Lipid Bilayer Of Plasma Membrane Research Articles

Principles of Tissue Engineering With Nonthermal Irreversible Electroporation

Nonthermal irreversible electroporation (NTIRE) is an emerging tissue ablation modality that may be ideally suited in developing a decellularized tissue graft. NTIRE utilizes short electric pulses that produce nanoscale defects in the cell membrane lipid bilayer. The electric parameters can be chosen in such a way that Joule heating to the tissue is minimized and cell death occurs solely due to loss in cell homeostasis. By coupling NTIRE with the body’s response, the cells can be selectively ablated and removed, leaving behind a tissue scaffold. Here, we introduce two different methods for developing a decellularized arterial scaffold. The first uses an electrode clamp that is applied to the outside of a rodent carotid artery and the second applies an endovascular minimally invasive approach to apply electric fields from the inner surface of the blood vessels. Both methods are first modeled using a transient finite element analysis of electric and thermal fields to ensure that the electric parameters used in this study will result in minimal thermal damage. Experimental work demonstrates that both techniques result in not only a decellularized arterial construct but an endothelial regrowth is evident along the lumen 7 days after treatment, indicating that the extracellular matrix was not damaged by electric and thermal fields and is still able to support cell growth.

Proteomics Characterization of Extracellular Space Components in the Human Aorta

The vascular extracellular matrix (ECM) is essential for the structural integrity of the vessel wall and also serves as a substrate for the binding and retention of secreted products of vascular cells as well as molecules coming from the circulation. Although proteomics has been previously applied to vascular tissues, few studies have specifically targeted the vascular ECM and its associated proteins. Thus, its detailed composition remains to be characterized. In this study, we describe a methodology for the extraction of extracellular proteins from human aortas and their identification by proteomics. The approach is based on (a) effective decellularization to enrich for scarce extracellular proteins, (b) successful solubilization and deglycosylation of ECM proteins, and (c) relative estimation of protein abundance using spectral counting. Our three-step extraction approach resulted in the identification of 103 extracellular proteins of which one-third have never been reported in the proteomics literature of vascular tissues. In particular, three glycoproteins (podocan, sclerostin, and agrin) were identified for the first time in human aortas at the protein level. We also identified extracellular adipocyte enhancer-binding protein 1, the cartilage glycoprotein asporin, and a previously hypothetical protein, retinal pigment epithelium (RPE) spondin. Moreover, our methodology allowed us to screen for proteolysis in the aortic samples based on the identification of proteolytic enzymes and their corresponding degradation products. For instance, we were able to detect matrix metalloproteinase-9 by mass spectrometry and relate its presence to degradation of fibronectin in a clinical specimen. We expect this proteomics methodology to further our understanding of the composition of the vascular extracellular environment, shed light on ECM remodeling and degradation, and provide insights into important pathological processes, such as plaque rupture, aneurysm formation, and restenosis.

Desmosterol, the main sterol in rabbit semen: distribution among semen subfractions and its role in the in vitro spermatozoa acrosome reaction and motility.

Sterols are essential components of the cell membrane lipid bilayer that include molecules such as cholesterol and desmosterol, which are significantly found in the spermatozoa of several animal species. However, the presence of desmosterol in rabbit semen has never been investigated. The aims of this study were to characterize the sterol composition of subfractions of ejaculated rabbit semen and evaluate the in vitro effects of sterol on the spermatozoa acrosome reaction and motility. Two sterols, occurring prevalently in the free form (94.3%), were identified in whole semen collected from 10 fertile New Zealand White rabbits, specifically desmosterol (58.5% of total sterols) and cholesterol (35.9% of total sterols). Desmosterol was the predominant sterol found in all subfractions of rabbit semen, varying from 56.7% (in the prostatic secretory granules, PSGs) to 63.8% (in the seminal plasma). Spermatozoa contained an intermediate proportion of desmosterol (59.8%), which was asymmetrically distributed between the heads (52.0% of the total content of sterols) and the tails (81.8%). Results showed that both desmosterol and cholesterol can be transferred from the PSGs to the spermatozoa and are equally effective in inhibiting in vitro spermatozoa capacitation at a concentration higher than 1 mg L(-1). In contrast, neither desmosterol nor cholesterol had a significant effect on spermatozoa motility. Thus, it was concluded that, the various fractions of rabbit seminal fluid differ from each other in sterol composition and quantity, probably due to their different functional properties, and these fractions may undergo significant sterol changes depending on the stage of spermatozoa capacitation.

