crystals is changed insignificantly. This result was associated with the invariability of the distance of the lipophilic layers of formed lamellar liquid crystals by PHDS molecules. On the basis of video-enhanced microscopic studies, it was established that in two-phase region of the phase diagram, lamellar liquid crystals coexist with water in the form of vesicles. [ABSTRACT FROM AUTHOR]

Background: Δ9-Tetrahydrocannabinol (Δ9-THC) is a principal psychoactive extract of Cannabis sativa and has been traditionally used as palliative medicine for neuropathic pain. Cannabidiol (CBD), an extract of hemp species, has recently attracted increased attention as a cancer treatment, but Δ9-THC is also requiring explored pharmacological application. Objective: This study evaluated the pharmacological effects of Δ9-THC in two human colorectal cancer cell lines. We investigated whether Δ9-THC treatment induces cell death in human colorectal cancer cells. Methods: We performed an MTT assay to determine the pharmacological concentration of Δ9-THC. Annxein V and Western blot analysis confirmed that Δ9-THC induced apoptosis in colorectal cancer cells. Metabolic activity was evaluated using MitoTracker staining and ATP determination. We investigated vesicle formation by Δ9-THC treatment using GW9662, known as a PPARγ inhibitor. Results: The MTT assay showed that treatment with 40 μM Δ9-THC and above inhibited the proliferation of colorectal cancer cells. Multiple intracytoplasmic vesicles were detected upon microscopic observation, and fluorescence-activated cell sorting analysis showed cell death via G1 arrest. Δ9-THC treatment increased the expression of cell death marker proteins, including p53, cleaved PARP-1, RIP1, and RIP3, suggesting that Δ9-THC induced the death of colorectal cancer cells. Δ9-THC treatment also reduced ATP production via changes in Bax and Bcl-2. Δ9-THC regulated intracytoplasmic vesicle formation by modulating the expression of PPARγ and clathrin, adding that antiproliferative activity of Δ9-THC was also affected. Conclusion: In conclusion, Δ9-THC regulated two functional mechanisms, intracellular vesicle formation and cell death. These findings can help to determine how cannabinoids can be used most effectively to improve the efficacy of cancer treatment. [ABSTRACT FROM AUTHOR]

The epsin‐related adaptor proteins Ent3p and Ent5p participate in budding of clathrin coated vesicles in transport between trans‐Golgi network and endosomes in yeast. Transport of the arginine permease Can1p was analyzed, which recycles between plasma membrane and endosomes and can be targeted to the vacuole for degradation. ent3∆ cells accumulate Can1p‐GFP in endosomes. Can1p‐GFP is transported faster to the vacuole upon induction of degradation in ent5∆ cells than in wild type cells. The C‐terminal domain of Ent5p was sufficient to restore recycling of the secretory SNARE GFP‐Snc1p between plasma membrane and TGN in ent3∆ ent5∆ cells. The SNARE Tlg2p was identified as interaction partner of the Ent5p ENTH domain by in vitro binding assays and the interaction site on Ent5p was mapped. Tlg2p functions in transport from early endosomes to the trans‐Golgi network and in homotypic fusion of these organelles. Tlg2p is partially shifted to denser fractions in sucrose density gradients of organelles from ent5∆ cells while distribution of Kex2p is unaffected demonstrating that Ent5p acts as cargo adaptor for Tlg2p in vivo. Taken together we show that Ent3p and Ent5p have different roles in transport and function as cargo adaptors for distinct SNAREs. [ABSTRACT FROM AUTHOR]

Ascoviruses are large, enveloped DNA viruses that induce remarkable changes in cellular architecture during which the cell is partitioned into numerous vesicles for viral replication. Previous studies have shown that these vesicles arise from a process resembling apoptosis yet which differs after nuclear lysis in that mitochondria are not degraded but are modified by the virus, changing in size, shape, and motility. Moreover, infection does not provoke an obvious innate immune response. Thus, we used in vivo RNA sequencing to determine whether infection by the Spodoptera frugiperda ascovirus 1a (SfAV-1a) modified expression of host mitochondrial, cytoskeletal, and innate immunity genes. We show that transcripts from many mitochondrial genes were similar to those from uninfected controls, whereas others increased slightly during vesicle formation, including those for ATP6, ATP8 synthase, and NADH dehydrogenase subunits, supporting electron microscopy (EM) data that these organelles were conserved for virus replication. Transcripts from 58 of 106 cytoskeletal genes studied increased or decreased more than 2-fold postinfection. More than half coded for mitochondrial motor proteins. Similar increases occurred for innate immunity transcripts and their negative regulators, including those for Toll, melanization, and phagocytosis pathways. However, those for many antimicrobial peptides, such as moricin, increased more than 20-fold. In addition, transcripts for gloverin-3, spod_x_tox, Hdd23, and lebocin, also antimicrobial, increased more than 20-fold. Interestingly, a phenoloxidase inhibitor transcript increased 12-fold, apparently to interfere with melanization. SfAV-1a destroys most fat body cells by 7 days postinfection, so innate immunity gene transcripts apparently occur in remaining cells in this tissue and possibly other major tissues, namely, epidermis and tracheal matrix.IMPORTANCE Ascoviruses are large DNA viruses that infect insects, inducing a cellular pathology that resembles apoptosis but which differs by causing enormous cellular hypertrophy followed by cleavage of the cell into numerous viral vesicles for replication. Previous EM studies suggest that mitochondria are important for vesicle formation. Transcriptome analyses of Spodoptera frugiperda larvae infected with SfAV-1a showed that mitochondrial transcripts were similar to those from uninfected controls or increased slightly during vesicle formation, especially for ATP6, ATP8 synthase, and NADH dehydrogenase subunits. This pattern resembles that for chronic disease-inducing viruses, which conserve mitochondria, differing markedly from viruses causing short-term viral diseases, which degrade mitochondrial DNA. Though mitochondrial transcript increases were low, our results demonstrate that SfAV-1a alters host mitochondrial expression more than any other virus. Regarding innate immunity, although SfAV-1a destroys most fat body cells, certain immunity genes were highly upregulated (greater than 20-fold), suggesting that these transcripts may originate from other tissues.

