Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins

In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)–based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis.

Overview of mathematical approaches used to model bacterial chemotaxis I: the single cell

Mathematical modeling of bacterial chemotaxis systems has been influential and insightful in helping to understand experimental observations. We provide here a comprehensive overview of the range of mathematical approaches used for modeling, within a single bacterium, chemotactic processes caused by changes to external gradients in its environment. Specific areas of the bacterial system which have been studied and modeled are discussed in detail, including the modeling of adaptation in response to attractant gradients, the intracellular phosphorylation cascade, membrane receptor clustering, and spatial modeling of intracellular protein signal transduction. The importance of producing robust models that address adaptation, gain, and sensitivity are also discussed. This review highlights that while mathematical modeling has aided in understanding bacterial chemotaxis on the individual cell scale and guiding experimental design, no single model succeeds in robustly describing all of the basic elements of the cell. We conclude by discussing the importance of this and the future of modeling in this area.

Modelling 2,4-dichlorophenol bioavailability and bioaccumulation by the freshwater fingernail clam Sphaerium corneum using artificial particles and humic acids

The complex and variable composition of natural sediments makes it very difficult to predict the bioavailability and bioaccumulation of sediment-bound contaminants. Several approaches have been proposed to overcome this problem, including an experimental model using artificial particles with or without humic acids as a source of organic matter. For this work, we have applied this experimental model, and also a sample of a natural sediment, to investigate the uptake and bioaccumulation of 2,4-dichlorophenol (2,4-DCP) by Sphaerium corneum. Additionally, the particle-water partition coefficients (K-d) were calculated. The results showed that the bioaccumulation of 2,4-DCP by clams did not depend solely on the levels of chemical dissolved, but also on the amount sorbed onto the particles and the characteristics and the strength of that binding. This study confirms the value of using artificial particles as a suitable experimental model for assessing the fate of sediment-bound contaminants. (c) 2006 Elsevier Ltd. All rights reserved.

Quantitative model for the kinetics of lyotropic phase transitions involving changes in monolayer curvature

We present a kinetic model for transformations between different self-assembled lipid structures. The model shows how data on the rates of phase transitions between mesophases of different geometries can be used to provide information on the mechanisms of the transformations and the transition states involved. This can be used, for example, to gain an insight into intermediate structures in cell membrane fission or fusion. In cases where the monolayer curvature changes on going from the initial to the final mesophase, we consider the phase transition to be driven primarily by the change in the relaxed curvature with pressure or temperature, which alters the relative curvature elastic energies of the two mesophase structures. Using this model, we have analyzed previously published kinetic data on the inter-conversion of inverse bicontinuous cubic phases in the 1-monoolein-30 wt% water system. The data are for a transition between QII(G) and QII(D) phases, and our analysis indicates that the transition state more closely resembles the QII(D) than the QII(G) phase. Using estimated values for the monolayer mean curvatures of the QII(G) and QII(D) phases of -0.123 nm(-1) and -0.133 nm(-1), respectively, gives values for the monolayer mean curvature of the transition state of between -0.131 nm(-1) and -0.132 nm(-1). Furthermore, we estimate that several thousand molecules undergo the phase transition cooperatively within one "cooperative unit", equivalent to 1-2 unit cells of QII(G) or 4-10 unit cells of QII(D).

An engineering model of the human colon

The development and performance of a three-stage tubular model of the large human intestine is outlined. Each stage comprises a membrane fermenter where flow of an aqueous polyethylene glycol solution on the outside of the tubular membrane is used to control the removal of water and metabolites (principally short chain fatty acids) from, and thus the pH of, the flowing contents on the fermenter side. The three stage system gave a fair representation of conditions in the human gut. Numbers of the main bacterial groups were consistently higher than in an existing three-chemostat gut model system, suggesting the advantages of the new design in providing an environment for bacterial growth to represent the actual colonic microflora. Concentrations of short chain fatty acids and Ph levels throughout the system were similar to those associated with corresponding sections of the human colon. The model was able to achieve considerable water transfer across the membrane, although the values were not as high as those in the colon. The model thus goes some way towards a realistic simulation of the colon, although it makes no pretence to simulate the pulsating nature of the real flow. The flow conditions in each section are characterized by low Reynolds numbers: mixing due to Taylor dispersion is significant, and the implications of Taylor mixing and biofilm development for the stability, that is the ability to operate without washout, of the system are briefly analysed and discussed. It is concluded that both phenomena are important for stabilizing the model and the human colon.