Amphiphile regulation of ion channel function by changes in the bilayer spring constant

Many drugs are amphiphiles that, in addition to binding to a particular target protein, adsorb to cell membrane lipid bilayers and alter intrinsic bilayer physical properties (e.g., bilayer thickness, monolayer curvature, and elastic moduli). Such changes can modulate membrane protein function by altering the energetic cost (DeltaG(bilayer)) of bilayer deformations associated with protein conformational changes that involve the protein-bilayer interface. But amphiphiles have complex effects on the physical properties of lipid bilayers, meaning that the net change in DeltaG(bilayer) cannot be predicted from measurements of isolated changes in such properties. Thus, the bilayer contribution to the promiscuous regulation of membrane proteins by drugs and other amphiphiles remains unknown. To overcome this problem, we use gramicidin A (gA) channels as molecular force probes to measure the net effect of amphiphiles, at concentrations often used in biological research, on the bilayer elastic response to a change in the hydrophobic length of an embedded protein. The effects of structurally diverse amphiphiles can be described by changes in a phenomenological bilayer spring constant (H(B)) that summarizes the bilayer elastic properties, as sensed by a bilayer-spanning protein. Amphiphile-induced changes in H(B), measured using gA channels of a particular length, quantitatively predict changes in lifetime for channels of a different length--as well as changes in the inactivation of voltage-dependent sodium channels in living cells. The use of gA channels as molecular force probes provides a tool for quantitative, predictive studies of bilayer-mediated regulation of membrane protein function by amphiphiles.

Effect of flavonoids on the Aβ(25-35)-phospholipid bilayers interaction

Amyloid-β peptide (Aβ) is the major component of amyloid deposits found in the brain tissue of Alzheimer patients. The tendency of amyloid peptide to form amyloid plaques is known to be related to the features of the plasma membrane. Flavonoids, a group of naturally occurring molecules, exert beneficial properties to human health thanks to their antioxidant property; this property depends on their capacity to interact and permeate the cell membrane lipid bilayer. In the present research we report an Electron Paramagnetic Resonance (EPR) investigation of 2-dioleoyl- sn-glycero-3-phosphocholine (DOPC) membranes interacting with the β-amyloid fragment Aβ(25-35), in the presence of flavonoids rutin, quercetin, naringin and naringenin. Our results, evidencing a flavonoid-dependent rigidifying effect of the bilayer, may provide the molecular basis to explain the known neuroprotective effect of flavonoid compounds.

Editorial [ Hot topic: Aquaporins and Nervous System: from Bench to Bedside (Guest Editors: Rita Rezzani and Luigi F. Rodella)