Abstract Intracellular vesicle trafficking is the fundamental process to maintain the homeostasis of membrane-enclosed organelles in eukaryotic cells. These organelles transport cargo from the donor membrane to the target membrane through the cargo containing vesicles. Vesicle trafficking pathway includes vesicle formation from the donor membrane, vesicle transport, and vesicle fusion with the target membrane. Coat protein mediated vesicle formation is a delicate membrane budding process for cargo molecules selection and package into vesicle carriers. Vesicle transport is a dynamic and specific process for the cargo containing vesicles translocation from the donor membrane to the target membrane. This process requires a group of conserved proteins such as Rab GTPases, motor adaptors, and motor proteins to ensure vesicle transport along cytoskeletal track. Soluble N-ethyl-maleimide-sensitive factor (NSF) attachment protein receptors (SNARE)-mediated vesicle fusion is the final process for vesicle unloading the cargo molecules at the target membrane. To ensure vesicle fusion occurring at a defined position and time pattern in eukaryotic cell, multiple fusogenic proteins, such as synaptotagmin (Syt), complexin (Cpx), Munc13, Munc18 and other tethering factors, cooperate together to precisely regulate the process of vesicle fusion. Dysfunctions of the fusogenic proteins in SNARE-mediated vesicle fusion are closely related to many diseases. Recent studies have suggested that stimulated membrane fusion can be manipulated pharmacologically via disruption the interface between the SNARE complex and Ca2+ sensor protein. Here, we summarize recent insights into the molecular mechanisms of vesicle trafficking, and implications for the development of new therapeutics based on the manipulation of vesicle fusion.

Extracts of phytocannabinoids from Cannabis sativa have been studied for therapeutic purposes. Although nonpsychoactive CBD has been studied as a promising anticancer drug because it induces apoptosis in many cancer cells, it is also known to induce several physiological changes. In this study, we clarify the functional role it plays in the morphological characteristics of intracellular vesicle formation as well as apoptosis in A549 human lung cancer cells. CBD treatment shows growth inhibition at concentrations above 20 μM, but FACS analysis shows low efficacy in terms of cell death. Microscopic observations suggest that multiple vesicles were detected in the cytoplasmic region of CBD-treated A549 cells. CBD treatment upregulates apoptosis-related proteins, such as p53, PARP, RIP1, RIP3, Atg12, and Beclin, indicating that CBD regulates several types of cell death. CBD treatment also induced E-cadherin, PPARγ, clathrin, β-adaptin, and Tsg101, also known to be cellular-differentiation inducers or vesicle-formation components. Treatment combining CBD with GW9662, a PPARγ inhibitor, reduced CBD-induced cytoplasmic vesicle formation. This indicates that PPARγ regulates the vesicle-formation mechanism. However, CBD-treated E-cad KO clones did not show this regulatory mechanism. These results elucidate the pharmacological and molecular networks associated with CBD in PPARγ-dependent vesicle formation and the induction of apoptosis. [ABSTRACT FROM AUTHOR]

Sangiliyandi Gurunathan,1 Min-Hee Kang,1 Muhammad Qasim,2 Khalid Khan,3 Jin-Hoi Kim1 1Department of Stem Cell and Regenerative Biotechnology, Konkuk University, Seoul, 05029, Korea; 2Center of Bioengineering and Nanomedicine, Department of Food Science, University of Otago, Dunedin, 9054, New Zealand; 3Science and Technology KPK, Peshawar, PakistanCorrespondence: Sangiliyandi Gurunathan; Jin-Hoi KimDepartment of Stem Cell and Regenerative Biotechnology, Konkuk University, Seoul, 05029, KoreaTel +82 2 450 3687Fax +82 2 544 4645Email gsangiliyandi@yahoo.com; jhkim541@konkuk.ac.krAbstract: Extracellular vesicles (EVs) are a heterogeneous group of membrane-limited vesicles and multi-signal messengers loaded with biomolecules. Exosomes and ectosomes are two different types of EVs generated by all cell types. Their formation depends on local microdomains assembled in endocytic membranes for exosomes and in the plasma membrane for ectosomes. Further, EV release is a fundamental process required for intercellular communication in both normal physiology and pathological conditions to transmit/exchange bioactive molecules to recipient cells and the extracellular environment. The unique structure and composition of EVs enable them to serve as natural nanocarriers, and their physicochemical properties and biological functions can be used to develop next-generation nano and precision medicine. Knowledge of the cellular processes that govern EVs biology and membrane trafficking is essential for their clinical applications. However, in this rapidly expanding field, much remains unknown regarding EV origin, biogenesis, cargo sorting, and secretion, as well as EV-based theranostic platform generation. Hence, we present a comprehensive overview of the recent advances in biogenesis, membrane trafficking, and functions of EVs, highlighting the impact of nanoparticles and oxidative stress on EVs biogenesis and release and finally emphasizing the role of EVs as nanotherapeutic agents.Keywords: extracellular vesicle, membrane trafficking, vesicle formation, cargo sorting and fusion, nanotherapeutics

Background. The study of surface activity, micelle formation, phase behavior and the identification of the nature of the structure of aggregates in solutions of surfactants with a double lipophilic chain is urgent. The study of the micelle-forming ability and phase behavior of surfactants is of practical importance, because their use in many industries is associated with the properties of micellar solutions. Purpose. Investigation of the surface activity and micelle-forming ability of the sodium salt of hexyl ester of decilensuccinic acid (D-12) and the phase behavior of the binary system AD-12 / water, the structure of aggregates in this system. Methodology. The critical micelle concentration was determined using the data on the surface tension of surfactant solutions. The phase state of the binary system was determined by visual observations and using a polarizing microscope. The type of liquid crystal was determined based on the microscopic texture. Originality. Due to the presence of hydrocarbon chains of the surfactant D-12, the lamellar liquidcrystalline phase prevails in a binary system with water on the phase diagram in a wide range. It was found that it coexists with water in the form of vesicles in the two-phase region of the phase diagram. Findings. A phase diagram of the D-12 / water binary system was obtained depending on the concentration of the components and the temperature of the system. At a surfactant concentration above the critical one, D-12 forms a normal micellar solution. At higher concentrations, D-12 molecules form lamellar liquid crystals. It was found that due to the double hydrocarbon chain of the D-12 molecule, the lamellar liquid crystal phase exists in a wide range of the phase diagram. It has been shown that, upon addition of water, the cross-sectional area of D-12 molecules in lamellar liquid crystals changes insignificantly, which is associated with the invariability of the distance between the lipophilic layers of liquid crystals. [ABSTRACT FROM AUTHOR]

Extracts of phytocannabinoids from Cannabis sativa have been studied for therapeutic purposes. Although nonpsychoactive CBD has been studied as a promising anticancer drug because it induces apoptosis in many cancer cells, it is also known to induce several physiological changes. In this study, we clarify the functional role it plays in the morphological characteristics of intracellular vesicle formation as well as apoptosis in A549 human lung cancer cells. CBD treatment shows growth inhibition at concentrations above 20 μM, but FACS analysis shows low efficacy in terms of cell death. Microscopic observations suggest that multiple vesicles were detected in the cytoplasmic region of CBD-treated A549 cells. CBD treatment upregulates apoptosis-related proteins, such as p53, PARP, RIP1, RIP3, Atg12, and Beclin, indicating that CBD regulates several types of cell death. CBD treatment also induced E-cadherin, PPARγ, clathrin, β-adaptin, and Tsg101, also known to be cellular-differentiation inducers or vesicle-formation components. Treatment combining CBD with GW9662, a PPARγ inhibitor, reduced CBD-induced cytoplasmic vesicle formation. This indicates that PPARγ regulates the vesicle-formation mechanism. However, CBD-treated E-cad KO clones did not show this regulatory mechanism. These results elucidate the pharmacological and molecular networks associated with CBD in PPARγ-dependent vesicle formation and the induction of apoptosis.