Development of a fermentation system to model sessile bacterial populations in the human colon

A fermentation system was designed to model the human colonic microflora in vitro. The system provided a framework of mucin beads to encourage the adhesion of bacteria, which was encased within a dialysis membrane. The void between the beads was inoculated with faeces from human donors. Water and metabolites were removed from the fermentation by osmosis using a solution of polyethylene glycol (PEG). The system was concomitantly inoculated alongside a conventional single-stage chemostat. Three fermentations were carried out using inocula from three healthy human donors. Bacterial populations from the chemostat and biofilm system were enumerated using fluorescence in situ hybridization. The culture fluid was also analysed for its short-chain fatty acid (SCFA) content. A higher cell density was achieved in the biofilm fermentation system (taking into account the contribution made by the bead-associated bacteria) as compared with the chemostat, owing to the removal of water and metabolites. Evaluation of the bacterial populations revealed that the biofilm system was able to support two distinct groups of bacteria: bacteria growing in association with the mucin beads and planktonic bacteria in the culture fluid. Furthermore, distinct differences were observed between populations in the biofilm fermenter system and the chemostat, with the former supporting higher populations of clostridia and Escherichia coli. SCFA levels were lower in the biofilm system than in the chemostat, as in the former they were removed via the osmotic effect of the PEG. These experiments demonstrated the potential usefulness of the biofilm system for investigating the complexity of the human colonic microflora and the contribution made by sessile bacterial populations.

Relation between neuronal morphology and electrophysiology in the Kainate lesion model of Alzheimer's Disease

This paper describes a computational and statistical study of the influence of morphological changes on the electrophysiological response of neurons from an animal model of Alzheimer's Disease (AD). We combined experimental morphological data from rat hippocampal CA1 pyramidal cells with a well-established model of active membrane properties. Dendritic morphology and the somatic response to simulated current clamp conditions were then compared for cells from the control and the AD group. The computational approach allowed us to single out the influences of neuromorphology on neuronal response by eliminating the effects of active channel variability. The results did not reveal a simple relationship between morphological changes associated with AD and changes in neural response. However, they did suggest the existence of more complex than anticipated relationships between dendritic morphology and single-cell electrophysiology.

Permeate flux modeling of membrane distillation

This paper concerns the modeling of membrane distillation. The model developed has been used to predict permeate fluxes using different initial operating conditions. PVDF and PTFE membranes were successfully used in a flat plate module to experimentally confirm the theoretical results. The correlation between theory and experiment was close for both membranes. The PTFE membranes produced higher fluxes than PVDF. A Versapor membrane was also used for this work. This membrane is a composite, with a thin porous layer on a support layer. It was found not to be suitable for membrane distillation. A comparison of the heat flux was also carried out. Again, there was good correlation between theory and experiment

Intracellular or intercellular localization of the polar pathway of penetration across stratum corneum