Water is the single most abundant substance in cells and organisms and is an important molecule involved in several biochemical processes present in living cells. In humans 60-70% of body weight is water which equilibrates across the lipid bilayer in cell membranes. Forty years ago, a small number of scientists argued that specialized water-selective pores are necessary to explain the high water permeability of red blood cells and renal tubules. Therefore, the molecular identification of a 28 kDa integral membrane protein in these cells has characterized a new stream of research. Aquaporins (AQPs) are membrane proteins that transport water and, in some cases, also small solutes such as glycerol and urea. Each subtype has its own cellular distribution and distinct regulatory mechanisms of their expression. Their classical role in facilitating trans-epithelial fluid transport is well understood, as in the urinary concentrating mechanism and gland fluid secretion, while the molecular mechanisms to regulate water permeability in the nervous system are still unclear. Maintenance of the ionic and osmotic composition and volume of interstitial, glial and neuronal compartments within the nervous system is essential for normal function. Small changes in intracellular or extracellular ion or solute composition can dramatically modify bi-directional water pathway between the brain and blood vessels and alter cerebrospinal fluid formation, neural signal transduction and information processing. To date, only some AQP isoforms (AQP1, 3, 4, 5, 8, 9) have been reported in the central nervous system being identified in choroidal cells (AQP1), astrocytes (AQP1, 3, 4, 5, 8, 9), oligodendrocytes (AQP8), neurons (AQP1, 5, 8), tanycytes (AQP9) and ependymal cells (AQP1, 4, 9). In contrast to numerous studies of AQP localization and function in the central nervous system, little information is available on the expression and function of AQPs in peripheral nervous system. This issue includes six review articles in which the authors report and explore the recent findings about the involvement of AQPs both in peripheral and central nervous system. The paper by B. Buffoli summarizes the data about the structure, regulation and function of AQPs, giving more importance to their involvement in the nervous system and underlying the development of new methods for diagnosis and therapy diseases. The review of R. Albertini and R. Bianchi is focused on the different isoforms of AQP protein that have been identified in glial cells in central and peripheral nervous system and in reactive microglial. The chapter supports the idea that AQPs are involved in water homeostasis during different glial cell functions, such as differentiation, metabolism and excitability of neurons. F. Bonomini and R. Rezzani emphasize the role of some AQPs present in glial cells in the maintenance or/and in the regulatory mechanisms of blood brain barrier. On the basis of the role of AQPs in brain edema, a personal account of the role of AQPs is then presented by C. Loreto and E. Reggio, who summarized the implication of different isoforms of these proteins in relation with vascular diseases and nervous system. In the literature, there is a lot of evidence that indicates a correlation between the expression of AQPs and the development of neurodegenerative diseases in which preservation of brain homeostasis is at risk. The review of E. Foglio and L.F. Rodella was to consider this topic concentrating on some neurodegenerative diseases, such as Neuromyielitis Optica, Alzheimer’s Diseases, Parkinson’s Diseases, Amyotrophic lateral sclerosis, Transmissible Spongiform Encephalopathies. Recent evidence suggests a novel role of AQPs in pain transmission both in the central and peripheral nervous system. In this issue, E. Borsani reports the modulation of AQPs both in inflammatory and neuropathic pain considering different animal models and knock-out animals. In the future, the numerous ongoing studies will certainly reveal other multifunctional roles of these proteins in humans. These roles might be exploited clinically by the development of drugs to alter AQP expression or function that could serve in the treatment of different diseases associated to peripheral and central nervous system.

Abstract 5115: The role of Cdc42-interacting protein 4 (CIP4) in osteosarcoma tumorigenesis

Abstract Osteosarcoma (OS) remains a devastating disease and requires better understanding of its biology for the development of novel therapeutic approaches. Tumor growth and metastases formation require an ability of cells to have a flexible cytoskeletal organization. Cdc42-interacting protein 4 (CIP4) is one of the scaffolding cytoskeleton-regulatory protein, whose role in tumors remains unknown. In the current study we investigated the role of CIP4 in OS tumorigenesis. We transfected highly aggressive mouse OS cells, DLM8 with the plasmid that downregulated expression of CIP4 protein (DLM8/shCIP4). We found that the growth rate of DLM8/shCIP4 clones was reduced by 30% when compared with non-transfected DLM8 cells or DLM8 cells transfected with control plasmid (DLM8/sh). When DLM8/shCIP4 cells were injected into mice subcutaneously they formed tumors that grew significantly slower than DLM8 or DLM8/sh control cells (p<0.02). We also found that inhibition of CIP4 expression in DLM8 cells decreased their motility and invasion properties in vitro, indicating on its ability to impair metastases growth. Fluorescent immunostaining for actin in DLM8/shCIP4 cells revealed lower perinuclear actin stress filaments formation and lower actin bridge to the lipid bilayer of the lipid cell membrane when compared with wild-type DLM8 or DLM8/sh. We also observed that downregulation of CIP4 changed cell membrane morphology in DLM8 cells - they had less protrusions and smoother cell surface than control cells. All these findings indicate that CIP4 may also play a role in metastatic behavior of OS cells and needs further investigation. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5115.