Vesicle structures primarily embody spherical capsules composed of a single or multiple bilayers, entrapping a pool of aqueous solution in their interior. The bilayers can be synthesised by phospholipids or other amphiphiles (surfactants, block copolymers, etc.). Vesicles with broad-spectrum applications in numerous scientific disciplines, including biochemistry, biophysics, biology, and various pharmaceutical industries, have attracted widespread attention. Consequently, a multitude of protocols have been devised and proposed for their fabrication. In this review, with a motivation to derive the basic conditions for the formation of vesicles, the associated thermodynamic and kinetic aspects are comprehensively appraised. Contextually, an all-purpose overview of the underlying thermodynamics of bilayer/membrane generation and deformation, including the chemical potential of aggregates, geometric packing and the concept of elastic properties, is presented. Additionally, the current review highlights the probable, inherent mechanisms of vesicle formation under distinct modes of manufacturing. We lay focus on vesicle formation from pre-existing bilayers, as well as from bilayers, which form when lipids from an organic solvent are transferred into an aqueous medium. Furthermore, we outline the kinetic effects on vesicle formation from the lamellar phase, with and without the presence of shearing force. Wherever required, the experimental and/or theoretical outcomes, the driving forces for vesicle size selection, and various scaling laws are also reviewed, all of which facilitate an overall improved understanding of the vesicle formation mechanisms. [ABSTRACT FROM AUTHOR]

Protein kinase D (PKD) is a family of serine/threonine kinases that is required for the structural integrity and function of the Golgi complex. Despite its importance in the regulation of Golgi function, the molecular mechanisms regulating PKD activity are still incompletely understood. Using the genetically encoded PKD activity reporter G-PKDrep we now uncover a Rho signaling network comprising GEF-H1, the RhoGAP DLC3, and the Rho effector PLCε that regulate the activation of PKD at trans-Golgi membranes. We further show that this molecular network coordinates the formation of TGN-derived Rab6-positive transport carriers delivering cargo for localized exocytosis at focal adhesions.

Approximately one-third of all eukaryotic proteins are delivered to their destination by trafficking within the endomembrane system. Such cargo proteins are incorporated into forming membrane vesicles on donor compartments and delivered to acceptor compartments by vesicle fusion. How cargo proteins are sorted into forming vesicles is still largely unknown. Here we review the roles of small GTPases of the ARF/SAR1 family, their regulators designated ARF guanine-nucleotide exchange factors (ARF-GEFs) and ARF GTPase-activating proteins (ARF-GAPs) as well as coat protein complexes during membrane vesicle formation. Although conserved across eukaryotes, these four functional groups of proteins display plant-specific modifications in composition, structure and function. [ABSTRACT FROM AUTHOR]

Terrestrial geothermal fields and oceanic hydrothermal vents are considered as candidate environments for the emergence of life on Earth. Nevertheless, the ionic strength and salinity of oceans present serious limitations for the self-assembly of amphiphiles, a process that is fundamental for the formation of first protocells. Consequently, we systematically characterized the efficiency of amphiphile assembly, and vesicular stability, in terrestrial geothermal environments, both, under simulated laboratory conditions and in hot spring water samples (collected from Ladakh, India, an Astrobiologically relevant site). Combinations of prebiotically pertinent fatty acids and their derivatives were evaluated for the formation of vesicles in aforesaid scenarios. Additionally, the stability of these vesicles was characterized over multiple dehydration-rehydration cycles, at elevated temperatures. Among the combinations that were tested, mixtures of fatty acid and its glycerol derivatives were found to be the most robust, also resulting in vesicles in all of the hot spring waters that were tested. Importantly, these vesicles were stable at high temperatures, and this fatty acid system retained its vesicle forming propensity, even after multiple cycles of dehydration-rehydration. The remaining systems, however, formed vesicles only in bicine buffer. Our results suggest that certain prebiotic compartments would have had a selective advantage in terrestrial geothermal niches. Significantly, our study highlights the importance of validating results that are obtained under ‘buffered' laboratory conditions, by verifying their plausibility in prebiotically analogous environments. [ABSTRACT FROM AUTHOR]

The purpose of this article is to describe a case of drug-induced cutaneous toxicity observed in cynomolgus monkeys and to introduce approaches attempted in order to elucidate mechanisms. The test article was a small molecule with a ubiquitously distributed target, especially in rapidly dividing cells, and which modulated cell cycle regulation. After 7 consecutive days of oral dosing, animals developed multifocal skin lesions. The lesions were characterized clinically by vesicles and scabs formation and were distributed mainly in thin-skinned areas of the body including the cheek, chest, abdomen, and inner limbs. Microscopically, the lesions were confirmed as epidermal vesicle formation and ulceration. Immunohistochemical staining revealed that the levels within the epidermis where separation (vesicle formation) occurred were not consistent. The differential diagnoses for vesicular skin lesions and our efforts to elucidate the mechanism of toxicity using in-house database searches and immunohistochemistry are discussed. To the best of our knowledge, similar cutaneous toxicity has not been reported previously, although there are reports of other types of cutaneous toxicities. Understanding the mechanisms of the toxicity is very important when assessing human relevancy during drug development. Our investigative approach can be utilized when unusual skin toxicities are observed in the future. [ABSTRACT FROM AUTHOR]

Crowding of asymmetrically-distributed membrane proteins has been recently recognized as an important factor in remodeling of biological membranes, for example during transport vesicle formation. In this paper, we theoretically analyze the effect of protein crowding on membrane bending and examine its dependence on protein size, shape, transmembrane asymmetry and lateral confinement. We consider three scenarios of protein lateral organization, which are highly relevant for cellular membranes in general: freely diffusing membrane proteins without lateral confinement, the presence of a diffusion barrier and interactions with a vesicular coat. We show that protein crowding affects vesicle formation even if the proteins are distributed symmetrically across the membrane and that this effect depends significantly on lateral confinement. The largest crowding effect is predicted for the proteins that are confined to the forming vesicle by a diffusion barrier. We calculate the bending properties of a crowded membrane and find that its spontaneous curvature depends primarily on the degree of transmembrane asymmetry, and its effective bending modulus on the type of lateral confinement. Using the example of COPII vesicle formation from the endoplasmic reticulum, we analyze the energetic cost of vesicle formation. The results provide a novel insight into the effects of lateral and transmembrane organization of membrane proteins, and can guide data interpretation and future experimental approaches. [ABSTRACT FROM AUTHOR]