A pharmacokinetic hypothesis of stratum corneum with two parallel pathways, lipophilic and porous hydrophilic, is not well documented yet. Still questionable is the localization of the pores, and the present experiments were designed to elucidate the contribution of extracellular lipids and intracellular keratin to the structure of this pathway. Percutaneous penetration of baclofen, a model zwitterion, was studied in vitro using human cadaver skin. Aqueous or ethanolic saturated solutions of the drug (Cs = 4.6 and 0.4 mg/ mL, respectively) were applied on the skin that was pretreated with: methanol/chloroform (Me/Ch) or acetone-chloroform (Ac/Ch) (1:1) mixtures, or with these solvents followed by 0.2% solution of sodium lauryl sulfate (SLS). As controls, baclofen penetration through the intact full-thickness skin was determined, and the fluxes were 0.18 ±0.08 and 0.14 ±0.07 µg/cm2/h for aqueous and ethanolic solutions, respectively. When Me/Ch was used for 1 h, an expected increase of the penetration was observed, but the lag time, Tlag, was still nearly 20 h. When the less polar mixture, Ac/Ch, was used, no flux enhancement was observed, and with ethanol as the vehicle, decreased penetration was even noted. No effect on baclofen penetration was observed when SLS was used for 1 h after delipidization of the skin was done with either the Me/Ch or Ac/Ch mixture. The results suggest that the polar pathway may be located intercellularly and comprises aqueous regions surrounded by polar lipids, which create the walls of such microchannels.

Identification of critical residues in the active site of porcine membrane-bound aminopeptidase P

The membrane-bound form of mammalian aminopeptidase P (AP-P; EC 3.4. 11.9) is a mono-zinc-containing enzyme that lacks any of the typical metal binding motifs found in other zinc metalloproteases. To identify residues involved in metal binding and catalysis, sequence and structural information was used to align the sequence of porcine membrane-bound AP-P with other members of the peptidase clan MG, including Escherichia coli AP-P and methionyl aminopeptidases. Residues predicted to be critical for activity were mutated and the resultant proteins were expressed in COS-1 cells. Immunoelectrophoretic blot analysis was used to compare the levels of expression of the mutant proteins, and their ability to hydrolyze bradykinin and Gly-Pro-hydroxyPro was assessed. Asp449, Asp460, His523, Glu554, and Glu568 are predicted to serve as metal ion ligands in the active site, and mutagenesis of these residues resulted in fully glycosylated proteins that were catalytically inactive. Mutation of His429 and His532 also resulted in catalytically inactive proteins, and these residues, by analogy with E. coli AP-P, are likely to play a role in shuttling protons during catalysis. These studies indicate that mammalian membrane-bound AP-P has an active-site configuration similar to that of other members of the peptidase clan MG, which is compatible with either a dual metal ion model or a single metal ion in the active site. The latter model is consistent, however, with the known metal stoichiometry of both the membrane-bound and cytosolic forms of AP-P and with a recently proposed model for methionyl aminopeptidase.

Investigation of flux in flat-plate modules for membrane distillation

A theoretical model for predicting the behaviour of membrane distillation by incorporating mass and heat transfer equations has been used to find permeate fluxes, and has been validated experimentally. The model accurately predicts mass and heat transfer. The main work studied the effect of module design using a flat-plate module in laminar flow conditions. Areas of investigation included the use of channels across the membrane surface, decreasing the available membrane surface area, and widening the inlet and outlet channels. The work showed that widening the channels increased the flux. Increased flux was also obtained by the use of channels on the permeate side, though not on the feed side.

Arenavirus budding resulting from viral-protein-associated cell membrane curvature

Viral replication occurs within cells, with release (and onward infection) primarily achieved through two alternative mechanisms: lysis, in which virions emerge as the infected cell dies and bursts open; or budding, in which virions emerge gradually from a still living cell by appropriating a small part of the cell membrane. Virus budding is a poorly understood process that challenges current models of vesicle formation. Here, a plausible mechanism for arenavirus budding is presented, building on recent evidence that viral proteins embed in the inner lipid layer of the cell membrane. Experimental results confirm that viral protein is associated with increased membrane curvature, whereas a mathematical model is used to show that localized increases in curvature alone are sufficient to generate viral buds. The magnitude of the protein-induced curvature is calculated from the size of the amphipathic region hypothetically removed from the inner membrane as a result of translation, with a change in membrane stiffness estimated from observed differences in virion deformation as a result of protein depletion. Numerical results are based on experimental data and estimates for three arenaviruses, but the mechanisms described are more broadly applicable. The hypothesized mechanism is shown to be sufficient to generate spontaneous budding that matches well both qualitatively and quantitatively with experimental observations.