Wound repair in type I alveolar epithelial cells involves lysosome secretion followed by initiation of actin repolymerization

Stress failure of alveolar epithelial cells is a common feature of ventilator‐injured lungs. While some injured cells undergo necrosis, others repair their plasma membrane (PM) wounds through non‐secretory exocytosis of intracellular vesicles (e.g. lysosomes) to and cytoskeleton remodeling at and around the wound. We measured the timing of lysosomal trafficking and F‐actin reorganization following micropuncture injury in primary type I alveolar epithelial cells (ATI).ATI were isolated from female rats and loaded with Calcein AM, N‐rhodamine phosphatidylethanolamine (lysosome membrane label) or transfected with LifeAct (GFP tagged actin label). Changes in fluorescence intensity were indicators of time to wound repair (cessation of calcein fluorescence decay following injury), lysosomal trafficking to the wound site, and actin remodeling.ATI repaired wounds within 27.9±3.9s. Recruitment of lysosomes to the wound site was detected at 2.9±1.5s following injury and preceded actin polymerization by 23.7±5.4s. Failure to repair PM wounds was associated with a weak or absent actin polymerization response.We conclude that local actin polymerization is a critical step in PM wound repair and postulate that adhesive interactions between PM lipids and the remodeled actin cytoskeleton serve as a driving force for wound closure by promoting lateral PM lipid bilayer flow.Support: HL63178

The Current Knowledge of Invertebrate Aquaporin Water Channels with Particular Emphasis on Insect AQPs

SummaryAquaporins (AQPs) or water channels are some of the most ubiquitous integral membrane proteins, and are present in all living organisms. Their presence in the lipid bilayer of cell membranes considerably increases their permeability to water and, in some cases, to other small solutes. All AQPs, identified thus far, share the same structure, comprising of six transmembrane segments and two conserved regions forming the pore. Depending on the transported solutes, AQPs can be divided into two classes: ‘classical’ aquaporins (permeable only to water) and aquaglyceroporins (permeable also to glycerol and/or other solutes). Many subtypes of AQPs coexist in a single organism. Localization of particular subtypes of AQPs is tissue-specific. AQPs have been well characterized in almost all vertebrate classes. However, little is known about their counterparts in invertebrates. Most of the water channels characterized in invertebrates are found in insects. Therefore, the knowledge of aquaporins in invertebrates is generally limited to the information concerning water channels in this class of organism. Insects are characterized by an astonishing variety of physiological adaptations, notable in their feeding strategies or survival strategies in hostile environments. An example of such, is feeding on blood, or tolerating extreme cold or drought. It is likely that many of these adaptation patterns emerged due to the expression and regulation of particular aquaporins. Here we review the current state of knowledge of invertebrate AQPs (of insects and nematodes) and compare their structure and function with mammalian water channels

Rapid Binding and Transmembrane Diffusion of Pepducins in Phospholipid Bilayers

Pepducins are GPCR-targeted lipopeptides designed to anchor in the cell membrane lipid bilayer and modulate the receptor/G protein signal transduction pathway via an allosteric mechanism. It is thus presumed that pepducins cross the plasma membrane by some mechanism, possibly passive diffusion. The goal of this research is to study the biophysical transport properties of pepducins in model membranes. We utilized fluorescent probes that measure the binding (fluorescein phosphatidylethanolamine - FPE) and diffusion (pH probe - pyranine) of charged ligands across the lipid bilayer of large unilamellar vesicles (LUV) comprised of egg-phosphatidylcholine. We tested pepducins with a palmitate or myristate linked to the N-terminal of the peptide sequence (KKSRALF). The GPCR target for these pepducins is the protease activator receptor 1 (PAR1). Addition of pepducins (0.16-5.0 mol%) to LUVs labeled in the outer leaflet with FPE or containing entrapped pyranine produced a fast (<2s) and dose-dependent increase in the fluorescence of both probes. The fast response of FPE, resulting from the insertion of positive charges (lysine and arginines residues) into the outer leaflet, demonstrated rapid partitioning into the membrane. The increase in pyranine fluorescence indicated alkalinization of the intravesicular compartment, probably due to protonation of the lysine residues. In order for this to be detected, the pepducin must cross the membrane. The peptide alone (not acylated) did not cause any change in the fluorescence of either FPE or pyranine. These data are consistent with favorable partitioning of pepducins into the membrane and rapid passive diffusion to the sites of their action at the cytosolic leaflet of the plasma membrane.