Terrestrial geothermal fields and oceanic hydrothermal vents are considered as candidate environments for the emergence of life on Earth. Nevertheless, the ionic strength and salinity of oceans present serious limitations for the self-assembly of amphiphiles, a process that is fundamental for the formation of first protocells. Consequently, we systematically characterized the efficiency of amphiphile assembly, and vesicular stability, in terrestrial geothermal environments, both, under simulated laboratory conditions and in hot spring water samples (collected from Ladakh, India, an Astrobiologically relevant site). Combinations of prebiotically pertinent fatty acids and their derivatives were evaluated for the formation of vesicles in aforesaid scenarios. Additionally, the stability of these vesicles was characterized over multiple dehydration-rehydration cycles, at elevated temperatures. Among the combinations that were tested, mixtures of fatty acid and its glycerol derivatives were found to be the most robust, also resulting in vesicles in all of the hot spring waters that were tested. Importantly, these vesicles were stable at high temperatures, and this fatty acid system retained its vesicle forming propensity, even after multiple cycles of dehydration-rehydration. The remaining systems, however, formed vesicles only in bicine buffer. Our results suggest that certain prebiotic compartments would have had a selective advantage in terrestrial geothermal niches. Significantly, our study highlights the importance of validating results that are obtained under ‘buffered’ laboratory conditions, by verifying their plausibility in prebiotically analogous environments.

Vesicles were formed in aqueous mixtures of gemini anionic surfactant, O,O′-bis(sodium 2-alkylcarboxylate)- p-dibenzenediol (referred to as CϕC, m = 10, 12), and single-chain cationic surfactant, alkyltrimethylammonium bromides CNBr ( n = 8, 10, 12). This was confirmed by measurements of solution turbidity, dynamic light scattering (DLS) and freeze-fractured TEM. The improvement of the CϕC molecular geometry by CNBr was considered to have been due to vesicle formation. As the temperature was raised, these vesicles were found to associate together and DLS measurements showed a rapid increase of the aggregate size at the critical temperature T. The mechanism of vesicle aggregation was assumed to be due to the temperature-induced configuration transition of a few CϕC molecules in the vesicles from cis- into trans-form. Thus, a few tails of CϕC stretched toward the solution. When the vesicles approached, these projecting tails interacted hydrophobically, thereby non-covalently attaching one vesicle to another. This should benefit from the special molecular structure of CϕC with a long, rigid spacer. [ABSTRACT FROM AUTHOR]

The influence of a polyampholyte, i.e., poly( N, N′-diallyl- N, N′-dimethyl-altmaleamic carboxylate) (PalH), on the lamellar liquid crystalline (LC) system sodium dodecyl sulfate (SDS)/decanol/water was investigated by means of microdifferential scanning calorimetry, small-angle X-ray diffraction (SAXS), and cryo-scanning electron microscopy. After incorporating PalH into the lamellar liquid crystalline system, SAXS measurements show that three different LC phases exist: i.e., a swelling, slightly swelling, and non-swelling one. At pH 4, the positively charged polymer with an extended conformation can directly adsorb at the anionic head groups of the surfactant and more compact vesicles are formed at room temperature. At pH 9, the electrostatic interactions between the polyampholyte (in a more coiled conformation) and the sulfate head groups of the SDS are leveled off and incompact vesicles are formed at room temperature. That means in presence of the polyampholyte the morphology of the LC phase, i.e., the supramolecular vesicle structure, can be tuned by varying the pH and/or the temperature. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Polyhedral oligomeric silsesquioxane (POSS) molecules contain a silicon/oxygen nano-size cage substituted with organic groups. 2-(Propylcarbamoyl)terephthalic acid heptaisobutyl polyhedral oligomeric silsesquioxane (POSS DCA) is a novel mono-tethered POSS compound which is not commercially available yet and can be regarded as a single-tail surfactant. In contrast to theoretically possible micellization of amphiphilic POSS compounds very rare interest is paid by scientific community to this issue. POSS DCA vesiculation occurred at a very low concentration (0.09 mM) in a mixture of acetone, ethanol and 1-propanol according to the experiments. Critical packing parameter of POSS DCA is bigger than one and it must self-assemble into inverted-micelle morphology in a non-polar solvent. The vesicle formation for POSS DCA in a relatively polar solvent system was mainly attributed to formation of stable various-size intermolecular bridges of ethanol or 1-propanol. Also, π- π interaction between aromatic rings is another driving force. Vesicles can trap both hydrophilic and hydrophobic solvents and change composition of a solvent mixture. For the first time, we studied this property for POSS DCA vesicles in different concentrations by a gas chromatographic method. Significant increase in surface tension was attributed to the changes in solvent system composition due to vesicle formation and variation in vesicles morphology. [ABSTRACT FROM AUTHOR]

A Cu2+-induced vesicle phase was prepared from a mixture of 3-aminopropylmethyltrisiloxane and CuCl2or Cu(NO3)2in aqueous solutions. Cu2+coordinated with 3-aminopropylmethyltrisiloxane resulting in the formation of molecular bilayers, because Cu2+can firmly bind to the amine groups of 3-aminopropylmethyltrisiloxane which reduced the area of the head-group. In this system, no counterions in aqueous solution exist because of the Cu2+-ligand coordination, and the bilayer membranes are not shielded by salts. The vesicle formation of 3-aminopropylmethyltrisiloxane induced by Cu2+ions was determined by transmission electron microscopy (TEM) images, dynamic light scattering (DLS) measurements, and x-ray diffraction (XRD). [ABSTRACT FROM PUBLISHER]

The Golgi complex processes secretory proteins and lipids, carries out protein sorting and signaling, and supports growth and composition of the plasma membrane. Golgi complex size likely is regulated to meet the demands of each function, and this may involve differential changes of its distinct subdomains. Nevertheless, the primary size change is elongation of the Golgi ribbon-like network as occurs during Golgi complex doubling for mitosis and during differentiation involving upregulated secretion. One hypothesis states that Golgi complex size is set by the abundance of secretory cargo and Golgi complex components that, through binding vesicle coat complexes, drive vesicle coat formation to alter Golgi complex influx and efflux. Regulation of transport factors controlling Golgi membrane traffic is also observed and may control Golgi complex size, but more work is needed to directly link these events to Golgi complex size regulation, especially during differentiation of specialized cell types. [ABSTRACT FROM AUTHOR]