A mathematical model of the sterol regulatory element binding protein 2 cholesterol biosynthesis pathway

Cholesterol is one of the key constituents for maintaining the cellular membrane and thus the integrity of the cell itself. In contrast high levels of cholesterol in the blood are known to be a major risk factor in the development of cardiovascular disease. We formulate a deterministic nonlinear ordinary differential equation model of the sterol regulatory element binding protein 2 (SREBP-2) cholesterol genetic regulatory pathway in an hepatocyte. The mathematical model includes a description of genetic transcription by SREBP-2 which is subsequently translated to mRNA leading to the formation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), a main precursor of cholesterol synthesis. Cholesterol synthesis subsequently leads to the regulation of SREBP-2 via a negative feedback formulation. Parameterised with data from the literature, the model is used to understand how SREBP-2 transcription and regulation affects cellular cholesterol concentration. Model stability analysis shows that the only positive steady-state of the system exhibits purely oscillatory, damped oscillatory or monotic behaviour under certain parameter conditions. In light of our findings we postulate how cholesterol homestasis is maintained within the cell and the advantages of our model formulation are discussed with respect to other models of genetic regulation within the literature.

The role of lipid composition on the interaction between a tryptophan-rich protein and model bacterial membranes

The interaction between tryptophan-rich puroindoline proteins and model bacterial membranes at the air-liquid interface has been investigated by FTIR spectroscopy, surface pressure measurements and Brewster angle microscopy. The role of different lipid constituents on the interactions between lipid membrane and protein was studied using wild type (Pin-b) and mutant (Trp44 to Arg44 mutant, Pin-bs) puroindoline proteins. The results show differences in the lipid selectivity of the two proteins in terms of preferential binding to specific lipid head groups in mixed lipid systems. Pin-b wild type was able to penetrate mixed layers of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) head groups more deeply compared to the mutant Pin-bs. Increasing saturation of the lipid tails increased penetration and adsorption of Pin-b wild type, but again the response of the mutant form differed. The results provide insight as to the role of membrane architecture, lipid composition and fluidity, on antimicrobial activity of proteins. Data show distinct differences in the lipid binding behavior of Pin-b as a result of a single residue mutation, highlighting the importance of hydrophobic and charged amino acids in antimicrobial protein and peptide activity.

Integration of steroid hormone initiated membrane action to genomic function in the brain

Estrogen is a ligand for the estrogen receptor (ER), which on binding 17beta-estradiol, functions as a ligand-activated transcription factor and regulates the transcription of target genes. This is the slow genomic mode of action. However, rapid non-genomic actions of estrogen also exist at the cell membrane. Using a novel two-pulse paradigm in which the first pulse rapidly initiates non-genomic actions using a membrane-limited estrogen conjugate (E-BSA), while the second pulse promotes genomic transcription from a consensus estrogen response element (ERE), we have demonstrated that rapid actions of estrogen potentiate the slower transcriptional response from an ERE-reporter in neuroblastoma cells. Since rapid actions of estrogen activate kinases, we used selective inhibitors in the two-pulse paradigm to determine the intracellular signaling cascades important in such potentiation. Inhibition of protein kinase A (PKA), PKC, mitogen activated protein kinase (MAPK) or phosphatidylinositol 3-OH kinase (PI-3K) in the first pulse decreases potentiation of transcription. Also, our data with both dominant negative and constitutive mutants of Galpha subunits show that Galpha(q) initiates the rapid signaling cascade at the membrane in SK-N-BE(2)C neuroblastoma cells. We discuss two models of multiple kinase activation at the membrane Pulses of estrogen induce lordosis behavior in female rats. Infusion of E-BSA into the ventromedial hypothalamus followed by 17beta-estradiol in the second pulse could induce lordosis behavior, demonstrating the applicability of this paradigm in vivo. A model where non-genomic actions of estrogen couple to genomic actions unites both aspects of hormone action.