A statistical model for multidimensional irreversible electroporation cell death in tissue

BackgroundIrreversible electroporation (IRE) is a minimally invasive tissue ablation technique which utilizes electric pulses delivered by electrodes to a targeted area of tissue to produce high amplitude electric fields, thus inducing irreversible damage to the cell membrane lipid bilayer. An important application of this technique is for cancer tissue ablation. Mathematical modelling is considered important in IRE treatment planning. In the past, IRE mathematical modelling used a deterministic single value for the amplitude of the electric field required for causing cell death. However, tissue, particularly cancerous tissue, is comprised of a population of different cells of different sizes and orientations, which in conventional IRE are exposed to complex electric fields; therefore, using a deterministic single value is overly simplistic.MethodsWe introduce and describe a new methodology for evaluating IRE induced cell death in tissue. Our approach employs a statistical Peleg-Fermi model to correlate probability of cell death in heterogeneous tissue to the parameters of electroporation pulses such as the number of pulses, electric field amplitude and pulse length. For treatment planning, the Peleg-Fermi model is combined with a numerical solution of the multidimensional electric field equation cast in a dimensionless form. This is the first time in which this concept is used for evaluating IRE cell death in multidimensional situations.ResultsWe illustrate the methodology using data reported in literature for prostate cancer cell death by IRE. We show how to fit this data to a Fermi function in order to calculate the critical statistic parameters. To illustrate the use of the methodology, we simulated 2-D irreversible electroporation protocols and produced 2-D maps of the statistical distribution of cell death in the treated region. These plots were compared to plots produced using a deterministic model of cell death by IRE and the differences were noted.ConclusionsIn this work we introduce a new methodology for evaluation of tissue ablation by IRE using statistical models of cell death. We believe that the use of a statistical model rather than a deterministic model for IRE cell death will improve the accuracy of treatment planning for cancer treatment with IRE.

Induction and regulation ofTrypanosoma bruceiVSG-specific antibody responses

The review addresses how infection with Trypanosoma brucei affects the development, survival and functions of B lymphocytes in mice. It discusses (1) the contributions of antibodies to trypanosome clearance from the bloodstream, (2) how B lymphocytes, the precursors of antibody producing plasma cells, interact with membrane form variable surface glycoprotein (VSG), i.e. with monovalent antigen that is free to diffuse within the lipid bilayer of the trypanosome plasma membrane and consequently can cross-link B cell antigen specific receptors by indirect processes only and (3) the extent and underlying causes of dysregulation of humoral immune responses in infected mice, focusing on the impact of wild type and GPI-PLC⁻/⁻ trypanosomes on bone marrow and extramedullary B lymphopoiesis, B cell maturation and survival.

Molecular dynamics of leucine and dopamine transporter proteins in a model cell membrane lipid bilayer