Abstract: Arf GAP proteins are a versatile and diverse group of proteins. They control the activity of the GTP-binding proteins of the ARF family by inducing the hydrolysis of GTP that is bound to Arf proteins. The best-studied role of Arf GAPs is in intracellular traffic. In this review, we will focus mainly on the Arf GAPs that play a role in vesicle formation, Arf GAP1, Arf GAP2 and Arf GAP3 and their yeast homologues, Gcs1p and Glo3p. We discuss the roles of Arf GAPs as regulators and effectors for Arf GTP-binding proteins. [Copyright &y& Elsevier]

Background and Aims: Optimization of osmotic dehydration in different plant cells has been investigated through the variation of parameters such as the nature of the sugar used, the concentration of osmotic solutions and the processing time. In micro-organisms such as the yeast, Saccharomyces cerevisiae, the exposure of a cell to a slow increase in osmotic pressure preserves cell viability after rehydration, while sudden dehydration involves a lower rate of cell viability, which could be due to membrane vesiculation. The aim of this work is to study cytoplasmic vesicle formation in onion epidermal cells (Allium cepa) as a function of the kinetics of osmotic pressure variation in the external medium. [ABSTRACT FROM PUBLISHER]

Abstract: A biosurfactant produced by Pseudomonas aeruginosa strain SP4 was previously reported as a mixture of 11 types of rhamnolipid compounds. Among them, the major component in the biosurfactant was characterized as l-rhamnosyl-3-hydroxydecanoyl-3-hydroxydecanoate, or monorhamnolipid (Rha–C10–C10). In this present study, solution properties of the biosurfactant were investigated in a phosphate-buffer saline (PBS) solution (pH 7.4) by using surface tension, turbidity, electrical conductivity, and dynamic light scattering (DLS) measurements. It was found that spherical biosurfactant vesicles of various sizes (ranging from 50 to larger than 250nm) were spontaneously formed at a biosurfactant concentration greater than its critical micelle concentration (CMC), which was 200mg/l. The encapsulation efficiency (E%) of the biosurfactant vesicles was preliminarily studied by using Sudan III, a water-insoluble dye, as a model hydrophobic substance. The obtained results showed that the vesicle formed in the PBS solution at a biosurfactant concentration of 1280mg/l could entrap about 10% of the initial hydrophobic dye concentration. The effects of salt and alcohol on the vesicle formation of the biosurfactant and its encapsulation efficiency were also observed by using sodium chloride (NaCl) and ethanol (C2H5OH), respectively. In the presence of either NaCl or C2H5OH, the vesicle size was reduced from larger than 250nm to 50–250nm. The encapsulation efficiency of the biosurfactant vesicle was slightly influenced by the addition of NaCl, but was significantly increased, up to nearly 30%, in the presence of C2H5OH. [Copyright &y& Elsevier]

Our understanding of flippase-mediated lipid translocation and membrane vesiculation, and the involvement of P-type ATPases in these processes is just beginning to emerge. The results obtained so far demonstrate significant complexity within this field and point to major tasks for future research. Most importantly, biochemical characterization of P4-ATPases is required in order to clarify whether these transporters indeed are capable of catalyzing transmembrane phospholipid flipping. The β-subunit of P4-ATPases shows unexpected similarities between the β- and γ-subunits of the Na+/K+-ATPase. It is likely that these proteins provide a similar solution to similar problems, and might have adopted similar structures to accomplish these tasks. No P4-ATPases have been identified in the endoplasmic reticulum and it remains an intriguing possibility that, in this compartment, P5A-ATPases are functional homologues of P4-ATPases. [ABSTRACT FROM AUTHOR]

The coat protein complex II (COPII) is essential for vesicle formation from the endoplasmic reticulum (ER) and is composed of two heterodimeric subcomplexes, Sec23p/Sec24p and Sec13p/Sec31p, and the small guanosine triphosphatase Sar1p. In an effort to identify novel factors that may participate in COPII vesicle formation, we isolated SMY2, a yeast gene encoding a protein of unknown function, as a multicopy suppressor of the temperature-sensitive sec24-20 mutant. We found that even a low-copy expression of SMY2 was sufficient for the suppression of the sec24-20 phenotypes, and the chromosomal deletion of SMY2 led to a severe growth defect in the sec24-20 background. In addition, SMY2 exhibited genetic interactions with several other genes involved in the ER-to-Golgi transport. Subcellular fractionation analysis showed that Smy2p was a peripheral membrane protein fractionating together with COPII components. However, Smy2p was not loaded onto COPII vesicles generated in vitro. Interestingly, coimmunoprecipitation between Smy2p and the Sec23p/Sec24p subcomplex was specifically observed in sec23-1 and sec24-20 backgrounds, suggesting that this interaction was a prerequisite for the suppression of the sec24-20 phenotypes by overexpression of SMY2. We propose that Smy2p is located on the surface of the ER and facilitates COPII vesicle formation through the interaction with Sec23p/Sec24p subcomplex. [ABSTRACT FROM AUTHOR]

Abstract: Ascoviruses (AVs) are double-stranded DNA viruses causing a fatal disease in lepidopteran host larvae. A unique feature of AV infection is cleavage of host cells into membrane bound vesicles containing the virions. A recent study showed that a caspase from Spodoptera frugiperda AV (SfAV) is directly involved in initiation of apoptosis and eventually cell cleavage. Results shown here indicate that Heliothis virescens AV does not induce apoptosis in host cells. HvAV codes for a caspase-like protein but no apoptosis was observed when the gene was expressed in vitro. RNAi studies indicated that the gene is essential for virus replication. [Copyright &y& Elsevier]

Abstract: A study of the self-organization of nonionic surfactant span 60 (sorbitan mono stearate) in presence of fatty alcohol (stearyl, cetyl and lauryl) is presented. When ethanolic solution of the surfactant–fatty alcohol (1:1) mixture is added in water spontaneous large unilamellar vesicles (LUV) are formed which may potentially be useful vehicles for drug delivery purposes. Vesicular suspension has been characterized by transmission electron microscopy, dynamic light scattering, confocal laser scanning microscopy, dye entrapment and release studies. Surface tension measurement indicates the suitability of fatty alcohols towards spontaneous vesicle formation from span 60. [Copyright &y& Elsevier]

Abstract: In coated vesicle formation, coat protein recruitment needs to be spatially and temporally controlled. The coating process involves conformational changes of the coat protein complexes that activate them for interaction with cargo or machinery components and coat polymerization. Here we discuss mechanisms that have emerged recently from studies of the clathrin adaptor and the COPI systems. [Copyright &y& Elsevier]