The dopamine transporter (DAT) operates via facilitated diffusion, harnessing an inward Na(+) gradient to drive dopamine from the extracellular synaptic cleft to the neuron interior. The DAT is relevant to central nervous system disorders such as Parkinson disease and attention-deficit hyperactivity disorder and is the primary site of action for the abused psychostimulants cocaine and amphetamines. Crystallization of a DAT homolog, the bacterial leucine transporter LeuT, provided the first reliable 3-D DAT template. Here, the LeuT crystal structure and the DAT molecular model have been combined with their respective substrates, leucine and dopamine, in lipid bilayer molecular dynamics simulations toward tracking substrate movement along the protein's substrate/ion permeation pathway. Specifically, movement of residue pairs that comprise the "external gate" was followed as a function of substrate presence. The transmembrane (TM) 1 arginine-TM 10 aspartate strut formed less readily in DAT compared with LeuT, with or without substrate present. For LeuT but not DAT, the addition of substrate enhanced the chances of forming the TM 1-10 bridge. Also, movement of the fourth extracellular loop EL-4 in the presence of substrate was more pronounced for DAT, the EL-4 unwinding to a degree. The overall similarity between the LeuT and DAT molecular dynamics simulations indicated that LeuT was a legitimate model to guide DAT structure-function predictions. There were, nevertheless, differences significant enough to allow for DAT-unique insights, which may include how cocaine, methylphenidate (Ritalin, NIDA Drug Supply, Rockville, MD), and other DAT blockers are not recognized as substrates even though they can access the primary substrate binding pocket. Proteins 2010. (c) 2009 Wiley-Liss, Inc.

Nuclear translocation of the epidermal growth factor receptor family membrane tyrosine kinase receptors.

Integral membrane proteins contain a hydrophobic transmembrane domain and mainly locate in the plasma membrane lipid bilayer. The receptor tyrosine kinases (RTK) of the epidermal growth factor receptor (EGFR) superfamily, including ErbB-1, ErbB-2, ErbB-3, and ErbB-4, constitute an important group of such membrane proteins, which have a profound impact on cancer initiation, progression, and patient outcome. Although studies of their functions have conventionally focused on their membrane-associated forms, documented observations of the presence of these membrane receptors and their functioning partners in the nucleus have reshaped the intracellular geography and highlight the need to modify the central dogma. The ErbB proteins in the membrane can translocate to the nucleus through different mechanisms. Nuclear RTKs regulate a variety of cellular functions, such as cell proliferation, DNA damage repair, and signal transduction, both in normal tissues and in human cancer cell. In addition, they play important roles in determining cancer response to cancer therapy. Nuclear presence of these ErbB proteins is emerging as an important marker in human cancers. An integrated picture of the RTK-centered signaling transduction network extending from the membrane-cytoplasm boundary to the nuclear compartment is looming in the foreseeable horizon for clinical application.

Mechanisms of Alveolar Epithelial Translocation of a Defined Population of Nanoparticles

To explore mechanisms of nanoparticle interactions with and trafficking across lung alveolar epithelium, we utilized primary rat alveolar epithelial cell monolayers (RAECMs) and an artificial lipid bilayer on filter model (ALBF). Trafficking rates of fluorescently labeled polystyrene nanoparticles (PNPs; 20 and 100 nm, carboxylate (negatively charged) or amidine (positively charged)-modified) in the apical-to-basolateral direction under various experimental conditions were measured. Using confocal laser scanning microscopy, we investigated PNP colocalization with early endosome antigen-1, caveolin-1, clathrin heavy chain, cholera toxin B, and wheat germ agglutinin. Leakage of 5-carboxyfluorescein diacetate from RAECMs, and trafficking of (22)Na and (14)C-mannitol across ALBF, were measured in the presence and absence of PNPs. Results showed that trafficking of positively charged PNPs was 20-40 times that of negatively charged PNPs across both RAECMs and ALBF, whereas translocation of PNPs across RAECMs was 2-3 times faster than that across ALBF. Trafficking rates of PNPs across RAECMs did not change in the presence of EGTA (which decreased transepithelial electrical resistance to zero) or inhibitors of endocytosis. Confocal laser scanning microscopy revealed no intracellular colocalization of PNPs with early endosome antigen-1, caveolin-1, clathrin heavy chain, cholera toxin B, or wheat germ agglutinin. Leakage of 5-carboxyfluorescein diacetate from alveolar epithelial cells, and sodium ion and mannitol flux across ALBF, were not different in the presence or absence of PNPs. These data indicate that PNPs translocate primarily transcellularly across RAECMs, but not via known major endocytic pathways, and suggest that such translocation may take place by diffusion of PNPs through the lipid bilayer of cell plasma membranes.

Partitioning of Poly(amidoamine) Dendrimers between n-Octanol and Water

Dendritic nanomaterials are emerging as key building blocks for a variety of nanoscale materials and technologies. Poly(amidoamine) (PAMAM) dendrimers were the first class of dendritic nanomaterials to be commercialized. Despite numerous investigations, the environmental fate, transport, and toxicity of PAMAM dendrimers is still not well understood. As a first step toward the characterization of the environmental behavior of dendrimers in aquatic systems, we measured the octanol-water partition coefficients (logK(ow)) of a homologous series of PAMAM dendrimers as a function of dendrimer generation (size), terminal group and core chemistry. We find that the logK(ow) of PAMAM dendrimers depend primarily on their size and terminal group chemistry. For G1-G5 PAMAM dendrimers with terminal NH2 groups, the negative values of their logK(ow) indicate that they prefer to remain in the water phase. Conversely, the formation of stable emulsions at the octanol-water (O/ W) interface in the presence of G6-NH2 and G8-NH2 PAMAM dendrimers suggest they prefer to partition at the O/W interface. In all cases, published studies of the cytotoxicity of Gx-NH2 PAMAM dendrimers show they strongly interact with the lipid bilayers of cells. These results suggest that the logK(ow) of a PAMAM dendrimer may not be a good predictor of its affinity with natural organic media such as the lipid bilayers of cell membranes.

Cholesterol Regulation of Membrane Protein Function and Sorting By Changes in Lipid Bilayer Elastic Properties

Cholesterol regulates the function of numerous membrane proteins. This regulation may depend on specific cholesterol-protein interactions, as well as on cholesterol-induced changes in the collective properties of the cell membrane lipid bilayer. We have studied the role of cholesterol-induced changes in the bilayer elastic properties on the function of: voltage dependent sodium channels, N-type calcium channels and GABAA receptors. For all three channel types, the effects of cholesterol, which increases lipid stiffness (measured using gramicidin channels as molecular force transducers), are opposite those induced by amphiphiles that decrease bilayer stiffness. Similar correlations exist for a number of other membrane proteins. These findings strongly suggest that cholesterol can regulate membrane protein function by altering the bilayer elastic properties.Cholesterol, similarly, has been proposed to regulate membrane protein sorting between different cellular membranes, by altering the energetic cost of the bilayer deformation associated with a mismatch between the length of a protein trans-membrane segment and the thickness of the bilayer hydrophobic core. We have investigated the energetic feasibility of such a sorting mechanism, using the continuum theory of liquid crystal deformations. The relative importance of a cholesterol-induced increase in bilayer thickness vs. an increase in the bilayer elastic moduli was evaluated. We find that cholesterol-induced sorting of membrane proteins indeed should be feasible - primarily due to the increase in the bilayer elastic moduli. Subsequent experimental studies support this conclusion.

EnteropathogenicEscherichia coliSubverts Phosphatidylinositol 4,5-Bisphosphate and Phosphatidylinositol 3,4,5-Trisphosphate upon Epithelial Cell Infection

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] are phosphoinositides (PIs) present in small amounts in the inner leaflet of the plasma membrane (PM) lipid bilayer of host target cells. They are thought to modulate the activity of proteins involved in enteropathogenic Escherichia coli (EPEC) infection. However, the role of PI(4,5)P(2) and PI(3,4,5)P(3) in EPEC pathogenesis remains obscure. Here we show that EPEC induces a transient PI(4,5)P(2) accumulation at bacterial infection sites. Simultaneous actin accumulation, likely involved in the construction of the actin-rich pedestal, is also observed at these sites. Acute PI(4,5)P(2) depletion partially diminishes EPEC adherence to the cell surface and actin pedestal formation. These findings are consistent with a bimodal role, whereby PI(4,5)P(2) contributes to EPEC association with the cell surface and to the maximal induction of actin pedestals. Finally, we show that EPEC induces PI(3,4,5)P(3) clustering at bacterial infection sites, in a translocated intimin receptor (Tir)-dependent manner. Tir phosphorylated on tyrosine 454, but not on tyrosine 474, forms complexes with an active phosphatidylinositol 3-kinase (PI3K), suggesting that PI3K recruited by Tir prompts the production of PI(3,4,5)P(3) beneath EPEC attachment sites. The functional significance of this event may be related to the ability of EPEC to modulate cell death and innate immunity.