Abstract: Several chlamydial antigens have been detected in the infected epithelial cell cytosol and on the host cell surface prior to their presumed natural release at the end of the 72–96h developmental cycle. These extra-inclusion antigens are proposed to influence vital host cell functions, antigen trafficking and presentation and, ultimately, contribute to a prolonged inflammatory response. To begin to dissect the mechanisms for escape of these antigens from the chlamydial inclusion, which are enhanced on exposure to antibiotics, polarized endometrial epithelial cells (HEC-1B) were infected with Chlamydia trachomatis serovar E for 36h or 48h. Infected cells were then exposed to chemotactic human polymorphonuclear neutrophils not loaded or pre-loaded in vitro with the antibiotic azithromycin. Viewed by electron microscopy, the azithromycin-mediated killing of chlamydiae involved an increase in chlamydial outer membrane blebbing followed by the appearance of the blebs in larger vesicles (i) everting from but still associated with the inclusion as well as (ii) external to the inclusion. Evidence that the vesicles originated from the chlamydial inclusion membrane was shown by immuno-localization of inclusion membrane proteins A, F, and G on the vesicular membranes. Chlamydial heat shock protein 60 (chsp60) copies 2 and 3, but not copy 1, were released from RB and incorporated into the everted inclusion membrane vesicles and delivered to the infected cell surface. These data represent direct evidence for one mechanism of early antigen delivery, albeit membrane-bound, beyond the confines of the chlamydial inclusion. [Copyright &y& Elsevier]

The core complex of Coat Protein I (COPI), known as coatomer, is sufficient to induce coated vesicular-like structures from liposomal membrane. In the context of biological Golgi membrane, both palmitoyl-coenzyme A (p-coA) and ARFGAP1, a GTPase-activating protein (GAP) for ADP-Ribosylation Factor 1, also participate in vesicle formation, but how their roles may be linked remains unknown. Moreover, whether COPI vesicle formation from Golgi membrane requires additional factors also remains unclear. We now show that Brefeldin-A ADP-Ribosylated Substrate (BARS) plays a critical role in the fission step of COPI vesicle formation from Golgi membrane. This role of BARS requires its interaction with ARFGAP1, which is in turn regulated oppositely by p-coA and nicotinamide adenine dinucleotide, which act as cofactors of BARS. Our findings not only identify a new factor needed for COPI vesicle formation from Golgi membrane but also reveal a surprising mechanism by which the roles of p-coA and GAP are linked in this process. [ABSTRACT FROM AUTHOR]

Abstract: Vesicles are self-assembled spherical aggregates of amphiphilic molecules with rich structural variety. They have attracted increasing attention due to their possible applications in pharmaceutics and cosmetic fields, but also from a more fundamental point of view. In this work, we present vesicle formation from mixtures of commercial surfactants. We show how mixing of two non-ionic surfactants can lead to vesicle formation by adjusting the packing parameter of the mixture. High-frequency viscosity data can be described in terms of an effective hard sphere system applying the Lionberger–Russel model. Thus, we were able to estimate the hydrodynamic volume of the vesicles and hence their average size, which agrees well with the structures observed by transmission electron microscopy. Finally, we show that oil-in-water emulsions prepared with the adjusted surfactant mixture have two orders of magnitude higher viscosities compared to similar emulsions stabilised with an anionic surfactant, demonstrating the significant impact of the vesicles on the rheological and hence creaming behaviour of emulsions. [Copyright &y& Elsevier]

We have identified two LIM domain proteins, LimF and ChLim, from Dictyostelium that interact with each other and with the small, Rab5-related, Rab21 GTPase to collectively regulate phagocytosis. To investigate in vivo functions, we generated cell lines that lack or overexpress LimF and ChLim and strains that express activating or inhibiting variants of Rab21. Overexpression of LimF, loss of ChLim, or expression of constitutively active Rab21 increases the rate of phagocytosis above that of wild type. Conversely, loss of LimF, overexpression of ChLim, or expression of a dominant-negative Rab21 inhibits phagocytosis. Our studies using cells carrying multiple mutations in these genes further indicate that ChLim antagonizes the activating function of Rab21-GTP during phagocytosis; in turn, LimF is required for Rab21-GTP function. Finally, we demonstrate that ChLim and LimF localize to the phagocytic cup and phago-lysosomal vesicles. We suggest that LimF, ChLim, and activated Rab21-GTP participate as a novel signaling complex that regulates phagocytic activity. [ABSTRACT FROM AUTHOR]

Hermansky–Pudlak syndrome (HPS) is an autosomal recessive disorder characterized by oculocutaneous albinism, a bleeding disorder, and ceroid lipofuscinosis in the lungs and gut. HPS is genetically heterogeneous and the most common variant, HPS type 1, is caused by mutations in HPS1 gene. The protein encoded by HPS1 is considered to facilitate the trafficking of melanocyte-specific gene products into the premelanosome. We report the ultrastructural findings in a melanocytic nevus seen in a 17-y-old Japanese female patient with HPS1 who is a compound heterozygote of HPS1 mutations, including a novel mutation. Electron microscopy of a pinkish papule corresponding to the melanocytic nevus revealed markedly aberrant, immature melanosomes, large membranous structures, and giant melanosomes in the vicinity of trans-Golgi network, the characteristic abnormalities because of protein trafficking defects in HPS1. These ultrastructural features were far more clearly demonstrated in the nevus cells than in the epidermal melanocytes. Thus, ultrastructural analysis of nevus cells may be an additional diagnostic tool for HPS1 and could give us important clues to further understanding of the pathomechanisms of HPS. [ABSTRACT FROM AUTHOR]

Caveolin is the principal component of caveolae in vivo. In addition to a structural role, it is believed to play a scaffolding function to organize and inactivate signaling molecules that are concentrated on the cytoplasmic surface of caveolar membranes. The large GTPase dynamin has been shown to mediate the scission of caveolae from the plasma membrane, although it is unclear if dynamin interacts directly with caveolin or via accessory proteins. Therefore, the goal of this study was to test whether dynamin associates with caveolae via a direct binding to the caveolin 1 (Cav1) protein. Immunoelectron microscopy of lung endothelium or a cultured hepatocyte cell line stained with antibodies for Dyn2 and Cav1 shows that these proteins co-localize to caveolae. To further define this interaction biochemically, in vitro experiments were performed using glutathione-S-transferase (GST)-Dyn2 and GST-Cav1 fusion proteins, which demonstrated a direct interaction between these proteins. This interaction appears to be mediated by the proline-arginine-rich domain (PRD) of Dyn2, as a GST-PRD fragment binds Cav1 while GST-Dyn2ΔPRD does not. Further, in vitro binding studies using two Dyn2 spliced forms and Cav1 peptides immobilized on paper identify specific domains of Cav1 that bind Dyn2. Interestingly, these Cav1-binding domains differ markedly between two spliced variant forms of Dyn2. In support of these distinctive physical interactions, we find that the different Dyn2 forms, when expressed as GTPase-defective mutants, exert markedly different inhibitory effects on caveolae internalization, as assayed by cholera toxin uptake. These studies provide the first evidence for a direct interaction between dynamin and the caveolin coat, and demonstrate a selectivity of one Dyn2 form toward the caveolae-mediated endocytosis. [Copyright &y& Elsevier]

Bacteria may possess various kinds of cytoskeleton. In general, bacterial cytoskeletons may play a role in the control and preservation of the cell shape. Such functions become especially evident when the bacteria do not possess a true wall and are nevertheless elongated (e.g. Mycoplasma spp.) or under extreme cultivation conditions whereby loss of the entire bacterial cell wall takes place. Bacterial cytoskeletons may control and preserve the cell shape only if a number of preconditions are fulfilled. They should be present not only transiently, but permanently, they should be located as a lining close to the inner face of the cytoplasmic membrane, enclosing the entire cytoplasm, and they should comprise structural elements (fibrils) crossing the inner volume of the cell in order to provide the necessary stability for the lining. Complete loss of the cell wall layers had earlier been observed to occur during extensive production of bacterial starch-degrading enzymes in an optimized fermentation process by a Gram-positive bacterium. Even under these conditions, the cells had maintained their elongated shape and full viability. Which of the various kinds of bacterial cytoskeleton might have been responsible for shape preservation? Only one of them, the primary or basic cytoskeleton turns out to fulfil the necessary preconditions listed above. Its structural features now provided a first insight into a possible mechanism of formation of membrane blebs and vesicles as observed in the Gram-positive eubacterium Thermoanaerobacterium thermosulfurogenes EM1, and the putative role of the cytoskeletal web in this process. Copyright © 2003 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Syndapins are potential links between the cortical actin cytoskeleton and endocytosis because this family of dynamin-associated proteins can also interact with the Arp2/3 complex activator N-WASP. Here we provide evidence for involvement of N-WASP inter- actions in receptor-mediated endocytosis. We reveal that the observed dominant-negative effects of N-WASP are dependent exclusively on the prolinerich domain, the binding interface of syndapins. Our results therefore suggest that syndapins integrate N-WASP functions in endocytosis. Both proteins co-localize in neuronal cells. Consistent with a crucial rote for syndapins in endocytic uptake, co-overexpression of syndapins rescued the endocytosis block caused by N-WASP. An in vivo reconstitution of the syndapin-N-WASP interaction at cellular membranes triggered local actin polymerization. Depletion of endogenous N-WASP by sequestering it to mitochondria or by introducing anti-N-WASP antibodies impaired endocytosis. Our data suggest that syndapins may act as important coordinators of N-WASP and dynamin functions during the different steps of receptor-mediated endocytosis and that local actin polymerization induced by syndapin-N-WASP interactions may be a mechanism supporting clathrincoated vesicle detachment and movement away from the plasma membrane. [ABSTRACT FROM AUTHOR]

Protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus is mediated by transport vesicles coated with the coat protein complex II (COPII). In the process of searching for novel factors that participate in the formation of COPII-coated vesicles (COPII vesicles), we isolated high-copy suppressors of a sec24-20 mutant defective in COPII vesicle formation from the ER at the restrictive temperature. Unexpectedly, one of them was identified as HAC1, a gene encoding the basic leucine-zipper type transcription factor Hac1p. Hac1p is essential for a signaling cascade activated by ER stress, termed the unfolded protein response (UPR) pathway, that leads from the ER to the nucleus. Overexpression of another UPR-related gene IRE1, which encodes an ER-resident transmembrane protein kinase/ribonuclease, also suppressed the growth defect of the sec24-20 mutant in a HAC1-dependent manner. Moreover, overexpression of IRE1 specifically suppressed growth defects of other sec mutants defective in COPII vesicle formation. These findings suggest that the activation of the UPR affects ER-to-Golgi transport via stimulation of COPII vesicle formation from the ER. [Copyright &y& Elsevier]

Hermansky–Pudlak syndrome (HPS) consists of oculocutaneous albinism, a platelet storage pool deficiency and, in patients with HPS1 gene mutations, a progressive, fatal pulmonary fibrosis. We investigated the safety and efficacy of an antifibrotic agent, pirfenidone (800 mg, t.i.d.), in treating 21 adult Puerto Rican HPS patients, including 20 homozygous for the same HPS1 mutation. Patients were examined every 4 months for up to 44 months in a randomized, placebo-controlled trial, with rate of change in pulmonary function values as outcome parameters. Using the complete data set of 130 patient admissions, a repeated measures model showed that 11 pirfenidone-treated patients lost FVC at a rate 5% of predicted (∼400 mL) per year slower than 10 placebo-treated patients (p=0.001). A random coefficients model showed no significant difference. However, using data restricted to patients with an initial FVC >50% of predicted, both models showed the pirfenidone group losing FVC (p<0.022), FEV1 (p<0.0007), TLC (p<0.001), and DLCO (p<0.122) at a rate ∼8%/year slower than the placebo group. Clinical and laboratory side effects were similar in the two groups. Pirfenidone appears to slow the progression of pulmonary fibrosis in HPS patients who have significant residual lung function. [Copyright &y& Elsevier]

Shape changes in the endoplasmic reticulum control fundamental cell processes including nuclear envelope assembly in mitotic cells, calcium homeostasis in cytoplasmic domains of secreting and motile cells, and membrane traffic in the early secretion apparatus between the endoplasmic reticulum and Golgi. Opposing forces of assembly (membrane fusion) and disassembly (membrane fragmentation) ultimately determine the size and shape of this organelle. This review examines some of the regulatory mechanisms involved in these processes and how they occur at specific sites or subcompartments of the endoplasmic reticulum.Key words: rough endoplasmic reticulum, smooth endoplasmic reticulum, shape changes, assembly, membrane fusion, organelle size, vesicle formation.Les changements de forme du réticulum endoplasmique sont associés à plusieurs évènements importants pour la physiologie de la cellule incluant l'assemblage de l'enveloppe nucléaire dans les cellules post-mitotiques, la libération du calcium dans les cellules mobiles et le trafic membranaire entre le réticulum endoplasmique et l'appareil de Golgi lors de la sécrétion. La forme et la taille du réticulum endoplasmique dépendent des forces opposées de la formation (fusion membranaire) et de la déformation (fragmentation). Cette revue examine la régulation moléculaire impliquée dans ces phénomènes et indique comment elles ont lieu à des sites spécifiques du réticulum endoplasmique.Mots clés : réticulum endoplasmique rugueux, réticulum endoplasmique lisse, changement de forme, assemblage, fusion membranaire, taille d'un organite, formation de vésicules. [ABSTRACT FROM AUTHOR]