Molecular Dynamics Study on the Biophysical Interactions of Seven Green Tea Catechins with Lipid Bilayers of Cell Membranes

Molecular dynamics simulations were performed to study the interactions of bioactive catechins (flavonoids) commonly found in green tea with lipid bilayers, as a model for cell membranes. Previously, multiple experimental studies rationalized catechin's anticarcinogenic, antibacterial, and other beneficial effects in terms of physicochemical molecular interactions with the cell membranes. To contribute toward understanding the molecular role of catechins on the structure of cell membranes, we present simulation results for seven green tea catechins in lipid bilayer systems representative of HepG2 cancer cells. Our simulations show that the seven tea catechins evaluated have a strong affinity for the lipid bilayer via hydrogen bonding to the bilayer surface, with some of the smaller catechins able to penetrate underneath the surface. Epigallocatechin-gallate (EGCG) showed the strongest interaction with the lipid bilayer based on the number of hydrogen bonds formed with lipid headgroups. The simulations also provide insight into the functional characteristics of the catechins that distinguish them as effective compounds to potentially alter the lipid bilayer properties. The results on the hydrogen-bonding effects, described here for the first time, may contribute to a better understanding of proposed multiple molecular mechanisms of the action of catechins in microorganisms, cancer cells, and tissues.

Advances in developing screening methods and improving aluminum resistance in common bean and Brachiaria

We summarize progress made towards the development of tools and knowledge that aid the genetic improvement of aluminum (Al) resistance in two of CIAT’s (International Center for Tropical Agriculture) commodity crops; common bean ( Phaseolus vulgaris L.) and brachiariagrasses ( Brachiaria spp . Grisib ). Approximately 40% of common bean production is on acid soils where Al toxicity limits root development. Improving Al resistance, therefore, is an important breeding objective. We compared a group of 53 common bean genotypes with differing levels of adaptation to acid soils to identify Al resistant genotypes using a hydroponic screening method. Four different root traits (percent inhibition of root elongation, percent increase of average root diameter, total root length per plant and total number of root tips per plant) were identified as useful indicators for Al resistance. Application of this method to 30 accessions of scarlet runner bean (P. coccineus L.) identified three Al-resistant genotypes that could be used for introgressing Al resistance into cultivated germplasm . Work is in progress to characterize the physiological mechanisms underpinning Al resistance in common bean. This will facilitate developing simplified screening procedures and identifying quantitative trait loci ( QTLs ) contributing to Al resistance. Compared to common bean, brachiariagrasses tend to be better adapted to acid soils. Yet edaphic adaptation is an important component of CIAT’s and EMBRAPA’s ( Empresa Brasiliera de Pesquisa Agropecuaria ) breeding programs because one of the three parental genotypes is poorly adapted. We developed and incorporated into our breeding program a solution-culture technique that uses rooted stem cuttings to screen for Al resistance and root vigor, both of which are segregating in breeding populations. Physiological research into the remarkably high level of Al resistance of signalgrass (B. decumbens Stapf ) has ruled out a significant contribution of organic-acid secretion at root apices (a widespread Al-exclusion mechanism in plants). Instead, Al resistance appears to be a facet of a more generic resistance mechanism that prevents intoxication by inorganic cations , possibly as a result of the change in composition of the lipid bilayer of root cell plasma membranes. Once taken up into roots, Al appears to be complexed by low-molecular-weight ligands such as citrate. Other adaptive traits may counteract secondary effects of Al toxicity on phosphorus (P) nutrition through stimulation of P uptake or use efficiency.