The object of the research was to investigate the influence of a semiflexible polyanion (carboxymethylcellulose) in the absence and presence of a more flexible cationic polyelectrolyte [poly(diallyldimethyl-ammonium chloride)] on structure formation in liquid-crystalline model systems consisting of sodium dodecyl sulfate (SDS)/ decanol/water. Small-angle X-ray measurements in combination with electron microscopic investigations show the adsorption of the polycation on the SDS head groups. These polymer-modified lamellae form multilamellar vesicles. The semiflexible polyanion was embedded into the liquid crystal without macroscopic phase separation and multivesicular vesicles were formed on the supramolecular level. In combination the oppositely charged polyelectrolytes induce the formation of multivesicular structures where two lamellar structures coexist. [ABSTRACT FROM AUTHOR]

In the aqueous mixtures of sodium alkylcarboxylate and alkyltrimethylammonium bromide, large unilamelar vesicles can be formed spontaneously or by sonication as the total carbon number in the HC chains is 19 (or larger). Vesicle formation can be influenced by changes of pH, molar ratio of the two surfactant components, and the polar head group of cationic surfactant. Micelles may coexist with the vesicles in these mixed systems. The larger hydrodynamic radius (∼200 nm) and aggregation number (∼800) illustrate that the shape of the micelle in 1:1 CHCOONa−CHN(CH)Br is rod-like. In some mixed systems, the micelles can be transformed into stable vesicles by sonication - a phenomenon revealed for the first time. The surface-chemical properties of these catanionic surfactant solutions and the stabilities of vesicle have been studied systematically. [ABSTRACT FROM AUTHOR]

Following electro-fusion of plant protoplasts the volume of the fused cell is the sum of the volumes of the parent cells. As shown for mesophyll protoplasts from leaves of Kalanchoë daigremontiana, the excess in membrane material arising from the reduction in membrane area is removed-at least to a larger extent - by the formation of vesicles which are visible in the light microscope. These vesicles, which may have been formed by the fusion of sub-microscopic vesicles, are observed in the contact zone of the fusing cells. The mechanism of the formation of vesicles during electro-fusion is discussed. [ABSTRACT FROM AUTHOR]

Three marine bacteria were examined for physiological and morphological changes in the initial phase of starvation. It was found that the starvation process was induced in a similar way irrespective of whether the cells were suspended in nutrient and energy free artificial seawater (NSS) or NSS supplemented with nitrogen and phosphorus. An initial phase of increased activity was consistent with a decreased response to added nutrients. Recovery from starvation exhibited the same response in both these starvation regimes, measured throughout the starvation period. Cells in nitrogen or phosphorus deprived starvation regimes, showed a high and rapid increased activity, followed by a delayed and more pronounced decline in respiratory activity. The initial phase of starvation also included a loss of poly-β-hydroybutyrate as observed by transmission electron microscopy (TEM). Two bacterial strains showed formation of small vesicles on the outer cell layer when examined by TEM. This formation and release of vesicles was related to the continuous size reduction during starvation survival. The results are discussed in terms of defining the mechanisms of initial cellular responses to nutrient deprivation. [ABSTRACT FROM AUTHOR]

In the human gastric carcinoma cell line EPG85-257P (parent) induction of resistance to daunorubicin (DAU) was achieved by selection with stepwise increased concentrations of the drug. The new variant was named EPG85-257DAU and was shown to overexpress the mdr1 gene product 170 kDa P-glycoprotein (P-Gp) as demonstrated by immunocytochemistry and mdr1-specific RT-PCR. To investigate the intracellular pathway of DAU the subcellular distribution of this autofluorescent drug was studied in the resistant cells and compared to its chemosensitive counterpart EPG85-257P. When sensitive cells were exposed to DAU the drug rapidly accumulated in the nucleus until cell death. No redistribution of DAU to the cytoplasm was observed. In resistant cells exposed to the drug DAU also accumulated in the nucleus but to a lesser extent than in parent cells. Following exposure, nuclear fluorescence was observed to decrease over a time period of up to 48 h. Six hours after DAU exposure formation of fluorescent vesicle formation started in the perinuclear region and increased continuously. After 48 h nuclear fluorescence was no longer detectable and DAU was located exclusively in vesicles. During this period the vesicles moved from the region of origin to the cell periphery. A pulse chase experiment showed, that vesicles may contain DAU derived from the nucleus. Treatment of EPG85-257DAU cells with DAU in conjunction with the chemosensitizer cyclosporin A (CsA) increased nuclear fluorescence without impairing vesicle formation. Disruption of microtubules by nocodazole led to an accumulation of vesicles in the perinuclear region indicating that microtubules are involved in vesicular transport. Treatment of EPG85-257DAU cells with the actin disruptor cytochalasin B led to accumulation of vesicles in the cell periphery indicating that actin may be involved in exocytosis. Uptake and efflux of DAU and rhodamin (RH) were determined in sensitive and resistant cells using a fluorescence activated cell sorter. Uptake of both compounds was distinctly lower in resistant than in sensitive cells. When resistant cells preloaded for 2 h with RH subsequently were incubated in drug free medium the substance was rapidly released indicating transmembrane transport by P-Gp. In contrast, despite expression of P-Gp in resistant cells no considerable release of DAU was observed for up to 2 h under the same experimental protocol. This indicates that in resistant cells intracellular DAU at least in part may be inaccessible for P-Gp and that vesicular drug transport appears to contribute to DAU resistance by removing intracellular DAU via exocytosis. [ABSTRACT FROM AUTHOR]

A cell membrane patch isolated on a patch clamp pipette incorporates in addition to the phospholipid bilayer, an extracellular matrix and cytoskeletal components. The significance of the extracellular matrix for the patch formation was studied in aortic smooth muscle and cerebellar granule cells grow in the presence of an inhibitor of proteoglycan synthesis, β- d -xyloside. The xyloside improved the seal success rate, and after patch excision membrane vesicles were formed instead of inside-out patches. When amphotericin B was included in the pipette solution, perforated outside-out vesicles were formed in 96% of cells. The findings suggest, that membrane patches are supported by the extracellular matrix or by structures that relate to this matrix. [ABSTRACT FROM AUTHOR]